U.S. patent application number 15/570272 was filed with the patent office on 2018-10-11 for antimicrobial therapy.
The applicant listed for this patent is The Regents of the University of California. Invention is credited to Richard L. Gallo, Teruaki Nakatsuji.
Application Number | 20180289751 15/570272 |
Document ID | / |
Family ID | 57218441 |
Filed Date | 2018-10-11 |
United States Patent
Application |
20180289751 |
Kind Code |
A1 |
Nakatsuji; Teruaki ; et
al. |
October 11, 2018 |
ANTIMICROBIAL THERAPY
Abstract
Methods and compositions comprising hogocidin peptides
(SH-lantibiotics), derivatives and variants are provided. Also
provided are methods and compositions comprising probiotic
compositions utilizing strains of S. hominis and S. epidermidis
that produce hogocidin, hogocidin-like peptides, or other
inhibitors of skin pathogens. Methods of treatment for microbial
skin infections and atopic dermatitis are also provided.
Inventors: |
Nakatsuji; Teruaki; (San
Diego, CA) ; Gallo; Richard L.; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of California |
Oakland |
CA |
US |
|
|
Family ID: |
57218441 |
Appl. No.: |
15/570272 |
Filed: |
May 5, 2016 |
PCT Filed: |
May 5, 2016 |
PCT NO: |
PCT/US2016/031067 |
371 Date: |
October 27, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62157248 |
May 5, 2015 |
|
|
|
62300274 |
Feb 26, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 35/741 20130101;
A61P 31/04 20180101; A61K 9/06 20130101; A61P 17/00 20180101; C07K
14/31 20130101; A61K 2035/115 20130101; A61K 38/1729 20130101; A61K
38/164 20130101; A61K 9/0014 20130101; A61K 9/14 20130101; A61K
35/744 20130101 |
International
Class: |
A61K 35/741 20060101
A61K035/741; A61K 9/00 20060101 A61K009/00; A61P 31/04 20060101
A61P031/04; A61P 17/00 20060101 A61P017/00; A61K 9/06 20060101
A61K009/06; A61K 9/14 20060101 A61K009/14; A61K 38/16 20060101
A61K038/16; A61K 38/17 20060101 A61K038/17; C07K 14/31 20060101
C07K014/31 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED R&D
[0002] This invention was made with Government support under Grant
No. R01AI083358, Grant No. AR067547 and Grant No.
HHSN-272201000020C, all awarded by the National Institutes of
Health. The Government has certain rights in the invention.
Claims
1. A composition comprising a thickened topical formulation of one
or more probiotic bacterial strains and optionally, a prebiotic
compound, a protectant, humectant, emollient, abrasive, salt,
and/or surfactant; wherein the one or more probiotic bacterial
strain comprises one or more bacterial strains of the genus
Staphylococcus; wherein the one or more probiotic bacterial strains
produces a peptide having a sequence selected from the group
consisting of SEQ ID NO: 2, 4, 28, 30, 32, 34, 36, 38, 40, 42, 44,
46, 48, 50, 52, 54, and 55 and any combination thereof and wherein
such peptide is optionally post-translationally modified; and
wherein the composition is formulated for the topical treatment of
disorders of dysbiosis of the skin, scalp, or mucosae.
2. The composition according to claim 1, wherein the one or more
probiotic bacterial strain comprises Staphylococcus epidermidis,
Staphylococcus hominis or a combination of Staphylococcus
epidermidis and Staphylococcus hominis.
3. The composition according to claim 2, wherein the one or more
probiotic bacterial strain comprises Staphylococcus epidermidis
strains MO34, MO38, A11, AMT1, AMT5-C5, and/or AMT5-G6.
4. The composition according to claim 2, wherein one or more
probiotic bacterial strains comprises Staphylococcus hominis
strains A9, C2, AMT2, AMT3, AMT4-C2, AMT4-G1, and/or AMT4-D12.
5. The composition according to claim 2, wherein each probiotic
bacterial strain demonstrates a Fatty Acid Methyl Ester profile
corresponding to one of those shown in any of FIG. 11, 12, 13, 14,
15, 16, 17, 18, or 19.
6. (canceled)
7. The composition according to claim 1, wherein the one or more
probiotic bacterial strains is provided in a live form.
8. The composition according to claim 1, wherein the one or more
probiotic bacterial strains is provided in a lyophilized or
freeze-dried or spray dried form.
9. The composition according to claim 8, wherein the probiotic
bacterium can be reconstituted into a live form.
10. A method of treating skin or mucosal infections, atopic
dermatitis, psoriasis, mastitis, acne, or other disorders related
to skin dysbiosis in humans or other mammals by applying to the
skin or mucosa an effective amount of the composition of claim 1 to
a subject in need thereof.
11. The method according to claim 10, wherein the composition is
applied topically.
12. The method according to claim 10, wherein the composition is
formulated as a cream, ointment, unguent, spray, powder, oil,
thickened formulation or poultice.
13. A composition comprising one or more of a hogocidin peptide,
derivative or variant, an SH-lantibiotic peptide, an
SH-antimicrobial, an SE-lantibiotic peptide, and/or an SE
antimicrobial; wherein the hogocidin peptide comprise a sequence
that is at least 95% identical to SEQ ID NO:2 or 4, or a
biologically active fragment thereof having antimicrobial activity;
and wherein the composition further comprises one or more
thickeners, solvents, emulsifiers, or pharmaceutically acceptable
carriers or excipients.
14. The composition according to claim 13, further comprising a
cathelicidin peptide, derivative or variant.
15. The composition according to claim 13, wherein the peptide
comprises one or more D-amino acids or non-naturally occurring
amino acids.
16. A composition according to claim 13, wherein the hogocidin
peptide, SH-lantibiotic peptide, SH-antimicrobial, SE-lantibiotic
peptide, or SE antimicrobial is produced in situ by one or more of
Staphylococcus hominis strain A9, Staphylococcus hominis strain C2,
Staphylococcus hominis strain AMT2, Staphylococcus hominis strain
AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis
strain AMT4-G1, Staphylococcus hominis strain AMT4-D12,
Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis
strain SE-A11, Staphylococcus epidermidis strain AMT5-C5,
Staphylococcus epidermidis strain AMT5-G6 and Staphylococcus
epidermidis strain M034.
17. A composition according to claim 13, wherein the peptide is
formulated for topical administration.
18. A composition according to claim 17, wherein the formulation
comprises a lotion, ointment cream, powder, unguent, oil, or
spray.
19. A composition according to claim 13, wherein the hogocidin
peptide, derivative or variant comprises a sequence selected from
SEQ ID NO:2 or SEQ ID NO:4.
20. The composition according to claim 13, wherein the one or more
of a hogocidin peptide, derivative or variant, an SH-lantibiotic
peptide, an SH-antimicrobial, an SE-lantibiotic peptide, an SE
antimicrobial, and a cathelicidin peptide, derivative or variant is
provided as an extract or lysate of Staphylococcus hominis strain
A9, Staphylococcus hominis strain C2, Staphylococcus hominis strain
AMT2, Staphylococcus hominis strain AMT3, Staphylococcus hominis
strain AMT4-C2, Staphylococcus hominis strain AMT4-G1,
Staphylococcus hominis strain AMT4-D12, Staphylococcus epidermidis
strain AMT1, Staphylococcus epidermidis strain SE-A11,
Staphylococcus epidermidis strain AMT5-C5, Staphylococcus
epidermidis strain AMT5-G6 and Staphylococcus epidermidis strain
MO34.
21. A method for treating skin or mucosal infection or atopic
dermatitis in a subject comprising contacting the subject with an
effective amount of a composition of claim 13.
22. The method according to claim 21, wherein the contacting is by
topical administration or optionally by contacting the subject with
one or more of SH-lantibiotic or bacteriocin-producing
Staphylococcus hominis strains A9, C2, AMT2, AMT3, AMT4-C2,
AMT4-G1, AMT4-D12 and Staphylococcus epidermidis strains AMT5-G6
and MO34.
23. A recombinant vector comprising a polynucleotide encoding a
polypeptide that is at least 95% identical to SEQ ID NO:2 or 4, or
a biologically active fragment thereof having antimicrobial
activity.
24. The recombinant vector of claim 23, wherein the vector
comprises a polynucleotide that encodes a polypeptide of SEQ ID
NO:2 or 4.
25. The recombinant vector of claim 23, wherein the vector
comprises a polynucleotides that encodes a polypeptide of SEQ ID
NO:2 from about amino acid 32 to about amino acid 61.
26. The recombinant vector of claim 23, wherein the vector
comprises a polynucleotide that encodes a polypeptide of SEQ ID
NO:4 from about amino acid 29 to about amino acid 66.
27. The recombinant vector of claim 23, wherein the vector
comprises a polynucleotide that is at least 95% identical to SEQ ID
NO:1 or 3 and encodes a polypeptide of SEQ ID NO:2 or 4,
respectively.
28. (canceled)
29. A host cell engineered to express a recombinant vector of claim
23.
30. The host cell of claim 29, wherein the host cell is a
non-pathogenic attenuated host cell.
31. A recombinant polypeptide produced by the host cell of claim
29.
32. A composition comprising the host cell of claim 29.
33. A composition comprising the host cell of claim 30.
34. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under 35 U.S.C. .sctn. 119
from Provisional Application Ser. No. 62/157,248, filed May 5,
2015, and Provisional Application Ser. No. 62/300,274, filed Feb.
26, 2016, the disclosures of which are incorporated herein by
reference.
REFERENCE TO SEQUENCE LISTING
[0003] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled Sequence ST25.txt, created May 4, 2016, which is 45
Kb in size. The information in the electronic format of the
Sequence Listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0004] The disclosure relates to methods and compositions for
treating infection, and modulating skin and mucosal microflora to
treat diseases or disorders that are related to or exacerbated by
dysbiosis.
BACKGROUND
[0005] Small, cationic antimicrobial peptides (AMPs) are naturally
occurring antibiotics of the innate immune system. AMPs are widely
distributed in animals and plants and are among the most ancient
host defense factors. Their spectrum of activity includes
Gram-positive and Gram-negative bacteria as well as fungi and
certain infective agents. As resistance of pathogenic microbes to
conventional antibiotics increases, researchers are exploring these
endogenous antibiotics as a potential source or new therapies
against variety of infectious diseases.
[0006] Patients with atopic dermatitis (AD) have recurrent skin
infections by Staphylococcus aureus (SA) and dysbiosis of their
cutaneous microbiome. The increased susceptibility to SA has been
associated with diminished innate immune defense including abnormal
barrier function and decreased induction of antimicrobial peptides
(AMPs) such as cathelicidin and .beta.-defensins.
[0007] Symptoms of atopic dermatitis, also referred to as eczema or
atopic eczema include: dry skin that forms a rash; scaly, swollen,
and red skin; rash on the face, or inside the knees, elbows, or
wrists; blisters that ooze; changes in skin color after repeated
episodes; thickened, cracked, dry, scaly skin or skin that looks
leathery in patches; and severe itchiness (pruritis), especially at
night, along with raw, sensitive, swollen skin from scratching.
Atopic dermatitis (eczema) signs and symptoms vary widely from
person to person and may further include: red to brownish-gray
patches, especially on the hands, feet, ankles, wrists, neck, upper
chest, eyelids, inside the bend of the elbows and knees, and, in
infants, the face, scalp, back of the head, ears, legs, feet, arms,
hands and buttocks; small, raised bumps, which may leak fluid and
crust over when scratched. Atopic dermatitis most often begins
before age 5 and may persist into adolescence and adulthood. For
some people, it flares periodically and then clears up for a time,
even for several years. The skin changes brought about by atopic
dermatitis can facilitate high susceptibility of these patients to
colonization and infections by Staphylococcus aureus.
[0008] Dysbiosis comprises an imbalance in the cutaneous or mucosal
flora, including the nasal, oral, ophthalmic, urogenital,
intestinal flora, wherein species such as S. aureus become
overrepresented and other species become underrepresented.
Generally, in a healthy flora, nonpathogenic bacteria may secrete
inhibitors or simply occupy all available niches, thus either
directly inhibiting or indirectly excluding pathogens that would
otherwise be able to establish infectious states or foster the
development of disease or disease-like states, such as atopic
dermatitis.
SUMMARY
[0009] The disclosure provides compositions and methods for the
treatment of disorders related to dysbiosis of the skin. These
disorders, associated with imbalances in the normal skin flora and
overgrowth of skin pathogens such as S. aureus, result in skin
infections, atopic dermatitis, and psoriasis, among other
conditions. The disclosure provides compositions and methods for
treating these disorders by restoring the healthy cutaneous flora
utilizing antimicrobial peptides derived from residents of the
healthy cutaneous flora, or by directly administering probiotic
compositions containing strains that are derived from a healthy
cutaneous flora, or as the rare surviving florae cultured from the
skin of diagnosed patients with a floral dysbiosis, and that are
capable of either killing or inhibiting the growth of pathogenic
species on the skin or species associated with a disease-like
microbial imbalance.
[0010] Specifically, the disclosure provides a thickened topical
composition comprising one or more probiotic bacterial strains,
preferably of the genus Staphylococcus, and more preferably
comprising the disclosed strains of Staphylococcus hominis and
Staphylococcus epidermidis. These strains can be isolated from
healthy cutaneous florae, or as the surviving florae cultured from
the skin of diagnosed patients with a floral dysbiosis, by the
methods disclosed herein, and may be identified by the secreted
peptide sequences, fatty acid methyl ester profiles, and/or
antimicrobial peptide codon organizations disclosed herein. The
probiotic strains of the disclosure may be provided in live form,
in freeze-dried form, or in a reconstitutable form. The disclosure
further provides a composition comprising strains of Staphylococcus
epidermidis and Staphylococcus hominis as described herein which
may be formulated for topical administration to the skin, scalp, or
mucosae. The disclosure further provides a composition wherein the
probiotic bacterial strains comprise one or more of S. epidermidis
strains Staphylococcus epidermidis strains M034, M038, A11, AMT1,
AMT5-C5, and/or AMT5-G6 and/or Staphylococcus hominis strains A9,
C2, AMT2, AMT3, AMT4-C2, AMT4-G1, and/or AMT4-D12.
[0011] The compositions of the disclosure may also comprise
conditioned culture medium, or isolated antimicrobial compounds
derived from the strains described herein, such as the peptides
designated here as Hogocidins. The disclosure contemplates the use
of heterologously expressed or synthetic hogocidins, hogocidin
derivatives, or hogocidin-like peptides. The disclosure also
provides a composition comprising a hogocidin peptide, derivative
or variant and a cathelicidin peptide, derivative or variant. The
disclosure further provides a composition of any of the foregoing
embodiments, wherein the peptide comprises one or more D-amino
acids, one or more non-naturally occurring amino acids, and/or one
or more post-translational modifications. The disclosure provides a
composition of any of the foregoing embodiments, wherein the
peptide is substantially purified from other peptides. The
disclosure provides a composition of any of the foregoing
embodiments, wherein the peptide is partially purified from other
peptides. The disclosure provides a composition wherein the peptide
is present in a crude extract. The disclosure provides a
composition of any of the foregoing embodiments in a formulation
for topical administration.
[0012] The disclosure provides a composition of any of the
foregoing embodiments, wherein the formulation comprises a lotion,
ointment or spray or cream or oil suspension, but not limited to
these formats.
[0013] The disclosure provides a composition of any of the
foregoing embodiments, wherein the hogocidin peptide, derivative or
variant comprises a sequence selected from SEQ ID NO:2, SEQ ID
NO:4, SEQ ID NO:2 or 4 comprising a non-natural amino acid, SEQ ID
NO:2 or 4 comprising a D-amino acid, or SEQ ID NO:2 or 4 comprising
a fusion construct.
[0014] The disclosure also provides a method for inhibiting the
spread and/or reducing the risk of infection with a microbe
comprising contacting the microbe with an effective amount of a
composition of the disclosure. In one embodiment, the contacting is
in vivo. In another embodiment, the contacting in vivo is by
topical administration. The disclosure further provides a method of
treating skin or mucosal infections, atopic dermatitis, psoriasis,
acne, or other disorders related to skin dysbiosis by applying to
the skin or mucosa an effective amount of the compositions
disclosed herein to a subject in need thereof.
[0015] The disclosure provides a method of treating atopic
dermatitis comprising contacting a subject having or suspected of
having atopic dermatitis with an effective amount of a probiotic
composition comprising one or more of the bacterial strains
disclosed herein.
[0016] The disclosure provides a method of treating atopic
dermatitis comprising contacting a subject having or suspected of
having atopic dermatitis with an effective amount of a hogocidin
peptide, derivative or variant.
[0017] The disclosure provides a method of treating atopic
dermatitis or dysbiosis of the skin by contacting the affected area
with a composition comprising bacterial strains that secrete
hogocidin, firmocidin, SH-lantibiotic peptide, SH-antimicrobial,
SE-lantibiotic peptide, or SE antimicrobial such as Staphylococcus
hominis strain A9, Staphylococcus hominis strain C2, Staphylococcus
hominis strain AMT2, Staphylococcus hominis strain AMT3,
Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis
strain AMT4-G1, Staphylococcus hominis strain AMT4-D12,
Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis
strain SE-A11, Staphylococcus epidermidis strain AMT5-C5, and
Staphylococcus epidermidis strain AMT5-G6. The disclosure provides
methods and compositions as described above which further comprise
a cathelicidin peptide.
[0018] The disclosure provides a composition comprising a thickened
topical formulation of one or more probiotic bacterial strains and
optionally, a prebiotic compound, a protectant, humectant,
emollient, abrasive, salt, and/or surfactant; wherein the one or
more probiotic bacterial strain comprises one or more bacterial
strains of the genus Staphylococcus; and wherein the composition is
formulated for the topical treatment of disorders of dysbiosis of
the skin, scalp, or mucosae. In one embodiment, the one or more
probiotic bacterial strain comprises Staphylococcus epidermidis,
Staphylococcus hominis or a combination of Staphylococcus
epidermidis and Staphylococcus hominis. In a further embodiment,
the one or more probiotic bacterial strain comprises Staphylococcus
epidermidis strains MO34, MO38, A11, AMT1, AMT5-C5, and/or AMT5-G6.
In another embodiment, one or more probiotic bacterial strains
comprises Staphylococcus hominis strains A9, C2, AMT2, AMT3,
AMT4-C2, AMT4-G1, and/or AMT4-D12. In yet another embodiment, each
probiotic bacterial strain demonstrates a Fatty Acid Methyl Ester
profile corresponding to one of those shown in any of FIG. 11, 12,
13, 14, 15, 16, 17, 18, or 19. In another embodiment, the one or
more probiotic bacterial strains produces a peptide having a
sequence selected from the group consisting of SEQ ID NO: 2, 4, 28,
30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 55 and any
combination thereof and wherein such peptide is optionally
post-translationally modified. In another embodiment, the one or
more probiotic bacterial strains is provided in a live form. In
still another embodiment, the one or more probiotic bacterial
strains is provided in a lyophilized or freeze-dried or spray dried
form. In a further embodiment, the probiotic bacterium can be
reconstituted into a live form.
[0019] The disclosure also provides a method of treating skin or
mucosal infections, atopic dermatitis, psoriasis, mastitis, acne,
or other disorders related to skin dysbiosis in humans or other
mammals by applying to the skin or mucosa an effective amount of a
composition as described herein and in the preceding paragraph. In
one embodiment, the composition is applied topically. In a further
embodiment, the composition is formulated as a cream, ointment,
unguent, spray, powder, oil, thickened formulation or poultice. The
disclosure also provides a composition comprising one or more of a
hogocidin peptide, derivative or variant, an SH-lantibiotic
peptide, an SH-antimicrobial, an SE-lantibiotic peptide, and/or an
SE antimicrobial; and further comprising one or more thickeners,
solvents, emulsifiers, or pharmaceutically acceptable carriers or
excipients. In one embodiment, the composition further comprises a
cathelicidin peptide, derivative or variant. In yet a further or
alternate embodiment, the hogocidin peptide, derivative or variant,
the SH-lantibiotic peptide, and/or the SE-lantibiotic peptide
comprises one or more D-amino acids or non-naturally occurring
amino acids. In yet a further embodiment, the hogocidin peptide,
SH-lantibiotic peptide, SH-antimicrobial, SE-lantibiotic peptide,
or SE antimicrobial is produced in situ by one or more of
Staphylococcus hominis strain A9, Staphylococcus hominis strain C2,
Staphylococcus hominis strain AMT2, Staphylococcus hominis strain
AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus hominis
strain AMT4-G1, Staphylococcus hominis strain AMT4-D12,
Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis
strain SE-A11, Staphylococcus epidermidis strain AMT5-C5,
Staphylococcus epidermidis strain AMT5-G6 and Staphylococcus
epidermidis strain M034. In yet another embodiment of any of the
foregoing, the peptide is formulated for topical administration. In
yet a further embodiment, the formulation comprises a lotion,
ointment cream, powder, unguent, oil, or spray. In another
embodiment of any of the foregoing the hogocidin peptide,
derivative or variant comprises a sequence selected from SEQ ID
NO:2 or SEQ ID NO:4 or an active fragment thereof having
antimicrobial activity (e.g., a mature form). In yet another
embodiment, the one or more of a hogocidin peptide, derivative or
variant, an SH-lantibiotic peptide, an SH-antimicrobial, an
SE-lantibiotic peptide, an SE antimicrobial, and a cathelicidin
peptide, derivative or variant is provided as an extract or lysate
of Staphylococcus hominis strain A9, Staphylococcus hominis strain
C2, Staphylococcus hominis strain AMT2, Staphylococcus hominis
strain AMT3, Staphylococcus hominis strain AMT4-C2, Staphylococcus
hominis strain AMT4-G1, Staphylococcus hominis strain AMT4-D12,
Staphylococcus epidermidis strain AMT1, Staphylococcus epidermidis
strain SE-A11, Staphylococcus epidermidis strain AMT5-C5,
Staphylococcus epidermidis strain AMT5-G6 and Staphylococcus
epidermidis strain M034.
[0020] The disclosure also provides a method for treating skin or
mucosal infection or atopic dermatitis in a subject comprising
contacting the subject with an effective amount of a composition
comprising one or more of a hogocidin peptide, derivative or
variant, an SH-lantibiotic peptide, an SH-antimicrobial, an
SE-lantibiotic peptide, and optionally, a cathelicidin peptide,
derivative or variant. In one embodiment, the contacting is by
topical administration or optionally by contacting the subject with
one or more of SH-lantibiotic or bacteriocin-producing
Staphylococcus hominis strains A9, C2, AMT2, AMT3, AMT4-C2,
AMT4-G1, AMT4-D12 and Staphylococcus epidermidis strains AMT5-G6
and M034. The disclosure also provides a recombinant vector
comprising a polynucleotide encoding a polypeptide that is at least
95% identical to SEQ ID NO:2 or 4, or a biologically active
fragment thereof having antimicrobial activity. In one embodiment,
the vector comprises a polynucleotide that encodes a polypeptide of
SEQ ID NO:2 or 4. In yet another embodiment, the vector comprises a
polynucleotides that encodes a polypeptide of SEQ ID NO:2 from
about amino acid 32 to about amino acid 61. In a further
embodiment, the vector comprises a polynucleotide that encodes a
polypeptide of SEQ ID NO:4 from about amino acid 29 to about amino
acid 66. In another embodiment, the vector comprises a
polynucleotide that is at least 95% identical to SEQ ID NO:1 or 3
and encodes a polypeptide of SEQ ID NO:2 or 4, respectively. In yet
another embodiment of the foregoing embodiments, the vector
comprises a fragment of SEQ ID NO:1 or 3. In yet a further
embodiment of any of the foregoing, the vector is an expression
vector.
[0021] The disclosure also provides a host cell engineered to
express a recombinant vector of the disclosure. In one embodiment,
the host cell is a non-pathogenic attenuated host cell.
[0022] The disclosure also provide a recombinant polypeptide
produced by the host cell of the disclosure. In another embodiment,
the recombinant polypeptide is purified from a host cell
culture.
[0023] The disclosure also provides a composition comprising the
host cell of disclosure.
[0024] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1A-D. The ratio of culturable Staphylococcus compared
to Staphylococcus DNA is higher in lesional skin of atopic
dermatitis. FIG. 1A: Culturable total Staphylococcus spp. were
counted on a selective mannitol salt agar plate from 49 subjects
with atopic dermatitis (AD) and 30 subjects without AD. FIG. 1B:
CFU results for growth of S. aureus are shown from 30 non-atopic
subjects and 49 atopic dermatitis patients. FIG. 1C: Total
Staphylococcus spp. DNA abundance was determined by quantitative
PCR (qPCR) on DNA from 14 non-atopic and 37 atopic subjects.
Relative CFU (rCFU) was determined by comparison to a standard of
known CFUs of S. epidermidis (ATCC12228). FIG. 1D: The ratio of
live Staphylococcus spp. CFU to relative abundance of
Staphylococcus determined by DNA was calculated at each
corresponding skin site.
[0026] FIG. 2A-C. Atopic dermatitis skin is colonized by
coagulase-negative Staphylococcus with a low frequency of
antimicrobial activity. FIG. 2A: Coagulase-negative Staphylococcus
(CONS) with antimicrobial activity against S. aureus was determined
by a high-throughput assay of individual culture isolates and the
proportion (%) of total colonies that inhibited growth of S. aureus
was determined. FIG. 2B: The proportion of CoNS with antimicrobial
activity was determined from the same subjects at day-1, day-7 and
day-14. FIG. 2C: The ratio of live Staphylococcus spp. to abundance
of Staphylococcus DNA was determined from the same subjects as in
FIG. 2B. *P<0.05, ****P<0.0001. 11 atopic and 11 non atopic
subjects were randomly selected for analysis in Panels B and C.
[0027] FIG. 3A-B. Antimicrobial coagulase-negative Staphylococcus
correlate with the absence of S. aureus colonization. FIG. 3A: The
proportion of antimicrobial CoNS in each sample is plotted against
the abundance of S. aureus cultured from each subject. Quadrants
are divided based on frequency of antimicrobial CoNS (>50% or
<50%) and detection of live S. aureus (<1 CFU/cm.sup.2 or
>1 CFU/cm.sup.2). The proportion (%) of subjects in each
quadrant to total subjects is shown. FIG. 3B: The frequency of
antimicrobial CoNS in S. aureus-culture negative subjects (white)
and S. aureus-culture positive subjects (solid) are shown. Data are
mean.+-.SE for 29 non-atopic subjects and 41 nonlesional or 40
lesional sites of atopic subjects.
[0028] FIG. 4A-B. Diverse bacterial species have antimicrobial
activity. Species of antimicrobial or non-antimicrobial CoNS were
identified by DNA sequencing of full length 16S rRNA from randomly
isolated colonies with and without antimicrobial activity. FIG. 4A:
Proportions of CoNS species identified with antimicrobial activity
from 5 non-atopic subjects. FIG. 4B: Proportions of CoNS species
identified from antimicrobial and non-antimicrobial colonies
isolated from subjects with atopic dermatitis. Up to 48 CoNS
isolates were sequenced from each individual. The relative
proportion of colonies with antimicrobial (solid) and
non-antimicrobial CoNS (white) from each AD subject is shown by pie
chart.
[0029] FIG. 5A-B. Identification of antimicrobial peptides from a
coagulase-negative Staphylococcus strain within the skin microbiome
(SH-A9). FIG. 5A. Amino acid sequence and predicted mono and
di-sulfide bonds from two antimicrobial peptides purified from S.
hominis isolated from non-atopic skin. Peptides are named
Hogocidin-.alpha. (SEQ ID NO:2 from aa 32-61) and Hogocidin-.beta.
(SEQ ID NO:4 aa 29-66) (SH-lantibiotic .alpha. and .beta.). The
calculated molecular weights of a hypothetical mature form of
Hogocidin-.alpha. [3152.52 (M+H)] and Hogocidin-.beta. [3548.04
(M+H)] are identical to the observed molecular masses [m/z 3152.22
and 3547.71 (M+H), respectively]. FIG. 5B shows dose response
curves for the antimicrobial activity of Hogocidin-.alpha. and
Hogocidin-.beta. against S. aureus. Co-incubation with an
antimicrobial peptide produced by human skin (LL-37) shows
synergistic activity. Data represent mean.+-.SE of triplicate
assays. Arrow shows minimal inhibitory concentration (MIC) of each
AMP, defined as 3-log reduction of viable bacteria in comparison to
control. Dha, 2,3-didehydroalanine; Dhb,
(Z)-2,3-didehydrobutyrine.
[0030] FIG. 6. Abundance of S. aureus DNA on non-atopic skin,
nonlesional and lesional site of atopic skin. Fourteen and 37 DNA
swabs were obtained from 30 normal and 50 atopic dermatitis
patients recruited, respectively. The abundance of S. aureus DNA
was determined by qPCR with species-specific primers targeting the
S. aureus-specific femA gene. Relative CFU (rCFU) of S. aureus DNA
was determined by comparison with known CFUs of S. aureus
(ATCC35556). Density of live bacteria or bacterial DNA was
normalized into the area swabbed. AD: atopic dermatitis.
[0031] FIG. 7A-B. Purification and mass spectrometric analysis of
AMPs produced by S. hominis isolated from non-atopic skin (SH-A9).
AMPs were purified by HPLC using CapcelPac C8 column from culture
supernatant of a representative antimicrobial isolate of S. hominis
(FIG. 7A). The last step of 5 purification steps is shown. The
insert panel represents antimicrobial activity of each fraction on
radial diffusion assay against S. aureus. Fractions with
antimicrobial activity were characterized by MALDI-TOF-MS (FIG.
7B).
[0032] FIG. 8. Representation of amino acids losses in
genome-guided MALDI-TOF/TOF analysis for Hogocidin-.beta.
(SH-lantibiotic-.beta.). Amino acid sequence of purified
Hogocidin-.beta. was obtained from amino acids losses in MS/MS
fragmentation spectrum of precursor mass 3547.7 m/z. Dha,
2,3-didehydroalanine; Dhb, (Z)-2,3-didehydrobutyrine.
[0033] FIG. 9A-B. Organization of gene clusters encoding Hogocidin
precursors and lantibiotic biosynthetic genes in a S. hominis
strain isolated from non-atopic skin (SH-A9). FIG. 9A shows the
order of lantibiotic precursors (A1 and A2; SH-lantibiotic-.alpha.
and .beta.) and biosynthetic genes (C, T and M) on the S. hominis
SH-A9 genome. FIG. 9B lists hypothetical gene, gene locus and
putative function. This S. hominis strain contains multiple copies
of the lantibiotic-related gene clusters.
[0034] FIG. 10 shows the high-throughput methodology used in the
disclosure.
[0035] FIG. 11A-B. Chromatograms showing results of FAME analysis
of S. epidermidis strains MO-34 and MO-38, which were identified by
the methods given in the present disclosure.
[0036] FIG. 12A-B. Chromatograms showing results of FAME analysis
of S. hominis strains A9 and C2, which were identified by the
methods given in the present disclosure.
[0037] FIG. 13A-B. Chromatograms showing results of FAME analysis
of S. epidermidis strains A11 and AMT1-A9, which were identified by
the methods given in the present disclosure.
[0038] FIG. 14A-B. Chromatograms showing results of FAME analysis
of S. hominis strains AMT2-A11 and AMT3-A12, which were identified
by the methods given in the present disclosure.
[0039] FIG. 15A-B. Chromatograms showing results of FAME analysis
of S. hominis strains AMT4-C2 and AMT4-G1, which were identified by
the methods given in the present disclosure.
[0040] FIG. 16A-B. Chromatograms showing results of FAME analysis
of S. hominis strains AMT4-D12 and S. epidermidis AMT5-C5, which
were identified by the methods given in the present disclosure.
[0041] FIG. 17A-B. Chromatograms showing results of FAME analysis
of S. epidermidis strain AMT5-G6, which was identified by the
methods given in the present disclosure, and S. hominis strain C4,
which does not produce hogocidin.
[0042] FIG. 18A-B. Chromatograms showing results of FAME analysis
of S. hominis strains C5 and C6, which do not produce
hogocidin.
[0043] FIG. 19A. Chromatogram showing results of FAME analysis of
S. epidermidis strain MO1, which does not produce SE-lantibiotics
or SE-antimicrobials.
[0044] FIG. 20A-E. Transplantation of antimicrobial CoNS reduces
survival of S. aureus on the skin. FIG. 20A: Effect of S. hominis
on survival of S. aureus on pigskin. Live S. hominis A9 that
produces hogocidin (1.times.10 CFU/cm.sup.2) was compared to
controls including UV-killed and washed A9 strain, strains of live
S. hominis that do not produce AMP activity (C4, C5 and C6) or
vehicle cream alone with the pigskin assay. Data represent
mean.+-.s.e.m. of five independent assays. FIG. 20B: Effect of
bacterial transplantation on survival of S. aureus on mouse skin.
S. aureus was applied at 1.times.10.sup.5 CFU/cm.sup.2 on the
shaved dorsal skin of mice. After two hours the control (vehicle
alone), active S. hominis (A9), or inactive strains (C4, C5 and C6)
were applied at equal concentrations (1.times.10.sup.5
CFU/cm.sup.2). S. aureus recovery 20 hrs after application of CoNS
or controls is shown. Data represent mean.+-.s.e.m. of six mice.
FIG. 20C: Work flow for autologous human microbiome transplant
(AMT) on AD subjects colonized with S. aureus. FIG. 20D:
Characterization of CoNS clones used for AMT. Antimicrobial class
of each clone was identified by whole genome sequencing. FIG. 20E:
Effect of transplantation of antimicrobial CoNS on the survival of
S. aureus on the skin of subjects with AD. S. aureus survival was
measured by colony counting of swabs taken before transplant
(baseline) and 24 hrs after treatment. Difference in S. aureus
between vehicle and AMT arms is shown as A % S. aureus CFU.
Inactive strains have no effect.
[0045] FIG. 21A-C: Hypothetical antimicrobial genes identified in
anti-S. aureus strains AMT1-A9 (FIG. 21A), AMT2-A12 (FIG. 21B), and
AMT3-A12 (FIG. 21C) used for autologous microbiome transplant.
Whole genome sequence of active CoNS clones were obtained by miSeq
and analyzed on the RAST Server (rast.nmpdr.org) to identify
antimicrobial class.
[0046] FIG. 22A-B: Hypothetical antimicrobial genes identified in
anti-S. aureus strains AMT4-C2 (FIG. 22A) and AMT4-G1 (FIG. 22B)
used for autologous microbiome transplant. Whole genome sequence of
active CoNS clones were obtained by miSeq and analyzed on the RAST
Server (rast.nmpdr.org) to identify antimicrobial class.
[0047] FIG. 23A-C: Hypothetical antimicrobial genes identified in
anti-S. aureus strains AMT4-D12 (FIG. 23A), AMT5-C5 (FIG. 23B), and
AMT5-G6 (FIG. 23C) used for autologous microbiome transplant. Whole
genome sequence of active CoNS clones were obtained by miSeq and
analyzed on the RAST Server (rast.nmpdr.org) to identify
antimicrobial class.
[0048] FIG. 24 shows dose-dependent killing curve of antimicrobial
peptide purified from culture supernatant of Staphylococcus
epidermidis A11 strain isolated from normal skin. Indicated
bacterial species (1.times.10.sup.5 CFU/mL) were incubated with
various concentrations of purified Staphylococcus epidermidis A11
antimicrobial peptide (comprising SEQ ID NO:55) in 50%
Muller-Hinton Broth/50% PBS at 37.degree. C. for 24 hrs.
Propionibacterium acnes were incubated in 100%
Reinforsed-Clostridial Media under an anaerobic condition. S.
epiA11, Staphylococcus epidermidis A11 strain; S. epi12228,
Staphylococcus epidermidis ATCC12228 strain; S. homA9,
Staphylococcus hominis A9 strain; S. aur113, Staphylococcus aureus
113 strain; P.ac, Propionibacterium acnes ATCC6919 strain; E. coli,
Escherichia coli ATCC25922; P. aeruginosa, Pseudomonas aeruginosa
ATCC14213.
[0049] FIG. 25A-B shows data related to an antimicrobial peptide
purified from culture supernatant of a clinical isolate strain of
Staphylococcus epidermidis (A11 strain) by HPLC (A). Active
fraction (Fraction 33-34) was analyzed by MALDO-TOF mass to
estimate molecular weight of active antimicrobial peptide (B).
Observed molecular weight was 3484.90 (m/z). N-terminal sequencing
of the peptide is provided in SEQ ID NO:55.
DETAILED DESCRIPTION
[0050] As used herein and in the appended claims, the singular
forms "a," "and," and "the" include plural referents unless the
context clearly dictates otherwise. Thus, for example, reference to
"a probe" includes a plurality of such cells and reference to "the
cell" includes reference to one or more cells and equivalents
thereof known to those skilled in the art, and so forth.
[0051] Also, the use of "or" means "and/or" unless stated
otherwise. Similarly, "comprise," "comprises," "comprising"
"include," "includes," and "including" are interchangeable and not
intended to be limiting.
[0052] It is to be further understood that where descriptions of
various embodiments use the term "comprising," those skilled in the
art would understand that in some specific instances, an embodiment
can be alternatively described using language "consisting
essentially of" or "consisting of."
[0053] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood to one of
ordinary skill in the art to which this disclosure belongs.
Although any methods and reagents similar or equivalent to those
described herein can be used in the practice of the disclosed
methods and compositions, the exemplary methods and materials are
now described.
[0054] All publications mentioned herein are incorporated herein by
reference in full for the purpose of describing and disclosing the
methodologies, which are described in the publications, which might
be used in connection with the description herein. With respect to
any term that is presented in one or more publications that is
similar to, or identical with, a term that has been expressly
defined in this disclosure, the definition of the term as expressly
provided in this disclosure will control in all respects.
[0055] Atopic dermatitis is a common, chronic skin disorder
characterized by dysfunction of the epidermal barrier and relapsing
skin inflammation. The severity of this disease is associated with
dysbiosis of the skin microbiome and the high susceptibility of
these patients to colonization and infections by Staphylococcus
aureus.
[0056] A unifying model for the etiology of atopic dermatitis has
emerged with recognition that immunity is co-dependent upon
functions provided by epithelia. For example, the production of
antimicrobial peptides (AMPs) provides direct disinfectant activity
against invading pathogens. In healthy skin, AMPs such as
cathelicidins and .beta.-defensins are increased after injury.
However, the skin of patients with atopic dermatitis has a
decreased capacity to produce certain AMPs and this is associated
with an increased rate of infection by S. aureus, a pathogen that
should be killed by these AMPs. S. aureus further exacerbates
symptoms of atopic dermatitis and leads to immune dysfunction such
as TH2 lymphocyte skewing, reduced AMPs, exacerbated allergic
reactions and disruption of the skin barrier.
[0057] Prior studies of patients with atopic dermatitis have shown
that the bacterial flora present on these patients is different
than the bacteria found on the skin of non-atopic subjects. The
microbiome of patients with atopic dermatitis is less diverse and
typically has a higher abundance of Staphylococcal species. Without
intending to be bound by any particular theory, it has been
hypothesized that dysbiosis of the skin microbiome could contribute
to the pathophysiology of this disease. Specifically, the diverse
community of microorganisms that normally comprise the microbiome
have been suggested to contribute to cutaneous homeostasis. For
example, in mice, Staphylococcus epidermidis can control
inflammation after injury, influence T-cell development and induce
expression of AMPs. Furthermore, the microbiome may produce its own
AMPs that could synergize with AMPs produced by host cells.
Therefore, in addition to the deleterious effects of colonization
by S. aureus, dysbiosis of the microbiome in atopic dermatitis
could contribute to disease by loss of their beneficial
functions.
[0058] Existing antibiotic therapies non-specifically kill
bacteria, which impacts the homeostasis of the resident microflora.
Imbalanced microflora contribute to the pathogenesis of skin
inflammatory diseases, such as atopic dermatitis, rosacea and acne
vulgaris etc. This disclosure provides compositions and
formulations for disinfecting surfaces or treating infections but
does not pose the safety risks of non-specific antibiotics. Further
the disclosure provides for probiotic approaches wherein subjects
may be provided with live S. hominis or S. epidermidis strains
which may produce the necessary antimicrobial compounds in situ
while simultaneously restoring the characteristics of a healthy
cutaneous flora.
[0059] Staphylococcus hominis (S. hominis) is a major constituent
of the microflora of healthy human skin. Recent studies indicate
that S. epidermidis protect human skin by preventing pathogenic
infections by producing phenol-soluble modulins (PSMs) and small
molecule antibiotic, named "Firmocidin", which function as
additional antimicrobial compounds on normal human skin (see, e.g.,
U.S. Pat. Publ. No. 2013/0331384A1, the disclosure of which is
incorporated herein by reference). In addition, lipoteichoic acid
produced by S. epidermidis benefits human skin by suppressing skin
inflammation during wound repair. The present disclosure provides
the use of live S. epidermidis and/or S. hominis cells or cultures
to restore or enhance the normal cutaneous flora to support wound
healing and prevent infection and restore skin barrier
function.
[0060] A limitation of DNA sequencing is that it is unable to
distinguish between viable and dead organisms, in the methods of
the disclosure microbial abundance was directly evaluated in atopic
and non-atopic subjects by both culture and DNA quantification
techniques. Surprisingly, the relative capacity to culture live
bacteria compared to measurements of DNA differed greatly between
non-atopic and atopic dermatitis patients. Approximately 10 times
more bacterial DNA relative to CFUs of cultured bacteria was
detected in non-atopic skin compared to atopic lesional skin. These
observations suggested a lower survival rate of bacteria on
non-atopic skin than atopic lesional skin.
[0061] One explanation for the lower survival rate of bacteria on
non-atopic skin is a more effective surface antimicrobial activity.
AMPs such as LL-37 and hBDs-2 and -3 have lower levels of
expression in inflamed skin of atopic patients than inflamed skin
of normal subjects, but these AMP expressions are low in
non-inflamed skin. Therefore, the increased capacity of the
non-inflamed normal skin to kill bacteria is not likely due to the
expression of these host AMPs. The high frequency of antimicrobial
CoNS observed on non-atopic skin suggests that these resident
bacteria are important to resist colonization by pathogens.
Supporting this, S. aureus colonization was only detected in
subjects with a low frequency of CoNS strains with antimicrobial
activity. CoNS that could inhibit biofilm formation have also been
observed in the nasal mucosa and inhibited nasal colonization by S.
aureus. The observation of the lack of direct antimicrobial
activity derived from the community of bacteria residing on the
skin of patients with atopic dermatitis defines a previously
unknown defect in the innate defense system of these
individuals.
[0062] A high-throughput screen for antimicrobial activity on over
7500 individual isolates of coagulase-negative Staphylococcus
(CONS) cultured from the skin swabs of 30 healthy control subjects
and 50 lesional and nonlesional sites of Atopic Dermatitis (AD)
patients identified several CoNS isolates with antimicrobial
activity. Healthy subjects had a high frequency of CoNS isolates
with antimicrobial activity against Staphylococcus aureus
(75.26.+-.6.59%) whereas the bacteria isolated from AD nonlesional
and lesional skin had significantly less activity [22.83.+-.5.10%,
15.76.+-.4.10%, respectively (p<0.0001)]. Notably, subjects with
a low frequency of antimicrobial CoNS isolates were also colonized
by SA. 16S rRNA sequencing revealed that antimicrobial activity was
detected in diverse strains of CONS, such as S. epidermidis, S.
hominis, S. warneri, and S. capitis. Two prokaryotic AMPs with
molecular weights of 3152.2 Da and 3550.7 Da were identified using
HPLC, protein sequencing by MALDI-TOF-MS2 and genome sequencing.
Additionally, as shown in Nakatsuji, T. et al. (2016), Nature
Medicine Submitted Manuscript No. NMED-A78395A, submitted Mar. 29,
2016, which is incorporated herein by reference in its entirety, by
applying functional CoNS isolates to ex vivo model systems, animal
models, and through autologous transplants in human subjects, it
was shown that the application of CoNS strains produce reductions
in S. aureus levels in infected skin. For example, application of
this antimicrobial CoNS isolate to mouse skin colonized by SA was
effective in reducing SA survival by >90% above that seen when
AD CoNS strains were applied. These findings suggest that the
microbiome is the first line of defense against SA and that
dysbiosis in AD has a major functional association to SA
colonization.
[0063] Several CoNS species were identified that produced
antimicrobial activity against S. aureus. Some laboratory strains
of S. epidermidis and S. warneri were previously described to
produce lantibiotics that could inhibit growth of other bacteria,
but these were not detected in the subject population. Furthermore,
several of the CoNS species isolated based on their antimicrobial
activity were not previously suspected to have antimicrobial
function. To better understand these, two previously unknown
lantibiotics were identified that had potent activity against S.
aureus and were highly synergistic with the host AMPs LL-37. The
gene encoding these lantibiotics was prevalent in non-atopic
individuals. This discovery illustrates the potential in further
analysis of the host-defense function of the healthy human skin
microbiome and may provide a genetic approach to predicting the
activity of the microbiome. Metagenomic sequencing and correlation
with functional screening of the microbiome could be of great
benefit in the treatment of patients with atopic dermatitis and
other skin diseases.
[0064] The disclosure provides evidence that the community of
bacteria residing on normal human skin provides an important shield
against S. aureus. Again, without intending to be bound by any
particular theory, dysfunction in this microbiome-mediated
antimicrobial defense system may enable colonization of the skin by
S. aureus in atopic dermatitis and further exacerbation of the
disease. This observation suggests that strategies of
bacteriotherapy of the skin may be useful as a method to suppress
S. aureus without use of pharmaceutically derived antibiotics.
Given the complex nature of this disease, the ideal therapeutic
approach to atopic dermatitis should include targeting both repair
of the intrinsic epidermal barrier and optimizing the immune
defense functions provided by the microbiome.
[0065] The disclosure also describes novel antimicrobial peptides
(AMPs) from culture supernatant of a clinical isolate of S.
hominis. These AMPs are referred to herein as Hogocidin-.alpha. and
Hogocidin-.beta.. Hogocidins exert antimicrobial and bactericidal
activity against Staphylococcus aureus (S. aureus), but do not
inhibit the growth of commensal bacteria on the skin such as S.
epidermidis. Therefore, the disclosure represents provides
antibiotics with potent but selective activity against pathogens,
and high safety profile as they are found normally in the human
skin microbiome, as well as probiotic approaches to treating these
conditions.
[0066] The term "antimicrobial" as used herein means that the
peptide destroys, or inhibits or prevents the growth or
proliferation of, a microbe (e.g., a bacterium, fungus, and/or
virus). Likewise, the term "antiviral" as used herein means that a
peptide destroys, or inhibits or prevents the growth or
proliferation of a virus or a virus-infected cell. The term
"anti-tumor" as used herein means that a peptide prevents, inhibits
the growth of, or destroys, a tumor cell(s). Similarly, the term
"antifungal" means that a peptide prevents, destroys, or inhibits
the growth of a fungus.
[0067] As used herein, "probiotic" refers to the process of
providing live or attenuated microbial cultures, or lysates,
lyophiles or extracts of such cultures, in order to supplement or
replace elements of a healthy cutaneous or mucosal flora. An
attenuated vector for delivery to the skin can be include a virus
or bacteria that has been genetically modified to (a) make the
vector non-pathogenic, (b) have reduced pathogenicity, (c) be
replication defective, or (d) to be non-antigenic. Other
attenuation are known in the art. The attenuation is typically
performed by knocking out a gene or disrupting a gene coding
sequence or expression control element such that the attention of
(a)-(c) or (d) is accomplished. Such techniques are known in the
art and numerous such attenuated bacterial and viral vectors are
known.
[0068] The "hogocidins" are composed of two distinct domains: an
N-terminal "prosequence" domain and the C-terminal domain of the
mature hogocidin. The mature hogocidin-.alpha. comprises a sequence
of SEQ ID NO:2 from about amino acid 32 to about amino acid 61
(e.g., beginning at about amino acid 30, 31, 32 or 33 of SEQ ID
NO:2 and extending to about amino acid 59, 60 or 61 of SEQ ID
NO:2). It will be readily apparent to one of skill in the art that
the pre-pro form of hogocidin-.alpha. is about 61 amino acids in
length and is post-translationally process to provide the mature
form. Based upon the expression system and organism, the mature
form may be processed slightly differently depending upon the
proteases present. Moreover, it will also be readily apparent that
the pre-pro form of hogocidin-.alpha. can be used in the methods,
compositions and kits of the disclosure, wherein prior to or after
administration the pre-pro form can be processed in vitro or in
vivo.
[0069] Similarly, the mature form of hogocidin-(comprises a
sequence of SEQ ID NO:4 from about amino acid 29 to about amino
acid 66 (e.g., beginning at about amino acid 27, 28, 29 or 30 of
SEQ ID NO:4 and extending to about amino acid 64, 65, or 66 of SEQ
ID NO:4). It will be readily apparent to one of skill in the art
that the pre-pro form of hogocidin-(is about 66 amino acids in
length and is post-translationally process to provide the mature
form. Based upon the expression system and organism, the mature
form may be processed slightly differently depending upon the
proteases present. Moreover, it will also be readily apparent that
the pre-pro form of hogocidin-(can be used in the methods,
compositions and kits of the disclosure, wherein prior to or after
administration the pre-pro form can be processed in vitro or in
vivo.
[0070] The polypeptide comprising SEQ ID NO:2 is typically cleaved
following amino acid number 31 of SEQ ID NO:2, however, one of
skill in the art will recognize that depending upon the enzyme
used, the expression system used and/or the conditions under which
proteolytic cleavage of the polypeptide takes place, the cleavage
site may vary from 1 to 3 amino acid in either direction of amino
acid number 31 of SEQ ID NO:2.
[0071] The polypeptide comprising SEQ ID NO:4 is typically cleaved
following amino acid number 28 of SEQ ID NO:4, however, one of
skill in the art will recognize that depending upon the enzyme
used, the expression system used and/or the conditions under which
proteolytic cleavage of the polypeptide takes place, the cleavage
site may vary from 1 to 3 amino acid in either direction of amino
acid number 31 of SEQ ID NO:4.
[0072] Although the genetic code is well understood by one of skill
in the art and it is routine in generating polynucleotides encoding
a desired polypeptides sequence; the disclosure also provides
polynucleotides encoding the polypeptides of the disclosure. For
example, the disclosure provides SEQ ID NO:1 and 3, which encode
the polypeptides of SEQ ID NO:2 and 4.
[0073] As used herein, the term "hogocidin peptide" refers to the
mature form of hogocidins comprising a chain of amino acids that is
about 30 to about 50 amino acids in length and comprises a sequence
as set forth in SEQ ID NO:2 or 4 or post-translationally modified
versions thereof:
SEQ ID NO:2 (from aa 32 to aa 61)--KCSWWNASCHLGNNGKICTVSHECAAGCNL
SEQ ID NO:4 (from aa 29 to aa
66)--ATPTITTSSATCGGIIVAASAAQCPTLACSSRCGKRKK.
[0074] In one embodiment, the method provides hogocidin derivatives
comprising (a) peptides that are at least 90% identical to a
hogocidin peptide of SEQ ID NO:2 or 4 and having antimicrobial
activity; (b) mature forms of (a) that have been
post-translationally processed; (c) fragments of hogocidin peptides
that are about 15-40 amino acids in length and have antimicrobial
activity; (d) fusion proteins comprising (a)-(c) above and having
antimicrobial activity; (e) peptides comprising any of (a), (b),
(c) or (d) wherein one or more amino acids comprise a D-amino acid
and the peptide has antimicrobial activity; and (f) retroinverso
peptides of any of the foregoing and having antimicrobial peptides.
In some further embodiments, the method provides hogocidin
derivatives comprising lanthionine or methyllanthionine residues,
or hogocidin derivatives modified such that they contain
lanthionine or methyllanthionine residues. It is not necessary that
the analog, derivative, variation, or variant have activity
identical to the activity of the hogocidin peptide from which the
analog, derivative, conservative variation, or variant is derived
so long as it has some antimicrobial activity. In another
embodiment, the disclosure provides a hogocidin polypeptide
comprising at least one conservative amino acid difference compared
to polypeptide of SEQ ID NO:2 or 4.
[0075] The disclosure also provides a polypeptide comprising a
sequence of SEQ ID NO:55 at the N-terminal end and wherein the
polypeptide has antimicrobial activity and observed molecular
weight was 3484.90 (m/z). In a further embodiment, the polypeptide
is produced by S. epidermidis A11.
[0076] The disclosure also provides compositions comprising a
pharmaceutically acceptable excipient and comprising a
substantially pure hogocidin peptide or derivative. The disclosure
also provides compositions comprising a probiotic formulation which
includes one or more hogocidin- or firmocidin-producing bacterial
strains.
[0077] The term "purified" as used herein refers to a peptide that
is substantially free of other proteins, lipids, and
polynucleotides (e.g., cellular components with which an in vivo
produced peptide would naturally be associated). Typically, the
peptide is at least 70%, 80%, or most commonly 90% pure by weight.
As described more fully below, the composition can further comprise
a cathelicidin peptide or derivative thereof.
[0078] A "variant" is an antimicrobial peptide (e.g., a hogocidin
peptide of the disclosure) that is an altered form of a referenced
antimicrobial peptide. For example, the term "variant" includes an
antimicrobial peptide produced by the method disclosed herein in
which at least one amino acid (e.g., from about 1 to 10 amino
acids) of a reference peptide is substituted with another amino
acid. The term "reference" peptide means any of the antimicrobial
peptides of the disclosure (e.g., a polypeptide consisting of SEQ
ID NO:2 and 4 or a mature form thereof), from which a variant,
derivative, analog, or conservative variation is derived. Included
within the term "derivative" is a hybrid peptide that includes at
least a portion of each of two antimicrobial hogocidin peptides.
Derivatives can be produced by adding one or a few (e.g., 1 to 5)
amino acids to an antimicrobial peptide without completely
inhibiting the antimicrobial activity of the peptide. In addition,
C-terminal derivatives, e.g., C-terminal methyl esters, can be
produced and are encompassed by the disclosure.
[0079] The disclosure also includes peptides that are conservative
variations of those peptides as exemplified herein. The term
"conservative variation" as used herein denotes a polypeptide in
which at least one amino acid is replaced by another, biologically,
chemically, or structurally similar residue. Examples of
conservative variations include the substitution of one hydrophobic
residue, such as isoleucine, valine, leucine, alanine, cysteine,
glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine
or methionine for another, or the substitution of one polar residue
for another, such as the substitution of arginine for lysine,
glutamic for aspartic acid, or glutamine for asparagine, and the
like. Neutral hydrophilic amino acids that can be substituted for
one another include asparagine, glutamine, serine and threonine.
Structurally conservative variations include the substitution of
alanine for serine (and vice versa), isoleucine for threonine (and
vice versa), arginine for lysine (and vice versa), and the
replacement of any of tyrosine, phenylalanine, tryptophan, and
histidine for any other member of that group. The term
"conservative variation" also encompasses a peptide having a
substituted amino acid in place of an unsubstituted parent amino
acid; typically, antibodies raised to the substituted polypeptide
also specifically bind the unsubstituted polypeptide.
[0080] As used herein, a "SH-lantibiotic" refers to a compound
comprising a post-translationally modified peptide produced by S.
hominis which optionally contains one or more lanthionine or
methyllanthionine moieties, and shows antimicrobial activity
against one or more non-S. hominis species.
[0081] As used herein, a "SH-antimicrobial" refers to a compound
comprising a non-lantibiotic compound produced or secreted by S.
hominis, which may optionally comprise a non-lantibiotic peptide,
and which shows antimicrobial activity against one or more non-S.
hominis species.
[0082] As used herein, a "SE-lantibiotic" refers to a compound
comprising a post-translationally modified peptide produced by S.
epidermidis which optionally contains one or more lanthionine or
methyllanthionine moieties, and shows antimicrobial activity
against one or more non-S. epidermidis species. In one embodiment,
the peptide comprises a sequence of SEQ ID NO:55.
[0083] As used herein, a "SE-antimicrobial" refers to a compound
comprising a non-lantibiotic compound produced or secreted by S.
epidermidis, which may optionally comprise a non-lantibiotic
peptide, and which shows antimicrobial activity against one or more
non-S. epidermidis species.
[0084] Hogocidin peptide variants of the disclosure can be
identified by screening a large collection, or library, of random
peptides or peptides of interest using, for example, one of a
number of animal models such as CRAMP knockout mice that display
increased susceptibility to skin infections. Hogocidin peptide
variants can be, for example, a population of peptides related in
amino acid sequence to SEQ ID NO:2 and 4 by having various
substitutions based upon such sequences.
[0085] Peptide libraries include, for example, tagged chemical
libraries comprising peptides and peptidomimetic molecules. Peptide
libraries also comprise those generated by phage display
technology. Phage display technology includes the expression of
peptide molecules on the surface of phage as well as other
methodologies by which a protein ligand is or can be associated
with the nucleic acid, which encodes it. Methods for the production
of phage display libraries, including vectors and methods of
diversifying the population of peptides, which are expressed, are
known in the art (see, for example, Smith and Scott, Methods
Enzymol. 217:228 257 (1993); Scott and Smith, Science 249:386 390
(1990); and Huse, WO 91/07141 and WO 91/07149). These or other
known methods can be used to produce a phage display library, from
which the displayed peptides can be cleaved and assayed for
antibacterial activity. If desired, a population of peptides can be
assayed for activity, and an active population can be subdivided
and the assay repeated in order to isolate an active peptide from
the population. Other methods for producing peptides useful in the
disclosure include, for example, rational design and mutagenesis
based on the amino acid sequences of a hogocidin peptide as set
forth in SEQ ID NO:2 or 4, for example.
[0086] A hogocidin peptide variant can be a peptide mimetic, which
is a non-amino acid chemical structure that mimics the structure
of, for example, a hogocidin peptide of SEQ ID NO:2 or 4 (or a
mature form thereof) yet retains antimicrobial/antibacterial
activity. Such a mimetic generally is characterized as exhibiting
similar physical characteristics such as size, charge or
hydrophobicity in the same spatial arrangement found in the
hogocidin peptide counterpart. A specific example of a peptide
mimetic is a compound in which the amide bond between one or more
of the amino acids is replaced, for example, by a carbon-carbon
bond or other bond well known in the art (see, for example, Sawyer,
Peptide Based Drug Design, ACS, Washington (1995)).
[0087] The amino acids of a hogocidin peptide, variant or
peptidomimetic of the disclosure are selected from the twenty
naturally occurring amino acids, including, unless stated
otherwise, L-amino acids and D-amino acids. The use of D-amino
acids are particularly useful for increasing the life of a protein
or peptide. Polypeptides incorporating D-amino acids are resistant
to proteolytic digestion. The term amino acid also refers to
compounds such as chemically modified amino acids including amino
acid analogs, naturally occurring amino acids that are not usually
incorporated into proteins such as norleucine, and chemically
synthesized compounds having properties known in the art to be
characteristic of an amino acid, provided that the compound can be
substituted within a peptide such that it retains its biological
activity. For example, glutamine can be an amino acid analog of
asparagine, provided that it can be substituted within an active
fragment of a hogocidin peptide, variant and the like such that it
retains its antimicrobial/antibacterial activity. Other examples of
amino acids and amino acids analogs are listed in Gross and
Meienhofer, The Peptides: Analysis, Synthesis, Biology, Academic
Press, Inc., New York (1983). An amino acid also can be an amino
acid mimetic, which is a structure that exhibits substantially the
same spatial arrangement of functional groups as an amino acid but
does not necessarily have both the "-amino" and "-carboxyl" groups
characteristic of an amino acid.
[0088] Polypeptides and peptides of the disclosure can be
synthesized by commonly used methods such as those that include
t-BOC or FMOC protection of alpha-amino groups. Both methods
involve stepwise synthesis in which a single amino acid is added at
each step starting from the C terminus of the polypeptide or
peptide (See, Coligan, et al., Current Protocols in Immunology,
Wiley Interscience, 1991, Unit 9). Polypeptide and peptides of the
disclosure can also be synthesized by the well-known solid phase
peptide synthesis methods such as those described by Merrifield, J.
Am. Chem. Soc., 85:2149, 1962) and Stewart and Young, Solid Phase
Peptides Synthesis, Freeman, San Francisco, 1969, pp. 27 62). If
desired, the peptides can be quantitated by the solid phase Edman
degradation.
[0089] Using a synthesizer a hogocidin peptide (i.e., the mature
form of SEQ ID NO:2 or 4 can be generated specifically without the
need for post-translational processing.
[0090] The disclosure also includes isolated polynucleotides (e.g.,
DNA, cDNA, or RNA) encoding the polypeptide and peptides of the
disclosure. Included are polynucleotides that encode analogs,
mutants, conservative variations, and variants of the polypeptides
and peptides described herein. The term "isolated" as used herein
refers to a polynucleotide that is substantially free of proteins,
lipids, and other polynucleotides with which an in vivo-produced
polynucleotides naturally associated. Typically, the polynucleotide
is at least 70%, 80%, or 90% isolated from other matter, and
conventional methods for synthesizing polynucleotides in vitro can
be used in lieu of in vivo methods.
[0091] As used herein, "polynucleotide" refers to a polymer of
deoxyribonucleotides or ribonucleotides, in the form of a separate
fragment or as a component of a larger genetic construct (e.g., by
operably linking a promoter to a polynucleotide encoding a peptide
of the disclosure). Numerous genetic constructs (e.g., plasmids and
other expression vectors) are known in the art and can be used to
produce the peptides of the disclosure in cell-free systems or
prokaryotic or eukaryotic (e.g., yeast, insect, or mammalian)
cells. By taking into account the degeneracy of the genetic code,
one of ordinary skill in the art can readily synthesize
polynucleotides encoding the peptides of the disclosure. The
polynucleotides of the disclosure can readily be used in
conventional molecular biology methods to produce the peptides of
the disclosure.
[0092] DNA encoding the hogocidin peptides, derivatives of variants
thereof of the disclosure can be inserted into an "expression
vector." The term "expression vector" refers to a genetic construct
such as a plasmid, virus or other vehicle known in the art that can
be engineered to contain a polynucleotide encoding a polypeptide of
the disclosure. Such expression vectors are typically plasmids that
contain a promoter sequence that facilitates transcription of the
inserted genetic sequence in a host cell. The expression vector
typically contains an origin of replication, and a promoter, as
well as genes that allow phenotypic selection of the transformed
cells (e.g., an antibiotic resistance gene). Various promoters,
including inducible and constitutive promoters, can be utilized in
the disclosure. Typically, the expression vector contains a
replicon site and control sequences that are derived from a species
compatible with the host cell.
[0093] Transformation or transfection of a host cell with a
polynucleotide of the disclosure can be carried out using
conventional techniques well known to those skilled in the art. For
example, where the host cell is E. coli, competent cells that are
capable of DNA uptake can be prepared using the CaCl.sub.2,
MgCl.sub.2 or RbCl methods known in the art. Alternatively,
physical means, such as electroporation or microinjection can be
used. Electroporation allows transfer of a polynucleotide into a
cell by high voltage electric impulse. Additionally,
polynucleotides can be introduced into host cells by protoplast
fusion, using methods well known in the art. Suitable methods for
transforming eukaryotic cells, such as electroporation and
lipofection, also are known.
[0094] "Host cells" encompassed by of the disclosure are any cells
in which the polynucleotides of the disclosure can be used to
express the hogocidin peptides, derivatives or variants of the
disclosure. The term also includes any progeny of a host cell. Host
cells, which are useful, include bacterial cells, fungal cells
(e.g., yeast cells), plant cells and animal cells. For example,
host cells can be a higher eukaryotic cell, such as a mammalian
cell, or a lower eukaryotic cell, such as a yeast cell, or the host
cell can be a prokaryotic cell, such as a bacterial cell.
Introduction of the construct into the host cell can be effected by
calcium phosphate transfection, DEAE-Dextran mediated transfection,
or electroporation (Davis, L., Dibner, M., Battey, I., Basic
Methods in Molecular Biology (1986)). As representative examples of
appropriate hosts, there may be mentioned: fungal cells, such as
yeast; insect cells such as Drosophila S2 and Spodoptera Sf9;
animal cells such as CHO, COS or Bowes melanoma; plant cells, and
the like. The selection of an appropriate host is deemed to be
within the scope of those skilled in the art from the teachings
herein. In one embodiment, a host cell can comprise a bacterial
cell present in a normal bacterial flora of the skin that has been
engineered to express or over express a hogocidin peptide or other
antimicrobial peptide of the disclosure. These engineered bacterial
cells can then be used as a probiotic such that they are applied to
skin.
[0095] Host cells can be eukaryotic host cells (e.g., mammalian
cells). In one embodiment, the host cells are mammalian production
cells adapted to grow in cell culture. Examples of such cells
commonly used in the industry are CHO, VERO, BHK, HeLa, CV1
(including Cos; Cos-7), MDCK, 293, 3T3, C127, myeloma cell lines
(especially murine), PC12 and W138 cells. Chinese hamster ovary
(CHO) cells are widely used for the production of several complex
recombinant proteins, e.g. cytokines, clotting factors, and
antibodies (Brasel et al., Blood 88:2004 2012 (1996); Kaufman et
al., J. Biol Chem 263: 6352 6362 (1988); McKinnon et al., J Mol
Endocrinol 6:231 239 (1991); Wood et al., J. Immunol 145:3011 3016
(1990)). The dihydrofolate reductase (DHFR)-deficient mutant cell
lines (Urlaub et al., Proc Natl Acad Sci USA 77:4216 4220 (1980))
are the CHO host cell lines commonly used because the efficient
DHFR selectable and amplifiable gene expression system allows high
level recombinant protein expression in these cells (Kaufman, Meth
Enzymol 185:527 566 (1990)). In addition, these cells are easy to
manipulate as adherent or suspension cultures and exhibit
relatively good genetic stability. CHO cells and recombinant
proteins expressed in them have been extensively characterized and
have been approved for use in clinical manufacturing by regulatory
agencies.
[0096] Polynucleotides encoding the polypeptide and peptides of the
disclosure can be isolated from a cell (e.g., a cultured cell), or
they can be produced in vitro. A DNA sequence encoding a hogocidin
peptide of interest can be obtained by: 1) isolation of a
double-stranded DNA sequence from genomic DNA; 2) chemical
manufacture of a polynucleotide such that it encodes the hogocidin
peptide of interest; or 3) in vitro synthesis of a double-stranded
DNA sequence by reverse transcription of mRNA isolated from a donor
cell (i.e., to produce cDNA). Among the standard procedures for
isolating cDNA sequences of interest is the formation of plasmid or
phage containing cDNA libraries that are derived from reverse
transcription of mRNA in donor cells that have a high level of
genetic expression. When used in combination with polymerase chain
reaction technology, even rare gene products can be cloned.
[0097] Any of various art-known methods for protein purification
can be used to isolate the peptides of the disclosure. For example,
preparative chromatographic separations and immunological
separations (such as those employing monoclonal or polyclonal
antibodies) can be used. Carrier peptides can facilitate isolation
of fusion proteins that include the peptides of the disclosure.
Purification tags can be operably linked to a hogocidin peptide of
the disclosure. For example, glutathione-S-transferase (GST) allows
purification with a glutathione agarose affinity column. When
either Protein A or the ZZ domain from Staphylococcus aureus is
used as the tag, purification can be accomplished in a single step
using an IgG-sepharose affinity column. The pOprF-peptide, which is
the N-terminal half of the P. aeruginosa outer membrane protein F,
can readily be purified because it is the prominent protein species
in outer membrane preparations. If desired, the fusion peptides can
be purified using reagents that are specifically reactive with
(e.g., specifically bind) the hogocidin peptide of the fusion
peptide. For example, monoclonal or polyclonal antibodies that
specifically bind the hogocidin peptide can be used in conventional
purification methods. Techniques for producing such antibodies are
well known in the art.
[0098] A fusion construct comprising a polypeptide linked to a
hogocidin peptide of the disclosure can be linked at either the
amino or carboxy terminus of the peptide. Typically, the
polypeptide that is linked to the hogocidin peptide is sufficiently
anionic such that the hogocidin peptide has a net charge that is
neutral or negative. The anionic polypeptide can correspond in
sequence to a naturally occurring protein or can be entirely
artificial in design. Functionally, the polypeptide linked to a
hogocidin peptide (the "carrier polypeptide") may help stabilize
the hogocidin peptide and protect it from proteases, although the
carrier polypeptide need not be shown to serve such a purpose.
Similarly, the carrier polypeptide may facilitate transport of the
fusion peptide. Examples of carrier polypeptides that can be
utilized include anionic pre-pro peptides and anionic outer
membrane peptides. Examples of carrier polypeptides include
glutathione-S-transferase (GST), protein A of Staphylococcus
aureus, two synthetic IgG-binding domains (ZZ) of protein A, outer
membrane protein F of Pseudomonas aeruginosa, protein transduction
domains and the like. The disclosure is not limited to the use of
these polypeptides; others suitable carrier polypeptides are known
to those skilled in the art. In another aspect, a linker moiety
comprising a protease cleavage site may be operably linked to a
hogocidin peptide or variant of the disclosure. For example, the
linker may be operable between to domains of a fusion protein
(e.g., a fusion protein comprising a hogocidin peptide and a
carrier polypeptide). Because protease cleavage recognition
sequences generally are only a few amino acids in length, the
linker moiety can include the recognition sequence within flexible
spacer amino acid sequences, such as GGGGS (SEQ ID NO: 6). For
example, a linker moiety including a cleavage recognition sequence
for Adenovirus endopeptidase could have the sequence
GGGGGGSMFGGAKKRSGGGGGG (SEQ ID NO: 7). If desired, the spacer DNA
sequence can encode a protein recognition site for cleavage of the
carrier polypeptide from the hogocidin peptide. Examples of such
spacer DNA sequences include, but are not limited to, protease
cleavage sequences, such as that for Factor Xa protease, the
methionine, tryptophan and glutamic acid codon sequences, and the
pre-pro defensin sequence. Factor Xa is used for proteolytic
cleavage at the Factor Xa protease cleavage sequence, while
chemical cleavage by cyanogen bromide treatment releases the
peptide at methionine or related residues. In addition, the fused
product can be cleaved by insertion of a codon for tryptophan
(cleavable by o-iodosobenzoic acid) or glutamic acid (cleavable by
Staphylococcus protease). Insertion of such spacer DNA sequences is
not a requirement for the production of functional hogocidin
peptides, such sequences can enhance the stability of the fusion
peptide. The pre-pro defensin sequence is negatively charged, so
accordingly, it is envisioned within the disclosure that other DNA
sequences encoding negatively charged peptides also can be used as
spacer DNA sequences to stabilize the fusion peptide.
[0099] The disclosure also provides a method for inhibiting the
growth of a bacterium by contacting the bacterium with an
inhibiting effective amount of a peptide of the disclosure. The
term "contacting" refers to exposing the bacterium to the peptide
so that the peptide can inhibit, kill, or lyse bacteria. The
disclosure also provides a method for inhibiting skin disease or
disorder and/or bacterial infection comprising placing on or within
a subject a probiotic formulation comprising bacteria which secrete
a peptide or antimicrobial molecule such that the growth of the
pathogen or undesirable microbe is inhibited or prevented.
Contacting of an organism with a hogocidin peptide of the
disclosure can occur in vitro, for example, by adding the peptide
to a bacterial culture to test for susceptibility of the bacteria
to the peptide, or contacting a bacterially contaminated surface
with the peptide. Alternatively, contacting can occur in vivo, for
example by administering the peptide to a subject afflicted with a
bacterial infection or susceptible to infection. Further,
contacting can occur by exposing the bacterium to a probiotic
formulation comprising bacterial strains that produce the hogocidin
peptide, or other peptide or non-peptide inhibitors of bacterial
growth. In vivo contacting includes both parenteral as well as
topical. "Inhibiting" or "inhibiting effective amount" refers to
the amount of peptide that is sufficient to cause, for example, a
bacteriostatic or bactericidal effect. Bacteria that can be
affected by the peptides of the disclosure include both
gram-negative and gram-positive bacteria. For example, bacteria
that can be affected include Staphylococcus aureus, Streptococcus
pyogenes (group A), Streptococcus sp. (viridans group),
Streptococcus agalactiae (group B), S. bovis, Streptococcus
(anaerobic species), Streptococcus pneumoniae, and Enterococcus
sp.; Gram-negative cocci such as, for example, Neisseria
gonorrhoeae, Neisseria meningitidis, and Branhamella catarrhalis;
Gram-positive bacilli such as Bacillus anthracis, Bacillus
subtilis, P. acne Corynebacterium diphtheriae and Corynebacterium
species which are diptheroids (aerobic and anerobic), Listeria
monocytogenes, Clostridium tetani, Clostridium difficile,
Escherichia coli, Enterobacter species, Proteus mirablis and other
sp., Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella,
Shigella, Serratia sp., and Campylobacter jejuni. Infection with
one or more of these bacteria can result in diseases such as
bacteremia, pneumonia, meningitis, osteomyelitis, endocarditis,
sinusitis, arthritis, urinary tract infections, tetanus, gangrene,
colitis, acute gastroenteritis, impetigo, acne, acne posacue, wound
infections, born infections, fascitis, bronchitis, and a variety of
abscesses, nosocomial infections, and opportunistic infections.
Fungal organisms may also be affected by the hogocidin peptides of
the disclosure and include dermatophytes (e.g., Microsporum canis
and other Microsporum sp.; and Trichophyton sp. such as T. rubrum,
and T. mentagrophytes), yeasts (e.g., Candida albicans, C.
Tropicalis, or other Candida species), Saccharomyces cerevisiae,
Torulopsis glabrata, Epidermophyton floccosum, Malassezia furfur
(Pityropsporon orbiculare, or P. ovale), Cryptococcus neoformans,
Aspergillus fumigatus, Aspergillus nidulans, and other Aspergillus
sp., Zygomycetes (e.g., Rhizopus, Mucor), Paracoccidioides
brasiliensis, Blastomyces dermatitides, Histoplasma capsulatum,
Coccidioides immitis, and Sporothrix schenckii. The method for
inhibiting the growth of bacteria can also include contacting the
bacterium with the peptide in combination with one or more
antibiotics.
[0100] A peptide(s) of the disclosure can be administered to any
host, including a human or non-human animal, in an amount effective
to inhibit growth of a bacterium, virus, or fungus. Thus, the
peptides are useful as antimicrobial agents, antiviral agents,
and/or antifungal agents. The bacterial strains that produce the
peptides are useful as probiotic agents.
[0101] Any of a variety of art-known methods can be used to
administer the peptide to a subject. For example, the peptide of
the disclosure can be administered parenterally by injection or by
gradual infusion over time. The peptide can be administered
intravenously, intraperitoneally, intramuscularly, subcutaneously,
intracavity, topically or transdermally. In another embodiment, a
hogocidin peptide of the disclosure may be formulated for topical
administration (e.g., as a lotion, cream, spray, gel, oil
suspension, or ointment). Examples of formulations in the market
place include topical lotions, creams, soaps, wipes, powders,
devices like gauze pads to cover wounds, and the like. It may be
formulated into liposomes to reduce toxicity or increase
bioavailability or stability. Other methods for delivery of the
peptide include oral methods that entail encapsulation of the
peptide in microspheres or proteinoids, aerosol delivery (e.g., to
the lungs), or transdermal delivery (e.g., by iontophoresis or
transdermal electroporation). Other methods of administration will
be known to those skilled in the art.
[0102] Preparations for parenteral administration of a peptide of
the disclosure include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils (e.g., olive
oil), and injectable organic esters such as ethyl oleate. Examples
of aqueous carriers include water, saline, and buffered media,
alcoholic/aqueous solutions, and emulsions or suspensions. Examples
of parenteral vehicles include sodium chloride solution, Ringer's
dextrose, dextrose and sodium chloride, lactated Ringer's, and
fixed oils. Intravenous vehicles include fluid and nutrient
replenishers, electrolyte replenishers (such as those based on
Ringer's dextrose), and the like. Preservatives and other additives
such as, other antimicrobial, anti-oxidants, chelating agents,
inert gases and the like also can be included.
[0103] The disclosure provides a method for inhibiting a topical
bacterial or fungal-associated disorder by contacting or
administering a therapeutically effective amount of a peptide or
skin-probiotic of the disclosure to a subject who has, or is at
risk of having, such a disorder. The term "inhibiting" means
preventing or ameliorating a sign or symptoms of a disorder (e.g.,
a rash, sore, and the like). Examples of disease signs that can be
ameliorated include an increase in a subject's blood level of TNF,
fever, hypotension, neutropenia, leukopenia, thrombocytopenia,
disseminated intravascular coagulation, adult respiratory distress
syndrome, shock, and organ failure. Examples of subjects who can be
treated in the disclosure include human or animal subjects at risk
for, or those suffering from, a toxemia, such as endotoxemia
resulting from a gram-negative bacterial infection, venom
poisoning, or hepatic failure. Other examples include subjects
having a dermatitis as well as those having skin infections such as
mastitis and especially bovine mastits, or injuries subject to
infection with gram-positive or gram-negative bacteria or a fungus.
Examples of candidate patients include those suffering from
infection by E. coli, Hemophilus influenza B, Neisseria
meningitides, staphylococci, or pneumococci. Other patients include
those suffering from gunshot wounds, renal or hepatic failure,
trauma, burns, immunocompromising infections (e.g., HIV/SIV/FIV
infections), hematopoietic neoplasias, multiple myeloma,
Castleman's disease or cardiac myxoma. Those skilled in the art of
medicine can readily employ conventional criteria to identify
appropriate subjects for treatment in accordance with the
disclosure.
[0104] The term "therapeutically effective amount" as used herein
for treatment of a subject afflicted with a disease or disorder
means an amount of hogocidin peptide sufficient to ameliorate a
sign or symptom of the disease or disorder. For example, a
therapeutically effective amount can be measured as the amount
sufficient to decrease a subject's symptoms of dermatitis or rash
by measuring the frequency of severity of skin sores. Typically,
the subject is treated with an amount of hogocidin peptide
sufficient to reduce a symptom of a disease or disorder by at least
50%, 90% or 100%. Generally, the optimal dosage of the peptide will
depend upon the disorder and factors such as the weight of the
patient, the type of bacterial or fungal infection, the weight,
sex, and degree of symptoms. Nonetheless, suitable dosages can
readily be determined by one skilled in the art. Typically, a
suitable dosage is 0.5 to 40 mg peptide/kg body weight, e.g., 1 to
8 mg peptide/kg body weight.
[0105] If desired, a suitable therapy regime can combine
administration of a peptide(s) or probiotic composition of the
disclosure with one or more additional therapeutic agents (e.g., an
inhibitor of TNF, an antibiotic, and the like). The peptide(s),
other therapeutic agents, and/or antibiotic(s) can be administered,
simultaneously, but may also be administered sequentially. Suitable
antibiotics include aminoglycosides (e.g., gentamicin),
beta-lactams (e.g., penicillins and cephalosporins), quinolones
(e.g., ciprofloxacin), and novobiocin. Generally, the antibiotic is
administered in a bactericidal amount. However, the peptide
provides for a method of increasing antibiotic activity. Typically,
the hogocidin peptide and antibiotic are administered within 48
hours of each other (e.g., 2 to 8 hours, or may be administered
simultaneously). A "bactericidal amount" is an amount sufficient to
achieve a bacteria-killing blood concentration in the subject
receiving the treatment. In accordance with its conventional
definition, an "antibiotic," as used herein, is a chemical
substance that, in dilute solutions, inhibits the growth of, or
kills microorganisms. Also encompassed by this term are synthetic
antibiotics (e.g., analogs) known in the art.
[0106] The peptides of the disclosure can be used, for example, as
preservatives or sterilants of materials susceptible to microbial
or viral contamination. For example, the peptides can be used as
preservatives in processed foods (e.g., to inhibit organisms such
as Salmonella, Yersinia, Listeria and Shigella). If desired, the
peptides can be used in combination with antibacterial food
additives, such as lysozymes. The peptides and/or probiotics of the
disclosure also can be used as a topical agent, for example, to
inhibit Pseudomonas or Streptococcus or kill odor-producing
microbes (e.g., Micrococci). The optimal amount of a hogocidin
peptide of the disclosure for any given application can be readily
determined by one of skill in the art.
[0107] The hogocidins and/or probiotics of the disclosure are also
useful in promoting wound repair and tissue regeneration. Matrix
metalloproteinases (MMPS) are inflammatory enzymes that degrade
proteins in various tissues. Recent scientific research has shown
elevated levels of proteases (e.g., MMPs) in chronic wound exudate,
the fluid that bathes the wound bed. These excess proteases cause
degradation of important extracellular matrix proteins and
inactivation of vital growth factors that are essential in the
wound healing process. This may contribute to a sub-optimal healing
environment resulting in delayed wound healing.
[0108] Compositions provided herein can be used concurrently with
other antibacterial agents including sulfa drugs such as
sulfamethizole, sulfisoxazole, sulfamonomethoxine, sulfamethizole,
salazosulfapyridine, silver sulfadiazine and the like; quinoline
antibacterial agents such as nalidixic acid, pipemidic acid
trihydrate, enoxacin, norfloxacin, ofloxacin, tosufloxacin
tosilate, ciprofloxacin hydrochloride, lomefloxacin hydrochloride,
sparfloxacin, fleroxacin and the like; antiphthisics such as
isoniazid, ethambutol (ethambutol hydrochloride), p-aminosalicylic
acid (calcium p-aminosalicylate), pyrazinamide, ethionamide,
protionamide, rifampicin, streptomycin sulfate, kanamycin sulfate,
cycloserine and the like; antiacidfast bacterium drugs such as
diaphenylsulfone, rifampicin and the like; antiviral drugs such as
idoxuridine, acyclovir, vidarabine, ganciclovir and the like;
anti-HIV agents such as zidovudine, didanosine, zalcitabine,
indinavir sulfate ethanolate, ritonavir and the like;
antispirocheteles; antibiotics such as tetracycline hydrochloride,
ampicillin, piperacillin, gentamicin, dibekacin, kanendomycin,
lividomycin, tobramycin, amikacin, fradiomycin, sisomycin,
tetracycline, oxytetracycline, rolitetracycline, doxycycline,
ampicillin, piperacillin, ticarcillin, cephalothin, cephapirin,
cephaloridine, cefaclor, cephalexin, cefroxadine, cefadroxil,
cefamandole, cefotoam, cefuroxime, cefotiam, cefotiam hexetil,
cefuroxime axetil, cefdinir, cefditoren pivoxil, ceftazidime,
cefpiramide, cefsulodin, cefinenoxime, cefpodoxime proxetil,
cefpirome, cefozopran, cefepime, cefsulodin, cefinenoxime,
cefinetazole, cefminox, cefoxitin, cefbuperazone, latamoxef,
flomoxef, cefazolin, cefotaxime, cefoperazone, ceftizoxime,
moxalactam, thienamycin, sulfazecin, aztreonam or a salt thereof,
griseofulvin, lankacidin-group and the like.
[0109] In humans, there are several classes of known antimicrobial
peptides (AMPs) including .alpha.-defensins, .beta.-defensins, and
cathelicidins. Cathelicidins are found in several mammalian
species. Production of cathelicidins is induced in response to
epithelial wounding or infectious challenge, or suppressed by the
virulence mechanisms of certain bacterial pathogens, e.g., Shigella
dysenteriae. Cathelicidin expression is also differentially
effected in certain chronic inflammatory disorders. In psoriasis,
cathelicidin levels are elevated and secondary infection is rare,
whereas in atopic dermatitis cathelicidin expression is deficient
and bacterial or viral superinfection is common. Therapeutic
benefits of cathelicidin have been demonstrated experimentally,
including decreased bacterial colonization of skin wounds following
topical administration and improved pulmonary bacterial clearance
with cathelicidin overexpression through viral gene transfer. The
hogocidin peptides of the disclosure show a synergistic effect with
cathelicidins. Thus, in some embodiments a formulation, composition
and method comprise both a hogocidin and cathelicidin. In some
embodiments, a topical formulation (e.g., a lotion, ointment or
aerosol spray) can comprise both a cathelicidin and hogocidin
peptide (or derivatives thereof).
[0110] Cathelicidin proteins are composed of two distinct domains:
an N-terminal "cathelin-like" or "prosequence" domain and the
C-terminal domain of the mature AMP. The C-terminal domains of
cathelicidins were among the earliest mammalian AMPs to show
potent, rapid, and broad-spectrum killing activity. The term
"cathelin-like" derives from the similarity of the N-terminal
sequence with that of cathelin, a 12 kDa protein isolated from
porcine neutrophils that shares similarity with the cystatin
superfamily of cysteine protease inhibitors.
[0111] Cathelicidins are expressed in neutrophils and myeloid bone
marrow cells and most epithelial sources, and were the first AMPs
discovered in mammalian skin due to their presence in wound fluid.
In the neutrophil, cathelicidins are synthesized as full length
precursor and targeted to the secondary granules where they are
stored. Upon stimulation, the full-length cathelicidin protein is
proteolytically processed to unleash the microbiocidal activity of
the C-terminal peptide from the cathelin-like domain.
[0112] The C-terminal 37 amino acids of human cathelicidin (LL-37)
has been characterized. LL-37 was originally referred to as FALL39,
named for the first 4 N-terminal amino acids of this domain and the
total number of residues (i.e., 39). LL-37 is a peptide predicted
to contain an amphipathic alpha helix and lacks cysteine, making it
different from all other previously isolated human peptide
antibiotics of the defensin family, each of which contain 3
disulfide bridges. Full length human cathelicidin (sometimes
referred to as full length LL-37) comprises the cathelin-like
precursor protein and the C-terminal LL-37 peptide, thus comprising
170 amino acids (SEQ ID NO:5).
[0113] The polypeptide comprising SEQ ID NO:5 has a number of
distinct domains. For example, a signal domain comprising a
sequence as set forth from about 1 to about 29-31 of SEQ ID NO:5 is
present. The signal domain is typically cleaved following amino
acid number 30 of SEQ ID NO:5, however, one of skill in the art
will recognize that depending upon the enzyme used, the expression
system used and/or the conditions under which proteolytic cleavage
of the polypeptide takes place, the cleavage site may vary from 1
to 3 amino acid in either direction of amino acid number 30 of SEQ
ID NO:5. Another domain comprises the N-terminal domain, referred
to as the cathelin-like domain. The cathelin-like domain comprises
from about amino acid 29 (e.g., 29-31) to about amino acid 128
(e.g., 128-131) of SEQ ID NO:5. Yet another domain of SEQ ID NO:5
comprises the C-terminal domain referred to as LL-37. The LL-37
domain comprises from about amino acid 128 (e.g., 128-134) to amino
acid 170 of SEQ ID NO:5. LL-37 comprises the amino acid sequence
set forth in SEQ ID NO:5.
TABLE-US-00001 (SEQ ID NO: 5)
MKTQRNGHSLGRWSLVLLLLGLVMPLAIIAQVLSYKEAVLRAIDGINQR
SSDANLYRLLDLDPRPTMDGDPDTPKPVSFTVKETVCPRTTQQSPEDCD
FKKDGLVKRCMGTVTLNQARGSFDISCDKDNKRFALLGDFFRKSKEKIG
KEFKRIVQRIDDFLRNLVPRTES
[0114] The mechanisms by which cationic human antimicrobial
peptides kill bacteria and fungi are generally through binding of
the peptide to the microbial cell membrane, after which the
membrane's proton gradient and integrity are lost.
[0115] Vitamin D3 (or its analogs) with hogocidin (and in some
embodiments in combination with a cathelicidin) can be administered
systemically to treat systemic infections, in particular pneumonia,
sepsis and TB. It may also be applied topically to treat infectious
skin disorders. It may be used in combination therapy with
antibiotics or to treat immunocompromised patients such as HIV
positive individuals. In combination with immune stimulating
approaches, it may therapeutically address cancer.
[0116] The compositions and methods of the disclosure may also
comprise treating disorders of skin dysbiosis by administration of
an antimicrobial compound or an organism secreting an antimicrobial
compound, or administration of a probiotic composition comprising
organisms that support skin health. In some embodiments, the
composition includes a second active agent (e.g., an antibiotic,
vitamin D3, cathelicidin etc.).
[0117] In some embodiments, the compositions described herein
comprise a probiotic organism. In further embodiments, the
probiotic organism is a bacterium. In further embodiments, the
bacterium comprises a component of the normal skin flora. In
further embodiments, the bacterium comprises a strain of
Staphylococcus hominis. In other embodiments, the bacterium
comprises a strain of Staphylococcus epidermidis. In other
embodiments, the probiotic organism comprises a mixture of strains.
In some embodiments, the mixture of strains comprises multiple
strains of S. hominis. In other embodiments, the mixture of strains
comprises multiple strains of S. epidermidis. In other embodiments,
the mixture of strains comprises one or more strains of S. hominis
and one or more strains of S. epidermidis. In some embodiments, the
composition comprises one or more strains in addition to S. hominis
and/or S. epidermidis. In some further embodiments, the additional
strain or strains comprise one or more strains from the genus
Staphylococcus, Lactobacillus or Lactococcus. For example, specific
formulations may comprise Staphylococcus hominis or Staphylococcus
epidermidis, in particular, Staphylococcus hominis strain A9,
Staphylococcus hominis strain C2, Staphylococcus hominis strain
AMT2, Staphylococcus hominis strain AMT3, Staphylococcus hominis
strain AMT4-C2, Staphylococcus hominis strain AMT4-G1,
Staphylococcus hominis strain AMT4-D12, Staphylococcus epidermidis
strain AMT1, Staphylococcus epidermidis strain SE-A11,
Staphylococcus epidermidis strain AMT5-C5, and/or Staphylococcus
epidermidis strain AMT5-G6. Such formulations typically comprise
sufficient quantities of bacterial cells as to provide a final
density of 10.sup.3-10.sup.6 CFU/cm.sup.2 when applied to the skin
of a subject. Such formulations may comprise concentrations of from
about 10.sup.4 to about 10.sup.7 CFU/g, or alternatively, from 10
to about 10.sup.5 CFU/g, or alternatively, from about 10.sup.5 to
about 10.sup.9 CFU/g. Such formulations may comprise multiple
strains of S. hominis and/or S. epidermidis, and may further
comprise Lactococcus lactis, Lactobacillus plantarum, Lactobacillus
rhamnosus, Lactobacillus acidophilus, and/or other such species or
strains as are known in the art to form a part of the normal
healthy cutaneous or mucosal flora. In some embodiments, S. hominis
strains as described above comprise 100% of the bacterial cells in
a formulation. In some further embodiments, S. hominis comprises
90-100%, 85-95%, 70-80%, 75-85%, 60-70%, 65-75%, 50-60%, 55-65%,
40-50%, 45-55%, 30-40%, 35-45%, 20-30%, 25-35%, 10-20%, 15-20%,
1-10%, 5-15%, or less than 1% of the bacterial cells in a given
formulation, wherein the remainder of the colony forming units are
provided by S. epidermidis, Lactococcus lactis, Lactobacillus
plantarum, Lactobacillus rhamnosus, Lactobacillus acidophilus,
and/or other such strains as are known in the art to form a part of
the normal healthy cutaneous or mucosal flora. In some embodiments,
S. epidermidis strains as described above comprise 100% of the
bacterial cells in a formulation. In some further embodiments, S.
epidermidis comprises 90-100%, 85-95%, 70-80%, 75-85%, 60-70%,
65-75%, 50-60%, 55-65%, 40-50%, 45-55%, 30-40%, 35-45%, 20-30%,
25-35%, 10-20%, 15-20%, 1-10%, 5-15%, or less than 1% of the
bacterial cells in a given formulation, wherein the remainder of
the colony forming units are provided by S. hominis, Lactococcus
lactis, Lactobacillus plantarum, Lactobacillus rhamnosus,
Lactobacillus acidophilus, and/or other such strains as are known
in the art to form a part of the normal healthy cutaneous or
mucosal flora. In some embodiments, bacteria other than S. hominis
or S. epidermidis comprise about 50% or less of the bacterial cells
in the formulation. In some embodiments said bacteria comprise less
than 50%, less than 40%, less than 30%, less than 20%, less than
10%, less than 5%, or less than 1% of the bacterial cells within a
given formulation. In some embodiments, bacteria other than S.
hominis or S. epidermidis may comprise Lactococcus lactis,
Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus
acidophilus, and/or other such species or strains as are known in
the art to form a part of the normal healthy cutaneous or mucosal
flora. In some embodiments, the formulations comprise about 60% S.
hominis of the strains listed above and about 40% S. epidermidis of
the strains listed above. In some embodiments, the formulations
comprise about 50% S. hominis of the strains listed above and about
50% S. epidermidis of the strains listed above. In some
embodiments, the formulations comprise about 40% S. hominis of the
strains listed above and about 60% S. epidermidis of the strains
listed above. In some embodiments, the formulations comprise about
70% S. hominis of the strains listed above and about 30% S.
epidermidis. In some embodiments, the formulations comprise about
30% S. hominis of the strains listed above and about 70% S.
epidermidis of the strains listed above. In some embodiments, the
formulations comprise about 80% S. hominis of the strains listed
above and about 20% S. epidermidis of the strains listed above. In
some embodiments, the formulations comprise about 20% S. hominis of
the strains listed above and about 80% S. epidermidis of the
strains listed above. In some embodiments, the formulations
comprise about 90% S. hominis of the strains listed above and about
10% S. epidermidis of the strains listed above. In some
embodiments, the formulations comprise greater than about 90% S.
hominis of the strains listed above and less than about 10% S.
epidermidis of the strains listed above. In some embodiments, the
formulations comprise less than about 10% S. hominis of the strains
listed above and greater than about 90% S. epidermidis of the
strains listed above.
[0118] As used herein, an autologous transplant refers to the
transplantation of bacterial strains from one site to another on
the same subject or to the same site, regardless of whether the
strains are cultured prior to administration or not. In some
embodiments, the bacterial strains obtained from the subject are
expanded in culture and then transplanted back to the subject.
[0119] As used herein, an allogeneic transplant refers to the
transplantation of bacterial strains from one subject to another
subject, or to the administration to a subject of a composition
comprising bacterial strains that were not collected from upon or
within their own body.
[0120] Such collection can be carried out by swabbing, scraping,
wiping, or cutting and removing tissue on which resides one of the
bacterial strains as described herein; optionally growing and
isolating single colonies from agar plates or otherwise using
methods known in the art; optionally growing expanded cultures of
the isolated bacteria or crude swabs, wipes, scrapes, tissue, or
other isolate in liquid or solid culture according to methods known
in the art; optionally harvesting bacteria from said expanded
culture by centrifugation, filtration, gravity settling, scraping,
or by other means known in the art; formulating the bacteria or the
crude isolate with a thickener, carrier, or excipient; and
contacting the subject in an area determined to be in need of the
transplant, with said formulation.
[0121] As used herein, a prebiotic compound comprises a
polysaccharide, hydrolysate, salt, herbal extract, or any other
compound sufficient to foster the growth of an associated probiotic
strain when used in combination with that strain, such as yeast
hydrolysate in concentrations of less than about 40% (w/w),
microcrystalline cellulose in concentrations of less than about 10%
(w/w), and/or sucrose in concentrations of less than about 10%
(w/w). Other examples of prebiotics that may be adapted for use
with cutaneous bacteria include inulin, glucooligosaccharides,
isomaltooligosaccharides, lactosucrose, polydextrose, soybean
oligosaccharides, and xylooligosaccharides, and those disclosed in
Gibson, G. R. and Roberfroid, M, (Eds.) Handbook of Prebiotics, CRC
press (2008); Roberfroid, M., J. Nutr. 137(3):830S-837 (2007) and
Slavin, J. Nutrients 5(4):1417-1435 (2013), each of which is
incorporated herein by reference in its entirety.
[0122] In some embodiments the method comprises contacting a
subject with a probiotic and/or prebiotic composition as described
herein. In some embodiments, such contact comprises an autologous
transplant. In some embodiments such contact comprises an
allogeneic transplant, wherein elements of the cutaneous or mucosal
flora are transplanted to a first subject in need thereof from a
second subject (the donor). For example, in some embodiments,
bacterial strains as disclosed above are identified and isolated
from a second subject, amplified in an appropriate culture medium
under such conditions as are known in the art to be conducive to
bacterial growth, followed by harvest of the bacterial cells,
mixing of the harvested cells at a predetermined concentration
according to the disclosure with a predetermined formulation, and
application of the mixture to the affected area of the first
subject. In some embodiments such composition comprises a
standardized formulation, such as a formulation in which the
concentrations of ingredients are fixed and are not varied from
subject to subject. In some embodiments, the formulation is
developed individually for each subject, based on criteria
including but not limited to: the composition of the subject's own
cutaneous or mucosal flora; the subject's disease state and
treatment history; the nature and severity of the subject's
condition; the nature and severity of concurrent cutaneous or
mucosal infections; the presence of other antimicrobial compounds
including systemic antibiotics within the subject's body; and other
criteria such as are known to or would readily be apparent to those
of skill in the art.
[0123] In some embodiments, the composition comprises a cream,
ointment, oil suspension or unguent wherein the probiotic bacteria
as described above are incorporated within a moisturizer or
emulsion such as those described below and in Nakatsuji, T. et al.
(2016), Nature Medicine Submitted Manuscript No. NMED-A78395A,
submitted Mar. 29, 2016. In some embodiments, the composition
comprises a patch or poultice wherein the bacteria are combined
with a suitable excipient and are incorporated within a fabric, gel
matrix, or polymer sheet. Suitable excipients and carriers for
topical administration are known in the art and include thickeners,
emulsifiers, fatty acids, polysaccharides, polyols, and polymers
and copolymers, including, without limitation, alginate,
microcrystalline cellulose, polylactic acid, polylactic-co-glycolic
acid, petrolatum, and numerous others known in the art.
[0124] In some embodiments, the composition comprises a bacterial
culture medium, a conditioned bacterial culture medium, and/or a
bacterial culture. In some embodiments, the composition comprises a
filtrate or supernatant of a bacterial culture medium. In some
embodiments, the composition comprises a lyophilized culture
medium. In some embodiments, the composition comprises a
lyophilized conditioned culture medium produced from a filtrate or
supernatant of a bacterial culture medium.
[0125] In some embodiments, the method as described herein
comprises supporting the health of the skin of a subject. In
further embodiments, the method comprises providing a treatment for
skin dysbiosis and disorders derived therefrom. In some embodiments
the method comprises providing a treatment for bacterial infection
of the skin. In some embodiments, the treatment comprises the steps
of: identifying a subject with skin dysbiosis, bacterial infection,
mastitis, burn or other wound, atopic dermatitis, psoriasis, or
other chronic skin condition; and administering to the site of the
condition in need of treatment the probiotic compositions as
disclosed herein. Determination of the appropriate mode of
administration of a given formulation (ointment, gel, patch, etc.)
can be done by one of ordinary skill in the art of treating skin
infections. In some further embodiments, the probiotic compositions
are re-applied at regularly timed intervals. In some embodiments,
the probiotic compositions are reapplied every three days. In some
embodiments, the probiotic compositions are reapplied every two
days. In some embodiments, the probiotic compositions are reapplied
every two days. In some embodiments, the probiotic compositions are
reapplied daily. In some embodiments, the probiotic compositions
are reapplied more than once per day. In some embodiments, the
probiotic compositions are reapplied weekly. In some embodiments,
the probiotic compositions are only applied a single time.
[0126] In some embodiments, the method comprises providing a
treatment for Staphylococcus aureus, including methicillin or
oxacillin resistant S. aureus, infections. In some further
embodiments, the method comprises the steps of: diagnosing an S.
aureus infection; and applying to the site of the infection the
probiotic and/or prebiotic compositions as disclosed herein,
wherein such compositions are capable of killing or inhibiting the
growth of S. aureus, either by production of antimicrobial
compounds, by competition for resources within the cutaneous or
mucosal biota, or by other means. Determination of the appropriate
mode of administration of a given formulation (ointment, gel,
patch, etc.) can be done by one of ordinary skill in the art of
treating skin infections. In some further embodiments, the
probiotic compositions are re-applied at regularly timed intervals
(e.g., daily, every two days, every three days, weekly, etc.). It
will be apparent to one of ordinary skill in the art that in other
embodiments, similar or identical steps can be applied to provide a
treatment for Pseudomonas aeruginosa infections or infections
derived from bacteria of the genus Pseudomonas, Staphylococcus,
Propionibacterium, Streptococcus, or Vibrio, or uncharacterized
pathogens. In some embodiments, the method comprises providing a
treatment for infections with unknown or uncharacterized pathogens.
In some embodiments, the method comprises providing a treatment for
polymicrobial infections. In some embodiments, the method comprises
administering such treatment to a burn or wound. In some
embodiments, the method comprises providing a treatment for a
chronic skin condition. In some embodiments, the condition is
atopic dermatitis, psoriasis, or other chronic skin condition.
[0127] The following examples are intended to illustrate but not
limit the disclosure. While they are typical of those that might be
used, other procedures known to those skilled in the art may
alternatively be used.
EXAMPLES
Example 1
Isolation of CoNS Strains and Antimicrobial Peptides
[0128] Adult patients with atopic dermatitis and age-matched
nonatopic subjects were recruited. Demographic data are shown in
Table 1. All experiments involving human subjects were carried out
according to the IRB protocols approved by the facility.
TABLE-US-00002 TABLE 1 Clinical characteristics of atopic
dermatitis and nonatopic subjects. Atopic subjects Non-atopic
subjects (N = 50)* (N = 30) Facility (%) 1 54.00 63.33 2 46.00
36.67 Age Mean .+-. SD 33.80 .+-. 2.02 33.87 .+-. 1.49 BMI Mean
.+-. SD 25.10 .+-. 1.55 23.40 .+-. 1.15 Not recorded (%) 30.00
63.33 Gender (%) Male 42.00 56.67 Female 58.00 43.33 Race (%)
Caucasian 50.00 60.00 Asian 32.00 33.33 Hispanic 6.00 6.67
African-American 8.00 0.00 Other 4.00 0.00 EASI Score <=6 26.00
N/A (%) >6 48.00 N/A Not recorded 26.00 N/A *No live
Staphylococcus and Staphylococcus DNA were detected in 1 subject
with atopic dermatitis out of 50 patients recruited. Therefore the
data are reported for 49 atopic subjects. BMI, body mass index;
EASI, eczema area and severity index; NJH, National Jewish Health;
SD, standard deviation
[0129] Measurements of Bacterial Abundance.
[0130] Collection of live surface bacteria and bacterial DNA was
done from a pre-measured area (.about.3.times.10 cm) of lesional
skin on the antecubital fossa, and nonlesional skin of the upper
arm at least 2 cm separated from the lesional site. Similar
collections were obtained from non-atopic subjects at identical
skin sites. Skin was rubbed with swabs pre-moistened with tryptic
soy broth (TSB) for collecting live bacteria or with Tris-EDTA
buffer for collecting bacterial DNA. Live bacteria samples were
inoculated on a mannitol salt agar with egg yolk to distinguish
coagulase negative staphylococcus (CONS). Total genomic DNA was
extracted with QIAamp DNA micro kit (Qiagen) and DNA abundance
determined by quantitative real-time PCR (qPCR) with species- or
genus-specific primers. No live Staphylococcus and Staphylococcus
DNA were detected in 1 subject with atopic dermatitis out of 50
patients recruited. Therefore data are reported for 49 AD
subjects.
[0131] Quantification of Bacterial DNA.
[0132] To collect bacterial DNA, pre-measured areas similar to
those used for bacterial culture were rubbed with a swab
pre-moistened with Tris-EDTA buffer containing 0.1% TritonX-100 and
0.05% Tween-20 (w/v). Bacterial cells were lysed by proteinase K,
followed by purified achromopeptidase (Wako Chemical) and
Ready-Lyse.RTM. (epicenter Inc.). Total genomic DNA was purified
with QIAamp DNA micro kit (Qiagen) and eluted with 50 .mu.L of
elution buffer. The abundance of bacterial DNA was determined by
quantitative real-time PCR (qPCR) with species- or genus-specific
primers (Table 2). To determine relative CFU (rCFU) of
Staphylococcus spp. DNA, a standard curve was generated with
genomic DNA extracted from standards derived from known CFU of S.
epidermidis (ATCC12228). The specificity of all primer pairs was
confirmed by melting curve analysis and comparison with standard
curves.
TABLE-US-00003 TABLE 2 Sequences of PCR primers References or
Primer name Sequence (5'-3') Target gene accession# S. aureus-
AACTGTTGGCCACTATGAGT S. aureus- femA-2F (SEQ ID NO: 8) specific S.
aureus- CCAGCATTACCTGTAATCTCG sequence femA-2R (SEQ ID NO: 9) S.
epidermidis- TCAGCAGTTGAAGGACAGAT S. epidermidis- sodA-F (SEQ ID
NO: 10) specific S. epidermidis- CCAGAACAATGAATGGTTAAGG sequence
sodA-R (SEQ ID NO: 11) g-Staph-F TTTGGGCTACACACGTGCTACAATGGACAA
Staphylococcus- (SEQ ID NO: 12) genus specific g-Staph-R
AACAACTTTATGGGATTTGCWTGA 16S sequence (SEQ ID NO: 13) Univ16S-
AGAGTTTGGATCMTGGCTCAG Universal 27-F (SEQ ID NO: 14) sequence of
Univ16S- AAGGAGGTGWTCCARCC bacterial 16S 1525-R (SEQ ID NO: 15)
rRNA S. epidermidis- GATTCAGGAGCTGAACCAAGA S. epidermidis X07840
epiA-F (SEQ ID NO: 16) epidermin S. epidermidis-
TTGAAGCCCTGCCAATCTAA epiA-R (SEQ ID NO: 17) S. epidermidis-
CTGATGAACTTGAACCTCAAACTG S. epidermidis L23967 pepA-F (SEQ ID NO:
18) PeP5 S. epidermidis- GACACTGTAAATAAACGCGTAGC pepA-R (SEQ ID NO:
19) S. epidermidis- GCAACTAGACAGGTATGTCCTAAA S. epidermidis Y14023
eciA-F (SEQ ID NO: 20) epicidin280 S. epidermidis-
CATCTAAGATTAAATGAGGGTGGTT eciA-R (SEQ ID NO: 21) S. epidermidis-
TAAGTCCGCAATCTGCTAGTG S. epidermidis U20348 elkA-F (SEQ ID NO: 22)
epilancin K7 S. epidermidis- CAGTAATATTGCAACCGCATGT elkA-R (SEQ ID
NO: 23) Hogocidin- ATGAGTAAATTAGAACTACTTAATG S. hominis This
.alpha.-F (SEQ ID NO: 24) Hogocidin-.alpha. study Hogocidin-
TTATAAATTACATCCTGCTGCACAC .alpha.-R (SEQ ID NO: 25)
[0133] Screening for Antimicrobial Activity.
[0134] Up to 84 individual colonies of CoNS isolates from each skin
site were randomly picked and transferred to a 96-well cluster tube
containing TSB. Each plate also received a non-antimicrobial strain
of S. epidermidis (ATCC1457) as negative control, a known
antimicrobial strain of Staphylococcus hominis (see below) as
positive control, and blank wells without bacteria. CoNS were
cultured at 37.degree. C. overnight with shaking. Growth was
evaluated by OD.sub.600. Bacteria were removed by centrifugation
followed by sterile filtration with a 0.22 .mu.m membrane. The
antimicrobial activity released from each colony was evaluated by
mixing with 1.times.10.sup.4 colony-forming units (CFU) of S.
aureus (ATCC35556). Antimicrobial strains were defined as those
that suppressed S. aureus growth after 22 hrs to less than 50%
(I.sub.50) of growth seen in negative controls. Insufficient CoNS
colonies were grown from some of the subjects recruited. Therefore
data are reported for 29 non-atopic subjects and 41 nonlesional and
40 lesional sites of atopic subjects. All CoNS isolates were stored
frozen for species identification. Full-length 16S rRNA genes were
amplified from 48 representative colonies with universal 16S
primers, 27-F and 1525-R. Amplicons were sequenced from both ends
by Sanger method.
[0135] Purification of AMPs Produced by S. hominis.
[0136] Sterile conditioned media from a representative
antimicrobial S. hominis strain isolated from a healthy subject was
used to further identify molecules with antimicrobial activity on
normal skin that were in low abundance on atopics. Activity was
precipitated by ammonium sulfate (70% saturation), dissolved in
H.sub.2O and applied on a Sep-Pak cartridge (Waters Co). Active
fractions were eluted with 30% acetonitrile in H.sub.2O and
subjected to HiTrap.RTM. SP (GE Healthcare Life Sciences)
separation and activity eluted at 125 mM NaCl. Third step HPLC
purification was done with CapCel Pak C8 (5 .mu.m, 300 .ANG.,
4.6.times.250 mm) (Shiseido Co.) with a linear gradient of
acetonitrile from 5% to 50% in 0.1% (v/v) TFA at 0.8 mL/min.
[0137] Identification of AMPs Produced by S. hominis.
[0138] Antimicrobial activity was purified from sterile conditioned
media of a representative antimicrobial S. hominis strain isolated
from a non-atopic subject. The secondary structure of the purified
active molecules was determined by MALDI-TOF/TOF, Edman terminal
sequencing, and genome sequencing.
[0139] Mass Spectrometry.
[0140] Mass spectra of HPLC-purified AMPs from S. hominis were
recorded using a MALDI-TOF/TOF Bruker Autoflex.TM. Speed instrument
(Bruker Daltonics) controlled by the flexcontrol software (Bruker
Daltonics). Mass spectrometric analyses were performed in positive
ion reflectron mode using cyano-4-hydroxycinnamic acid as a matrix
(CHCA) 10 mg/mL (Sigma-Aldrich) dissolved in 50% acetonitrile (ACN)
and 0.1% trifluoroacetic acid (TFA). Full scan mass spectra were
acquired in positive ion reflectron mode for mass range 1000-4000
m/z. Each mass spectrum is the result of 750 averaged laser shots
with the laser intensity set around 65% of full laser intensity and
a detector gain enhanced at 8.times.4 GS/s (as selected within the
Bruker Flex Control software). MALDI-MS/MS spectra of manually
selected ion m/z 3547, with a window range of 5 Da, were acquired
using TOF/TOF collision-induced dissociation. Each MS/MS spectrum
is the result of 1000 averaged laser shots with the laser intensity
set around 60% selected within the software and a detector gain
enhanced at 10.times.4 GS/s. Resulting mass spectra were analyzed
using flex analysis software (Bruker Daltonics). Spectra were
calibrated to PepMix internal standard solutions.
[0141] N-Terminal Protein Sequencing.
[0142] The N-terminal amino acid sequence of purified
Hogocidin-.alpha. (Fraction 30, FIG. 7A) was analyzed by 15 cycles
of Edman degradation on Procise.RTM. 494HT Protein Sequence system
(Applied Biosystems). A predicted mature form of Hogocidin-.alpha.
is shown in FIG. 5A.
[0143] Protein Sequencing by MALDI-TOF/TOF Analysis.
[0144] Because the N-terminal region of Hogocidin-.beta. from S.
hominis (Fraction 32, FIG. 8) contains modified amino acids,
sequence could not be obtained by Edman degradation. Therefore,
whole protein sequence of this AMP was obtained based on genome
guided MALDI-TOF/TOF analysis. The analysis of nucleotide sequence
of S. hominis was performed on antibiotics and secondary metabolite
analysis shell--AntiSMASH platform in order to identify secondary
metabolites biosynthesis gene clusters. AntiSMASH results provided
one gene cluster for lantipeptides with a potential candidate at
locus 2050-2250 (FIG. 9A). NCBI BlastP analyses of genes involved
in synthesis and modification show high homology to the class 2
lantipeptide. A core peptide with sequence
ATPTITTSSATCGGIIVAASAAQCPTLACSSRCGKRKK (SEQ ID NO:4 from aa 29 to
66) cleaved from leader peptide with a GG cleavage site, common for
type 2 lantibiotics. Combining genome mining and MS/MS
fragmentation predicted a mature form of Hogocidin-.beta. (FIG.
5A).
[0145] Genome Sequencing.
[0146] Because the protein sequences of AMPs from S. hominis did
not match to any molecules found in the existing genome database,
whole genome sequence of the antimicrobial strain of S. hominis was
performed. Genomic DNA was purified using UltraClean.RTM. Microbial
DNA Isolation kit (MO Bio). Whole genome DNA sequencing libraries
were constructed using the Nextera-XT DNA Sample Prep Kit
(Illumina) following the vendor's protocol. The final library was
sequenced by paired end sequencing (300.times.300) on an Illumina
MiSeq.TM.. Sequenced reads were de novo assembled using SPAdes
2.5.1 with k-mers of lengths 21, 33, 55, 77, and 127 and the flag
for "careful" turned on. On all of the produced scaffolds, a
six-frame translation was performed using translate Whole Genome
Multi Chromosome.pl ([http://]proteomics.ucsd.edu/Downloads/). This
output was then queried for a match to the peptide fragment
identified via mass spectrometry.
[0147] Antimicrobial Assays.
[0148] Radial diffusion assay was performed using S. aureus
(ATCC35556) strain to test antimicrobial activity of purified
fractions. Briefly, melted TSB agar (10 mL) was mixed with S.
aureus (1.times.10.sup.6 CFU) and poured in a 10 cm petri dish. Two
to four .mu.L of test samples was applied in a small well punched
on the agar plate. Plates were incubated at 37.degree. C. overnight
to allow visible growth of bacteria. Antibacterial activity was
indicated by the clear zone (no bacterial growth) around the well.
Antimicrobial activity of Hogocidins was evaluated by incubating S.
aureus (1.times.10.sup.5 CFU/mL) with 2-fold serial dilutions of
purified Hogocidins in half strength Muller-Hinton broth (MHB) in
PBS at 37.degree. C. for 24 hrs. After incubation, the number of
viable bacteria was measured by counting CFU after spreading
10-fold serial dilutions of bacteria on TSB agar plates. MBC was
determined as a 3-log reduction (99.9%) of viable bacteria after 24
hour incubation.
[0149] Statistical Analysis.
[0150] Paired t-tests were used to compare lesional to non-lesional
samples within atopic subjects and independent t-tests were used to
compare non-atopic to atopic samples. Longitudinal mixed models of
frequency of antimicrobial CoNS and the ratio of live
Staphylococcus to Staphylococcus DNA over time were also fit. Each
model included lesion type, visit, and their interaction term as
fixed effects, while a compound symmetry structure was used to
account for correlation between samples obtained from the same
subject at multiple time points. Frequency of antimicrobial CoNS
used a cumulative logit link and multinomial distribution of
categorized percentages (<=20, 21-79, >=80) to account for a
bi-modal distribution. Statistical analyses were performed using
SAS (version 9.3) software.
[0151] Staphylococcus Survival is Increased in Atopic
Dermatitis.
[0152] To assess the relationship between the amount of
culturable/live Staphylococcus spp. and unculturable/dead
Staphylococcus spp. bacterial density was measured by both manual
colony counting and qPCR for genus-specific 16S ribosomal DNA.
Consistent with prior reports, more total Staphylococcus spp. and
S. aureus could be cultured from lesional skin on the forearms of
patients with atopic dermatitis than from nonlesional skin of these
patients or non-atopic subjects (FIGS. 1A and 1B). Measurements of
DNA abundance revealed a similar trend (FIGS. 1C and 6). However,
the results of these two independent techniques differed
significantly between atopic lesional skin and non-atopic skin. In
lesional skin of subjects with atopic dermatitis, culture and DNA
based results were similar (FIG. 1D). In contrast, in non-atopic
skin the ratio of cultured CFU to relative CFU based on DNA
abundance was approximately 0.1, suggesting a lower survival rate
of bacteria on the skin of non-atopic subjects.
[0153] Antimicrobial Activity of the Skin Microbiome.
[0154] Live CoNS from 30 non-atopic (2029 colonies) and 50 atopic
subjects (5695 colonies) were isolated to characterize their
influence on S. aureus survival. A majority of CoNS clones isolated
from nonatopic subjects (75.26.+-.35.49%) were observed to inhibit
S. aureus growth (FIG. 2A). In contrast, a minority of the CoNS
found on atopic skin possessed this activity [22.83.+-.32.64%
(nonlesional) and 15.76.+-.25.92% (lesional)]. This difference in
the antimicrobial function of CoNS isolated from each population
was stable and reproducible over time as seen following repeated
swabs over a 2 week period (FIG. 2B). The increased ratio of
culturable Staphylococcus to total Staphylococcus DNA was also
stable in this cohort over a 2 week period (FIG. 2C). These data
suggest that although the skin of patients with atopic dermatitis
supports growth of CoNS bacteria, it enables survival of strains
that differ in antimicrobial function from those found on
non-atopic skin.
[0155] As shown in FIG. 1, only 3% of non-atopic subjects were S.
aureus culture-positive (>1 CFU/cm.sup.2), whereas 57% of atopic
subjects cultured positive for S. aureus. To determine if the
antimicrobial activity detected from CoNS was related to S. aureus
survival the frequency of antimicrobial CoNS to measurements of
live S. aureus was compared (FIG. 3A). Strikingly, all patients
with live S. aureus had a low frequency of antimicrobial CONS. In
addition, the frequency was lower in S. aureus culture-positive
group (>1 CFU/cm.sup.2) than in S. aureus-negative group (FIG.
3B). These data suggest that antimicrobial CoNS were protective
against S. aureus colonization.
[0156] Identification of Antimicrobial Bacterial Species on the
Skin.
[0157] To further identify the CoNS species with antimicrobial
activity, a random subset of bacterial colonies were selected for
full-length 16S rRNA gene sequencing. In non-atopic skin, the
predominant species of antimicrobial CoNS were S. epidermidis or S.
hominis (FIG. 4A). In the atopic subjects, antimicrobial CoNS
members included Staphylococcus pasteuri, Staphylococcus warneri
Staphylococcus capitis, S. epidermidis and S. hominis. However, in
most subjects, similar species were identified within groups found
to have antimicrobial and non-antimicrobial function (FIG. 4B).
These observations show that antimicrobial activity is not
predictable at the species level. Overgrowth of functionally
inactive CoNS strains appears to occur in patients with atopic
dermatitis.
[0158] Peptides with Antimicrobial Activity Produced by the
Microbiome.
[0159] To determine what was responsible for the antimicrobial
activity detected in CoNS strains genetic and biochemical
approaches were used. Bacteriocins are a class of AMPs produced by
some CoNS species. However, no DNA encoding the known bacteriocins
epiA, pepA, eciA and elkA, was detected in non-atopic skin (n=14)
by PCR (Table 2, primer sequences). Accordingly, experiments were
performed to purify and identify the source of activity from a
representative colony of antimicrobial S. hominis isolated from a
non-atopic subject. Reverse-phase chromatography revealed two
independent peaks (3152.2 and 3547.7 Da) associated with
antimicrobial activity (FIG. 7). N-terminal amino acid sequencing
of the 3152.2 Da peptide was KCSWWNAA. The full sequence of the
3547.7 Da peptide was obtained by genome-guided MALDI-TOF/TOF
analysis (FIG. 8). Alignment of mass and amino acid sequence to the
genome sequence of this S. hominis strain revealed that these novel
AMPs were encoded within the gene cluster of lanM, lanC and lanT
homologs (FIG. 9), and consistent with identities as lantibiotics.
As these AMPs were previously unknown, they were named
Hogocidin-.alpha. (3152.2 Da) and -.beta. (3547.7 Da) from the
Japanese "Hogo" meaning "Protect." These newly described AMPs were
readily detectable by PCR in 50% of 14 non-atopic individuals. The
predicted secondary structures of mature Hogocidin-.alpha. and
Hogocidin-.beta. are shown in FIG. 5A. The minimal bactericidal
concentration (>99.9% killing) of purified Hogocidin-.alpha. and
-.beta. against S. aureus was 0.625 .mu.M and 1.25 .mu.M,
respectively (FIG. 5B), an activity more potent than conventional
AMPs produced on human skin. Importantly, co-incubation of each
Hogocidin peptide with the human skin cathelicidin AMP LL-37 showed
strong synergistic antimicrobial activity against S. aureus (FIG.
5B), suggesting AMPs derived from microbiome enhance capacity of
host innate immune defense to resist S. aureus.
Example 2
[0160] FAME Analysis of Bacterial Strains.
[0161] Because the lipid composition of whole bacterial cells
(predominantly the cell membranes), which can be represented by the
relative abundance of fatty acid methyl esters present in a
saponified and methylated sample of bacterial cell extracts, is
very nearly unique to each strain, the identified strains were
subjected to Fatty Acid Methyl Ester (FAME) analysis. Bacterial
strains were cultured and harvested according to standard
techniques. Cells were subjected to saponification and methylation
before being extracted into the mobile phase solvent for gas
chromatography. Samples were loaded and run according to the
instrument manufacturer's instructions. The resulting chromatograms
are shown in FIGS. 11 through 19.
Example 3
[0162] Commensal Bacteria Protect the Skin from Colonization by S.
aureus.
[0163] Having established an association between AMP-producing
commensal CoNS and colonization with S. aureus, and having
identified active peptides produced by these strains, experiments
were performed to test if the presence of these bacteria will
reduce colonization by S. aureus. Clinical CoNS isolates were
applied to the surface of sanitized pigskin on which defined
amounts of S. aureus had first been applied. A significant decrease
in survival of S. aureus was seen after a single application of S.
hominis A9 at a density consistent with estimates of the density of
bacteria on normal human skin (1.times.10.sup.5 CFU/cm.sup.2) (FIG.
20A) Application of S. hominis A9 that was killed and rinsed prior
to application, or use of other S. hominis strains that did not
show antimicrobial activity in culture did not affect S. aureus
survival. Similarly, a single application of active S. hominis to
the backs of mice on which defined amounts of S. aureus had been
applied reduced the survival of S. aureus on the skin (FIG. 20B).
In contrast, application of inactive strains at the similar density
did not inhibit S. aureus.
[0164] Finally, to directly test the capacity of functionally
screened and isolated commensal bacteria to inhibit S. aureus in
humans, experiments were performed to test the effect of
application of these bacteria to the skin of subjects with AD. As
previously shown, strains with antimicrobial activity were rare
within the total CoNS community of these subjects, but could be
identified if sufficient colonies were screened. 5 AD patients who
were S. aureus-culture positive were recruited to participate in
this study. CoNS clones with antimicrobial activity were identified
and expanded for planned reapplication to the subject (Autologous
Transplant). Each selected clone was sequenced and clusters of
lantibiotic-related genes were identified in 5 S. epidermidis or S.
hominis strains, and colicin V genes were seen in 2 S. epidermidis
strains and in 1 S. hominis strain (FIG. 21-23). In a double-blind
fashion, vehicle cream alone or bacteria formulated in cream were
applied once to the skin of each arm and then S. aureus measured 24
hrs later. Selected strains were applied to a total final
concentration of 1.times.10.sup.5 CFU/cm.sup.2, a density similar
to previous assessments of the abundance of bacteria on normal
human skin. A single application of these functionally defined and
autologously derived CoNS strain(s) significantly decreased S.
aureus CFU within 24 hrs in comparison to baseline (FIG. 20E).
Example 4
[0165] Transplantation of Antimicrobial CoNS on Ex Vivo Pig Skin
and Mice.
[0166] Frozen pig skin sheet was obtained from Loretta Tomlin
Animal Technologies (Livermore, Calif.) and sanitized by surgical
brush with 3% chloroxylenol. The skin sheet was cut into 2.5
cm.times.2.5 cm and rinsed with sterile PBS more than 20 times to
remove chloroxylenol residue. Back skin of C57BL6 female, 6
week-old mice that were randomly selected was shaved, treated with
depilatory cream and rinsed with water at least 24 hrs before
bacteria application. The shaved skin was cleaned with alcohol swab
twice to remove originally colonized bacteria. All experiments
involving live animal work were in accordance with the approval of
the Institutional Animal Care and Use Guidelines.
[0167] S. aureus (ATCC35556) (1.times.10.sup.5 CFU/cm.sup.2) were
epicutaneously challenged on the pig skin (2.5.times.2.5 cm) or
dorsal skin of mice (2.times.2 cm). S. hominis A9 strain isolated
from non-AD subject, that produce Sh-lantibiotics (hogocidins), or
S. hominis strains isolated from lesional skin of AD subject, which
did not produce antimicrobial activity in culture, was formulated
at 1.times.10.sup.7 CFU/g in skin moisturizer which was confirmed
not to affect bacteria viability. Either S. hominis A9 strain with
anti-S. aureus activity, UV-killed S. hominis A9, inactive strains
of S. hominis C4, C5 and C6 (1.times.10.sup.5 CFU/10 .mu.L), or
vehicle were subsequently applied on the surface of pig skin or
mouse dorsal skin for 20 hrs (FIGS. 20A and 20B). Purified
lantibiotic (0.5 nmol), conditioned media of S. hominis A9 (50
.mu.L) were applied to the surface of sanitized pig skin. Pig skin
was incubated at 30.degree. C. in a 6-well plate. Live bacteria
were harvested with a Catch-All Swab pre-wetted with TSB from the
skin surface as described above. Bacteria were suspended by vortex
swab head vigorously in 1 mL TSB. Ten-fold serial dilution of the
bacteria suspension was spread on a Baird-Parker agar with egg yolk
tellurite for selective count of S. aureus. S. aureus (a large
black colony with halo) were distinguished from S. hominis (a small
gray colony without halo) on the selective agar plate.
Example 5
[0168] Autologous Microbiome Transplant.
[0169] The approach of autologous microbiome transplant (AMT) for
patients with AD has been officially approved by US Food and Drug
Administration (FDA) and this protocol has been filed as an
investigational new drug application (IND) (UCSD Approval #15786).
At the screening visit, AD patients who are S. aureus carriers on
the lesional sites of both antecubital fossa were screened. In the
meantime, skin bacteria were obtained by swabbing from nonlesional
skin of upper arm of AD patient to screen CoNS strain producing
antimicrobial activity against S. aureus. Species of antimicrobial
CoNS isolates were identified by Sanger sequencing of the
full-length 16S rRNA gene. Glycerol stocks of CoNS isolates were
stored at -80.degree. C. until the second visit when patients
received transplant therapy. Each CoNS strain was individually
expanded in TSB overnight. Each CoNS strain was formulated at
1.times.10.sup.7 CFU/g in skin moisturizer which was confirmed not
to affect bacteria viability. Only a single S. epidermidis or S.
hominis strain with antimicrobial activity was isolated from 3
patients. In these cases, a single strain of CoNS was formulated.
Three and 2 antimicrobial S. hominis or S. epidermidis strains were
isolated from 2 AD patients (FIG. 20D). In these cases, an equal
CFU of each CoNS was formulated at the total concentration of
10.sup.7 CFU/g. At the second visit, involved area was measured and
the baseline CFU of live S. aureus on lesional sites of both
forearm was quantified as described above. One arm was treated with
AMT formulation at 10 mg/cm.sup.2 to get 1.times.10.sup.5
CFU/cm.sup.2 of CONS. The other arm received an equal amount of
moisturizer only. All treatment was conducted in double-blinded
fashion and unblinded after all results were analyzed. Subjects
avoided bathing, showering, exercising or applying any topical
products to their arms, and wore a clean and long-sleeved shirt to
avoid cross contamination of applied CoNS to the other arm until
the next visit. At the third visit, S. aureus CFU in involved area
was measured. Difference in S. aureus survival between vehicle and
AMT arms was calculated as [AMT (.chi. hrs)-Vehicle (.chi.
hrs)]/AMT (Baseline) to get A % S. aureus CFU (.chi.=0 hr or 24
hrs) (FIG. 20E).
[0170] Statistical Analysis.
[0171] For all experiments, at least three or more biological
replicates were used, and these are indicated in Figure legends.
For all mouse experiments, at least six mice were used per
treatment group. Therefore, for the reported differences, the
sample size used gave sufficient power for reliability. Paired
t-tests (two-tailed) were used to compare lesional to nonlesional
samples within AD subjects and independent t-tests (two-tailed)
were used to compare non-AD to AD samples. For non-normally
distributed variables such as CoNS with Sh-antibiotic-.alpha. (%),
non-parametric approaches such as Wilcoxon-Mann-Whitney tests for
non-AD to AD samples and Wilcoxon signed rank tests for lesional to
nonlesional samples within AD subjects were used. Longitudinal
mixed models of frequency of antimicrobial CoNS and the ratio of
live Staphylococcus to Staphylococcus DNA over time were also fit.
Each model included lesion type, visit, and their interaction term
as fixed effects, while a compound symmetry structure was used to
account for correlation between samples obtained from the same
subject at multiple time points. Frequency of antimicrobial CoNS
used a cumulative logit link and multinomial distribution of
categorized percentages (<=20, 21-79, >=80) to account for a
bi-modal distribution. Statistical analyses were performed using
SAS (version 9.3) software and R software (version 3.1.1).
Example 6
[0172] Allogeneic Transplant.
[0173] 10.sup.5 CFU/g of strains SH-A9, SH-C2, SE-A11, AMT1, AMT2,
AMT3, AMT4-C2, AMT4-G1, AMT4-D12, AMT5-C5, AMT5-G6 and/or SE-M034,
are formulated in skin moisturizer which is confirmed not to affect
bacteria viability. Subjects for treatment are identified based on
the existence of Atopic Dermatitis and/or active S. aureus
infection. Subjects are instructed to avoid bathing, showering,
exercising or applying any topical products to the affected area
for three days. Compliant patients show significant reductions in
S. aureus levels in the treated area after 7 days (>/=3 log
reduction in recoverable S. aureus colony counts). Compliant
patients show clinically observable reductions in symptoms of S.
aureus infection and/or atopic dermatitis continuing for at least
14 days after the initial treatment.
Example 6
[0174] Purification of AMPs Produced by S. epidermidis.
[0175] Sterile conditioned media from a representative
antimicrobial S. epidermidis A11 strain was used to further
identify molecules with antimicrobial activity on normal skin that
were in low abundance on atopics. Activity was precipitated by
ammonium sulfate (70% saturation), dissolved in H.sub.2O and
applied on a Sep-Pak cartridge (Waters Co). Active fractions were
eluted with 40% acetonitrile in H.sub.2O and subjected to
HiTrap.RTM. SP (GE Healthcare Life Sciences) separation and
activity eluted at 500 mM NaCl. Third step HPLC purification was
done with CapCel Pak C8 (5 .mu.m, 300 .ANG., 4.6.times.250 mm)
(Shiseido Co.) with a linear gradient of acetonitrile from 5% to
50% in 0.1% (v/v) TFA at 0.8 mL/min.
[0176] Identification of AMPs Produced by S. epidermidis.
[0177] Antimicrobial activity was purified from sterile conditioned
media of a representative antimicrobial S. epidermidis A11 strain
isolated from a non-atopic subject. The characteristics of the
purified active molecule was determined by MALDI-TOF/TOF and Edman
terminal sequencing.
[0178] Mass Spectrometry.
[0179] Mass spectra of HPLC-purified AMPs from S. epidermidis A11
were recorded using a MALDI-TOF/TOF Bruker Autoflex.TM. Speed
instrument (Bruker Daltonics) controlled by the flexcontrol
software (Bruker Daltonics). Mass spectrometric analyses were
performed in positive ion reflectron mode using
cyano-4-hydroxycinnamic acid as a matrix (CHCA) 10 mg/mL
(Sigma-Aldrich) dissolved in 50% acetonitrile (ACN) and 0.1%
trifluoroacetic acid (TFA). Full scan mass spectra were acquired in
positive ion reflectron mode for mass range 1000-6000 m/z. Each
mass spectrum is the result of 750 averaged laser shots with the
laser intensity set around 65% of full laser intensity and a
detector gain enhanced at 8.times.4 GS/s (as selected within the
Bruker Flex Control software). Resulting mass spectra were analyzed
using flex analysis software (Bruker Daltonics). Spectra were
calibrated to PepMix internal standard solutions.
[0180] N-Terminal Protein Sequencing.
[0181] The N-terminal amino acid sequence of purified (Fractions
33-34, FIG. 25A) was analyzed by 15 cycles of Edman degradation on
Procise.RTM. 494HT Protein Sequence system (Applied Biosystems).
The N-terminal sequence is provided in SEQ ID NO:55.
[0182] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Accordingly, other embodiments are within
the scope of the following claims.
Sequence CWU 1
1
551186DNAStaphylococcus hominisCDS(1)..(186) 1atg agt aaa tta gaa
cta ctt aat gaa tct aaa gca aat tat ctt gaa 48Met Ser Lys Leu Glu
Leu Leu Asn Glu Ser Lys Ala Asn Tyr Leu Glu 1 5 10 15 aaa ctt act
gat gaa aaa att gaa gaa acg gaa gca tac ggc ggt aaa 96Lys Leu Thr
Asp Glu Lys Ile Glu Glu Thr Glu Ala Tyr Gly Gly Lys 20 25 30 tgt
tct tgg tgg aat gca tca tgt cat tta gga aat aat ggg aaa att 144Cys
Ser Trp Trp Asn Ala Ser Cys His Leu Gly Asn Asn Gly Lys Ile 35 40
45 tgt aca gtt tct cat gag tgt gca gca gga tgt aat tta taa 186Cys
Thr Val Ser His Glu Cys Ala Ala Gly Cys Asn Leu 50 55 60
261PRTStaphylococcus hominis 2Met Ser Lys Leu Glu Leu Leu Asn Glu
Ser Lys Ala Asn Tyr Leu Glu 1 5 10 15 Lys Leu Thr Asp Glu Lys Ile
Glu Glu Thr Glu Ala Tyr Gly Gly Lys 20 25 30 Cys Ser Trp Trp Asn
Ala Ser Cys His Leu Gly Asn Asn Gly Lys Ile 35 40 45 Cys Thr Val
Ser His Glu Cys Ala Ala Gly Cys Asn Leu 50 55 60
3201DNAStaphylococcus hominisCDS(1)..(201) 3atg ttt agt aaa aat ttc
caa aga aat gaa aag atg gaa aat act ttg 48Met Phe Ser Lys Asn Phe
Gln Arg Asn Glu Lys Met Glu Asn Thr Leu 1 5 10 15 aaa aag gta agt
tca gct aat gat gtg aat gga gga gct aca ccg act 96Lys Lys Val Ser
Ser Ala Asn Asp Val Asn Gly Gly Ala Thr Pro Thr 20 25 30 att act
aca tct tca gca act tgt ggt ggt att att gtt gcg gca agt 144Ile Thr
Thr Ser Ser Ala Thr Cys Gly Gly Ile Ile Val Ala Ala Ser 35 40 45
gct gct cag tgt ccg aca tta gct tgc tct tct aga tgt gga aaa aga
192Ala Ala Gln Cys Pro Thr Leu Ala Cys Ser Ser Arg Cys Gly Lys Arg
50 55 60 aaa aaa taa 201Lys Lys 65 466PRTStaphylococcus hominis
4Met Phe Ser Lys Asn Phe Gln Arg Asn Glu Lys Met Glu Asn Thr Leu 1
5 10 15 Lys Lys Val Ser Ser Ala Asn Asp Val Asn Gly Gly Ala Thr Pro
Thr 20 25 30 Ile Thr Thr Ser Ser Ala Thr Cys Gly Gly Ile Ile Val
Ala Ala Ser 35 40 45 Ala Ala Gln Cys Pro Thr Leu Ala Cys Ser Ser
Arg Cys Gly Lys Arg 50 55 60 Lys Lys 65 5170PRTHomo sapiens 5Met
Lys Thr Gln Arg Asn Gly His Ser Leu Gly Arg Trp Ser Leu Val 1 5 10
15 Leu Leu Leu Leu Gly Leu Val Met Pro Leu Ala Ile Ile Ala Gln Val
20 25 30 Leu Ser Tyr Lys Glu Ala Val Leu Arg Ala Ile Asp Gly Ile
Asn Gln 35 40 45 Arg Ser Ser Asp Ala Asn Leu Tyr Arg Leu Leu Asp
Leu Asp Pro Arg 50 55 60 Pro Thr Met Asp Gly Asp Pro Asp Thr Pro
Lys Pro Val Ser Phe Thr 65 70 75 80 Val Lys Glu Thr Val Cys Pro Arg
Thr Thr Gln Gln Ser Pro Glu Asp 85 90 95 Cys Asp Phe Lys Lys Asp
Gly Leu Val Lys Arg Cys Met Gly Thr Val 100 105 110 Thr Leu Asn Gln
Ala Arg Gly Ser Phe Asp Ile Ser Cys Asp Lys Asp 115 120 125 Asn Lys
Arg Phe Ala Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu 130 135 140
Lys Ile Gly Lys Glu Phe Lys Arg Ile Val Gln Arg Ile Asp Asp Phe 145
150 155 160 Leu Arg Asn Leu Val Pro Arg Thr Glu Ser 165 170
65PRTArtificial SequenceSynthetic linker 6Gly Gly Gly Gly Ser 1 5
722PRTArtificial SequenceSynthetic linker 7Gly Gly Gly Gly Gly Gly
Ser Met Phe Gly Gly Ala Lys Lys Arg Ser 1 5 10 15 Gly Gly Gly Gly
Gly Gly 20 820DNAArtificial SequenceStaphylococcus aureus-specific
PCR primer 8aactgttggc cactatgagt 20921DNAArtificial
SequenceStaphylococcus aureus-specific PCR primer 9ccagcattac
ctgtaatctc g 211020DNAArtificial SequenceStaphylococcus
epidermidis-specific PCR primer 10tcagcagttg aaggacagat
201122DNAArtificial SequenceStaphylococcus epidermidis-specific PCR
primer 11ccagaacaat gaatggttaa gg 221230DNAArtificial
SequenceStaphylococcus-genus specific 16S sequence PCR primer
12tttgggctac acacgtgcta caatggacaa 301324DNAArtificial
SequenceStaphylococcus-genus specific 16S sequence PCR primer
13aacaacttta tgggatttgc wtga 241421DNAArtificial SequenceUniversal
sequence of bacterial 16S rRNA PCR primer 14agagtttgga tcmtggctca g
211517DNAArtificial SequenceUniversal sequence of bacterial 16S
rRNA PCR primer 15aaggaggtgw tccarcc 171621DNAArtificial
SequenceStaphylococcus epidermidis epidermn PCR primer 16gattcaggag
ctgaaccaag a 211720DNAArtificial SequenceStaphylococcus epidermidis
epidermn PCR primer 17ttgaagccct gccaatctaa 201824DNAArtificial
SequenceStaphylococcus epidermidis pep5 PCR primer 18ctgatgaact
tgaacctcaa actg 241923DNAArtificial SequenceStaphylococcus
epidermidis pep5 PCR primer 19gacactgtaa ataaacgcgt agc
232024DNAArtificial SequenceStaphylococcus epidermidis epicidin280
PCR primer 20gcaactagac aggtatgtcc taaa 242125DNAArtificial
SequenceStaphylococcus epidermidis epicidin280 PCR primer
21catctaagat taaatgaggg tggtt 252221DNAArtificial
SequenceStaphylococcus epidermidis epilancin K7 PCR primer
22taagtccgca atctgctagt g 212322DNAArtificial
SequenceStaphylococcus epidermidis epilancin K7 PCR primer
23cagtaatatt gcaaccgcat gt 222425DNAArtificial
SequenceStaphylococcus hominis Hogocidin-alpha PCR primer
24atgagtaaat tagaactact taatg 252525DNAArtificial
SequenceStaphylococcus hominis Hogocidin-alpha PCR primer
25ttataaatta catcctgctg cacac 25268PRTStaphylococcus hominis 26Lys
Cys Ser Asp Asp Asn Ala Ala 1 5 27138DNAStaphylococcus
hominisCDS(1)..(138) 27atg gat att ata aaa gta aat aaa aca gaa aga
atg aat gat aat aga 48Met Asp Ile Ile Lys Val Asn Lys Thr Glu Arg
Met Asn Asp Asn Arg 1 5 10 15 aaa att gta atg att ttt tct tta tac
gat aca ttt ttt aac gct act 96Lys Ile Val Met Ile Phe Ser Leu Tyr
Asp Thr Phe Phe Asn Ala Thr 20 25 30 aat aca cat aag cta aag agt
atg aag ctt aat gcg aaa taa 138Asn Thr His Lys Leu Lys Ser Met Lys
Leu Asn Ala Lys 35 40 45 2845PRTStaphylococcus hominis 28Met Asp
Ile Ile Lys Val Asn Lys Thr Glu Arg Met Asn Asp Asn Arg 1 5 10 15
Lys Ile Val Met Ile Phe Ser Leu Tyr Asp Thr Phe Phe Asn Ala Thr 20
25 30 Asn Thr His Lys Leu Lys Ser Met Lys Leu Asn Ala Lys 35 40 45
29168DNAStaphylococcus epidermidisCDS(1)..(168) 29atg gta tgg atc
cat ggt ggt ggt aac tta ggt ggt gct ggc tta gaa 48Met Val Trp Ile
His Gly Gly Gly Asn Leu Gly Gly Ala Gly Leu Glu 1 5 10 15 gat gct
ttt gat ggt aat act tta gct aaa cat aca tca aaa att aaa 96Asp Ala
Phe Asp Gly Asn Thr Leu Ala Lys His Thr Ser Lys Ile Lys 20 25 30
tat gtt ttt gga aat ttg caa cct gaa aac cat tat gat gat att gat
144Tyr Val Phe Gly Asn Leu Gln Pro Glu Asn His Tyr Asp Asp Ile Asp
35 40 45 ata ata atc tca aaa caa tta taa 168Ile Ile Ile Ser Lys Gln
Leu 50 55 3055PRTStaphylococcus epidermidis 30Met Val Trp Ile His
Gly Gly Gly Asn Leu Gly Gly Ala Gly Leu Glu 1 5 10 15 Asp Ala Phe
Asp Gly Asn Thr Leu Ala Lys His Thr Ser Lys Ile Lys 20 25 30 Tyr
Val Phe Gly Asn Leu Gln Pro Glu Asn His Tyr Asp Asp Ile Asp 35 40
45 Ile Ile Ile Ser Lys Gln Leu 50 55 31207DNAStaphylococcus
epidermidisCDS(1)..(207) 31atg att tca gtg ata ctg ccg atg gaa gaa
ata att att gca ata ata 48Met Ile Ser Val Ile Leu Pro Met Glu Glu
Ile Ile Ile Ala Ile Ile 1 5 10 15 aat aac gac tta ggc cat tta att
ttt gag aat aaa aaa aat agt ggg 96Asn Asn Asp Leu Gly His Leu Ile
Phe Glu Asn Lys Lys Asn Ser Gly 20 25 30 ttt tct ttt ttc ata ata
aaa cct ttc ata act aat att tat ttt cta 144Phe Ser Phe Phe Ile Ile
Lys Pro Phe Ile Thr Asn Ile Tyr Phe Leu 35 40 45 tca ggt att aaa
aaa att tta caa aaa cag aga aaa tat tat acg atg 192Ser Gly Ile Lys
Lys Ile Leu Gln Lys Gln Arg Lys Tyr Tyr Thr Met 50 55 60 cta ata
aaa gta taa 207Leu Ile Lys Val 65 3268PRTStaphylococcus epidermidis
32Met Ile Ser Val Ile Leu Pro Met Glu Glu Ile Ile Ile Ala Ile Ile 1
5 10 15 Asn Asn Asp Leu Gly His Leu Ile Phe Glu Asn Lys Lys Asn Ser
Gly 20 25 30 Phe Ser Phe Phe Ile Ile Lys Pro Phe Ile Thr Asn Ile
Tyr Phe Leu 35 40 45 Ser Gly Ile Lys Lys Ile Leu Gln Lys Gln Arg
Lys Tyr Tyr Thr Met 50 55 60 Leu Ile Lys Val 65
33210DNAStaphylococcus epidermidisCDS(1)..(210) 33atg aac ata tac
tta aaa gta att tta act tct tta ttt ttt gct tta 48Met Asn Ile Tyr
Leu Lys Val Ile Leu Thr Ser Leu Phe Phe Ala Leu 1 5 10 15 ata att
ttt att gta act tat ata acg act aag caa tgg gga aca tcg 96Ile Ile
Phe Ile Val Thr Tyr Ile Thr Thr Lys Gln Trp Gly Thr Ser 20 25 30
tta ggt ttt tca tct tta tca ttt atc ggt aac ttt att tac gat tat
144Leu Gly Phe Ser Ser Leu Ser Phe Ile Gly Asn Phe Ile Tyr Asp Tyr
35 40 45 tca acg aaa tta agt gat aaa aaa tat gaa aaa aga ata aat
agc aac 192Ser Thr Lys Leu Ser Asp Lys Lys Tyr Glu Lys Arg Ile Asn
Ser Asn 50 55 60 aaa aaa gat aaa ctt tag 210Lys Lys Asp Lys Leu 65
3469PRTStaphylococcus epidermidis 34Met Asn Ile Tyr Leu Lys Val Ile
Leu Thr Ser Leu Phe Phe Ala Leu 1 5 10 15 Ile Ile Phe Ile Val Thr
Tyr Ile Thr Thr Lys Gln Trp Gly Thr Ser 20 25 30 Leu Gly Phe Ser
Ser Leu Ser Phe Ile Gly Asn Phe Ile Tyr Asp Tyr 35 40 45 Ser Thr
Lys Leu Ser Asp Lys Lys Tyr Glu Lys Arg Ile Asn Ser Asn 50 55 60
Lys Lys Asp Lys Leu 65 35183DNAStaphylococcus
epidermidisCDS(1)..(183) 35atg aaa aat aac aaa aat tta ttt gat tta
gaa att aaa aaa gaa aca 48Met Lys Asn Asn Lys Asn Leu Phe Asp Leu
Glu Ile Lys Lys Glu Thr 1 5 10 15 agt caa aac act gat gaa ctt gaa
cct caa act gct gga cca gcg att 96Ser Gln Asn Thr Asp Glu Leu Glu
Pro Gln Thr Ala Gly Pro Ala Ile 20 25 30 aga gct tct gtg aaa caa
tgt cag aaa act ttg aaa gct acg cgt tta 144Arg Ala Ser Val Lys Gln
Cys Gln Lys Thr Leu Lys Ala Thr Arg Leu 35 40 45 ttt aca gtg tct
tgc aaa gga aaa aac gga tgt aaa tag 183Phe Thr Val Ser Cys Lys Gly
Lys Asn Gly Cys Lys 50 55 60 3660PRTStaphylococcus epidermidis
36Met Lys Asn Asn Lys Asn Leu Phe Asp Leu Glu Ile Lys Lys Glu Thr 1
5 10 15 Ser Gln Asn Thr Asp Glu Leu Glu Pro Gln Thr Ala Gly Pro Ala
Ile 20 25 30 Arg Ala Ser Val Lys Gln Cys Gln Lys Thr Leu Lys Ala
Thr Arg Leu 35 40 45 Phe Thr Val Ser Cys Lys Gly Lys Asn Gly Cys
Lys 50 55 60 37171DNAStaphylococcus hominisCDS(1)..(171) 37atg gaa
aac aaa aaa gat tta ttt gat tta gaa atc aaa aaa gat aat 48Met Glu
Asn Lys Lys Asp Leu Phe Asp Leu Glu Ile Lys Lys Asp Asn 1 5 10 15
atg gaa aat aat aat gaa tta gaa gct caa tct ctt ggt cct gca att
96Met Glu Asn Asn Asn Glu Leu Glu Ala Gln Ser Leu Gly Pro Ala Ile
20 25 30 aag gca act aga cag gta tgt cct aaa gca aca cgt ttt gtt
aca gtt 144Lys Ala Thr Arg Gln Val Cys Pro Lys Ala Thr Arg Phe Val
Thr Val 35 40 45 tct tgt aaa aaa agt gat tgt caa tag 171Ser Cys Lys
Lys Ser Asp Cys Gln 50 55 3856PRTStaphylococcus hominis 38Met Glu
Asn Lys Lys Asp Leu Phe Asp Leu Glu Ile Lys Lys Asp Asn 1 5 10 15
Met Glu Asn Asn Asn Glu Leu Glu Ala Gln Ser Leu Gly Pro Ala Ile 20
25 30 Lys Ala Thr Arg Gln Val Cys Pro Lys Ala Thr Arg Phe Val Thr
Val 35 40 45 Ser Cys Lys Lys Ser Asp Cys Gln 50 55
39135DNAStaphylococcus hominisCDS(1)..(135) 39atg aaa gtt gtt aaa
gaa aag aaa gaa ctt ttt gat ctt gac gtt aaa 48Met Lys Val Val Lys
Glu Lys Lys Glu Leu Phe Asp Leu Asp Val Lys 1 5 10 15 gta aat gcg
aga gac atg aat aat tca gaa tca ggt cca cct aat aca 96Val Asn Ala
Arg Asp Met Asn Asn Ser Glu Ser Gly Pro Pro Asn Thr 20 25 30 agt
tta ata tgg tgt acg gat gga tgc gct aaa cgg taa 135Ser Leu Ile Trp
Cys Thr Asp Gly Cys Ala Lys Arg 35 40 4044PRTStaphylococcus hominis
40Met Lys Val Val Lys Glu Lys Lys Glu Leu Phe Asp Leu Asp Val Lys 1
5 10 15 Val Asn Ala Arg Asp Met Asn Asn Ser Glu Ser Gly Pro Pro Asn
Thr 20 25 30 Ser Leu Ile Trp Cys Thr Asp Gly Cys Ala Lys Arg 35 40
41138DNAStaphylococcus hominisCDS(1)..(138) 41atg gat att ata aaa
gta aat aaa aca gaa aga atg aat gat aat aga 48Met Asp Ile Ile Lys
Val Asn Lys Thr Glu Arg Met Asn Asp Asn Arg 1 5 10 15 aaa att gta
atg att ttt tct tta tac gat aca ttt ttt aac gct act 96Lys Ile Val
Met Ile Phe Ser Leu Tyr Asp Thr Phe Phe Asn Ala Thr 20 25 30 aat
aca cat aag cta aag agt atg aag ctt aat gcg aaa taa 138Asn Thr His
Lys Leu Lys Ser Met Lys Leu Asn Ala Lys 35 40 45
4245PRTStaphylococcus hominis 42Met
Asp Ile Ile Lys Val Asn Lys Thr Glu Arg Met Asn Asp Asn Arg 1 5 10
15 Lys Ile Val Met Ile Phe Ser Leu Tyr Asp Thr Phe Phe Asn Ala Thr
20 25 30 Asn Thr His Lys Leu Lys Ser Met Lys Leu Asn Ala Lys 35 40
45 43201DNAStaphylococcus hominisCDS(1)..(201) 43atg agt aat aaa
gat tta gaa tta ttt aat aca gcc ggt gat tta ata 48Met Ser Asn Lys
Asp Leu Glu Leu Phe Asn Thr Ala Gly Asp Leu Ile 1 5 10 15 caa gaa
tta aaa gat ggt gac cta aat atc cat tta tat ggt gaa tcg 96Gln Glu
Leu Lys Asp Gly Asp Leu Asn Ile His Leu Tyr Gly Glu Ser 20 25 30
gaa att aga aaa aaa tct ttc tct caa aaa aca ggg aat gat ggg aaa
144Glu Ile Arg Lys Lys Ser Phe Ser Gln Lys Thr Gly Asn Asp Gly Lys
35 40 45 cat tgt aca att act tgg gaa tgt tct ata tgt cct act aaa
act tgt 192His Cys Thr Ile Thr Trp Glu Cys Ser Ile Cys Pro Thr Lys
Thr Cys 50 55 60 tgg tgc taa 201Trp Cys 65 4466PRTStaphylococcus
hominis 44Met Ser Asn Lys Asp Leu Glu Leu Phe Asn Thr Ala Gly Asp
Leu Ile 1 5 10 15 Gln Glu Leu Lys Asp Gly Asp Leu Asn Ile His Leu
Tyr Gly Glu Ser 20 25 30 Glu Ile Arg Lys Lys Ser Phe Ser Gln Lys
Thr Gly Asn Asp Gly Lys 35 40 45 His Cys Thr Ile Thr Trp Glu Cys
Ser Ile Cys Pro Thr Lys Thr Cys 50 55 60 Trp Cys 65
45117DNAStaphylococcus hominisCDS(1)..(117) 45atg gga act tca gag
gta aga aaa gga aaa gga ggc ggt ttt agt acc 48Met Gly Thr Ser Glu
Val Arg Lys Gly Lys Gly Gly Gly Phe Ser Thr 1 5 10 15 gta acc gtt
gta aca cca att gta ccg aca tcg aag tgt gcc tca att 96Val Thr Val
Val Thr Pro Ile Val Pro Thr Ser Lys Cys Ala Ser Ile 20 25 30 gta
aaa cca tgt aac aaa taa 117Val Lys Pro Cys Asn Lys 35
4638PRTStaphylococcus hominis 46Met Gly Thr Ser Glu Val Arg Lys Gly
Lys Gly Gly Gly Phe Ser Thr 1 5 10 15 Val Thr Val Val Thr Pro Ile
Val Pro Thr Ser Lys Cys Ala Ser Ile 20 25 30 Val Lys Pro Cys Asn
Lys 35 47165DNAStaphylococcus hominisCDS(1)..(165) 47atg act aaa
ata act aaa gat gat ttg aaa aag att aca gaa aat cgt 48Met Thr Lys
Ile Thr Lys Asp Asp Leu Lys Lys Ile Thr Glu Asn Arg 1 5 10 15 att
gaa gca cgt aca cat cca acc gtt gtt cct gta agt gct gct gta 96Ile
Glu Ala Arg Thr His Pro Thr Val Val Pro Val Ser Ala Ala Val 20 25
30 tgc gga gtt gct act aaa tta gta cca aca tcg aaa tgt gct tca att
144Cys Gly Val Ala Thr Lys Leu Val Pro Thr Ser Lys Cys Ala Ser Ile
35 40 45 gta aaa cca tgt aat aaa taa 165Val Lys Pro Cys Asn Lys 50
4854PRTStaphylococcus hominis 48Met Thr Lys Ile Thr Lys Asp Asp Leu
Lys Lys Ile Thr Glu Asn Arg 1 5 10 15 Ile Glu Ala Arg Thr His Pro
Thr Val Val Pro Val Ser Ala Ala Val 20 25 30 Cys Gly Val Ala Thr
Lys Leu Val Pro Thr Ser Lys Cys Ala Ser Ile 35 40 45 Val Lys Pro
Cys Asn Lys 50 49522DNAStaphylococcus hominisCDS(1)..(522) 49atg
act ata gat tta gta atg ata ctt att att ttg atg tat atg ata 48Met
Thr Ile Asp Leu Val Met Ile Leu Ile Ile Leu Met Tyr Met Ile 1 5 10
15 att ggt ttt aga aga ggc ctt tgg ctg aat agt ctt cat ttg tcg tct
96Ile Gly Phe Arg Arg Gly Leu Trp Leu Asn Ser Leu His Leu Ser Ser
20 25 30 aca ctt gtc tca cta ttc att gcg cat cgt ttt tac caa tat
ata tca 144Thr Leu Val Ser Leu Phe Ile Ala His Arg Phe Tyr Gln Tyr
Ile Ser 35 40 45 aaa caa atg att gtt ttt gtt cca ttt cct aaa aca
gtt gct ttt gac 192Lys Gln Met Ile Val Phe Val Pro Phe Pro Lys Thr
Val Ala Phe Asp 50 55 60 acg cac ttc gca ttt caa tac cat gat gta
caa caa cgt ttt gat act 240Thr His Phe Ala Phe Gln Tyr His Asp Val
Gln Gln Arg Phe Asp Thr 65 70 75 80 att gtg gca ttt tta tgt att gct
ttt ata agt aag ttg ctt tta tat 288Ile Val Ala Phe Leu Cys Ile Ala
Phe Ile Ser Lys Leu Leu Leu Tyr 85 90 95 ctt att att gta act ttt
gat aat ata gtg tca tat cat aat att cat 336Leu Ile Ile Val Thr Phe
Asp Asn Ile Val Ser Tyr His Asn Ile His 100 105 110 gtt aca agt cga
ata ttg gga agc gta tta ggt agt att gca agt gtg 384Val Thr Ser Arg
Ile Leu Gly Ser Val Leu Gly Ser Ile Ala Ser Val 115 120 125 att gta
ctg caa ctt gtt tta tat tta gta tct tta tat cct aat gaa 432Ile Val
Leu Gln Leu Val Leu Tyr Leu Val Ser Leu Tyr Pro Asn Glu 130 135 140
tgg att caa gaa agt tta aaa tac ggt tat tta agc cat att att cta
480Trp Ile Gln Glu Ser Leu Lys Tyr Gly Tyr Leu Ser His Ile Ile Leu
145 150 155 160 ttt aag atg ccg ttt tta tca tct tat ata cta aat tta
taa 522Phe Lys Met Pro Phe Leu Ser Ser Tyr Ile Leu Asn Leu 165 170
50173PRTStaphylococcus hominis 50Met Thr Ile Asp Leu Val Met Ile
Leu Ile Ile Leu Met Tyr Met Ile 1 5 10 15 Ile Gly Phe Arg Arg Gly
Leu Trp Leu Asn Ser Leu His Leu Ser Ser 20 25 30 Thr Leu Val Ser
Leu Phe Ile Ala His Arg Phe Tyr Gln Tyr Ile Ser 35 40 45 Lys Gln
Met Ile Val Phe Val Pro Phe Pro Lys Thr Val Ala Phe Asp 50 55 60
Thr His Phe Ala Phe Gln Tyr His Asp Val Gln Gln Arg Phe Asp Thr 65
70 75 80 Ile Val Ala Phe Leu Cys Ile Ala Phe Ile Ser Lys Leu Leu
Leu Tyr 85 90 95 Leu Ile Ile Val Thr Phe Asp Asn Ile Val Ser Tyr
His Asn Ile His 100 105 110 Val Thr Ser Arg Ile Leu Gly Ser Val Leu
Gly Ser Ile Ala Ser Val 115 120 125 Ile Val Leu Gln Leu Val Leu Tyr
Leu Val Ser Leu Tyr Pro Asn Glu 130 135 140 Trp Ile Gln Glu Ser Leu
Lys Tyr Gly Tyr Leu Ser His Ile Ile Leu 145 150 155 160 Phe Lys Met
Pro Phe Leu Ser Ser Tyr Ile Leu Asn Leu 165 170
51522DNAStaphylococcus epidermidisCDS(1)..(522) 51atg ctc att gat
ata gtt gtt ctt ctt att att tgt tac ttt ata gtg 48Met Leu Ile Asp
Ile Val Val Leu Leu Ile Ile Cys Tyr Phe Ile Val 1 5 10 15 ata ggg
ttt cgt aga ggt att tgg tta tcg ata ttg cac ttt gct tct 96Ile Gly
Phe Arg Arg Gly Ile Trp Leu Ser Ile Leu His Phe Ala Ser 20 25 30
tca att gta tct tta tat att gcg tca caa cat tat caa tcg att gcg
144Ser Ile Val Ser Leu Tyr Ile Ala Ser Gln His Tyr Gln Ser Ile Ala
35 40 45 caa cgt tta gtt gta ttt gtg cca ttt ccg aaa acg gtg gcg
ttt gat 192Gln Arg Leu Val Val Phe Val Pro Phe Pro Lys Thr Val Ala
Phe Asp 50 55 60 atg gtc tat act ata cct tat gat cat ttg caa tac
aga ttt gaa aaa 240Met Val Tyr Thr Ile Pro Tyr Asp His Leu Gln Tyr
Arg Phe Glu Lys 65 70 75 80 gtg ata gca ttt att ata ata ttt ggt atg
tgt aag ctt att ttg tat 288Val Ile Ala Phe Ile Ile Ile Phe Gly Met
Cys Lys Leu Ile Leu Tyr 85 90 95 cta gtt gtt gtt aca ttt gat aat
ata ata acg tat aaa aag ata cat 336Leu Val Val Val Thr Phe Asp Asn
Ile Ile Thr Tyr Lys Lys Ile His 100 105 110 tta gta agt cgg ata tcg
agt gtc gtt ttg agt atc ata tcg gtt ttt 384Leu Val Ser Arg Ile Ser
Ser Val Val Leu Ser Ile Ile Ser Val Phe 115 120 125 ata tat tta caa
att gga ctt tat tta tta tcg cta tat ccg cat tca 432Ile Tyr Leu Gln
Ile Gly Leu Tyr Leu Leu Ser Leu Tyr Pro His Ser 130 135 140 ttt ata
cag tac caa tta tct caa tcg cta gta agt cga gtt gtg att 480Phe Ile
Gln Tyr Gln Leu Ser Gln Ser Leu Val Ser Arg Val Val Ile 145 150 155
160 gaa caa att cct tat tta tca caa ttt att tta aat tta taa 522Glu
Gln Ile Pro Tyr Leu Ser Gln Phe Ile Leu Asn Leu 165 170
52173PRTStaphylococcus epidermidis 52Met Leu Ile Asp Ile Val Val
Leu Leu Ile Ile Cys Tyr Phe Ile Val 1 5 10 15 Ile Gly Phe Arg Arg
Gly Ile Trp Leu Ser Ile Leu His Phe Ala Ser 20 25 30 Ser Ile Val
Ser Leu Tyr Ile Ala Ser Gln His Tyr Gln Ser Ile Ala 35 40 45 Gln
Arg Leu Val Val Phe Val Pro Phe Pro Lys Thr Val Ala Phe Asp 50 55
60 Met Val Tyr Thr Ile Pro Tyr Asp His Leu Gln Tyr Arg Phe Glu Lys
65 70 75 80 Val Ile Ala Phe Ile Ile Ile Phe Gly Met Cys Lys Leu Ile
Leu Tyr 85 90 95 Leu Val Val Val Thr Phe Asp Asn Ile Ile Thr Tyr
Lys Lys Ile His 100 105 110 Leu Val Ser Arg Ile Ser Ser Val Val Leu
Ser Ile Ile Ser Val Phe 115 120 125 Ile Tyr Leu Gln Ile Gly Leu Tyr
Leu Leu Ser Leu Tyr Pro His Ser 130 135 140 Phe Ile Gln Tyr Gln Leu
Ser Gln Ser Leu Val Ser Arg Val Val Ile 145 150 155 160 Glu Gln Ile
Pro Tyr Leu Ser Gln Phe Ile Leu Asn Leu 165 170
531260DNAStaphylococcus epidermidisCDS(1)..(1260) 53atg att ggt aga
aaa aaa gaa acc ctt tta aaa aac gaa gtt att tct 48Met Ile Gly Arg
Lys Lys Glu Thr Leu Leu Lys Asn Glu Val Ile Ser 1 5 10 15 gcg ttt
act act ttt ttt acc tgc agt tat ata ata att gtt aat ggt 96Ala Phe
Thr Thr Phe Phe Thr Cys Ser Tyr Ile Ile Ile Val Asn Gly 20 25 30
ata ttg tta cat caa gca gga atg tct ttg tta tgg acg att ata gct
144Ile Leu Leu His Gln Ala Gly Met Ser Leu Leu Trp Thr Ile Ile Ala
35 40 45 act act cta gtt tgt tgc att agt tgc atc ctt ctt ggt ata
tat gct 192Thr Thr Leu Val Cys Cys Ile Ser Cys Ile Leu Leu Gly Ile
Tyr Ala 50 55 60 aat gtt cca cta att att ata cca gga atc ggt gaa
act att ttt ttt 240Asn Val Pro Leu Ile Ile Ile Pro Gly Ile Gly Glu
Thr Ile Phe Phe 65 70 75 80 act tat aca atc att aaa agt cat tac tat
aat tat cat gaa gcg cta 288Thr Tyr Thr Ile Ile Lys Ser His Tyr Tyr
Asn Tyr His Glu Ala Leu 85 90 95 gct att gtt ttg att tca ggt ttg
att ttc act ttt att gca tac aca 336Ala Ile Val Leu Ile Ser Gly Leu
Ile Phe Thr Phe Ile Ala Tyr Thr 100 105 110 ccg ttt gct aga gtt cta
gac aag tcc ata cca aag aat tta aaa gaa 384Pro Phe Ala Arg Val Leu
Asp Lys Ser Ile Pro Lys Asn Leu Lys Glu 115 120 125 gga ata act att
ggt ata ggt ctg ttt atg gcg ttt gtt gga cta caa 432Gly Ile Thr Ile
Gly Ile Gly Leu Phe Met Ala Phe Val Gly Leu Gln 130 135 140 aac agc
aaa ata att ata cca aac agg caa agt att gtt gag cta aac 480Asn Ser
Lys Ile Ile Ile Pro Asn Arg Gln Ser Ile Val Glu Leu Asn 145 150 155
160 cac ata aac att tat agt ggg tta gcg ata cta cta cta tta ttt gca
528His Ile Asn Ile Tyr Ser Gly Leu Ala Ile Leu Leu Leu Leu Phe Ala
165 170 175 att gtt ata ttt act tta ggg acc aag ttg gct ttc ttt tat
aca gta 576Ile Val Ile Phe Thr Leu Gly Thr Lys Leu Ala Phe Phe Tyr
Thr Val 180 185 190 att att ggt atc att ata tct ttt tta gct ggg att
ata aag gtg aaa 624Ile Ile Gly Ile Ile Ile Ser Phe Leu Ala Gly Ile
Ile Lys Val Lys 195 200 205 tat cat ttt tat aat ttt agt ttg cga tca
ata gta agc gag aat aat 672Tyr His Phe Tyr Asn Phe Ser Leu Arg Ser
Ile Val Ser Glu Asn Asn 210 215 220 att ttt agt tac agt ttt gat aaa
ata ggt cat ttt tct ttt tgg tct 720Ile Phe Ser Tyr Ser Phe Asp Lys
Ile Gly His Phe Ser Phe Trp Ser 225 230 235 240 tta gtg ttc tca ctt
act att ttg tta ctg ttt caa aat tta ggt aca 768Leu Val Phe Ser Leu
Thr Ile Leu Leu Leu Phe Gln Asn Leu Gly Thr 245 250 255 tta cat gga
ttg aaa att aat gat aaa gta aaa ttg tca aga att ttt 816Leu His Gly
Leu Lys Ile Asn Asp Lys Val Lys Leu Ser Arg Ile Phe 260 265 270 aaa
atg gtc ggt att act aac ata att tca agc tta ttt ggt gtg agt 864Lys
Met Val Gly Ile Thr Asn Ile Ile Ser Ser Leu Phe Gly Val Ser 275 280
285 tct aca gtt att gca gtc gaa agt tct act gca act cat tca gga gct
912Ser Thr Val Ile Ala Val Glu Ser Ser Thr Ala Thr His Ser Gly Ala
290 295 300 aaa aca gga aaa gta tct att ttt gta ggt ata atg ttt ctt
tta tct 960Lys Thr Gly Lys Val Ser Ile Phe Val Gly Ile Met Phe Leu
Leu Ser 305 310 315 320 ttg ttg ata atg ccc gtt att ata gca ata cct
agt tta gtt gta tca 1008Leu Leu Ile Met Pro Val Ile Ile Ala Ile Pro
Ser Leu Val Val Ser 325 330 335 cct atc tta ata att gtt ggc ggt tta
atg ttt act aat att aaa gaa 1056Pro Ile Leu Ile Ile Val Gly Gly Leu
Met Phe Thr Asn Ile Lys Glu 340 345 350 tta gat ttt aat gat atg act
gaa ttt att cct tgt tat ata aca att 1104Leu Asp Phe Asn Asp Met Thr
Glu Phe Ile Pro Cys Tyr Ile Thr Ile 355 360 365 ata atg ata cca ctt
act ttt gat att gca act gga atg gga ttt gga 1152Ile Met Ile Pro Leu
Thr Phe Asp Ile Ala Thr Gly Met Gly Phe Gly 370 375 380 ttt att tca
tat gtt cta att aat ttt gta tgc aaa aaa acc gaa cgt 1200Phe Ile Ser
Tyr Val Leu Ile Asn Phe Val Cys Lys Lys Thr Glu Arg 385 390 395 400
tta aat cca att tta ata att att gct tta ctt ttt aca ata aat tta
1248Leu Asn Pro Ile Leu Ile Ile Ile Ala Leu Leu Phe Thr Ile Asn Leu
405 410 415 gtt tta caa taa 1260Val Leu Gln 54419PRTStaphylococcus
epidermidis 54Met Ile Gly Arg Lys Lys Glu Thr Leu Leu Lys Asn Glu
Val Ile Ser 1 5 10 15 Ala Phe Thr Thr Phe Phe Thr Cys Ser Tyr Ile
Ile Ile Val Asn Gly 20 25 30
Ile Leu Leu His Gln Ala Gly Met Ser Leu Leu Trp Thr Ile Ile Ala 35
40 45 Thr Thr Leu Val Cys Cys Ile Ser Cys Ile Leu Leu Gly Ile Tyr
Ala 50 55 60 Asn Val Pro Leu Ile Ile Ile Pro Gly Ile Gly Glu Thr
Ile Phe Phe 65 70 75 80 Thr Tyr Thr Ile Ile Lys Ser His Tyr Tyr Asn
Tyr His Glu Ala Leu 85 90 95 Ala Ile Val Leu Ile Ser Gly Leu Ile
Phe Thr Phe Ile Ala Tyr Thr 100 105 110 Pro Phe Ala Arg Val Leu Asp
Lys Ser Ile Pro Lys Asn Leu Lys Glu 115 120 125 Gly Ile Thr Ile Gly
Ile Gly Leu Phe Met Ala Phe Val Gly Leu Gln 130 135 140 Asn Ser Lys
Ile Ile Ile Pro Asn Arg Gln Ser Ile Val Glu Leu Asn 145 150 155 160
His Ile Asn Ile Tyr Ser Gly Leu Ala Ile Leu Leu Leu Leu Phe Ala 165
170 175 Ile Val Ile Phe Thr Leu Gly Thr Lys Leu Ala Phe Phe Tyr Thr
Val 180 185 190 Ile Ile Gly Ile Ile Ile Ser Phe Leu Ala Gly Ile Ile
Lys Val Lys 195 200 205 Tyr His Phe Tyr Asn Phe Ser Leu Arg Ser Ile
Val Ser Glu Asn Asn 210 215 220 Ile Phe Ser Tyr Ser Phe Asp Lys Ile
Gly His Phe Ser Phe Trp Ser 225 230 235 240 Leu Val Phe Ser Leu Thr
Ile Leu Leu Leu Phe Gln Asn Leu Gly Thr 245 250 255 Leu His Gly Leu
Lys Ile Asn Asp Lys Val Lys Leu Ser Arg Ile Phe 260 265 270 Lys Met
Val Gly Ile Thr Asn Ile Ile Ser Ser Leu Phe Gly Val Ser 275 280 285
Ser Thr Val Ile Ala Val Glu Ser Ser Thr Ala Thr His Ser Gly Ala 290
295 300 Lys Thr Gly Lys Val Ser Ile Phe Val Gly Ile Met Phe Leu Leu
Ser 305 310 315 320 Leu Leu Ile Met Pro Val Ile Ile Ala Ile Pro Ser
Leu Val Val Ser 325 330 335 Pro Ile Leu Ile Ile Val Gly Gly Leu Met
Phe Thr Asn Ile Lys Glu 340 345 350 Leu Asp Phe Asn Asp Met Thr Glu
Phe Ile Pro Cys Tyr Ile Thr Ile 355 360 365 Ile Met Ile Pro Leu Thr
Phe Asp Ile Ala Thr Gly Met Gly Phe Gly 370 375 380 Phe Ile Ser Tyr
Val Leu Ile Asn Phe Val Cys Lys Lys Thr Glu Arg 385 390 395 400 Leu
Asn Pro Ile Leu Ile Ile Ile Ala Leu Leu Phe Thr Ile Asn Leu 405 410
415 Val Leu Gln 5510PRTStaphylococcus
epidermidisMISC_FEATURE(10)..(10)Xaa at 10 is V or L 55Lys Asn Gly
Ala Tyr Lys Ala Gln Gly Xaa 1 5 10
* * * * *
References