U.S. patent application number 10/231055 was filed with the patent office on 2003-05-22 for novel adherence factors of non pathogenic microorganisms and applications thereof for screening microorganisms for specific probiotic properties; novel pharmaceutical compositions and food additives comprising such microorganisms and adherence factors.
This patent application is currently assigned to NEDERLANDES ORGANISATIE VOOR TOEGEPAST- NATUURWETENSCHAPPELIJK ONDERZOEK (TNO). Invention is credited to Conway, Patricia Lynne, Leer, Robert Jan, Pouwels, Pieter Hendrik.
Application Number | 20030095978 10/231055 |
Document ID | / |
Family ID | 28456437 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030095978 |
Kind Code |
A1 |
Leer, Robert Jan ; et
al. |
May 22, 2003 |
Novel adherence factors of non pathogenic microorganisms and
applications thereof for screening microorganisms for specific
probiotic properties; novel pharmaceutical compositions and food
additives comprising such microorganisms and adherence factors
Abstract
A protein obtainable from a non pathogenic microorganism, said
protein having mucosa binding promoting activity and a molecular
weight of 20-40 kD is disclosed. Application of such a protein or a
peptide derived therefrom in a method of screening non pathogenic
microorganisms for a microorganism capable of specifically binding
mucosa, said method comprising detection in a manner known per se.
of the presence of a particular protein on or in a microorganism or
in a culture of microorganisms, said particular protein being the
already defined protein. Kits suitable for such a screening method
are also disclosed. Use of a component selected from the group of
components comprising a protein or peptide as defined an expression
vector comprising nucleic acid encoding such protein or peptide a
recombinant. microorganism or a part of said microorganism
expressing such protein or peptide, said part expressing mucosa
binding promoting activity a non pathogenic microorganism capable
of expressing such protein or peptide or a part of said
microorganism, said part expressing mucosa binding promoting
activity as pharmaceutically active component in a pharmaceutical
composition for prophylaxis and/or treatment of disease or illness
associated with a mucosa colonising pathogenic microorganism. Use
of such components as food additive and compositions comprising
such components are described.
Inventors: |
Leer, Robert Jan; (Xk
Voorburg, NL) ; Pouwels, Pieter Hendrik; (Aj
Rijswijk, NL) ; Conway, Patricia Lynne; (Le Perouse
Nsw, AU) |
Correspondence
Address: |
YOUNG & THOMPSON
745 SOUTH 23RD STREET 2ND FLOOR
ARLINGTON
VA
22202
|
Assignee: |
NEDERLANDES ORGANISATIE VOOR
TOEGEPAST- NATUURWETENSCHAPPELIJK ONDERZOEK (TNO)
SCHOEMAKERSTRAAT 97
VK DELFT
NL
NL-2628
|
Family ID: |
28456437 |
Appl. No.: |
10/231055 |
Filed: |
August 30, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10231055 |
Aug 30, 2002 |
|
|
|
09051755 |
Sep 30, 1998 |
|
|
|
09051755 |
Sep 30, 1998 |
|
|
|
PCT/NL96/00409 |
Oct 21, 1996 |
|
|
|
09051755 |
Sep 30, 1998 |
|
|
|
PCT/NL95/00367 |
Oct 20, 1995 |
|
|
|
Current U.S.
Class: |
424/185.1 ;
424/190.1; 530/350 |
Current CPC
Class: |
C07K 14/335 20130101;
Y02A 50/47 20180101; A61K 38/00 20130101; Y02A 50/30 20180101 |
Class at
Publication: |
424/185.1 ;
424/190.1; 530/350 |
International
Class: |
A61K 039/02; C07K
014/335 |
Claims
1. An isolated and purified protein or polypeptide having mucosa
binding activity and a molecular weight of 20-40 kD as measured by
non-gradient denaturing SDS-PAGE, using a calibration curve
obtained with standard proteins, and gel filtration chromatography,
relative to the standard curve, or a functionally equivalent
polypeptide thereof having mucosa binding activity, wherein said
protein or polypeptide comprises one or more of the following amino
acid sequences:
3 I) AASAVNSELVHK (SEQ ID No.21) II) ANFVPTK (SEQ ID No.22) III)
DTAIQSSYNK (SEQ ID No.23) IV) ISALFNK (SEQ ID No.24) V) IAGTGTNNA,
(SEQ ID No.25)
and wherein said mucosa binding activity comprises binding to a
receptor-which is present on the mucosa and is recognized by a
pathogenic microorganism:
2. A protein or polypeptide according to claim 1, wherein said
mucosa binding activity is equal to or exceeds that of L.
fermenltur 104R 29 kD adherence factor with the amino acid sequence
of SEQ ID No. 2.
3. A protein or polypeptide according to claim 1, wherein said
protein or polypeptide comprises SEQ ID No. 6.
4. A protein or polypeptide according to claim 1, wherein said
protein or polypeptide comprises SEQ ID No. 13.
5. A protein or polypeptide according to claim 1, wherein said
protein or polypeptide comprises SEQ ID No. 6 and SEQ ID No.
13.
6. A protein or polypeptide according to claim 1, wherein the
protein or polypeptide comprises one or more of the following amino
acid sequences:
4 II) ANFVPTK (SEQ ID No.22) III) DTAIQSSYNK (SEQ ID No.23) IV)
ISALFNK (SEQ ID No.24) V) IAGTGTNNA. (SEQ ID No.25)
7. A protein or polypeptide according to claim 1, wherein said
protein or polypeptide comprises two structurally identifiable
domains.
8. A protein or polypeptide according to claim 1, wherein said
protein or polypeptide comprises the amino acid sequence of L.
fermentum 104R adherence factor of 29 kD of SEQ ID No. 2.
9. A protein or polypeptide according to claim 1, obtainable by
binding to an antibody either polyclonal or monoclonal raised
against the protein or polypeptide with the amino acid sequence of
L. fermentum 104R adherence factor of 29 kD of SEQ ID No. 2.
10. A polypeptide fragment of a protein or polypeptide according to
claim 1, said fragment having mucosa binding activity, having a
length of less than 60 amino acids and comprising one or more of
the following amino acid sequences:
5 I) AASAVNSELVHK (SEQ ID No.21) II) ANFVPTK (SEQ ID No.22) III)
DTAIQSSYNK (SEQ ID No.23) IV) ISALENK (SEQ ID No.24) V) IAGTGTNNA.
(SEQ ID No.25)
11. A polypeptide fragment according to claim 1, saids fragment
having a length of less than 40 amino acids.
12. A recombinant protein or polypeptide comprising the amino acid
sequence of a protein or polypeptide according to claim 1.
13. A recombinant polypeptide fragment comprising the amino acid
sequence of a polypeptide fragment according to claim 10.
14. A fusion protein or polypeptide comprising a protein or
polypeptide having mucosa binding activity according to claim 1
attached to a drug, an immunomodulator or antigen.
15. A fusion polypeptide fragment comprising a polypeptide fragment
having mucosa binding activity according to claim 10 attached to a
drug, an immunomodulator or antigen.
16. A composition comprising a protein or polypeptide having mucosa
binding activity according to claim 1 as a pharmaceutically active
component and a pharmaceutically acceptable carrier in a
pharmaceutically acceptable dosage form.
17. A composition comprising a polypeptide fragment having mucosa
binding activity according to claim 10 as a pharmaceutically active
component and a pharmaceutically acceptable carrier in a
pharmaceutically acceptable dosage form.
18. Food product comprising a protein or polypeptide having mucosa
binding activity according to claim 1 as an additive.
19. Food product comprising a polypeptide fraqment having mucosa
binding activity according to claim 10 as an additive.
20. A method of screening non-pathogenic microorganisms for a
microorganism capable of specifically binding mucosa, said method
comprising detection of the presence of a particular protein or
polypeptide on or in a microorganism or in a culture of said
microorganism, said particular protein or polypeptide being a
protein or polypeptide according to claim 1.
Description
[0001] Novel adherence factors of non pathogenic microorganisms and
applications thereof for screening microorganisms for specific
probiotic properties; novel pharmaceutical compositions and food
additives comprising such microorganisms and adherence factors
SUMMARY OF THE INVENTION
[0002] This invention relates to the screening of bacteria, in
particular non pathogenic bacteria for those bacteria that can
adhere to specific sites of the mucosa called receptors. More
specifically the invention is directed at screening of non
pathogenic Gram positive bacteria in particular lactic acid
bacterial (LAB) species, more in particular bacteria of the genera
LactobaciZLus and Bifidobacteriur. A preference is expressed for
screening bacteria indigenous to farm animals. pets and humans.
[0003] The invention comprises a method of screening for a
particular group of adherence factors of the non pathogenic
bacteria not previously recognised. In particular the adherence
factors e-g. of Lactobacilli are of interest. This novel group of
adherence factors of non pathogenic bacteria comprises proteins
that are structurally related to virulence factors of certain
classes of pathogenic microorganisms.
[0004] The invention also relates to the application of bacteria
obtainable via the screening method of the invention. in particular
to Lactobacilli producing said adherence factors, application of
the adherence factors as such, application of parts of the bacteria
and application of parts. of an adherence -factor from the novel
group for various pharmaceutical applications. Such application may
comprise the treatment or prophylaxis of infections of the
gastrointestinal tract, the respiratory tract, urogenital tract,
the oral cavity or any other part of the body in particular any
internal part of the body that can be colonised by pathogenic
Imicroorganisms.
[0005] Another suitable example of application comprises the
targeting of specific compounds to cells of the mucosa. for example
with the aim to evoke a specific mucosal immune response against
said compound, or to modulate the immune response.
[0006] Novel microorganisms obtainable e.g. through recombinant DNA
technology expressing or overexpressing any of the novel adherence
factors or effective parts thereof are also included within the
invention.
[0007] The nucleic acid sequences encoding the adherence factors
and fragments of said sequences encoding mucosa binding expression
products are also part of the invention as are the recombinant
products resulting from expression of said nucleic acid
sequences.
[0008] Novel pharmaceutical compositions comprising the nucleic
acid or expression products thereof or microorganisms expressing or
overexpressing an adherence factor of the novel type also fall
within the scope of the invention.
BACKGROUND TO THE INVENTION
[0009] Pathogenic viruses and bacteria can adhere to specific sites
of the mucosa, called receptors and invade the underlying cells via
these receptors, resulting in illness or even the death of the host
organism. For public and animal health care it is essential that
effective and cheap means are available to prevent and/or cure
infectious diseases in humans and animals.
[0010] The mucosa form the porte d'entree of numerous pathogenic
bacteria. for example of Gram negative bacteria of the genera
Escherichia. CampZclbacter, Haemophilus, SlitgeZla, Vibria,
Pasteurella, Yersinia, SaZmoneILa, Gram positive bacteria like
Mycobacterium, Listeria, CLostridiurn. Sraphylococcus and viruses
like rotavirus, poliovirus, measles and many other microorganisms
well known to a person skilled in the art of microbial
infections.
[0011] Bacteria of the genus CampyZobacter for example can cause
severe enteritis in humans and animals after oral ingestion.
C.Jejuni is a major cause of diarrhoea in humans and occasionally
in animals, Beside diarrhoea, C.Jejuri can occasionally also cause
appendicitis, meningitis, abortion and urinary tract infection in
humans. In developed countries, persons of all ages are affected
and Campylobacrer infections are as common as infections caused by
Salmonella, Shigelal or Vibrfo cholerae.
[0012] Mycobacteria such as Mycobacterium tuberculosis and
Mycobacterium leprae also cause serious diseases such as
tuberculosis and lepra respectively. These bacteria cause the death
of many individuals in particular in the less well developed
countries. These microorganisms invade the body via the mucosa of
the respiratory tract,.
[0013] Pathogenic microorganisms can adhere to parts of the body
e.g. the gastro-intestinal tract, thereby initiating a disease. The
studies of the adhesion of pathogenic microorganisms to parts of
the body of a host organism have resulted in a wealth of data. From
these studies it has become clear that adhesion of pathogenic
bacteria can be mediated by proteins. Detailed information is
available about proteins from pathogenic bacteria that bind to
components of the extra cellular matrix, e.g. collagens.
fibronectin or proteoglycans. Particular examples are the
mycobacterial fibronectin-binding proteins, the fibronectin and
collagen-binding proteins of Streptococci and Staphylococci
specific enterobacterial fimbrial types. and surface proteins of
Yersinias and the A-protein of Aeromonas (for a review. see
Westerlund and Korhonen, Mol. Microbiol. 9:687-694 1993).
[0014] Information about the adhesion of Gram-positive, non
pathogenic bacteria to surfaces of a host organism is more limited,
in particular information regarding specific binding of mucosal
receptors by non pathogenic microorganisms is scarce.
[0015] It is common knowledge that the normal human
gastro-intestinal tract is colonized bar a varietal of non
pathogenic microorganisms including bacteria of the genera
LactObaciltus, Streptococcus, Enterococcus, Bifidobacteriutn,
Clostridium, Bacteroides, and others. These microorganisms form
part of the indigenous microflora of the human being. As such
considerable interest has been directed to elucidating the
mechanisms of adherence and the role of adhesion in
gastrointestinal colonisation. However the nechanisms of adhesion
of LAB. a well examined group of non pathogenic bacteria present in
gut microflora of humans and animals are in general niore complex
than those of the gastrointestinal pathogens (Hasty et al. Infect,
Immun. 60.2147-2152 1992).
[0016] Adhesion of non-pathogenic bacteria may be specific or
especific. Hvdrophobic and electrostatic adhesion mechanisms are
involved in non-specific adhesion. Specific adhesion is
characterized by a so-called "lock-and key mechanism", in which the
adherence factor binds to a specific receptor. Specific adhesion is
usually associated with the adhesion of microorganisms to receptors
on living tissues. Adherence factors or adhesins are. in general,
surface bound molecules. The adhesin can be firmly attached to the
surface of the bacterium or loosely bound. The receptor is a
component or structure on the surface of the cell where the
bacterium will bind by an active site of the adhesin (Rutter et al,
1984 Mechanisms of adhsions in "Microbial adhesion and aggregation"
Marshall, K. C. ed. pp 5-19, Springer-Verlag,. Berlin).
[0017] Lactic acid bacteria, particularly Lactobacillus and
Enteaococcus, are examples of non pathogenic Gram-positive bacteria
that play a key role in the establishment and maintenance of the
microflora of the gastro-intestinal tract of man and animals.
Lactobacillus species have been isolated from various regions of
the human gastro-intestinal tract (Molin et al, J. Appl. Bacteriol.
74, 314-323 1993).
[0018] The determinants supposedly responsible for the adhesion of
some strains have been studied, and certain structures are reported
to be involved in the mechanism of adhesion. However. because of
the complexity of the intestinal ecosystem, little is known about
why and how certain bacterial strains adhere to and colonize
specific regions of the gastro-intestinsl tract.
[0019] There is indeed great confusion in the literature about the
mechanisms of adhesion of Lactobacilli to the gastro-intestinal
mucosa. Fuller described the adhesion of Lactobacillus to chicken
crop epithelium and concluded that the adhesion was mediated by
polysaccharides (Fuller, J. Gen Microbiol. 87;245-250 1975).
However. Conway and Adams (J. Gen. microbial. 135,1167-1173 1989),
who found no evidence for a role of polysaccharides in the adhesion
of lactobacilli. suggested that other components may be involved.
Other researchers have indeed shown that lipotheichoic acids (LTA)
are very important in the adhesion of Lactobacilus and
Streptococcus and proposed that LTA is responsible for the
association of Streptococci with fibronectin and dental plaque
(Hogg and Manning, J. Appl. Bacteriol. 65:483-489; Vickerman and
Jones, Infect, Immun. 60:400104008 1992). Suegara et al (Infect.
Immun. 12:173-179),have described that proteinaceous material
mediates adhesion of lactobacilli to the rat stomach
epithelium.
[0020] In Current Microbiology, Vol. 28 (1994) p. 231-236 Aleljung
P. et al. describe purification of 2 collagen binding proteins of
L. reuteri NC1B 11951 which bind to collagen type I. One of 31 kD
with a sequence xSNKPIIVCSK*XV. One of 29 kD with a sequence
ASS*AVNSELV. The closest homology appeared to be with a trigger
factor of E. coli. TIG position 27-33 with a relative score of 79%.
It is also stated the CnBP of L. reuteri do not seemn to be S
Protein. a protein type which has been illustrated to be involved
in adhesion to chicken alimentary tract. They state "now
non-pathogenrc indigenous gut microflora are illustrated as binding
extracellular matrix binding protein". They do not however
illustrate in vitro or in vivo binding to mucosa or mucin. They
merely illustrate binding to a component as such which is known to
be present in mucosa. No illustration of binding to such component
in the form in which it is present in mucosa is provided. It is not
clear whether such binding to collagen when present in mucosa would
occur due to the fact that it is unclear where the binding site is
and whether such site is available or present for binding in
collagen when present in mucin or mucosa. No illustration of
non-pathogenic microorganism adhesion to ECM or mucosa is provided.
The article is largely speculative in nature.
[0021] Recently, Toba et al have shown that adhesion of
Lactobacillus crispatus to the extracellular matrix is mediated by
the S-layer protein (Toba et al. Appl. Environm. Microbiol. 61:
1995).
[0022] In EP 0 210 579 (with a priority date of November 1984) a
preparation is described containing a protein of a Mw of 14 kD
claimed to be the responsible compound for the enhancement of
bacterial adhesion to squameous epithelium in mice and pigs. The
preparation containing the 14 kD protein was obtained by
cultivating Lactobacfltus fermentum in a medium rich in sugars and
amino acids. From EP 0 210 579 it is not clear whether the adhesion
promoting factor is specific for non-pathogenic bacteria or also
may enhance the adhesion of pathogens that normally do not adhere.
It is also not clear from EF 0 210 579 whether or not the adhesion
promoting factor enhances adhesion to specific sites (receptors) or
to a-specific sites. Moreover, EP 0 210 579 does not make clear
what the origin of the 14 kD protein is. It remains uncertain
whether the 14 kD protein is synthesized by L. fermentum as such or
is generated from medium components by an activity of L. fermentum.
Thus both identity and applicability of the 14 kD protein remain
obscure in the publication.
[0023] A number of later publications also suggest different
proteinaceous components being involved, however offer no
conclusive data.
[0024] WO 90/09393 of Conway and Kjelleberg for example describes a
fraction derived from L. crispatts 104 of over 30 kD exhibiting
enti-pathogenic activity The fraction of over 30 kD maintains it's
activity after treatment with pronase or trypsin, It is obtained by
growth of L. crispatie in complex medium. The application also
mentions that the corresponding fraction of 8000-30.000 did not
exhibit anti-pathogenic activity.
[0025] The application is silent on the exact nature of the
responsible component Go components. It suggests also inhibiting
the adhesion of pathogens to gastrointestinal epithelium of humans
and animals. They also indicate in this respect that adhesion of an
E. coli K88 strain to pig intestinal mucosa was inhibited by the
high molecular weight metabolites of Lactobacilli isolated from the
pig but not of lactobacilli from the mouse digestive tract.
Subsequent studies indicated that this was not the growth
inhibiting compound in casu and the mechanism of inhibition of
adhesion was to be investigated. Lactobacillus metabolites could
perhaps inhibit pathogen colonisation of the mucosal surface which
is a prerequisite for pathogenicity for many strains. Consequently
factors in addition to growth inhibition activities should also be
considered. No illustration is given of mnucosal binding
inhibition. What is illustrated is that Lactobacillus fermenrum KLD
inhibited growth of E. coli strains, Campilobacter jejeuni,
Salmonella sofia and Streprococcus faecium in vitro. The
supernatant derivable upon growth of the L. fermentum with glucose
e.g. BHI medium followed by dialysis and fractionation over
ultrafilters with a cut-off of MW of 10.000 and 30.000 is described
as being able to elicit such effect.
[0026] The most recent publication of the aforemetioned nature
being that of Blomberg L.; Henriksson A.; Conway P. (Appl. Env.
Microbiol. feb. 91, p499-502) in which a protein-mediated adhesion
mechanism of a Lactobactilus fermentum strain to mouse squamous
epithelium, said protein being present in a retentate fraction of
culture fluid with a MW higher than 250-000 is postulated. The
publication is silent on the nature of the protein and explicitly
states it had not been isolated and that the efficacy had to be
verified by further experiments.
[0027] In conclusion: Although the role of proteins and the nature
thereof in the adhesion of bacterial pathogens is undisputed and
well documented. the role of proteins in adhesion of non-pathogenic
bacteria is still at the least controversial and unclear.
[0028] Although many diseases can be treated with antibiotics or
drugs. there is a general tendency to limit the use of such
compounds, as more and more pathogenic organisms become resistent
to antibiotics and drugs. A very promising alternative to drugs for
treatment of intestinal diseases is the use of non-pathogenic
bacteria with probiotic properties.
[0029] Probiotics are defined as "mono or mixed cultures of living
organisms which, applied as dried cells or as a fermented product
to humans or animals, beneficially affect the host by improving the
properties of the indigenous microflora."
[0030] Some strains of Lactobacillus and Bifidobacterium strains,
reportedly, have probiotic properties. The beneficial effects have
been attributed to the lowering of the pH, a condition which
reduces the proliferation of Gram-negative pathogens like
Escheriehia coli. In addition. mdany species of lactic acid
bacteria produce oligopeptides with antimicrobial properties.
called bacteriocines. These compounds are bacteriostatic or
bacteriocidal for Gram-positive bacterial pathogens. like
Clostridium, Listeria etc.
[0031] Some Lactobacilli have been suggested as inhibiting adhesion
of pathogens in animals and in in vitro models, These inhibitory
effects are usually explained by non-specific steric hindrance of
the receptors for pathogens. In contrast, each pathogen has a
specific intestinal receptor (Falkow et al. Ann. Rev. Cell. Biol.
8. 333-363 1992).
[0032] Lactobacilli or preparations made with Lactobacilli are thus
widely used to treat intestinal and urinary tract disorders (see
e.g. WO 9 516 461; RU 2 000 116; WO 9 418 997; EP 0 577 903; GB 2
261 372; WO 9 301 823; WO 0 921-475; US 7 822 505; CA 1 298 556, EP
0 199 535; EP 0 210 579). The beneficial effects of such
preparations have been attributed to various factors, but the
properties and mode of action of such health stimulating compounds
have either not been disclosed or are at most mere postulations.
Answers regarding the mechanism of probiorics are crucial in order
to find novel enhanced probiotics and optimrise their use.
[0033] Considering the economic importance for food industries to
use starter strains which show a scientifically proven probiotic
effect, and the equally large interest of pharmaceutical companies
to use GRAS (Generally Recognised As Safe) organisms as carriers
for the development of mucosal vaccines. considerable effort is
spent in screening bacteria, in particular GRAS organisms. more in
particular Lactobacilli. for probiotic and/or immune modulating
properties. A major disadvantage of the present screening
programmes is that they are laborious, time-consuming and thus very
costly. No easy and reliable testsystem is available to screen for
probiotic OR immune modulating properties of bacterial strains.
OBJECT OF THE INVENTION
[0034] The objective of the invention is to overcome the above
mentioned difficulties. It is now proven unequivocally that a 29 kD
proteinaceous compound is responsible for specific adhesion or L.
fermetum to receptor sites in tne mucus of pigs and mice, and thus
methods to screen for other microorganisms that synthesize proteins
with a-simil-ar structure. and function are now provided. By
demonstrating that the adherence promoting entity of L. fermentum
is a protein of 29 kD which is structurally related to adherence
factors of certain pathogenic bacteria and by demonstrating the
nucleotide sequence of the gene encoding the adherence factor, the
present invention provides methods for the rapid screening of
microorganisms that contain a gene coding for an adhesin of the
novel type and methods for screening of microorganisms that produce
such an adherence factor using standard protein and nucleic acid
technologies,
[0035] By demonstrating for the first time a structural
relationship between virulence factors of pathogenic bacteria and
adherence factors of non-pathogenic bacteria, i.e. Lactobacilli,
the present invention provides methods to selectively and
specifically interfere with the adhesion of pathogens to receptors
on the mucosa of the gastrointestinal tract. of the urogenital
tract, of the oral cavity, of the respiratory tract and of the
nasal cavity and to screen microorganisms for the capacity to
interfere with adhesion of the aforementioned type of
pathogens.
[0036] The many applications now possible will be explained in more
detail below.
[0037] i) A more rapid and directed screening of bacteria for
bacteria with probiotic properties and/or immunomodulating
properties is now possible. The present invention allows rapidly
screening bacteria for the capacity to interfere with the adherence
of pathogens to mucosal receptors. In particular, the present
invention provides a method to screen microorganisms for the
presence of an adherence factor that enhances the specific adhesion
of non-pathogenic Gram positive bacteria, more in particular the
adhesion of lactobacilli, to bacterial receptor(s) of the mucosa of
the gaitro-intestinal tract, the urogenital tract, the respiratory
tract and the oral/nasal cavity of humans and animals. Preferably
the microorganisms to be screened will be microorganisms that are
non pathogenic in humans and animals. Such microorganisms will
preferably be indigenous to humans and/or animals, thus already
being able to withstand the environment in which they are to be
applied and also obviously not being toxic to the particular
species from which they are derived. Examples of suitable non
pathogenic microorganisms include bacteria of the genera
Lactobacillus, Streptococcus, Enterococcus, Bifidobacterium,
Clostridium and Bacteroides.
[0038] The screening can occur at protein and/or nucleic acid level
using standard technologies known per se for protein detection or
nucleic acid detection such as nucleic acid amplification and
hybridisation techniques and protein or peptide assays using
polypeptide or protein probes and/or antibodies specific for the
adherence factor or factors to be detected. The present invention
thus also provides a method to screen microorganisms for the
presence of nucleic acid that encodes proteins with the desired
adherence properties. Protein and nucleic acid assays are readily
carried out by persons skilled in the art once the relevant amino
acid and nucleic acid sequences have been determined as in the
instant case. Such information enables probes and primers to be
constructed when the nucleic acid sequence and/or the relevant
amino acid sequence of the protein has been determined and isolated
or synthesized as in the instant case. The isolation of the pure
protein and/or expression of the pure protein enables production of
antibodies in a manner known per se.
[0039] According to the invention, bacteria can be screened for the
presence of a protein falling within the definition of the novel
type of adherence factors of non pathogenic microorganisms as
detailed below or the presence of a DNA sequence encoding such a
protein or active part thereof. Application of the invention thus
in particular circumvents the laborious and costly route of
screening bacteria for the capacity to adhere to living tissue.
More in particular, application of the invention circumvents the
use of animals and/or human volunteers for screening purposes.
[0040] A method of screening non pathogenic microorganisms for a
microorganism capable of specifically binding mucose, said method
comprising detection in a manner known per se of the presence of a
particular protein on or in a microorganism on or in a culture of
microorganisms, said particular protein being a protein according
to any of claims 1-13 falls within the scope of protection.
Alternatively a method of screening non pathogenic microorganisms
for a microorganism capable of specifically binding mucose. said
method comprising detection in a manner known per se of the
presence of a particular gene on or in a microorganism on or in a
culture of microorganisms, said particular gene encoding a protein
according to any of claims 1-13 also falls within the scope of the
invention.
[0041] The invention also covers a kit suitable for detection of a
non pathogenic microorganism. capable of specifically binding
mucosa, said kit comprising a component capable of specifically
binding to a protein according to any of claims 1-13 such as an
antibody. In another embodiment the invention comprises a kit
suitable for detection of a non pathogenic microorganism capable of
specifically binding mucosa, said kit comprising a component
capable of specifically binding to a part of a nucleic acid
sequence encoding a protein according to any of claims 1-13 such as
a nucleic acid probe or primer.
[0042] ii) By applying the protein or polypeptide capable of
specifically binding mucosa to a human or animal or by applying a
microorganism capable of expressing such a protein or polypeptide
or a culture of such a Microorganism to a human or an animal it now
becomes possible to interfere with the adhesion of pathogenic
microorganisms to mucosa or mucin. In particular it becomes
possible to prevent or reduce adhesion by pathogenic microorganisms
to mucosa of the urogenital tract. gastro- intestinal tract,
respiratory tract and/or oral/nasal cavity of humans and animals.
Particularly interesting is that the invention offers a method to
efficiently and specifically interfere with the adhesion of certain
classes of pathogens to bacterial receptors of the mucosa and to
screen for microorganisms capable of interfering with adhesion of
certain classes of pathogens. Pathogens that may now be combatted
comprise both Gran positive and Gram negative microorganisms in
particular those that specifically bind mucosa receptors. Examples
of pathogens to be combatted comprise strains of the genera
Escherichia, Campytobacter, Haemophilus, Shigella, Vibrio,
Pasteurella, Yersinia, Salmonella, Mycobacterium, Listeria,
Clostridium, Staphylococcus and viruses like rotavirus, poliovirus
and measles.
[0043] The invention exploits the conclusion that the infectivity
of pathogens that adhere to a mucosal receptor through an adherence
factor similar to that of the adhesion protein of L. fermentum
104R. will be reduced by probiotic bacteria harbouring an adhesion
protein with a structure like that of the adhesion protein of L.
fermentum 104R, by specific interaction with the receptor, rather
than by the more general mechanism of steric hindrance.
[0044] According to the present invention, a strategy can be
devised to specifically inhibit adherence of certain pathogens
(those that adhere by means of an adherence factor that is
stucturally related to the adhesion protein of L. fermentum) , by
administering e.g. in food or feed or as pharmaceutical composition
such adhesion proteins or, microorganisms that produce such
adhesion proteins. The application can be topical, oral or
intravenous in anv dosage form normally applied for pharmaceutical
compositions and/or feed additives. The dosage form selected will
depend on the type of infectious pathogen to be combatted. The
dosage form may be solid or liquid. Certain standards with regard
to purity and hygiene i.e. sterility normally applicable for such
compositions must be adhered to. Such circumstances are well known
to a person skilled in the art.
[0045] A composition comprising a component selected from the group
of components comprising
[0046] a protein or peptide according to any of the claims 1-21
[0047] an expression vector according to claim 24 or 25
[0048] a recombinant microorganism according to claim 26 or 27 or a
part of said microorganism, said part expressing mucosa binding
promoting activity
[0049] a non pathogenic microorganism capable of expressing a
protein or peptide according to any of claims 1-21 or a part of
said microorganism, said part expressing Tucosa binding promoting
activity as pharmaceutically active component and a
pharmaceutically acceptable carrier in a pharnmaceutically
acceptable dosage form is covered by the invention. A composition
comprising the abovementioned components in a form suitable for use
as food additive is also envisaged to fall within the scope of the
invention. The use of a component selected from the group of
components comprising
[0050] a protein or peptide according to any of the claims 1-21
[0051] an expression vector according to claim 24 or 25
[0052] a recombinant micr-oorganism according to claim 26 or 27 or
a part of said microorganism. said part expressing mucosa binding
promoting activity
[0053] a non pathogenic a-.cicorganism capable of expressing a
protein or peptide according to any of claims 1-21 or a part of
said microorganism, said part expressing mucosa binding promoting
activity as pharmaceutically actie component in a pharmaceutical
composition for prophylaxis and/or treatment of disease or illness
associated with a mucosa colonising pathogenic microorganism also
falls within the scope of the invention.
[0054] As will be apparent from the above a method for improving
food products comprising addition of a product according to any of
claims 1-21 or 24-27 and/or a non pathogenic microorganism capable
of expressing a protein or peptide according to any of claims 1-21
or a part of said microorganism. said part expressing mucosa
binding promoting activity to the food product forms an embodiment
of the invention. Preferably such a method comprises addition of a
product according to any of claims 1-21 or 24-27 to the food
product.
[0055] Obviously a food product comprising a product according to
any of claims 1-21 or 24-27 and/or a non pathogenic microorganism
capable of expressing a protein or peptide according to any of
claims 1-21 or a part of said microorganism, said part expressing
mucosa binding promoting activity as additive is also covered. A
food product comprising a product according to any of claims 1-21
or 24-27 as additive is a particularly suitable embodiment.
[0056] A person skilled in the art will realise that the inhibiting
effect may also be obtained by addition of parts of the adherence
protein, e.g. peptides derived from the 29 kD adherence protein of
L. fermentum that are found to specifically bind to mucus and
mucin. The active peptides can either be synthesized chemically or
made micro-biologically by a genetically engineered microorganism.
Alternatively the protein can be produced by a non recombinant or
recombinant microorganism and subsequently e.g. via proteolytic
digestion and optionally separation of the proteolytic fragments
the desired polypeptide can be obtained. From analysis of the
adhesion factor of 29 kD and the adhesion factors of the pathogenic
organisms Echerichia coli and Helicobacter pylori strains and
cholera toxin a consensus sequence KKXXXK (Sequence id no 30) was
postulated wherein X stands for any amino acid and K stands for
lysine. The 29 kD protein according to the invention comprises
three such sequences. They are more or less evenly distributed over
the protein molecule at positions 47-52 (KKMGLX), 173-178 (KKNSTK)
and 223-238 (KKLSEX) of the mature protein, The numbering
corresponds to amino acids 54-59. 180-185 and 230-235 of sequence
id no. 2. of the sequence listing, in which the mature protein
commences with Ala at position 8. The presence of at least one of
the KKXXXK sequences. preferably two of these sequences in a
protein or peptide according to the invention is preferred. Most
desirably three such sequences are present. In a particular
embodiment the consensus sequence will be one of the natively
occurring amino acid sequences present in the 29 kD protein
disclosed above. Preferably sequences corresponding to those
present in their native environment will be used, such sequences
can however be arrived at through genetic engineering or synthetic
means generally known in the art such as through DNA synthesizers,
Merrifield synthesis and cloning technology as mentioned above.
Preferably such sequences will also be present in a sequence such
that the tertiary structure mimics that of the native protein. This
can be ascertained using computer technology in a manner known per
se. Such sequences are involved in binding to negatively charged
intestinal receptors.
[0057] Microorganisms that have the GRAS status, like Aspergillus,
Lactobacillus and Lactococcus are well suited for such purposes. A
person skilled in the art will realise that other microorganisms
can also be used for production of adherence factors or peptides
derived thereof. However it will be preferred for applications to
humans to employ GRAS organisms. Lists of GRAS organisms are
readily available to a person skilled in the field of foodstuffs
and/or pharmaceuticals and are incorporated herein by reference.
The US FDA for example maintains a list of such organisms.
[0058] The conclusion that proteins like the adhesion promoting
protein of L. fermentum 104R or microorganisms that produce a
protein with a structure similar to that of the adhesion promoting
protein of L. fermentum 104R will interfere with specific adhesion
of pathogens carrying an adhesion protein with a similar structure,
does not necessarily imply that such adhesion promoting proteins or
adhesion promoting protein producing microorganisms will not
interfere with the adhesion of pathogens that do not produce an
adherence factor with a similar structure. A person skilled in the
art will immediately realise that a corrolary of the use of
microorganisms with an adherence promoting protein like that of L.
fermentum 104R might be that adherence of such bacteria to a
specific receptor will also limit the adherence of pathogens with
adherence factors other than the L. fermentum-like adhesion factor,
by a general mechanism of steric hindrance. Thus the pathogenic
microorganisms that can be conmbatted do not only comprise
microorganisms that bind the mucosal receptor specifically bound by
the adherence factor from the non pathogenic organism.
[0059] iii) As the group of proteins exhibiting the desired
activity is now known and amino acid sequences and nucleic acid
sequences have been determined it is now possible to develop and/or
select microorganisms capable of improved production i.e.
overexpression of the desired protein or polypeptide. This can be
achieved via normal optimalisation of cultivation conditions, via
selection of strains expressing proteins with improved receptor
binding properties in a manner known per se.
[0060] It is also possible via genetic engineering to incorporate
the nucleic acid sequence or nucleic acid sequences in
microorganisms of choice that thus become capable of
(over)expression and preferably also secretion of mucosa binding
promoting component. Preferably the microorganism will be a GRAS
organism such as a lactic acid bacterium. It is also possible to
incorporate the encoding sequences or sequences such that they are
operably linked to regulating sequences that enable higher
expression than with the regulating sequence normally associated
with the encoding sequence. A number of high expression vectors are
known for various microorganisms in particular GRAS microorganisms
such as lactic acid bacteria. Recombinant microorganisms capable of
expressing or overexpressing the polypeptide or protein capable of
promoting the binding of mucosa of the novel group of adherence
factors from non pathogenic microorganisms or recombinant
expression vectors comprising the appropriate nucleic acid also
fall within the scope of the invention, The microorganism that is
genetically engineered may already express the adherence factor but
the microorganism may also be selected from a group that does not
natively express an adherence protein of the novel group. The
microorganism may simply be used as production plant for the
protein or polypeptide which mall subsequently be isolated and
applied as pharmaceutical or as food/feed additive, or the
microorganism itself may be used as pharmaceutical or as food/feed
additive, Preferably the protein or polypeptide producing
microorganism will be non pathogenic. In particular GRAS
microorganisms are preferred in order to enable applications of the
expression product and/or microorganisms as active component of a
pharmaceutical composition or food/feed additive.
[0061] The nucleic acid sequences may be incorporated onto a
plasmid vector or integrated into the chromosome in any embodiment
known per se in the recombinant DNA technology field. A large
number of transformation and expression vectors and tLechnologies
are known in the state-of-the-art and are currently also
commercially available. Preferred are food-grade transformation and
expression vectors and methods of transformation suitable for GRAS
microorganisms.
[0062] Preferably the microorganisms to be selected and/or
transformed have the following characteristics:
[0063] Survival of the environmental conditions at the location
where it must be active
[0064] Proliferation and/or colonisation at the location where it
is active
[0065] No immune reaction against the probiotic strain
[0066] No pathogenic, toxic, allergic, mutagenic or carcinogenic
reaction by the probiotic strain itself. its fermentation products
or its cell components after decease of the bacteria
[0067] Genetically stable, no plasmid transfer
[0068] Easy and reproducible production
[0069] Viable during processing and storage
[0070] In general terms a recombinant microorganism is claimed
comprising a nucleic acid sequence according to claim 23 and/or an
expression vector according to claim 24 or 25, said nucleic acid
sequence and/or expression vector being absent or in the
alternative being present in a lower copy number or being expressed
to a lower degree in the corresponding non recombinant
microorganism. In a further embodiment the invention comprises a
recombinant microorganism as just defined (according to claim 26),
said microorganism being a non pathogenic microorganism, preferably
indigenous to the microflora of a human or animal, more preferably
to the microflora of a human.
[0071] The invention also encompasses a nucleic acid sequence
encoding any of the proteins or peptides according to any of claims
1-21 and an expression vector comprising such a nucleic acid
sequence, operably linked to an expression regulating sequence,
said expression vector being capable of expressing the nucleic acid
in a non pathogenic microorganism such as a GRAS microorganism and
said expression vector preferably comprising nucleic acid derived
from a GRAS microorganism. In a further embodiment the expression
vector according to the invention is a vector, wherein the
expression regulating sequences are not naturally associated with
the gene encoding the adherence, factor from which the nucleic acid
sequence is derived.
[0072] iv) As the 3D structure, amino acid sequence and nucleic
acid-sequence on an adherence protein have now been ascertained and
the similarity between other protein groups has been determined it
lies within reach of a person skilled in the art to design a
protein or polypeptide exhibiting iimproved binding characteristics
and thus improved results in pharmaceutical applications or as
food/feed additive. The invention thus also covers mutant
polypeptides and proteins exhibiting better mucosa binding than the
protein with amino acid sequences of FIG. 3 and better mucosa
binding activity than any of polypeptides I-V as defined in the
experimental part of the subject description. The invention also
comprises equivalent sequences as available in nature and as
mutants i.e. nucleic acid sequenes encoding protein or polypeptide
having at least the mucosa binding activity of the 29 kD protein
and such proteins or polypeptides as well as their application in
any of the methods of the description and/or claims.
[0073] iv) Having discovered a group of proteins End peptides
capable of specifically binding mucosa it also becomes possible not
only to target the microorganism expressing the protein or peptide
to mucosa but also to use such microorganism as carrier for
targeting additional compounds such as drugs. immunomodulators or
antigens for eliciting an immune reponse to the mucosa. The
microorganism may be selected for already having this particular
characteristic or may be genetically engineered so that it
subsequently produces the desired drug, immunomodulator or antigen.
It also becomes possible to develop fusion proteins or peprides
comprising the mucosa binding promoting amino acid sequences and
additional desired amino acid sequences or molecules with the
characteristic activity Of choice that has to be targeted to the
mucosa. A whole line of new pharmaceutical compounds specifically
targeted to the mucosa can thus be developed. The invention covers
such novel microorganisms and molecules and applications thereof as
pharmaceutical compositions. The invention thus also covers a
method for targeting a bacterium that expresses a gene of interest.
for example a gene encoding an antigen of a pathogenic organism, to
specific receptors of the mucosa. thereby evoking a specific immune
response against the antigen and/or modulating an immune reponse.
The invention covers a fusion protein or peptide comprising a
protein or peptide according to any of the claims 1-21 attached to
a drug, immunomodulator or antigen of choice.
[0074] Use of a component selected from the group of components
comprising
[0075] a protein or peptide according to any of the claims 1-21
[0076] an expression vector according to claim 24 or 25
[0077] a recombinant microorganism according to claim 26 or 27 or a
part of said microorganism, said part expressing mucosa binding
promoting activity
[0078] a non pathogenic microorganism capable of expressing a
protein or peptide according to any of claims 1-21 or a part of
said microorganism, said part expressing mucosa binding promoting
activity as targeting component in a pharmaceutical composition for
targeting an additional pharmaceutically active component to
mucosa, said additional pharmaceutical component being physically
linked to the targeting component falls within the scope of the
invention.
[0079] The enhancement or specific adhesion of lactobacilli to
receptors of the mucosa, according to the invention, providing the
opportunity to specifically target bacteria carrying compounds of
interest, for example lactobacilli expressing an antigen of a
pathogenic organism or a human protein, to the cells of the mucosa,
thereby modulating the immune response against the antigen/human
protein is a preferred embodiment of the invention.
[0080] According to the invention, the adhesion capacity of
probiotic strains may be modulated by altering the properties of
the adhesion protein. Such properties may involve interaction of
the adhesion protein with the mucosal receptor or interaction with
other (accessory) proteins.
DETAILED DESCRIPTION OF THE INVENTION
[0081] According to the invention. use is made in particular of a
protein with a Mw of 29 kl of L. fermentum 104R, a strain isolated
from the porcine gastrointestinal tract and/or of the DNA sequence
encoding this adhesion protein, which had not been described sofar.
The novel protein has adhesion promoting activities. In particular
the adhesion promoting activity Comprises exhibiting binding to
mucosa or mucin. The adhesion protein is present on the surface and
is also shed off into the culture medium by L. fermentum 104R.
[0082] The invention more in particular exploits a special property
of the adhesion promoting protein, namely that it is structurally
similar to virulence proteins of several pathogenic bacteria, e.g.
to adherence factors from Campylobacter Jejune, Pasteurella
haemolytica and Mycobacterium. These features are documented in the
following paragraph. According to the invention the presence of
proteins with properties similar to those of the 29 kD protein can
be determined using the Western blot technique, a technique well
known to persons skilled in the art.
[0083] The adhesion promoting protein from L. fermentum 104R
belongs to a class of proteins, called Class III solute
transporters, of which the histidine transporter (HisJ), glutamine
transporter (GlnH) and the lysine, arginine and ornithirne
transporter (LAO) of Enterobacterfaceae are the prototypes. The 3-D
structure of two of these proteins, HisJ and LAO is known. The
amino acid sequence of the adhesion promoting protein of L.
fermentum 104R shows a striking similarity with on the one hand
adherence proteins of pathogens, Peb1 of C. jejuni and LapT of P.
hemolytica , and on the other hand with members of Class III solute
transporter proteins. like LAO and HisJ. Protein modelling has
shown that the predicted 3-D structure of the L. fermentum adhesin
is also similar to that of LAO and HisJ. Amino acids in proteins in
domain I of Class III solute transporters that are essential for
ligand binding are conserved among all members of this class of
proteins. These amino acids were also found at similar positions in
the adhesion promoting proteins of L. fermentum 104R and in the
virulence protein of C. Jejuni. In other words, the adhesion
promoting protein from L. fermentum 104R has a 3-D structure which
is similar to that of adherence factors of pathogens like C. jejuni
and P. haemolytica.
[0084] A protein belonging to the group of novel proteins as
defined according to the invention is defined as a protein
obtainable from a non pathogenic microorganism, said protein having
mucosa binding promoting activity and a molecular weight of 20-40
kD. Preferably the weight lies between 20-30 kD. Specific
embodiments are disclosed in claims 1-13. In particular a protein
according to the invention comprises one or more of the following
properties:
[0085] a) a molecular weight between 20 and 40 kD
[0086] b) an amino acid sequence exhibiting more than 20 %
identical amino acids and more than 40% similar amino acids with
the amino acid sequence of class III solute transporters and/or
virulence proteins Peb1 of C jejuni, LapT of P. haemolytica and
Mycobacterium tuberculosis or Mycobacterium leprae 85K complex
proteins A, B and C
[0087] c) promotes the specific binding to mucosal receptors also
used by any of C jejuni. P. haemolytica or Mycobactertum
[0088] d) has a 3D structure with 2 lobes like LAO or-HisJ
[0089] e) comprises one or more amino acid sequences that are 90%
or more similar to the following amino acid sequences
1 I) AASAVNSELVHK II) ANFVPTK III) DTAIQSSYNK IV) ISALPNK V)
IACTGTNNA,
[0090] preferable of the amino acid sequences II-V.
[0091] A specific embodiment is formed by the group of proteins
further characterised in that the protein exhibits the consensus
sequence illustrated in FIGS. 4 and 5. The proteins claimed as such
do not comprise virulence factors of pathogenic microorganisms or
Class III transporters, neither does the class of recombinant
proteins comprise recombinant virulence factors or recombinant
Class III reporters that could perhaps form state of the art at the
filing date of the subject patent application.
[0092] Preferably a protein belonging to the group of proteins
suitable for application according to the invention will exhibit
binding promoting activity for mucosel receptors used by any of C
jejuni, P. haemolytica or Mycobacterium higher or equal to that
exhibited by the 29 kD protein of L. fermentum 104 with the amino
acid sequence of FIG. 3 as can be determined by the mucosa binding
assay illustrated in the Example.
[0093] Since the nucleotide structure of the adhesion promoting
protein is known, non pathogenic microorganisms can also be
screened for the presence of DNA sequences encoding proteins with a
structure similar to that of the adhesion protein of L. fermentum
104R. The so called equivalent sequences which will encode a
protein or polypeptide exhibiting at least the same mucosa binding
activity. In particular such a nucleic acid sequence is a nucleic
acid sequence entoding the amino acid sequence of FIG. 2
corresponding to that of the 29 kD protein of L. fermentum 104R. A
nucleic acid sequence encoding the consensus amino acid sequence of
the FIGS. 4 and 5 as such also falls within the scope of the
invention. In particular a nucleic acid sequence encoding a protein
of 20-40 kD comprising the amino acid consensus sequence and
further corresponding to the sequence of the 29 kD sequence. the
only difference being in the presence of one or more mutations
resulting in substitution of amino acids by other similar amino
acids such that the hydropathy profiles remain similar and no
serious conformation change can be expected of the resulting
protein or polypeptide Falls within the scope of the invention.
Such sequences are known as those wherein conservative exchange of
amino acids has occurred in comparison to the sequence according to
sequence id, no 2. Also the invention comprises any nucleic acid
sequence capable of hybridising under stringent hybridisation
conditions when carrying out a blot assay in a manner known per se.
Such sequences thus comprise sequences encoded by nucleic acid
sequences derivable from other non pathogenic microorganisms
through cross hybridisation technology using oligonucleotide probes
encoding parts of the amino acid sequence according to sequence id
no 2, preferably probes in which the preferred codon usage of the
microorganism to be screened has been taken into account in a
manner known per se. Stringent hybridisation conditinns as
described forexample in Molecular Cloning, a Laboratory Manual,
Cold Spring Harbor Laboratory New York Maniatis. T. Fritsch, E. F.
and Sambrook, J. (1932) can be suitably applied to obtain such
equivalent sequences. The cited reference also provides information
regarding a number of other standard technologies mentioned
elsewhere in the description and is incorporated herein by
reference. In particular sequences from non pathogenic
microorganisms belonging to the genera mentioned previously in the
description are preferred. Also in a preferred embodiment at least
one consensus sequence according to sequence id no 30 will be
present. Alternatively or in addition one of the sequences of
polypeptides I-V will be present. A protein or polypeptide with the
amino acid sequence of the mature protein of sequence id no 2 in
which amino acids have been mutated, outside the consensus
sequences. having at least the mucosa binding activity of the
miature protein of sequence id no 2 is also comprised within the
invention. Also any sequence combining any of the above definitions
is also included within the scope of the invention and forms a
preferred embodiment. It is also possible that an equivalent
sequence is not derivable as such from a microorganism but can be
produced in an alternative manner e.g. recombinant DNA technology,
PCR etc. The above embodiments are also valid for such alternative
(mutant) sequences and fall within the scope of the invention.
Suitably a protein or polypeptide according to the invention will
be free of cell extract and other contaminating proteins.
Substantial purity is preferable i.e. more than 80% pure. The
purity being sufficient for application as pharmaceutical and food
additive and for achieving the activity required in the
applications according to the invention.
[0094] According to the present invention, bacteria may also be
screened for the presence of proteins like the L. fermenrum 104R
adhesion protein, that can adhere to non-living surfaces like
plastics or metal surfaces. such screening can occur as described
above using oligo probes based on the amino acid sequence of the 29
kD adhesion protein. Preferably such a probe will encode a part of
a consensus sequence of the FIGS. 4 and 5. In addition a suitable
probe will comprise a sequence encoding the consensus sequence of
sequence id no 30. The consensus sequence or the part thereof will
be at least 5 contiguous amino acids long and preferably the probe
will be comprised completely of consensus sequence. Use of a
combination of such probes is also possible in order to obtain a
sequence encoding a protein or polypeptide exhibiting as close an
identity as possible to tne 29 kD protein or active part thereof
required for mucosa binding activitv.
FURTHER DETAILS OF EMBODIMENTS OF THE INVENTION
[0095] i) Production and purificatin of adhesion protein from
Lactobacillus fermentum
[0096] In a preferred embodiment of, the present invention, the
adhesion promoting protein of L. fermentum 104R is produced by
cultivating bacteria in MRS broth or LDM medium (Conway and
Kjelleberg, J. Gen. Microbial. 135:1175-1186 1989) for 14 to 24
hours. The 29 kD adhesion protein is purified from the medium to
apparent homogeneity by ammonium sulphate precipitation.
gel-filtration and affinity chromatography. The adhesion promoting
activity is detected in the fractions by adhesion inhibition and
dot blot essays, and visualized by PAGE, SDS-PAGE and western blots
using horse radish peroxidase labelled mucus or mucin. The purified
protein has an estimated Mw of 29 kD, under non-denaturing
conditions as well as under reducing and denaturing conditions (non
gradient denaturing SDS-FAGE. using a calibration curve obtained
with standard proteins. and gel-filtration chromatography, relative
to the standard curve) and is sensitive to pronase, and therefore,
differs from the adhesion proteins described and/or implied in EP 0
210 579 and WO 90/09398, as well as those described by Conway and
Kjellenber& (J. Gen. Microbial. 135, 1175-1186), Blomberg et al
(Appl. Environm. Microbiol. 59, 34-39 1993) and Aleljung et al
(Current Microbiology vol 28 (1994) p. 231-236. The proteins
specifically disclosed as such in the cited references do not fall
within the scope of the protection of the protein or peptide
claims, The compositions specifically described as such in the
cited references do not fall within the scope of protection of the
composition claims. In particular application of the compositions
of WO 90/09398 described specifically as such for inhibition of
pathogens do not fall within the scope of the protection. Where
specifically is mentioned in this paragraph this implies in the
examples or following the materials and methods of the cited
references. The scope of generic diclosures of such references can
cover some aspects of the subject invention. which however
nevertheless forms a selection invention vis a vis said
reference.
[0097] The adhesion promoting protein could be extracted from the
cell surface of L. fermentum by treatment of the bacteria with 1 M
LiCl and low concentrations of lysozyme. The adhesion promoting
protein which had an affinity for both small intestine mucus and
gastric mucin from pigs or mice. was released into the culture
supernatant fluid after 24 h of growth.
[0098] ii) Screening of Thiciroortenismns for the presence of a L.
fermertum-like adhesion protein
[0099] In another preferred embodiment of the present invention,
lactobacilli are screened for the presence of an adhesion promoting
protein with properties similar to those of the adhesion promoting
protein from L. fermentum, by separating proteins from the culture
medium of afn overnight culture by SDS-PAGE, and Western blotting
using polyclonal antibodies raised in rabbits against purified
adhesion protein of L. fermentum 104R.
[0100] iii) Screening of microorganisms for the presence of a L.
fermentum-like adhesion protein encoding gene
[0101] In another preferred embodiment of the present invention,
DNA is isolated from microorganisms to be screened and subjected to
PCR analysis. using sets of primers that are based on the
nucleotide sequences of the L. fermentum 104R adhesion protein
encoding gene. The products formed are analysed by standard
molecular biological techniques as are described in handbooks (e.g,
as cited elsewhere in this description) or commercially available
kits.
[0102] iv) Synthesis of adhesion Prom other than L. fermentum
104R
[0103] In another specific embodiment of the present invention, the
gene encoding the adhesion protein from L. fermentum 104R or from
another selected strain. isolated by the aforementioned procedure.
is cloned behind a strong, preferably inducible promoter and
secretion signal encoding sequence. in a GRAS production organism
like Aspergillus niger, LactobacitLus etc. The culture medium is
either used as such and used as food/feed additive or
pharmaceutical composition, or the adhesion promoting protein is
first purified (by standard techniques) and then added to food/feed
preparations or pharmaceutical compositions. The nucleic acid
sequence mey be adjusted such that it encodes the identical amino
acid sequence of the 29 kD L. fermenrum 104R adherence protein of
FIG. 2 but has codons adjusted to the preferred codon usage of the
host in which it is incorporated. Detiails of preferred codon usage
are available from sources known to a person skilled in the art of
nucleic acid expression.
[0104] v) Production of peptides with adhesions prooting
properties
[0105] In another preferred embodiment of the present invention.
peptides derived From the L. fermentum 104R adhesion protein that
show adhesion promoting properties are synthesized chemically and
used as food/feed additive. Alternatively, DNA sequences. coding
for such peptides are cloned behind a strong, preferably inducible
promoter in a GRAS production organism like A. niger or
LactobacIilus etc. In cases where the peptide encoding sequences
are cloned behind a secretion signal encoding sequence and the
peptides are secreted into the medium, the medium can be used as
food/feed additive. In cases where the peptides are not secreted
into the medium. the entire organisms, or extracts made from such
organisms. can be used as food/feed additive. Alternatively the
desired proteins or polypeptides may be isolated e.g. using
chromotagraphy in a manner known per se for isolating protein or
polypeptide e.g. in combination with antibodies specific for the
protein or polypeptide to be isolated. An antibody or antibody
fragment capable of binding an epitope or protein or peptide
according to any of claims 1-20 falls within the scope of the
invention. Such an antibody may be a polyclonal antibodn (see
Example) or a monoclonal antibody. An antibody specifically
disclosed in any of the above cited references is excluded from the
scope of protection for antibody claims as such.
[0106] vi) Targeting of an antigen or human protein to mucosa
[0107] In another embodiment of the present invention, the ability
of adhesion protein to specifically adhere to mucosal tissue is
exploited to target an antigen of a pathogen to the mucosa to
enhance a mucosal immune response against the antigen. For this
purpose, microorganisms are constructed that are capable of
synthesizing the adhesion protein and the antigen of interest.
Alternatively. to modulate the immune response against humen
proteins for the sake of suppressing auto-immune responses,
microorganisms carrying a gene encoding a human protein are
genetically engineered in such a way that they synthesize an
adhesion protein with properties similar to those of the L.
fermentum adhesion protein.
EXAMPLES
[0108] i) Purification end chaiacterization of a surface protein
from Lactobacillus fermentum that binds to small intestine mucus
and gastric mucin from pig
[0109] Spent culture fluids from 14 or 24 cultures were collected
by centrifuging at 6000 g for 20 min end dialysing at 4.degree. C.
against ultra pure water. The retenate was concentrated by ultra
filtration through a 14 KDa molecular weight cut off membrane. The
high molecular weight fraction was freeze dried and stored at
4.degree. C. Spent culture fluid was also concentrated 10 times by
hollow fibre ultrafilter and ammonium sulphate was dissolved in the
concentrate (40, 60 and 100% of saturation at 4.degree. C.) The
precipitates were collected by centrifugation (18000 xg/30 min).
dissolved in ultra pure water and dialyzed against 0.1 M ammonium
bicarbonate. The solutions were freeze dried and kept at 40.degree.
C.
[0110] The freeze dried preparation from 24 hours spent culture
fluid concentrated by ultra filtration was dissolved in HEPES-Hanks
and filtered (0.22 .mu.m) to remove insoluble particles. A 4 ml
aliquot of the solution (2.1 mg of protein) was epplied to a
Sephadex G 200 in XK-26 column (Pharmacia-LKB, Uppsala Sweden) for
gel filtration chromatography. HEPES-Hanks buffer was used to
equilibrate the column and elute the sample. The fractions in each
280 nm-absorbing peak were assayed for the capacity to bind
HRP-mucin and HRP-crude mucus by dot blot assay and in the
inhibition of lactobacilli binding to crude mucus in microtiter
plates adhesion inhibition assay. The active fractions in each 280
nm absorbing peak were pooled, dialysed and freeze dried for
SDS-PAGE and western blot analysis.
[0111] Alternative purification
[0112] Mucin was covalently coupled to Activated CH-sepharose 4D
according to the instructions of the manufacturer (Pharmacia-LKB.
Biotechnology). A column C10/40 (30 ml bed volume) was packed with
this adsorbent and I equilibrated with HEPES-Hanks. L. fermentum
spent culture fluid, cell extracts. or active fractions from Gel
filtration chromatography were loaded throw the column. Column was
washed with two bed volumes of equilibrating buffer, then
successively washed with different solutions (0.1 M glycine pH 3,
0.1 M tris pH 8 and 0-2 M gradient of sodium chloride) at flow
rates of 6 ml h.sup.-1.
[0113] The adhesion promoting activity was detected in the
fractions by adhesion inhibition and dot blot assays. and was
visualized by PAGE, SOS-PAGE and western blots using horse radish
peroxidase labelled mucus or mucin. The adhesion promoting protein
could be extracted from the cell surface of L. fermentum by
treatment of the bacteria with 1 M Licl and low concentrations of
lysozyme. The adhesion promoting protein, which had an affinity for
both small intestine mucus and gastric mucin, was released into the
culture supernatant fluid after 24 h of growth. The active fraction
was characterized by assessing the presence of carbohydrates in
(periodic-acid Schiff stain procedure. SIGMA. and DIG glycan
detection kit, acehringer Mannheim.. Germany) and the heat
sensitivity of the active region of the adhesion promoting protein.
The adhesion promoting activity lacked carbohydrates and remained
completely biologially active. when LiCl cell extracts from L.
fermentum were heated for 5 min at 100.degree. C. and tested by dot
blot adhesion assay. The purified protein has an estimated Mw of 29
kD. under non-denaturing conditions as well as under reducing and
denaturing conditions (SDS-PAGE. using a calibration curve obtained
with standard proteins, and gel-filtration chromatography, relative
to the standard curve: FIG. 1).
[0114] The adhesion promoting protein was further characterized by
determination of the N-terninal amino acid sequence, showing the
following sequence:
AXXAVNXELV(V)(K)
[0115] When the adhesion promoting protein was digested with
modified porcine trypsin and the peptides formed were purified by
reverse-phase HPLC. a number of peptides were found to specifically
adhere to mucus and mucin, as measured by dot blot and muicn
adhesion assays. The aminoacid sequence of the peptides are;
I:ANPVPTK, II:DTAIQSSYNK. III:ISALFNK. IV:IIAG(T)G(T)NNA. In these
sequences X most likely represents serine (S).
[0116] ii) Cloning and sequencing of adherence factor encoding
protein
[0117] The adhesion promoting protein gene was cloned from a
genomic bank of L. fermentum 104R. To generate a probe with which
adhesion gene sequences could be identified, oligonucleotide
primers were synthesized, based on the aminoacid sequence data of
the sequenced peptides of the adhesion promoting protein. These
oligonucleotides were used in various combinations in PCR reaction.
Oligonucleotides 42 (sense;
5'-CTI.CCI.GTI.AAC/T.TCI.GAG/A.TTG/A.GT-3') and 105 (antisense;
5'-GCC.GGGA.TCC,TTT.G/A/T/CGT.G/TGG.G/TAC.G/AAA-G/ATT.G/A/TGC-3')
corresponding to the N terminal peptide and peptide I,
respectively, yielded a PCR product of 183 bp flanked by EcoRI and
BamiI sites, which. hybridized in a Southern blot with a 3.5 kb
SstI-PetI chromosomal L. fermentum fragment. The fragment was
cloned in pGEM3 in E. coli. The position of the adhesion er.coding;
gene was deteririned by restriction enzyme analysis and the
nucleotide sequence of the relevant part of the 3.5 kb fragment was
determined (FIG. 2). The predicted aminoacid sequence of the
adhesion protein is given in FIG. 3.
[0118] iii) Analysis of the amiioacid sequence of the L. fermentum
104R adhesion protein
[0119] Computer assisted analysis of the aminoacid sequence of the
L. fermentum 104R adhesion protein was carried out. FIG. 4 shows
that the protein shows striking similarity with the virulence
proteins Pebl from C. jejuni and LapT from P. haemolytica. FIG. 5
shows that the L. fermenrum adhesion protein also shows similarity
with Class III solute transporters. FIG. 6 shows that the adhesion
protein shows similarity to 85 K complex virulence proteins of
Mycobacterium leprae and Mycobacterium tuberculosis. Protein
modelling studies indicate that the predicted 3-D structure of the
adhesion protein of L. fermentum 104R is similar to that of LAO and
HisJ. These studies also indicate that Pebl has a 3-D structure
which is similar to that of LAO and HisJ.
[0120] iv) Determination or adhesion protein-like proteins in
Lactobacillus strains
[0121] Nearly 20 Lactobacillus strains were cultivated in LDM
medium. the culture medium was collected and the proteins separated
by SDS-PAGE. The presence of adhesion protein-like protein was
determined by Western blotting according to standard molecular
biological techniques. The results, which are presented in Table 1,
show that some Lactobacillus strains do produce an adhesion
protein-like protein whereas others don't.
BRIEF DESCRIPTION OF THE FIGURES
[0122] FIG. 1 SDS-PAGE and Western blot of the adhesion promoting
protein (APP) using ERP labelled mucus for blotting. A) Molecular
weight markers (lane 1): APP after affinity chromatography (lanes
2, 6 and 7), APP from native PAGE (lanes 3, 4 and 5). B) Molecular
weight markers (lane 1), APP from gel-filtration chromatography
(lanes 2 and 3)- Arrow in lane 3 indicates position of APP; C)
Western blot of APP from SDS-PAGE after (lare 2); 1 M LiCl
extraction of L. fermentum after 14 h of growth (lane 3); from PAGE
(lane 4)
[0123] FIG. 2 Nucleotide sequence of the adhesion promoting protein
of L. fermentum 104R. The open reading frame starts at nucleotide 1
and ends at nucleotide 734.
[0124] FIG. 3 Amino acid sequence of the adhesion promoting protein
of L. fermentum 104R.
[0125] FIG. 4 Comparison of the amino acid sequences of the
adhesion promoting protein of L. fermentum 104R, Pebl from C.
jejuni and LapT from P. haemolytica. A consensus sequence is given
below the sequences. Bold letters indicate identical aminoacids or
conserved substitutions.
[0126] FIG. 5 Comparison of the aminoacid sequences of the adhesion
promoting protein of L. fermentum 104R and Class III solute
transport proteins (Atunop, nopaline of Agrobacter tumefactens;
Atunct, octopine Agrobacter tumefaciens; GlnH, glutamine binding
protein of E. coli; HisJ, histidine binding protein. LAO, lysine.
arginine, ornithine binding protein of Salmonella thyphimurium, A
consensus sequence is given below the sequences. Aminoacids in
adhesion promoting protein that also occur in other proteins are
indicated in bold capital letters; colons indicate a conserved
substitution and asterics a less conserved substitution,
[0127] FIG. 6 Comparison of the aminoacid sequences of the adhesion
promoting protein of L. fermentum 104R and proteins of the 85K
complex of Mycobactexium. A consensus sequence is given below the
sequences. Aminoacids that are identical in adhesin and in one or
more Mycobacterium proteins are indicated in bold capital letters.
Conserved substitutions are indicated with a colon, and less
conserced substitutions with an asterisc.
2TABLE 1 Western blot of culture medium of Lactobacillus strains
using antibodies raised against L. fermentum 104R adhesion
promoting protein as a probe Signal L. gasseri NCK 89 + L. reuteri
ML1 ++ L. murinus + L. fermentum 2399 +/- L. plantarum ++ L.
fermentum KLD - L. animalis 364T + L. animalis 364 +/- L. casei
ATCC 393 +/- L. acidophilus NCK 65 - L. animalis 362 - L. plantarum
8014 +/- L. plantarum LP80 + L. brevis R3 + L. brevis ML12 + E.
coli - L. fermentum 104R ++ L. plantarum 256 ++
[0128]
Sequence CWU 1
1
30 1 747 DNA Artificial Sequence Description of Artificial
SequenceSequence to adhesion promoting protein L. fermentum 104R 1
ctgcaggaat cacaagtgtt tctgctgctt cagctgttaa ttcagaatta gttcataagg
60 gagaattaac aattggtctt gagggaacgt actctccgta ctcttatcgt
aaaaataaca 120 aattaactgg ctttgaagta gatcttggta aagcagttgc
taaaaagatg ggcttaaaag 180 ctaactttgt accaactaaa tgggattcgc
taattgccgg tcttggttca ggtaagtttg 240 atgtagtaat gaacaacatt
acacagacac ctgaacgggc caagcaatat aatttctcta 300 ccccatatat
caagtcccgg tttgcattaa ttgttcctac tgatagtaac atcaaaagct 360
tgaagaatat taaaggcaag aagattattg ctggtacggg aactaataat gcgaatgtgg
420 taaaaaaata taagggtaac cttacaccaa atggcgattt tgctagttcc
ttagatatga 480 tcaagcaagg tcgggctgcc gggacaatta actcccgtga
agcttggtac gcttacagca 540 agaagaacag tactaagggt ctcaagatga
ttgatgtttc tagtgaacaa gatccagcta 600 agatttcagc actttttaac
aagaaagata ctgctattca atcttcctac aacaaggcac 660 ttaaggaact
tcaacaagac ggaacagtca agaagctatc tgaaaagtac ttcggtgcag 720
atattactga ataattaaaa aagatct 747 2 244 PRT Artificial Sequence
UNSURE (244) Xaa is any amino acid. 2 Ala Gly Ile Thr Ser Val Ser
Ala Ala Ser Ala Val Asn Ser Glu Leu 1 5 10 15 Val His Lys Gly Glu
Leu Thr Ile Gly Leu Glu Gly Thr Tyr Ser Pro 20 25 30 Tyr Ser Tyr
Arg Lys Asn Asn Lys Leu Thr Gly Phe Glu Val Asp Leu 35 40 45 Gly
Lys Ala Val Ala Lys Lys Met Gly Leu Lys Ala Asn Phe Val Pro 50 55
60 Thr Lys Trp Asp Ser Leu Ile Ala Gly Leu Gly Ser Gly Lys Phe Asp
65 70 75 80 Val Val Met Asn Asn Ile Thr Gln Thr Pro Glu Arg Ala Lys
Gln Tyr 85 90 95 Asn Phe Ser Thr Pro Tyr Ile Lys Ser Arg Phe Ala
Leu Ile Val Pro 100 105 110 Thr Asp Ser Asn Ile Lys Ser Leu Lys Asn
Ile Lys Gly Lys Lys Ile 115 120 125 Ile Ala Gly Thr Gly Thr Asn Asn
Ala Asn Val Val Lys Lys Tyr Lys 130 135 140 Gly Asn Leu Thr Pro Asn
Gly Asp Phe Ala Ser Ser Leu Asp Met Ile 145 150 155 160 Lys Gln Gly
Arg Ala Ala Gly Thr Ile Asn Ser Arg Glu Ala Trp Tyr 165 170 175 Ala
Tyr Ser Lys Lys Asn Ser Thr Lys Gly Leu Lys Met Ile Asp Val 180 185
190 3 275 PRT Artificial Sequence UNSURE (1)..(18) Xaa is any amino
acid. 3 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 15 Xaa Xaa Ala Gly Ile Thr Ser Val Ser Ala Ala Ser Ala
Val Asn Ser 20 25 30 Glu Leu Val His Lys Gly Glu Leu Thr Ile Gly
Leu Glu Gly Thr Tyr 35 40 45 Ser Pro Tyr Ser Tyr Xaa Arg Lys Xaa
Asn Asn Lys Leu Thr Gly Phe 50 55 60 Glu Val Asp Leu Gly Lys Ala
Val Ala Lys Lys Met Xaa Xaa Xaa Gly 65 70 75 80 Leu Lys Ala Asn Phe
Val Pro Thr Lys Trp Asp Ser Leu Ile Ala Gly 85 90 95 Leu Gly Ser
Gly Lys Phe Asp Val Val Met Asn Asn Ile Thr Gln Thr 100 105 110 Pro
Glu Arg Ala Lys Gln Tyr Asn Phe Ser Thr Pro Tyr Ile Lys Ser 115 120
125 Arg Phe Ala Leu Ile Val Pro Thr Asp Ser Asn Ile Lys Ser Leu Lys
130 135 140 Asn Ile Lys Gly Lys Lys Ile Xaa Xaa Xaa Ile Ala Gly Thr
Gly Thr 145 150 155 160 Asn Asn Ala Asn Val Val Lys Lys Tyr Xaa Xaa
Xaa Lys Gly Asn Leu 165 170 175 Thr Pro Asn Gly Asp Phe Ala Ser Ser
Leu Asp Met Ile Lys Gln Gly 180 185 190 Arg Ala Ala Gly Thr Ile Asn
Ser Arg Glu Ala Trp Tyr Ala Tyr Ser 195 200 205 Lys Lys Asn Ser Thr
Lys Gly Leu Xaa Xaa Xaa Lys Met Ile Asp Val 210 215 220 Ser Ser Glu
Gln Asp Pro Ala Lys Ile Ser Ala Leu Phe Asn Lys Lys 225 230 235 240
Asp Thr Ala Ile Gln Ser Ser Tyr Asn Lys Ala Leu Lys Glu Leu Gln 245
250 255 Gln Asp Gly Thr Val Lys Lys Leu Ser Glu Lys Tyr Phe Gly Ala
Asp 260 265 270 Ile Thr Glu 275 4 275 PRT Artificial Sequence
UNSURE (1)..(20) Xaa is any amino acid. 4 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa
Met Lys Lys Thr Leu Leu Thr Leu Leu Phe Gly Cys 20 25 30 Val Val
Thr Ala Gln Ala Gln Asp Ile Ile Val Met Glu Pro Ser Tyr 35 40 45
Pro Pro Phe Glu Met Thr Glu Glu Xaa Lys Gly Glu Ile Ile Gly Phe 50
55 60 Asp Val Asp Ile Ala Asn Ala Ile Cys Lys Glu Met Xaa Xaa Xaa
Asn 65 70 75 80 Ala Asn Cys Thr Phe His Ser Gln Pro Phe Asp Ser Leu
Ile Gln Ser 85 90 95 Leu Lys Gln Lys Gln Phe Asp Ala Ala Ile Ser
Gly Met Gly Ile Thr 100 105 110 Glu Pro Arg Lys Lys Gln Val Leu Phe
Ser Glu Pro Tyr Phe Pro Ser 115 120 125 Ser Ala Ala Phe Ile Ala Lys
Lys Asp Thr Asp Phe Ala Lys Val Lys 130 135 140 Thr Ile Xaa Xaa Xaa
Gly Val Xaa Xaa Xaa Gln Asn Gly Thr Thr Tyr 145 150 155 160 Gln His
Tyr Leu Ala Lys Glu Lys Lys Xaa Xaa Xaa Glu Tyr Asn Val 165 170 175
Lys Ser Tyr Ala Ser Tyr Gln Asn Ala Ile Leu Asp Val Gln Asn Gly 180
185 190 Arg Ile Asp Ala Ile Phe Gly Asp Val Pro Val Leu Ala Glu Met
Ala 195 200 205 Arg Lys His Glu Gly Leu Asp Phe Val Gly Glu Lys Ile
Asn Asn Pro 210 215 220 Asn Tyr Phe Gly Asp Gly Leu Gly Ile Ala Thr
His Leu Xaa Xaa Lys 225 230 235 240 Asn Gln Val Leu Val Asp Gln Phe
Asn Ala Ala Leu Lys Thr Ile Lys 245 250 255 Glu Asn Gly Glu Tyr Gln
Lys Ile Tyr Asp Lys Trp Met Gly Gly Lys 260 265 270 Xaa Xaa Xaa 275
5 275 PRT Artificial Sequence UNSURE (205)..(213) Xaa is any amino
acid. 5 Met Val Phe Arg Lys Ser Leu Leu Lys Leu Ala Val Phe Ala Leu
Gly 1 5 10 15 Ala Cys Val Ala Phe Ser Asn Ala Asn Ala Ala Glu Gly
Lys Leu Glu 20 25 30 Ser Ile Lys Ser Lys Gly Gln Leu Ile Val Gly
Val Lys Asn Asp Val 35 40 45 Pro His Tyr Ala Leu Leu Asp Gln Ala
Thr Gly Glu Ile Lys Gly Phe 50 55 60 Glu Val Asp Val Ala Lys Leu
Leu Ala Lys Ser Ile Leu Gly Asp Asp 65 70 75 80 Lys Lys Ile Lys Leu
Val Ala Val Asn Ala Lys Thr Arg Gly Pro Leu 85 90 95 Leu Asp Asn
Gly Ser Val Asp Ala Val Ile Ala Thr Phe Thr Ile Thr 100 105 110 Pro
Glu Arg Lys Arg Ile Tyr Asn Phe Ser Glu Pro Tyr Tyr Gln Asp 115 120
125 Ala Ile Gly Leu Leu Val Leu Lys Glu Lys Lys Tyr Lys Ser Leu Ala
130 135 140 Asp Met Lys Gly Ala Asn Ile Gly Val Ala Gln Ala Ala Thr
Thr Lys 145 150 155 160 Lys Ala Ile Gly Glu Ala Ala Lys Lys Ile Gly
Ile Asp Val Lys Phe 165 170 175 Ser Glu Phe Pro Asp Tyr Pro Ser Ile
Lys Ala Ala Leu Asp Ala Lys 180 185 190 Arg Val Asp Ala Phe Ser Val
Asp Lys Ser Ile Leu Xaa Xaa Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa
Leu Gly Tyr Val Asp Asp Lys Ser Glu Ile Leu 210 215 220 Pro Asp Ser
Phe Glu Pro Gln Ser Tyr Gly Ile Val Thr Lys Lys Asp 225 230 235 240
Asp Pro Ala Phe Ala Lys Tyr Val Asp Asp Phe Val Lys Glu Xaa Xaa 245
250 255 His Lys Asn Glu Ile Asp Ala Leu Ala Lys Lys Trp Gly Leu Xaa
Xaa 260 265 270 Xaa Xaa Xaa 275 6 275 PRT Artificial Sequence
Description of Artificial Sequenceconsensus from se13-5 aligned aa
sequences 6 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Lys Gly Xaa Leu Xaa Ile
Gly Xaa Xaa Xaa Xaa Tyr 35 40 45 Xaa Pro Tyr Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe 50 55 60 Glu Val Asp Xaa Xaa Lys
Xaa Xaa Ala Lys Xaa Met Xaa Xaa Xaa Xaa 65 70 75 80 Xaa Lys Xaa Xaa
Xaa Val Xaa Xaa Xaa Xaa Asp Ser Leu Ile Xaa Xaa 85 90 95 Leu Xaa
Xaa Gly Xaa Phe Asp Xaa Val Xaa Xaa Xaa Xaa Thr Xaa Thr 100 105 110
Pro Glu Arg Xaa Lys Gln Tyr Asn Phe Ser Xaa Pro Tyr Xaa Xaa Ser 115
120 125 Xaa Xaa Ala Leu Ile Val Xaa Xaa Asp Xaa Xaa Xaa Lys Ser Leu
Lys 130 135 140 Xaa Ile Lys Gly Xaa Xaa Ile Xaa Xaa Xaa Xaa Ala Gly
Thr Xaa Xaa 145 150 155 160 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Xaa Xaa
Xaa Xaa Xaa Xaa Asn Xaa 165 170 175 Xaa Xaa Xaa Xaa Asp Xaa Xaa Ser
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly 180 185 190 Arg Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 195 200 205 Xaa Lys Xaa Xaa
Xaa Xaa Gly Xaa Xaa Xaa Xaa Lys Xaa Xaa Xaa Xaa 210 215 220 Xaa Xaa
Xaa Xaa Asp Pro Xaa Xaa Ile Xaa Xaa Xaa Xaa Xaa Lys Lys 225 230 235
240 Asp Xaa Ala Xaa Xaa Xaa Xaa Xaa Asn Xaa Ala Leu Lys Glu Ile Xaa
245 250 255 Xaa Xaa Gly Xaa Xaa Xaa Lys Leu Xaa Xaa Lys Xaa Xaa Gly
Xaa Xaa 260 265 270 Xaa Xaa Xaa 275 7 292 PRT Artificial Sequence
Description of Artificial Sequencealigned aa sequence of HisJ with
seq7-12 7 Xaa Xaa Xaa Xaa Xaa Xaa Met Lys Lys Leu Ala Leu Ser Leu
Ser Leu 1 5 10 15 Val Leu Ala Phe Ser Ser Ala Thr Ala Ala Phe Ala
Ala Ile Pro Gln 20 25 30 Lys Xaa Ile Arg Ile Gly Thr Asp Pro Thr
Tyr Ala Pro Phe Glu Ser 35 40 45 Lys Asn Ala Gln Gly Glu Leu Val
Gly Phe Asp Ile Asp Leu Ala Lys 50 55 60 Glu Leu Cys Lys Arg Ile
Asn Thr Gln Cys Thr Phe Val Glu Asn Pro 65 70 75 80 Leu Asp Ala Leu
Ile Pro Ser Leu Lys Ala Lys Lys Ile Asp Ala Ile 85 90 95 Met Ser
Ser Leu Ser Ile Thr Glu Lys Arg Gln Gln Glu Ile Ala Phe 100 105 110
Thr Asp Lys Leu Tyr Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Asp 115
120 125 Ser Arg Leu Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 130 135 140 Xaa Xaa Xaa Val Ala Lys Asn Ser Asp Ile Gln Pro Thr
Val Ala Ser 145 150 155 160 Leu Lys Gly Lys Arg Val Gly Val Leu Gln
Gly Thr Thr Gln Glu Thr 165 170 175 Phe Gly Asn Glu His Trp Ala Pro
Lys Gly Ile Glu Ile Val Ser Tyr 180 185 190 Gln Gly Gln Asp Asn Ile
Tyr Ser Asp Leu Thr Ala Xaa Gly Arg Ile 195 200 205 Asp Ala Ala Phe
Gln Asp Glu Val Ala Ala Ser Glu Gly Phe Leu Lys 210 215 220 Gln Pro
Val Gly Lys Asp Tyr Lys Phe Gly Gly Pro Ala Val Lys Asp 225 230 235
240 Glu Lys Leu Phe Gly Val Gly Thr Gly Met Gly Leu Arg Lys Glu Asp
245 250 255 Asn Glu Leu Arg Glu Ala Leu Asn Lys Ala Phe Ala Glu Met
Arg Ala 260 265 270 Asp Gly Thr Tyr Glu Lys Leu Ala Lys Lys Tyr Phe
Asp Phe Asp Val 275 280 285 Tyr Gly Gly Xaa 290 8 292 PRT
Artificial Sequence Description of Artificial Sequencealigned aa
sequence of LA0 with seq7-12 8 Xaa Xaa Xaa Xaa Xaa Xaa Met Lys Lys
Thr Val Leu Ala Leu Ser Leu 1 5 10 15 Leu Ile Gly Leu Gly Ala Thr
Ala Ala Ser Tyr Ala Ala Leu Pro Gln 20 25 30 Thr Xaa Val Arg Ile
Gly Thr Asp Thr Thr Tyr Ala Pro Phe Ser Ser 35 40 45 Lys Asp Ala
Lys Gly Glu Phe Ile Gly Phe Asp Ile Asp Leu Gly Asn 50 55 60 Glu
Met Cys Lys Arg Met Gln Val Lys Cys Thr Trp Val Ala Ser Asp 65 70
75 80 Phe Asp Ala Leu Ile Pro Ser Leu Lys Ala Lys Lys Ile Asp Ala
Ile 85 90 95 Ile Ser Ser Leu Ser Ile Thr Asp Lys Arg Gln Gln Glu
Ile Ala Phe 100 105 110 Ser Asp Lys Leu Tyr Ala Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Ala Asp 115 120 125 Ser Arg Leu Ile Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa Xaa Ala Ala Lys Gly
Ser Pro Val Gln Pro Thr Leu Glu Ser 145 150 155 160 Leu Lys Gly Lys
His Val Gly Val Leu Gln Gly Ser Thr Gln Glu Ala 165 170 175 Tyr Ala
Asn Asp Asn Trp Arg Thr Lys Gly Val Asp Val Val Ala Tyr 180 185 190
Ala Asn Gln Asp Leu Ile Tyr Ser Asp Leu Thr Ala Xaa Gly Arg Leu 195
200 205 Asp Ala Ala Leu Gln Asp Glu Val Ala Ala Ser Glu Gly Phe Leu
Lys 210 215 220 Gln Pro Ala Gly Lys Glu Tyr Ala Phe Ala Gly Pro Ser
Val Lys Asp 225 230 235 240 Lys Lys Tyr Phe Gly Asp Gly Thr Gly Val
Gly Leu Arg Lys Asp Asp 245 250 255 Thr Glu Leu Lys Ala Ala Phe Asp
Lys Ala Leu Thr Glu Leu Arg Gln 260 265 270 Asp Gly Thr Tyr Asp Lys
Met Ala Lys Lys Tyr Phe Asp Phe Asn Val 275 280 285 Tyr Gly Asp Xaa
290 9 292 PRT Artificial Sequence Description of Artificial
Sequencealigned aa sequence of Agrobacter tumefaciens nopaline 9
Met Lys Phe Phe Asn Leu Asn Ala Leu Ala Ala Val Val Thr Gly Val 1 5
10 15 Leu Leu Ala Ala Gly Pro Thr Gln Xaa Xaa Xaa Ala Lys Asp Tyr
Lys 20 25 30 Ser Xaa Ile Thr Ile Ala Thr Glu Gly Ser Tyr Ala Pro
Tyr Asn Phe 35 40 45 Lys Asp Ala Gly Gly Lys Leu Ile Gly Phe Asp
Ile Asp Leu Gly Asn 50 55 60 Asp Leu Cys Lys Arg Met Asn Ile Glu
Cys Lys Phe Val Glu Gln Ala 65 70 75 80 Trp Val Gly Ile Ile Pro Ser
Leu Thr Ala Gly Arg Tyr Asp Ala Ile 85 90 95 Met Ala Ala Met Gly
Ile Gln Pro Ala Arg Glu Lys Val Ile Ala Phe 100 105 110 Ser Arg Pro
Tyr Leu Leu Thr Pro Met Thr Phe Leu Thr Thr Ala Asp 115 120 125 Ser
Pro Leu Leu Lys Thr Gln Val Ala Ile Glu Asn Leu Pro Leu Asp 130 135
140 Asn Ile Ala Pro Glu Gln Lys Ala Glu Leu Asp Lys Phe Thr Lys Ile
145 150 155 160 Phe Glu Gly Val Lys Phe Gly Val Gln Ala Gly Thr Ser
His Glu Ala 165 170 175 Phe Met Xaa Lys Gln Met Met Pro Xaa Ser Val
Gln Ile Ser Thr Tyr 180 185 190 Asp
Thr Ile Asp Asn Val Val Met Asp Leu Lys Ala Xaa Gly Arg Ile 195 200
205 Asp Ala Ser Leu Xaa Ala Ser Val Ser Phe Leu Lys Pro Leu Thr Asp
210 215 220 Lys Pro Asp Asn Lys Asp Leu Lys Met Phe Gly Pro Arg Met
Thr Gly 225 230 235 240 Gly Xaa Leu Phe Gly Lys Gly Val Gly Val Gly
Ile Arg Lys Glu Asp 245 250 255 Ala Asp Leu Lys Ala Leu Phe Asp Lys
Ala Ile Asp Ala Ala Ile Ala 260 265 270 Asp Gly Thr Val Gln Lys Leu
Ser Gln Gln Trp Phe Gly Tyr Asp Ala 275 280 285 Ser Pro Lys Gln 290
10 292 PRT Artificial Sequence Description of Artificial
Sequencealigned aa sequence of Agrobacter tumefaciens octopine 10
Xaa Xaa Xaa Xaa Xaa Met Lys Leu Lys Thr Ile Leu Cys Ala Ala Leu 1 5
10 15 Leu Leu Val Ala Gly Gln Ala Ala Xaa Xaa Xaa Ala Gln Glu Xaa
Lys 20 25 30 Ser Xaa Ile Thr Ile Ala Thr Glu Gly Gly Tyr Ala Pro
Trp Asn Phe 35 40 45 Ser Gly Pro Gly Gly Lys Leu Asp Gly Phe Glu
Ile Asp Leu Ala Asn 50 55 60 Ala Leu Cys Glu Lys Met Lys Ala Lys
Cys Gln Ile Val Ala Gln Asn 65 70 75 80 Trp Asp Gly Ile Met Pro Ser
Leu Thr Gly Lys Lys Tyr Asp Ala Ile 85 90 95 Met Ala Ala Met Ser
Val Thr Pro Lys Arg Gln Glu Val Ile Gly Phe 100 105 110 Ser Ile Pro
Tyr Ala Ala Gly Ile Asn Gly Phe Ala Val Met Gly Asp 115 120 125 Ser
Lys Leu Ala Glu Met Pro Gly Leu Gly Glu Thr Tyr Ser Leu Asp 130 135
140 Ser Gln Ala Asp Ala Ala Lys Lys Ala Ile Ala Asp Ile Ser Ser Phe
145 150 155 160 Leu Asn Gly Thr Thr Val Gly Val Gln Gly Ser Thr Thr
Ala Ser Thr 165 170 175 Phe Leu Asp Lys Tyr Phe Lys Gly Xaa Ser Val
Asp Ile Lys Glu Tyr 180 185 190 Lys Ser Val Glu Glu His Asn Leu Asp
Leu Thr Ser Xaa Gly Arg Leu 195 200 205 Asp Ala Val Leu Xaa Ala Asn
Ala Thr Val Leu Ala Ala Ala Ile Glu 210 215 220 Lys Pro Glu Met Lys
Gly Ala Lys Leu Val Gly Pro Leu Phe Ser Gly 225 230 235 240 Gly Xaa
Glu Phe Gly Xaa Val Val Ala Val Gly Leu Arg Lys Glu Asp 245 250 255
Thr Ala Leu Lys Ala Asp Phe Asp Ala Ala Ile Lys Ala Ala Ser Glu 260
265 270 Asp Gly Thr Ile Lys Thr Leu Ser Leu Lys Trp Phe Lys Val Asp
Val 275 280 285 Thr Pro Gln Xaa 290 11 292 PRT Artificial Sequence
Description of Artificial Sequencealigned aa sequence of E. coli
GlnH 11 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Met Lys Ser Val Leu Lys Val Ser
Leu 1 5 10 15 Ala Ala Leu Thr Leu Ala Phe Ala Val Ser Ser His Ala
Ala Asp Lys 20 25 30 Lys Xaa Leu Val Val Ala Thr Asp Thr Ala Phe
Val Pro Phe Glu Phe 35 40 45 Lys Gln Xaa Gly Asp Lys Tyr Val Gly
Phe Asp Val Asp Leu Trp Ala 50 55 60 Ala Ile Ala Lys Glu Leu Lys
Leu Asp Tyr Glu Leu Lys Pro Met Asp 65 70 75 80 Phe Ser Gly Ile Ile
Pro Ala Leu Gln Thr Lys Asn Val Asp Leu Ala 85 90 95 Leu Ala Gly
Ile Thr Ile Thr Asp Glu Arg Lys Lys Ala Ile Asp Phe 100 105 110 Ser
Asp Gly Tyr Tyr Lys Ser Gly Leu Leu Val Met Val Lys Ala Asn 115 120
125 Asn Asn Asp Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
130 135 140 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Ser Val
Lys Asp 145 150 155 160 Leu Asp Gly Lys Val Val Ala Val Lys Ser Gly
Thr Gly Ser Val Asp 165 170 175 Tyr Ala Lys Ala Asn Ile Lys Thr Lys
Xaa Xaa Asp Leu Arg Gln Phe 180 185 190 Pro Asn Ile Asp Asn Ala Tyr
Met Glu Leu Gly Thr Asn Xaa Arg Ala 195 200 205 Asp Ala Val Leu His
Asp Thr Pro Asn Ile Leu Tyr Xaa Phe Ile Lys 210 215 220 Thr Ala Gly
Asn Gly Gln Phe Lys Ala Val Gly Asp Ser Leu Glu Ala 225 230 235 240
Gln Gln Tyr Xaa Xaa Xaa Xaa Xaa Gly Ile Ala Phe Pro Lys Gly Ser 245
250 255 Asp Glu Leu Arg Asp Lys Val Asn Gly Ala Leu Lys Thr Leu Arg
Glu 260 265 270 Asn Gly Thr Tyr Asn Glu Ile Tyr Lys Lys Trp Phe Gly
Thr Glu Pro 275 280 285 Lys Xaa Xaa Xaa 290 12 291 PRT Artificial
Sequence Description of Artificial Sequencealigned aa sequence of L
fermentum 104R adhesin with seq7-12 12 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Ala Gly Ile 1 5 10 15 Thr Ser Val Ser Ala
Ala Ser Ala Val Asn Ser Glu Leu Val His Lys 20 25 30 Gly Glu Leu
Thr Ile Gly Leu Glu Gly Thr Tyr Ser Pro Tyr Ser Tyr 35 40 45 Arg
Lys Xaa Asn Asn Lys Leu Thr Gly Phe Glu Val Asp Leu Gly Lys 50 55
60 Ala Val Ala Lys Lys Met Gly Leu Lys Ala Asn Phe Val Pro Thr Lys
65 70 75 80 Trp Asp Ser Leu Ile Ala Gly Leu Gly Ser Gly Lys Phe Asp
Val Val 85 90 95 Met Asn Asn Ile Thr Gln Thr Pro Glu Arg Ala Lys
Gln Tyr Asn Phe 100 105 110 Ser Thr Pro Tyr Ile Lys Ser Arg Phe Ala
Leu Ile Val Pro Thr Asp 115 120 125 Ser Asn Ile Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Lys Ser Leu Lys Asn 145 150 155 160 Ile Lys Gly
Lys Lys Ile Xaa Ile Ala Gly Thr Gly Thr Asn Asn Ala 165 170 175 Asn
Val Val Lys Lys Tyr Lys Gly Asn Leu Thr Pro Asn Gly Asp Phe 180 185
190 Ala Ser Ser Xaa Xaa Xaa Xaa Leu Asp Met Ile Lys Gln Gly Arg Ala
195 200 205 Xaa Ala Gly Thr Ile Asn Ser Arg Glu Ala Trp Tyr Ala Tyr
Ser Lys 210 215 220 Lys Asn Ser Thr Lys Gly Leu Lys Met Ile Asp Val
Ser Ser Glu Gln 225 230 235 240 Asp Xaa Xaa Xaa Pro Ala Lys Ile Ser
Ala Leu Phe Asn Lys Lys Asp 245 250 255 Thr Ala Ile Gln Ser Ser Tyr
Asn Lys Ala Leu Lys Glu Leu Gln Gln 260 265 270 Asp Gly Thr Val Lys
Lys Leu Ser Glu Lys Tyr Phe Gly Ala Asp Ile 275 280 285 Thr Glu Xaa
290 13 291 PRT Artificial Sequence Description of Artificial
Sequenceconsensus of aligned aa sequences 7-12 13 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 20 25 30
Xaa Xaa Leu Thr Ile Gly Xaa Glu Gly Thr Tyr Xaa Pro Tyr Ser Xaa 35
40 45 Xaa Xaa Xaa Xaa Xaa Lys Leu Xaa Gly Phe Glu Val Asp Leu Gly
Lys 50 55 60 Ala Xaa Ala Lys Lys Met Xaa Leu Lys Xaa Xaa Phe Val
Pro Xaa Xaa 65 70 75 80 Trp Asp Xaa Leu Ile Xaa Xaa Leu Xaa Xaa Gly
Lys Xaa Asp Xaa Xaa 85 90 95 Met Xaa Xaa Ile Thr Xaa Thr Pro Glu
Arg Xaa Lys Xaa Xaa Xaa Phe 100 105 110 Ser Xaa Pro Tyr Xaa Lys Ser
Xaa Xaa Xaa Xaa Xaa Val Xaa Xaa Asp 115 120 125 Ser Asn Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Ser Leu Lys Xaa 145 150 155 160
Xaa Lys Gly Lys Lys Xaa Xaa Xaa Xaa Gly Xaa Xaa Thr Xaa Xaa Ala 165
170 175 Xaa Xaa Xaa Lys Xaa Xaa Lys Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Phe 180 185 190 Ala Ser Xaa Xaa Xaa Xaa Xaa Leu Asp Xaa Xaa Xaa Xaa
Gly Arg Ala 195 200 205 Xaa Ala Xaa Xaa Xaa Xaa Ser Xaa Xaa Ala Xaa
Tyr Ala Xaa Xaa Lys 210 215 220 Lys Xaa Xaa Xaa Lys Gly Leu Lys Met
Xaa Xaa Xaa Ser Xaa Glu Xaa 225 230 235 240 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Phe Xaa Lys Xaa Asp 245 250 255 Thr Ala Xaa Xaa
Xaa Xaa Xaa Asn Lys Ala Leu Lys Glu Leu Xaa Gln 260 265 270 Asp Gly
Thr Val Lys Lys Leu Ser Xaa Lys Tyr Phe Gly Xaa Asp Xaa 275 280 285
Thr Xaa Xaa 290 14 364 PRT Artificial Sequence Description of
Artificial Sequencealigned aa sequence of Mycobacterium Mtu85c 14
Met Thr Phe Phe Glu Gln Val Arg Arg Leu Arg Ser Ala Ala Thr Thr 1 5
10 15 Leu Pro Arg Arg Val Ala Ile Ala Ala Met Gly Ala Val Leu Val
Tyr 20 25 30 Gly Leu Val Gly Thr Phe Gly Gly Pro Ala Thr Ala Gly
Ala Phe Ser 35 40 45 Arg Pro Gly Xaa Leu Pro Val Glu Tyr Leu Gln
Val Pro Ser Ala Xaa 50 55 60 Ser Met Gly Arg Asp Ile Lys Val Xaa
Gln Phe Gln Gly Gly Gly Pro 65 70 75 80 Xaa Xaa His Ala Val Tyr Leu
Leu Asp Gly Leu Arg Ala Gln Asp Asp 85 90 95 Tyr Xaa Xaa Asn Gly
Trp Asp Ile Asn Thr Pro Ala Phe Glu Glu Tyr 100 105 110 Tyr Gln Ser
Gly Xaa Leu Ser Val Ile Met Pro Val Gly Gly Gln Ser 115 120 125 Ser
Phe Tyr Thr Asp Trp Tyr Gln Pro Ser Gln Ser Asn Gly Gln Asn 130 135
140 Tyr Thr Tyr Lys Trp Glu Thr Xaa Phe Leu Thr Arg Glu Met Pro Ala
145 150 155 160 Trp Leu Gln Ala Asn Lys Gly Val Ser Pro Thr Gly Asn
Ala Ala Val 165 170 175 Gly Leu Xaa Xaa Xaa Xaa Xaa Xaa Ser Met Ser
Gly Gly Ser Xaa Xaa 180 185 190 Xaa Ala Leu Ile Leu Ala Ala Tyr Tyr
Pro Gln Gln Phe Pro Xaa Xaa 195 200 205 Xaa Tyr Ala Ala Ser Leu Ser
Gly Phe Leu Asn Pro Ser Glu Gly Trp 210 215 220 Trp Pro Thr Leu Ile
Gly Leu Ala Met Asn Asp Ser Gly Gly Tyr Asn 225 230 235 240 Ala Asn
Ser Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Lys Arg Asn 245 250 255
Asp Pro Met Val Gln Ile Pro Arg Leu Val Ala Asn Asn Thr Arg Ile 260
265 270 Trp Val Tyr Cys Gly Asn Gly Thr Pro Ser Asp Leu Gly Gly Asp
Asn 275 280 285 Ile Pro Ala Lys Phe Leu Glu Gly Leu Thr Leu Arg Thr
Asn Gln Thr 290 295 300 Phe Arg Asp Thr Tyr Ala Ala Asp Gly Gly Arg
Asn Gly Val Phe Asn 305 310 315 320 Phe Pro Pro Asn Gly Thr His Ser
Trp Pro Xaa Xaa Tyr Trp Asn Glu 325 330 335 Gln Leu Val Ala Met Lys
Ala Asp Ile Gln His Val Leu Asn Gly Ala 340 345 350 Thr Pro Pro Ala
Ala Pro Ala Ala Pro Ala Ala Xaa 355 360 15 364 PRT Artificial
Sequence Description of Artificial Sequencealigned aa sequence of
Mycobacterium Mlep85c 15 Met Lys Phe Leu Gln Gln Met Arg Lys Leu
Phe Gly Leu Ala Ala Lys 1 5 10 15 Phe Pro Ala Arg Leu Thr Ile Ala
Val Ile Gly Thr Ala Leu Leu Ala 20 25 30 Gly Leu Val Gly Val Val
Gly Asp Thr Ala Ile Ala Val Ala Phe Ser 35 40 45 Lys Pro Gly Xaa
Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Pro Xaa 50 55 60 Ser Met
Gly His Asp Ile Lys Ile Xaa Gln Phe Gln Gly Gly Gly Gln 65 70 75 80
Xaa Xaa His Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Glu Asp 85
90 95 Tyr Xaa Xaa Asn Gly Trp Asp Ile Asn Thr Pro Ala Phe Glu Glu
Tyr 100 105 110 Tyr His Ser Gly Xaa Leu Ser Val Ile Met Pro Val Gly
Gly Gln Ser 115 120 125 Ser Phe Tyr Ser Asn Trp Tyr Gln Pro Ser Gln
Gly Asn Gly Gln His 130 135 140 Tyr Thr Tyr Lys Trp Glu Thr Xaa Phe
Leu Thr Gln Glu Met Pro Ser 145 150 155 160 Trp Leu Gln Ala Asn Lys
Asn Val Leu Pro Thr Gly Asn Ala Ala Val 165 170 175 Gly Leu Xaa Xaa
Xaa Xaa Xaa Xaa Ser Met Ser Gly Ser Ser Xaa Xaa 180 185 190 Xaa Ala
Leu Ile Leu Ala Ser Tyr Tyr Pro Gln Gln Phe Pro Xaa Xaa 195 200 205
Xaa Tyr Ala Ala Ser Leu Ser Gly Phe Leu Asn Pro Ser Glu Gly Trp 210
215 220 Trp Pro Thr Met Ile Gly Leu Ala Met Asn Asp Ser Gly Gly Tyr
Asn 225 230 235 240 Ala Asn Ser Met Trp Gly Pro Ser Thr Asp Pro Ala
Trp Lys Arg Asn 245 250 255 Asp Pro Met Val Gln Ile Pro Arg Leu Val
Ala Asn Asn Thr Arg Ile 260 265 270 Trp Val Tyr Cys Gly Asn Gly Ala
Pro Asn Glu Leu Gly Gly Asp Asn 275 280 285 Ile Pro Ala Lys Phe Leu
Glu Ser Leu Thr Leu Ser Thr Asn Glu Ile 290 295 300 Phe Gln Asn Thr
Tyr Ala Ala Ser Gly Gly Arg Asn Gly Val Phe Asn 305 310 315 320 Phe
Pro Pro Asn Gly Thr His Ser Trp Pro Xaa Xaa Tyr Trp Asn Gln 325 330
335 Gln Leu Val Ala Met Lys Pro Asp Ile Gln Gln Ile Leu Asn Gly Ser
340 345 350 Asn Asn Asn Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360
16 364 PRT Artificial Sequence Description of Artificial
Sequencealigned aa sequence of Mycobacterium Mtu85b 16 Xaa Xaa Met
Thr Asp Val Ser Arg Lys Ile Arg Ala Xaa Xaa Xaa Xaa 1 5 10 15 Trp
Gly Arg Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro 20 25
30 Gly Leu Val Gly Leu Ala Gly Gly Ala Ala Thr Ala Gly Ala Phe Ser
35 40 45 Arg Pro Gly Xaa Leu Pro Val Glu Tyr Leu Gln Val Pro Ser
Pro Xaa 50 55 60 Ser Met Gly Arg Asp Ile Lys Val Xaa Gln Phe Gln
Ser Gly Gly Asn 65 70 75 80 Asn Ser Pro Ala Val Tyr Leu Leu Asp Gly
Leu Arg Ala Gln Asp Asp 85 90 95 Tyr Xaa Xaa Asn Gly Trp Asp Ile
Asn Thr Pro Ala Phe Glu Trp Tyr 100 105 110 Tyr Gln Ser Gly Xaa Leu
Ser Ile Val Met Pro Val Gly Gly Gln Ser 115 120 125 Ser Phe Tyr Ser
Asp Trp Tyr Ser Pro Ala Cys Gly Lys Ala Gly Cys 130
135 140 Gln Thr Tyr Lys Trp Glu Thr Xaa Phe Leu Thr Ser Glu Leu Pro
Gln 145 150 155 160 Trp Leu Ser Ala Asn Arg Ala Val Lys Pro Thr Gly
Ser Ala Ala Ile 165 170 175 Gly Leu Xaa Xaa Xaa Xaa Xaa Xaa Ser Met
Ala Gly Ser Ser Xaa Xaa 180 185 190 Xaa Ala Met Ile Leu Ala Ala Tyr
His Pro Gln Gln Phe Ile Xaa Xaa 195 200 205 Xaa Tyr Ala Gly Ser Leu
Ser Ala Leu Leu Asp Pro Ser Gln Gly Met 210 215 220 Gly Pro Ser Leu
Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr Lys 225 230 235 240 Ala
Ala Asp Met Trp Gly Pro Ser Ser Asp Pro Ala Trp Glu Arg Asn 245 250
255 Asp Pro Thr Gln Gln Ile Pro Lys Leu Val Ala Asn Asn Thr Arg Leu
260 265 270 Trp Val Tyr Cys Gly Asn Gly Thr Pro Asn Glu Leu Gly Gly
Ala Asn 275 280 285 Ile Pro Ala Glu Phe Leu Glu Asn Phe Val Arg Ser
Ser Asn Leu Lys 290 295 300 Phe Gln Asp Ala Tyr Asn Ala Ala Gly Gly
His Asn Ala Val Phe Asn 305 310 315 320 Phe Pro Pro Asn Gly Thr His
Ser Trp Glu Xaa Xaa Tyr Trp Gly Ala 325 330 335 Gln Leu Asn Ala Met
Lys Gly Asp Leu Gln Ser Ser Leu Xaa Gly Ala 340 345 350 Gly Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 17 364 PRT Artificial
Sequence Description of Artificial Sequencealigned aa sequence of
Mycobacterium Mlep85b 17 Xaa Xaa Met Ile Asp Val Ser Gly Lys Ile
Arg Ala Xaa Xaa Xaa Xaa 1 5 10 15 Trp Gly Arg Trp Leu Leu Val Gly
Ala Ala Ala Thr Xaa Xaa Leu Pro 20 25 30 Ser Leu Ile Ser Leu Ala
Gly Gly Ala Ala Thr Ala Ser Ala Phe Ser 35 40 45 Arg Pro Gly Xaa
Leu Pro Val Glu Tyr Leu Gln Val Pro Ser Glu Xaa 50 55 60 Ala Met
Gly Arg Thr Ile Lys Val Xaa Gln Phe Gln Asn Gly Gly Asn 65 70 75 80
Gly Ser Pro Ala Val Tyr Leu Leu Asp Gly Leu Arg Ala Gln Asp Asp 85
90 95 Tyr Xaa Xaa Asn Gly Trp Asp Ile Asn Thr Ser Ala Phe Glu Trp
Tyr 100 105 110 Tyr Gln Ser Gly Xaa Leu Ser Val Val Met Pro Val Gly
Gly Gln Ser 115 120 125 Ser Phe Tyr Ser Asp Trp Tyr Ser Pro Ala Cys
Gly Lys Ala Gly Cys 130 135 140 Thr Thr Tyr Lys Trp Glu Thr Xaa Phe
Leu Thr Ser Glu Leu Pro Lys 145 150 155 160 Trp Leu Ser Ala Asn Arg
Ser Val Lys Ser Thr Gly Ser Ala Val Val 165 170 175 Gly Leu Xaa Xaa
Xaa Xaa Xaa Xaa Ser Met Ala Gly Ser Ser Xaa Xaa 180 185 190 Xaa Ala
Leu Ile Leu Ala Ala Tyr His Pro Asp Gln Phe Ile Xaa Xaa 195 200 205
Xaa Tyr Ala Gly Ser Leu Ser Ala Leu Met Asp Ser Ser Gln Gly Ile 210
215 220 Glu Pro Gln Leu Ile Gly Leu Ala Met Gly Asp Ala Gly Gly Tyr
Lys 225 230 235 240 Ala Ala Asp Met Trp Gly Pro Pro Asn Asp Pro Ala
Trp Gln Arg Asn 245 250 255 Asp Pro Ile Leu Gln Ala Gly Lys Leu Val
Ala Asn Asn Thr His Leu 260 265 270 Trp Val Tyr Cys Gly Asn Gly Thr
Pro Ser Glu Leu Gly Gly Thr Asn 275 280 285 Val Pro Ala Glu Phe Leu
Glu Asn Phe Val His Gly Ser Asn Leu Lys 290 295 300 Phe Gln Asp Ala
Tyr Asn Gly Ala Gly Gly His Asn Ala Val Phe Asn 305 310 315 320 Leu
Asn Ala Asp Gly Thr His Ser Trp Glu Xaa Xaa Tyr Trp Gly Ala 325 330
335 Gln Leu Asn Ala Met Lys Pro Asp Leu Gln Asn Thr Leu Xaa Met Ala
340 345 350 Val Pro Arg Ser Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360
18 364 PRT Artificial Sequence Description of Artificial
Sequencealigned aa sequence of Mlep85a 18 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 20 25 30 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 50
55 60 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 65 70 75 80 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa 85 90 95 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa 100 105 110 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 130 135 140 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Leu Thr Ser Glu Leu Pro Gln 145 150 155 160 Tyr Leu
Gln Ser Asn Lys Gln Ile Lys Pro Thr Gly Ser Ala Ala Val 165 170 175
Gly Leu Xaa Xaa Xaa Xaa Xaa Xaa Ser Met Ala Gly Leu Ser Xaa Xaa 180
185 190 Xaa Ala Leu Thr Leu Ala Ile Tyr His Pro Asp Gln Phe Ile Xaa
Xaa 195 200 205 Xaa Tyr Val Gly Ser Met Ser Gly Leu Leu Asp Pro Ser
Asn Ala Met 210 215 220 Gly Pro Ser Leu Ile Gly Leu Ala Met Gly Asp
Ala Gly Gly Tyr Lys 225 230 235 240 Ala Ala Asp Met Trp Gly Pro Ser
Thr Asp Pro Ala Trp Lys Arg Asn 245 250 255 Asp Pro Thr Val Asn Val
Gly Thr Leu Ile Ala Asn Asn Thr Arg Ile 260 265 270 Trp Met Tyr Cys
Gly Asn Gly Lys Pro Thr Glu Leu Gly Gly Asn Asn 275 280 285 Leu Pro
Ala Lys Leu Leu Glu Gly Leu Val Arg Thr Ser Asn Ile Lys 290 295 300
Phe Gln Asp Gly Tyr Asn Ala Gly Gly Gly His Asn Ala Val Phe Asn 305
310 315 320 Phe Pro Asp Ser Gly Thr His Ser Trp Glu Xaa Xaa Tyr Trp
Gly Glu 325 330 335 Gln Leu Asn Asp Met Lys Pro Asp Leu Gln Gln Tyr
Leu Xaa Gly Ala 340 345 350 Thr Pro Gly Ala Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 355 360 19 364 PRT Artificial Sequence Description of
Artificial Sequencealigned aa sequence of adhesin seq14-19 19 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10
15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala
20 25 30 Gly Ser Thr Ser Val Ser Ala Ala Ser Ala Val Asn Ser Glu
Leu Val 35 40 45 His Lys Gly Glu Leu Thr Ile Gly Xaa Leu Glu Thr
Tyr Ser Pro Tyr 50 55 60 Ser Tyr Arg Lys Asn Asn Lys Leu Thr Gly
Phe Glu Val Asp Gly Lys 65 70 75 80 Xaa Xaa Xaa Ala Val Ala Lys Lys
Met Gly Leu Lys Ala Xaa Xaa Asn 85 90 95 Phe Val Pro Thr Lys Trp
Ser Leu Xaa Ile Ala Gly Leu Gly Xaa Xaa 100 105 110 Xaa Xaa Ser Gly
Lys Phe Asp Val Val Met Xaa Xaa Xaa Xaa Xaa Xaa 115 120 125 Xaa Xaa
Xaa Asn Asn Ile Thr Thr Pro Glu Arg Ala Lys Gln Xaa Xaa 130 135 140
Xaa Xaa Tyr Asn Phe Ser Thr Pro Tyr Ile Lys Ser Xaa Xaa Xaa Arg 145
150 155 160 Phe Leu Xaa Xaa Xaa Xaa Xaa Ile Val Pro Thr Asp Ser Asn
Ile Lys 165 170 175 Ser Leu Lys Asn Ile Lys Gly Lys Lys Ile Ala Gly
Thr Gly Thr Asn 180 185 190 Asn Ala Asn Val Val Lys Lys Tyr Lys Gly
Asn Leu Pro Asn Gly Asp 195 200 205 Phe Ala Ser Ser Leu Xaa Asp Met
Ile Lys Xaa Xaa Gln Gly Arg Xaa 210 215 220 Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Ala Ala Gly Ile Asn Ser 225 230 235 240 Arg Glu Ala
Trp Tyr Xaa Xaa Xaa Xaa Xaa Ala Tyr Ser Lys Lys Xaa 245 250 255 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Ser Thr Lys Xaa Xaa 260 265
270 Xaa Xaa Xaa Gly Leu Gly Ile Xaa Xaa Asp Val Ser Ser Glu Gln Asp
275 280 285 Pro Ala Lys Xaa Ile Ser Ala Leu Xaa Phe Asn Lys Asp Thr
Ala Ile 290 295 300 Gln Ser Ser Tyr Asn Xaa Xaa Xaa Xaa Xaa Lys Ala
Leu Lys Glu Leu 305 310 315 320 Gln Gln Asp Gly Val Lys Lys Leu Ser
Glu Lys Tyr Phe Gly Ala Asp 325 330 335 Ile Thr Glu Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 20 364 PRT Artificial Sequence
Description of Artificial Sequenceconsensus of aligned aa sequences
14-19 20 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Xaa Xaa 20 25 30 Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala
Xaa Xaa Xaa Xaa Xaa Xaa 35 40 45 Xaa Xaa Gly Xaa Leu Xaa Xaa Xaa
Xaa Leu Xaa Xaa Xaa Ser Pro Xaa 50 55 60 Ser Xaa Xaa Xaa Xaa Xaa
Lys Xaa Xaa Xaa Phe Xaa Xaa Xaa Gly Xaa 65 70 75 80 Xaa Xaa Xaa Ala
Val Xaa Xaa Xaa Xaa Gly Leu Xaa Ala Xaa Xaa Xaa 85 90 95 Xaa Xaa
Xaa Xaa Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 100 105 110
Xaa Xaa Ser Gly Xaa Xaa Xaa Val Val Met Xaa Xaa Xaa Xaa Xaa Xaa 115
120 125 Xaa Xaa Xaa Xaa Asn Xaa Xaa Xaa Pro Xaa Xaa Xaa Lys Xaa Xaa
Xaa 130 135 140 Xaa Xaa Tyr Xaa Xaa Xaa Thr Xaa Xaa Xaa Xaa Ser Xaa
Xaa Xaa Xaa 145 150 155 160 Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro
Thr Xaa Ser Xaa Xaa Xaa 165 170 175 Xaa Leu Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Ala Gly Xaa Xaa Xaa Xaa 180 185 190 Xaa Ala Xaa Xaa Xaa Xaa
Xaa Tyr Xaa Xaa Xaa Xaa Xaa Pro Xaa Xaa 195 200 205 Xaa Xaa Ala Xaa
Ser Leu Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gln Gly Xaa 210 215 220 Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Gly Xaa Asn 225 230 235
240 Xaa Xaa Xaa Xaa Trp Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa Xaa
245 250 255 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Asn Xaa Thr
Xaa Xaa 260 265 270 Xaa Xaa Xaa Xaa Gly Xaa Gly Xaa Xaa Xaa Asp Xaa
Xaa Xaa Xaa Xaa 275 280 285 Xaa Pro Ala Lys Xaa Xaa Xaa Xaa Leu Xaa
Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Gln Xaa Xaa Tyr Asn Xaa Xaa
Xaa Xaa Xaa Xaa Ala Xaa Xaa Xaa 305 310 315 320 Leu Xaa Xaa Asp Gly
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Gly Ala 325 330 335 Xaa Xaa Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345 350 Xaa
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355 360 21 12 PRT
Artificial Sequence Description of Artificial Sequencefragment of
adhesin sequence=peptide I 21 Ala Ala Ser Ala Val Asn Ser Glu Leu
Val His Lys 1 5 10 22 7 PRT Artificial Sequence Description of
Artificial Sequencefragment of adhesin sequence=peptide II 22 Ala
Asn Phe Val Pro Thr Lys 1 5 23 10 PRT Artificial Sequence
Description of Artificial Sequencefragment of adhesin
sequence=peptide III 23 Asp Thr Ala Ile Gln Ser Ser Tyr Asn Lys 1 5
10 24 7 PRT Artificial Sequence Description of Artificial
Sequencefragment of adhesin sequence=peptide IV 24 Ile Ser Ala Leu
Phe Asn Lys 1 5 25 10 PRT Artificial Sequence Description of
Artificial Sequencefragment of adhesin sequence=peptide V 25 Ile
Ala Gly Thr Gly Thr Asn Asn Ala Xaa 1 5 10 26 12 PRT Artificial
Sequence Description of Artificial SequenceN terminal fragment of
adhesin sequence 26 Ala Xaa Xaa Ala Val Asn Xaa Glu Leu Val Val Lys
1 5 10 27 11 PRT Artificial Sequence Description of Artificial
Sequencefragment of adhesin sequence 27 Ile Ile Ala Gly Thr Gly Thr
Asn Asn Ala Xaa 1 5 10 28 23 DNA Artificial Sequence Description of
Artificial Sequenceoligonucleotide 42 sense for N terminal adhesin
sequence 28 ctgcgtaact tcgagattga gta 23 29 23 DNA Artificial
Sequence Description of Artificial Sequenceoligonucleotide 105
antisense for peptide I 29 gccggtcctt tgtggacaat tgc 23 30 6 PRT
Artificial Sequence Description of Artificial Sequenceconsensus
fragment of adhesin sequence 30 Lys Lys Xaa Xaa Xaa Lys 1 5
* * * * *