Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1998-09-25
2002-06-18
Graser, Jennifer E. (Department: 1645)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C536S023700, C435S069300, C435S320100, C435S243000, C435S252300, C435S253400, C424S244100
Reexamination Certificate
active
06406883
ABSTRACT:
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention have been putatively identified as Lmb streptococcal adhesion mediator that promotes attachment of
Streptococcus agalactiae
cells to human laminin.
Laminin is a 900 kDa glycoprotein that is a major component of the basement membrane. It is composed of three distinct polypeptide chains (A, B1, B2) which reversibly assemble to form the macromolecular structure. Functions of laminin include the formation of the basement membrane by interaction with other basement membrane components and the development and maintenance of cellular organization.
S. agalactiae
has been demonstrated to damage the pulmonary epithelium (Nizei, Gibson et al. 1996) which leads to the exposure of underlying basement membrane structures. Adhesion to basement membrane components may be important for persistent bacterial colonization of damaged epithelium and invasion of bacteria into the bloodstream.
The adherence of bacteria to laminin may be important for the development of invasive group B streptococcal infection. Invasion of
S. agalactiae
into the blood stream as well as the entry into CSF, which occurs in the case of meningitis, requires the passage of bacteria through basement membranes. The interaction of bacterial surface proteins with laminin could be an important mechanism in this context. For
H. influenzae
adhesion and penetration of the basement membranes, which are open to circulation in the fenestrated endothelium of the choroid plexus, is discussed as the route of entry into the CSF (Virkola, Lähteenmäki et al. 1996).
Adhesion mediators of the LraI adhesion family mediate attachment of the streptococci pathogens to human laminin and contribute to streptococcal disease in humans. In particular, expression of cell surface receptors determines streptococcal adhesion properties which include binding to extracellular matrix proteins, epithelial cells, endothelial cells and to other bacterial organisms. The LraI (lipoprotein receptor antigen I) family of surface-associated lipoproteins is involved in co-aggregation of
Streptococcus gordonii
with
Actinomyces naeslundii
, the adherence of
S. sanguis
to the salivary pellicle, the binding of
S. parasanguis
to a platelet fibrin matrix (Viscount, Munro et al. 1997) (Jenkinson 1994) and the adherence of
S. pneumoniae
to type II pneumocytes (Berry and Paton 1996). Previously identified members of this family are PsaA from
Streptococcus pneumoniae
, FimA from
S. parasanguis
, SsaB from
S. sanguis
, EfaA from
Enterococcus faecalis
and ScbA from
S. crista
and ScaA from
S. gordonii
.
Genes of this family are located in ABC transporter type operons and thought to function as solute binding components in analogy to outer membrane proteins of gram-negative organisms (Jenkinson 1994) (Kolenbrander, Andersen et al. 1994). PsaA of
S. pneumoniae
and FimA of
S. parasanguis
have been shown to be essential for virulence in animal models (Viscount, Munro et al. 1997) (Berry and Paton 1996). In addition immunogenic properties were demonstrated for EfaA, FimA and PsaA indicating a potential use of these proteins as vaccine candidates.
Streptococcus agalactiae
(Group B streptococcus, GBS) is one of the most important neonatal pathogen causing septicemia or meningitis in up to 1.8 cases per 1000 live births. Even with antibiotic therapy mortality rates range between 5 and 30% (Weisman, Stoll et al. 1992). In addition recent studies report an increasing number of serious infections in adults (Farley 1995) (Farley, Harvey et al. 1993). Several virulence factors that contribute to the pathogenesis of the disease have been identified. These include the capsule (Viessels, Rubens et al. 1989), the CAMP factor (Fehrenbach, Jürgens et al. 1988) (Podbielski, Blankenstein et al. 1994), the hemolysin (Nizet, Gibson et al. 1996) and C-protein on the bacterial surface (Michel, Madoffet al. 1991).
The adherence of
S. agalactiae
to immobilized fibronectin has also been implicated in the pathogenesis of disease (Tamura and Rubens 1995). However, prior to the present invention, genetic determinants for the adherence of
S. agalactiae
to extracellular matrix proteins had not been identified.
In accordance with one aspect of the present invention, there are provided novel Lmb streptococcal adhesion mediator polypeptides, as well as active fragments, analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the Lmb streptococcal adhesion mediator polypeptides of the present invention including mRNAs, cDNAs, genomic DNAs as well as active analogs and fragments of such Lmb streptococcal adhesion mediator polypeptides.
In accordance with another aspect of the present invention there are provided isolated nucleic acid molecules encoding mature lmb polypeptides expressed by the DNA contained in ATCC strain Deposit No. 12400 utilizing the primes equences and procedures as described below, which maybe obtained by PCR, as construct of the article acid provided and a plasmid provided therefrom.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said Lmb streptococcal adhesion mediator polypeptides and subsequent recovery of said Lmb streptococcal adhesion mediator polypeptides.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such Lmb streptococcal adhesion mediator polypeptides as reagents for testing pharmaceutical antibiotics for their activity in deactivating or controlling the activity of the Lmb streptococcal adhesion mediator polypeptides as part of a screening process to identify pharmaceuticals for treating or controlling streptococcal infections.
In accordance with another aspect of the present invention the polynucleotides or epitopic fragments thereof are useful as in vitro agents for producing monoclonal antibodies useful in screening procedures for diagnosing streptococcus bacterial infections by identifying the presence of such bacteria in a specimen from a mammal suspected of having such an infection. Also, such polynucleotides or epitopic fragments are useful as reagents to test pharmaceutical chemicals for activity in suppressing the expression of such polynucleotides. Thus, the polynucleotides and polypeptides according to the invention are useful as in vitro agents for diagnostic and screening procedures for identifying and/or treating streptococcal infections in mammals.
In another aspect of the present invention, an antisense construct prepared through the use of antisense technology, may be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5′ coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of the streptococcal Imb genes. The antisense RNA oligonucleotide hybridizes to the MRNA in vivo and blocks translation of mRNA molecules into Lmb streptococcal adhesion mediator polypeptides (antisense -Okano, J. Neurochem., 56:5
Lütticken Rudolf
Podbielski Andreas
Rozdzinski Eva
Spellerberg Barbara
Graser Jennifer E.
Lillie Raymond J.
Olstein Elliot M.
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