Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-09-12
2004-02-10
Caputa, Anthony C. (Department: 1642)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C435S069100, C435S325000, C435S320100
Reexamination Certificate
active
06689878
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to the field of proteins and methods of using such, and more specifically to proteins and portions thereof that regulate the pathway by which toxins are formed by
Staphylococcus aureus.
BACKGROUND OF THE INVENTION
The Gram-positive, aerobic bacterial pathogen
Staphylococcus aureus
(
S. aureus
) inhabits the skin and mucous membrane of humans and other animals.
S. aureus
causes diseases ranging from minor skin infections to life-threatening infections such as pneumonia, endocarditis, meningitis, postoperative wound infections, septicemia, and toxic shock syndrome. Staphylococcus is one of the most common causes of hospital-acquired infections worldwide, and reports of its growing levels of resistance to last-resort antibiotics such as vancomycin threaten a serious international public health problem.
Pathogenic
S. aureus
causes disease mainly by the production of virulence factors such as hemolysins, enterotoxins, and toxic shock syndrome toxin. The toxins can interfere with the host's immune system and degrade tissue components such as proteins, nucleic acid, lipids, and polysaccharides. In culture, these toxins are produced only at higher cell densities (e.g., the post-exponential growth phase). At lower densities during the early exponential phase, the bacteria express surface molecules such as fibronectin binding-proteins, fibrinogen binding-proteins, and protein A, which facilitate adherence of the bacteria to host cells and thwart host immune defenses. This phenomenon of growth-phase variation allows the bacteria to adhere to host cells when in low numbers, but to “disengage” and spread when too crowded, thus allowing dissemination and spread of infection (Lowy
New Engl. J. Med.
339:520-32 (1998)).
The growth-phase associated regulation of
S. aureus
virulence factor synthesis is controlled by a quorom sensing mechanism. The control of virulence factor production is a complex process, which apparently involves multiple global regulatory loci. One of these regulatory loci, the agr locus, contains two divergent transcription units, RNAII and RNAIII, both of which are active only from the midexponential phase of growth and are autocatalytic (Novick et al.
Mol. Gen. Genet.
248:446058 (1995)). RNAIII, an RNA regulatory molecule encoded by the agr locus, upregulates genes encoding for toxic exomolecules while down regulating genes encoding for surface molecules, resulting in vivo in dissemination and disease (Novick et al.
EMBO J.
12:3967-75 (1993); Balaban et al.
Proc. Natl. Acad. Sci. USA
92:1619-23 (1995); Moerfeldt et al.
EMBO J.
14:4569-77 (1995)). The RNAII locus regulates the expression of RNAIII. The RNAII locus comprises four open reading frames (ORFs), agrA, agrB, agrC, and agrD. The agrA and agrC genes encode for a classical two-component signal transduction pathway, with agrC encoding a signal receptor and agrA the response regulator.
The autoinducers of RNAIII that have been described to date include the agr-independent RNAIII activating protein (RAP) (Balaban et al. (1995) supra; Balaban et al.
Science
280:438-40 (1998)), and the agrD-derived octapeptide pheromone (Ji et al.
Proc. Natl. Acad. Sci.
92:12055-9 (1995)), which binds and activates phosphorylation of AgrC (Lina et al.
Mol. Microbiol.
28:655-662 (1998), which in turn is thought to phosphorylate AgrA, leading to upregulation of RNAIII (Morfeldt et al.,
FEMS Microbiol. Lett.
143:195-201 (1996)). The agrD-derived octapeptide has also been shown to be part of a “bacterial interference” system that provides a mechanism for different
S. aureus
strains to compete with each other at an infection site (Ji et al.
Science
276:2027-30 (1997)). In this bacterial interference system, the octapeptide activates RNAIII transcription of the strain by which it is produced, while also acting as an inhibitor of RNAIII transcription of other strains of Staphyloccocus.
In addition to agr, the sar locus also plays a role in regulation of
S. aureus
virulence factor production. The sar locus comprises a sarA ORF preceded by a triple promoter region interspersed with two putative smaller ORFs (ORF3 and ORF4). The triple promoter system yields three overlapping sar transcripts (sarA, sarC and sarB) (Bayer et al.
J. Bacteriol.
178:4563-70 (1996)). The SarA protein has been shown to bind to the agr promoter region and to stimulate the transcription of RNAII (Chien et al.
J. Biol. Chem.
273:2645-52 (1998).
In vivo
S. aureus
first produce proteins that facilitate bacterial binding to host cells as well as the secreted autoinducer molecules. As the bacterial colony increases in density, the autoinducer molecules accumulate. Upon reaching a threshold concentration, the autoinducers activate RNAIII transcription, which in turn results in virulence factor production. The virulence factors damage and eventually destroy surrounding host cells, which serve as nutritive sources for the
S. aureus
bacteria and promoting further growth of the colony. Thus, inhibition of RNAIII by suppression of the autoinducers or their receptors is of particular interest in treatment or prevention of
S. aureus
-mediated disease. Several mechanisms for RNAIII inhibition have been identified, including inhibition of RNAIII by anti-RAP antibodies and by a peptide termed the RNAIII inhibiting peptide (RIP), which competes with RAP (Balaban et al. (1995) supra). Animals vaccinated with RAP or treated with RIP have been shown to be protected from an
S. aureus
infection (Balaban et al. (1998), supra).
Identification of additional components of the
S. aureus
virulence factor regulatory system is of great interest as such will provide additional therapeutic targets. The present invention provides a newly identified component of the system that regulates virulence factor production in
S. aureus.
REFERENCES
Roychoudhury, S. et al.
Proc. Natl. Acad. Sci. U.S.A.,
90: 965-969 (1993) described the identification of synthetic chemical compounds that block the expression of alginate, a virulence factor for the cystic fibrosis pathogen
Pseudomonas aeruginosa.
However, it has not been shown that the small molecules would have any effect on
S. aureus
, or offer potential clinical utility.
Balaban et al.
Proc. Natl. Acad. Sci. USA
92:1619-23 (1995) reports autocrine regulation of toxin synthesis by RIP and RAP.
Ji et al.
Proc. Natl. Acad. Sci. USA
(1995) describes an octapeptide that acts as an agr-dependent autoinducer of RNAIII.
PCT publication no. WO 96/106579 by Novick et al. describes peptides having a molecular weight of less than 3 kDa capable of inhibiting agr-RNAIII transcription in
S. aureus
. In addition, PCT publication no. WO 97/44349 by Novick et al. describes cyclic peptides of between six to twelve amino acids, including cysteine from the 28 position of the agrD region as capable, in vitro, of inhibiting agr-RNAIII transcription in
S. aureus.
Ji et al.
Science
276:2027-30 (1997) describe the phenomenon of bacterial interference mediated by octapeptides.
Balaban et al.
Science
280:438-440 (1998) describes inhibition of inhibit
S. aureus
infection in mice using RIP, synthetic peptides, and anti-RAP antibodies.
Novick et al.
Curr. Opin. Microbiol.
2:40-5 (1999) reviews virulence gene regulation in staphylococci and other gram-positive bacteria.
Mayville et al.
Proc. Natl. Acad. Sci. USA
96:1218-23 (1999) reports that the octapeptide contains a thiolactone moiety.
Sequence data from the genome of
S. aureas
and contig 876 were obtained from the Staphylococcus aureus Genome Sequencing Project website having an Internet address at www. genome.ou.edu/staph blast.html [at http://www.genome.ou.edu/staph_blast.html].
SUMMARY OF THE INVENTION
The present invention is directed to a protein isolated from
S. aureus
that is the target of RAP, called TRAP, which is characterized by a molecular weight of about 21 KDa, is capable of being phosphorylated by RAP, and comprises an amino acid sequence of SEQ ID NO:2. In addition, the present inve
Balaban Naomi
Goldkorn Tzipora
Caputa Anthony C.
Nickol Gary B.
Smith , Gambrell & Russell, LLP
Sundby Suzannah K.
The Regents of the University of California
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