The invention relates to a novel gene, waaP, which is involved in heptose modification in the lipopolysaccharide of bacterial membranes, its corresponding protein, WaaP, and a method of preventing or treating gram negative bacterial infections, particularly attenuating virulent gram negative pathogens including compounds for such attenuation.
The outer membrane of Gram-negative bacteria is a barrier to many antibiotics and host defense factors (Vaara, M., 1992). The outer leaflet of this membrane is comprised almost exclusively of lipopolysaccharide (LPS), a unique glycolipid with structural features essential to outer membrane stability. In Escherichia coli and Salmonella enterica, the LPS molecule is conceptually divided into three distinct regions: 1) a hydrophobic membrane anchor designated lipid A; 2) a short chain of sugar residues with multiple phosphoryl substituents, referred to as the core oligosaccharide (core OS); and 3) a structurally diverse polymer composed of oligosaccharide repeats, termed the O antigen. Lipid A and the core OS are synthesized together as a single unit (lipid A-core, FIG. 1), which serves as an acceptor for preformed O antigen to yield the completed LPS molecule (Whitfield, C. et al., 1997; Raetz, C. R. H. 1996).
Five distinct core OS structures have been identified in E. coli (core types K-12, R1, R2, R3, and R4), and two more are known for S. entarica (Holst, 0. and Brade, H., 1992; Olsthoorn, M. M. A., et al. 1998). The genes responsible for biosynthesis of the core OS in these bacteria are clustered on the chromosome in the waa (formerly rfa) locus near 81 min on the E. coli K-12 and S. Enterica linkage maps. Mutations in many of the glycosyl transferases encoded by this locus result in the production of LPS lacking O antigen (termed rough- or R-LPS) since O antigen cannot be ligated to an incomplete lipid A-core acceptor molecule. Strains which produce only R-LPS are more susceptible to complement-mediated serum killing than their wild-type counterparts (reviewed in Whitfield, C., 1994). Mutations in the waa locus which specifically affect the phosphoryl substitution of the core OS heptose region (FIG. 1) can significantly alter outer membrane permeability, giving rise to a pleiotropic phenotype called xe2x80x98deep roughxe2x80x99 (Helander, I. M. et al., 1989; Nikaido, H. 1996). Characteristics of the deep-rough phenotype include the following: 1) hypersensitivity to detergents and hydrophobic antibiotics, 2) sloughing of LPS from the outer membrane, 3) leakage of periplasmic proteins into the culture medium, and 4) a marked decrease in the protein content of the outer membrane (reviewed in Schnaitman, C. A. et al. 1993). The phosphoryl substituents in the heptose region are postulated to be so critical to outer membrane stability because their negative charge allows neighbouring LPS molecules to be crosslinked by divalent cations (Vaara, M. 1992; Nikaido, H. and Vaara, M., 1995).
Previous studies with S. enterica serovars Minnesota and Typhimurium (Helander, I. M., et al. 1989), and with E. coli K-12 (Parker, C. T., et al. 1992) have implicated waaP in the phosphorylation of both Heptose I (xe2x80x9cHepIxe2x80x9d) and Heptose II (xe2x80x9cHepIIxe2x80x9d) (see FIG. 1). Mutation of waaP in these organisms was also reported to cause characteristics of the deep-rough phenotype. Furthermore, in E. coli K-12, waaP has been implicated in the addition of HepIII (FIG. 1). Interpretation of these data, however, has been complicated by their reliance on strains with poorly-defined or polar mutations.
The R1 core is the most prevalent among clinical isolates of E. coli (Gibb, A. P. et al., 1992) and since its structure is known (Jansson, P. -E., et al. 1981) and the genetics of its outer portion have been resolved (Heinrichs, D. E., et al., 1998), studies were performed using the prototype E. coli R1 strain, F470. However, three genes in the waaQ operon of the E. coli R1 waa locus (FIG. 2B) have no clearly assigned function: waaQ, P, and Y.
The present inventors have made precise, non-polar insertion mutations in the waaQ, P, and Y genes of E. coli F470, and determined that the enzyme encoded by waaP is responsible for modification of the heptose (xe2x80x9cHepxe2x80x9d) in the heptose region (xe2x80x9cHep regionxe2x80x9d) by the phosphoryl substitution of the heptose units of the region. The inventors have also identified and isolated highly conserved homologs of the waaP gene from LPS core types R1, R3 and R4 from E. coli F470, F653, and F2513, respectively. The inventors have found that the activity of WaaP is to add the phosphoryl substituent to HepI, and that this activity is a prerequisite to the operation of WaaQ and WaaY (both of which are required for the phosphorylation of Hep II). Consequently, the identification and isolation of the waaP gene permits the determination of substances which affect modification of the Hep region, and particularly the modification of Hep I. These substances may be useful in attenuating virulent gram negative pathogens in a host infected by such pathogens.
Accordingly, the present invention provides an isolated nucleic acid molecule containing a sequence encoding the enzyme WaaP which is responsible for modification of the Hep region of LPS by the phosphoryl substitution of the Hep units of the region.
The nucleic acid sequence of waaP is shown in SEQ.ID.NO.:1 (from F470) or SEQ.ID.NO.:3 (from F653) or SEQ.ID.NO.:5 (from F2513). The corresponding amino acid sequence encoded by each one of these nucleic acid sequences of waaP is shown in SEQ.ID.NO.:2 or SEQ.ID.NO.:4 or SEQ.ID.NO.:6, respectively.
Accordingly, in one embodiment of the invention, an isolated nucleic acid molecule is provided having a sequence as shown in SEQ.ID. No.:1 or SEQ.ID.NO.: 3 or SEQ.ID.NO.:5.
Preferably, the purified and isolated nucleic acid molecule comprises
(a) a nucleic acid sequence as shown in SEQ. ID. NO.: 1 or SEQ.ID.NO.:3 or SEQ.ID.NO.:5, wherein T can also be U;
(b) nucleic acid sequences complementary to (a);
(c) nucleic acid sequences which are homologous to (a) or (b);
(d) a fragment of (a) to (c) that is at least 15 bases, preferably 20 to 30 bases, and which will hybridize to (a) to (d) under stringent hybridization conditions; or
(e) a nucleic acid molecule differing from any of the nucleic acids of (a) to (c) in codon sequences due to the degeneracy of the genetic code.
The invention further includes an isolated protein encoded by a nucleic acid molecule of the invention. Accordingly a preferred embodiment has the amino acid as shown in SEQ. ID. NO. 2 or SEQ.ID.NO.:4 or SEQ.ID.NO.:6.
The invention further provides a method of preventing or treating a gram negative bacterial infection comprising administering an effective amount of a substance that inhibits WaaP to an animal in need thereof.
According to one embodiment, there is provided a method for attenuating virulent gram negative pathogens comprising administering a sufficient amount of an inhibitor of WaaP to attenuate the pathogen. Preferably, the gram negative pathogen is selected from the group of pathogens including Salmonella enterica, Escherichia coli, Vibrio cholerae, Yersinia enterocolitica, Shigella flexneri, Shigella dysenteriae, and Pseudomonas aeruginosa serotypes 02, 05,16, 18 and 20, P. aeruginosa serotypes 03 or 06 and other members of the family Pseudomonadaceae.
Inhibitors of the invention include antibodies which inhibit the activity of the WaaP protein; antibodies which inhibit interaction of the WaaP substrate with the WaaP protein; and antisense oligonucleotide which inhibit translation of the waaP gene.
According to another embodiment, the present invention provides a method of assaying for inhibitors of WaaP, under appropriate conditions, comprising the steps of incubating a gram negative pathogen with a test substance which is suspected of affecting WaaP activity and determining the effect of the substance, preferably phosphorylation of Hep, by comparing to a control. Accordingly, using the methods of the present invention, inhibitors of WaaP in gram negative pathogens will be identified.
According to a further embodiment of the present invention, there is provided a method of treating or preventing a gram negative bacterial infection comprising administering an effective amount of a composition that inhibits WaaP activity, preferably in an animal in need thereof, wherein the composition comprises inhibitors of WaaP activity, preferably in a pharmaceutically acceptable diluent or carrier.
In another embodiment, the present invention provides a vaccine for preventing or treating a gram negative bacterial infection, preferably in an animal in need thereof, comprising an effective amount of a WaaP protein in admixture with a suitable diluent or carrier.
Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.