An avirulent, invasion plasmid insertion mutant of Shigea flexneri 5 (pHS1059) was restored to the virulence phenotype by transformation with a partial HindUI library of the wild-type invasion plasmid constructed in pBR322. Western immunoblot analysis of pHS1059 whole-cell lysates revealed that the synthesis of the invasion plasmid antigens VirG, IpaA, IpaB, IpaC, and IpaD was similar to that seen in the corresponding isogenic S. feneri 5 virulent strain, M9OT. IpaB and IpaC, however, were not present on the surface of pHS1059 as was found in M9OT, suggesting that the transport or presentation of the IpaB and IpaC proteins onto the bacterial surface was defective in the mutant. pHS1059 was complemented by pWR266, which carried contiguous 1.2-and 4.1-kb HinduI fragments of the invasion plasmid. pHS1059(pWR266) cells were positive in the HeLa cell invasion assay as well as colony immunoblot and enzyme-linked immunosorbent assays, using monoclonal antibodies to IpaB and IpaC. These studies established that the antigens were expressed on the surface of the transformed bacteria. In addition, water extraction of pHS1059 and pHS1059(pWR266) whole cells, which can be used to remove IpaB and IpaC antigens from the surface of wild-type M90T bacteria, yielded significant amounts of these antigens from pHS1059(pWR266) but not from pHS1059. Miniceli and DNA sequence analysis indicated that several proteins were encoded by pWR266, comprising the spa loci, which were mapped to a region approximately 18 kb upstream of the ipaBCDAR gene cluster. Subcloning and deletion analysis revealed that more than one protein was involved in complementing the Spa-phenotype in pHS1059. One of these proteins, Spa47, showed striking homology to ORF4 of the Bacilus subtUisflaA locus and the fliI gene sequence of SalmoneUa typhimurium, both of which bear strong resemblance to the a and 13 subunits of bacterial, mitochondrial, and chloroplast proton-translocating FoF, ATPases.

The oxazolidinones are a new class of synthetic antibiotics with good activity against gram-positive pathogenic bacteria. Experiments with a susceptible Escherichia coli strain, UC6782, demonstrated that in vivo protein synthesis was inhibited by both eperezolid (formerly U-100592) and linezolid (formerly U-100766). Both linezolid and eperezolid were potent inhibitors of cell-free transcription-translation in E. coli, exhibiting 50% inhibitory concentrations (IC50s) of 1.8 and 2.5 microM, respectively. The ability to demonstrate inhibition of in vitro translation directed by phage MS2 RNA was greatly dependent upon the amount of RNA added to the assay. For eperezolid, 128 microg of RNA per ml produced an IC50 of 50 microM whereas a concentration of 32 microg/ml yielded an IC50 of 20 microM. Investigating lower RNA template concentrations in linezolid inhibition experiments revealed that 32 and 8 microg of MS2 phage RNA per ml produced IC50s of 24 and 15 microM, respectively. This phenomenon was shared by the translation initiation inhibitor kasugamycin but not by streptomycin. Neither oxazolidinone inhibited the formation of N-formylmethionyl-tRNA, elongation, or termination reactions of bacterial translation. The oxazolidinones appear to inhibit bacterial translation at the initiation phase of protein synthesis.

The large invasion plasmid of Shigella flexneri M90T-W was used to generate recombinant plasmids carrying the ipa4, -B, -C, and -D genes, whose products are associated with the entry of the bacteria into colonic epithelial cells. Complete DNA sequences of ipaB, -C, and -D were determined. The proteins predicted (62, 42, and 37 kDa, respectively) from the nucleotide sequences lack a signalpeptide sequence. Hydrophilic segments of the IpaB and IpaC proteins were found to overlap known epitopic domains ofthese membrane antigens. Analysis of total RNA demonstrated that temperature control of ipa gene expression occurs at the level of transcription. Multiple mRNA bands were detected by using ipa gene fragments as hybridization probes, and a putative transcript map for the ipa genes was constructed. Comparison of this map with the DNA sequence reveals a complex system of ipa gene regulation.

Tn5-tagged invasion plasmid DNA (pWRllO) from Shigeglaflexneri serotype 5 (strain M9OT) was cloned into the expression vector Agtll. Recombinant phage (XgtllSfl) expressing pWR11O-encoded polypeptide antigens were identified by using rabbit antisera directed against S. flexneri M9OT invasion plasmid antigens. Antigens encoded by AgtllSfl recombinant phage were characterized by reacting affinity-purified antibodies, eluted from nitroceilulose-bound plaques of AgtllSfl recombinants, with virulent, wild-type S. flexneri M9OT polypeptides in Western blot analyses. AgtllSfl clones directing the synthesis of complete, truncated, and B-galactosidase fusion versions of three previously identified outer membrane polypeptides antigens) were isolated. A fourth polypeptide, similar in size to the 57-kDa antigen (ca. 58 kDa) but unrelated as determined by DNA homology and serological measurements, was also identified. Southern blot analysis of S. flexneri M9OT invasion plasmid DNA hybridized with AgtllSfl insert DNA probes was used to construct a map of invasion plasmid antigen genes (ipa) corresponding to the 57-kDa (ipaB), , and 39-kDa (ipaD) polypeptides. Genes ipaB, ipaC and ipaD mapped to contiguous 4.6-kilobase (kb) and 1.0-kb Hindm fragments contained within a larger (23-kb) Bam-H fragment. The ipall gene, which encodes the synthesis of the 58-kDa polypeptide, did not map in or near the ipaBCD gene cluster, suggesting a distinct location of ipaH on the invasion plasmid.The complex pathology of the dysenteric syndrome, caused by Shigella spp. and enteroinvasive Escherichia coli, is reflected in the diversity of genetic components controlling the virulence of these organisms. Both chromosomal and extrachromosomal loci that are essential for the expression of the virulent phenotype have been identified (21, 22; reviewed in reference 10). One aspect of this phenotype, the invasion of colonic epithelial cells, has its genetic components located on a large 120-to 140-megadalton (MDa) nonconjugative plasmid found in all Shigella and enteroinvasive E. coli strains (11,23,24). Loss of the plasmid is accompanied by loss of the invasive phenotype, as measured by in vitro infection of cultured mammalian cells, and by the inability of spontaneously cured shigellae to elicit keratoconjunctivitis (i.e., the Sereny reaction) in guinea pigs (21,23,24,27). Reintroduction of the invasion plasmid into a plasmid-free avirulent Shigella strain restores the invasive phenotype (23,24,30).A 37-kilobase (kb) region of the S. flexneri serotype 5 invasion plasmid cloned into the cosmid vector pJB8 restores the HeLa cell invasiveness of plasmid-cured Shigella spp. but does not restore the ability to cause a positive Sereny reaction (13). At least eight polypeptides, ranging in size from 12 to 140 kDa, have been identified as unique products of the invasion plasmid (7,8 temperature for the invasive phenotype (13, 14). These polypeptides, plus an additional 140-kDa outer membrane protein, are also important immunogens, since convalescentstage sera...

Gene products required for in vivo growth and survival of microbial pathogens comprise a unique functional class and may represent new targets for antimicrobial chemotherapy, vaccine construction, or diagnostics. Although some factors governing Staphylococcus aureus pathogenicity have been identified and studied, a comprehensive genomic analysis of virulence functions will be a prerequisite for developing a global understanding of interactions between this pathogen and its human host. In this study, we describe a genetic screening strategy and demonstrate its use in screening a collection of 6,300 S. aureus insertion mutants for virulence attenuation in a murine model of systemic infection. Ninety-five attenuated mutants were identified, reassembled into new pools, and rescreened using the same murine model. This effort identified 24 highly attenuated mutants, each of which was further characterized for virulence attenuation in vivo and for growth phenotypes in vitro. Mutants were recovered in numbers up to 1,200-fold less than wild type in the spleens of systemically infected animals and up to 4,000-fold less than wild type in localized abscess infections. Genetic analysis of the mutants identified insertions in 23 unique genes. The largest gene classes represented by these mutants encoded enzymes involved in small-molecule biosynthesis and cell surface transmembrane proteins involved in small-molecule binding and transport. Additionally, three insertions defined two histidine kinase sensor-response regulator gene pairs important for S. aureus in vivo survival. Our findings extend the understanding of pathogenic mechanisms employed by S. aureus to ensure its successful growth and survival in vivo. Many of the gene products we have identified represent attractive new targets for antibacterial chemotherapy.