Representative Shigella flexneri strains were studied to determine whether plasmids are involved in their virulence. All invasive S. flexneri strains, irrespective of serotype, were found to harbor a large plasmid of approximately 140 megadaltons in size, although some strains carried additional plasmid species. Spontaneous variants of strains of serotypes 1, 2, and 5 had lost this 140-megadalton plasmid and had concomitantly become avirulent, i.e., could neither invade HeLa cell monolayers nor produced keratoconjunctivitis in guinea pigs. To monitor plasmid transfer, the 140-megadalton plasmid of strain M90T (serotype 5) was tagged with the kanamycin resistance transposon Tn5. This tagged plasmid, pWR110, was not self-transmissible, but was mobilized by one of several different conjugative plasmids into avirulent derivatives of the heterologous serotypes 1 and 2 which had lost the comparable large plasmid. Transconjugants of both serotypes which had received pWR110 regained virulence. These data directly demonstrate that this large S. flexneri plasmid encodes or regulates some function(s) required for epithelial cell penetration.

Campylobacter jejuni strain 81-176 contains two, previously undescribed plasmids, each of which is approximately 35 kb in size. Although one of the plasmids, termed pTet, carries a tetO gene, conjugative transfer of tetracycline resistance to another strain of C. jejuni could not be demonstrated. Partial sequence analysis of the second plasmid, pVir, revealed the presence of four open reading frames which encode proteins with significant sequence similarity to Helicobacter pylori proteins, including one encoded by the cag pathogenicity island. All four of these plasmid-encoded proteins show some level of homology to components of type IV secretion systems. Mutation of one of these plasmid genes, comB3, reduced both adherence to and invasion of INT407 cells to approximately one-third that seen with wild-type strain 81-176. Mutation of comB3 also reduced the natural transformation frequency. A mutation in a second plasmid gene, a virB11 homolog, resulted in a 6-fold reduction in adherence and an 11-fold reduction in invasion compared to the wild type. The isogenic virB11 mutant of strain 81-176 also demonstrated significantly reduced virulence in the ferret diarrheal disease model. The virB11 homolog was detected on plasmids in 6 out of 58 fresh clinical isolates of C. jejuni, suggesting that plasmids are involved in the virulence of a subset of C. jejuni pathogens.Although Campylobacter jejuni is one of the major causes of bacterial diarrhea worldwide (51, 59), the details of its molecular pathogenesis are not well understood. The clinical symptoms of campylobacter infection can range from a mild, watery diarrhea to a dysentery-like illness with fecal blood and leukocytes (2). Although there are reports of numerous cytotoxins, only the cytolethal distending toxin, which arrests eukaryotic cells at the G 2 phase of the cell cycle (64), has been characterized in detail. There are numerous reports that C. jejuni strains can invade intestinal epithelial cells in vitro (20,21,24,29,30,38), although levels of invasion by different strains vary considerably (20,28,38,52). Strain 81-176, originally isolated from a diarrheal outbreak associated with raw-milk consumption (31), is one of the best-characterized strains of C. jejuni. This strain has been shown to cause an inflammatory diarrhea in two human feeding studies (8; D. Tribble, unpublished data) and to cause disease in experimental models using primates (40) and ferrets (19,67). Further, C. jejuni strain 81-176 invades INT407 cells at levels higher than those of most other C. jejuni strains (28, 38).Plasmids have been found in between 19 and 53% of C. jejuni strains (5, 9-11, 41, 53-58), and many of these have been reported to be R plasmids that are transmissible among Campylobacter spp. but not to Escherichia coli (53)(54)(55)(56)(57)(58)60). Despite the importance of plasmids to virulence in numerous other pathogens, it is generally believed that plasmids play no role in Campylobacter pathogenicity. This paradigm is based on the rather low level at which plasmids ...

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Bacterial invasion of six different human epithelial ceUl lines showed that some strains of the intestinal pathogen Campylobacterjejuni invaded intestinal cell lines at a level 102-104 times higher than reported previously for other Campylobacter strains. Separately, urinary tract isolates of Citrobacter freundii triggered a high-efficiency invasion of bladder cells. Use of multiple inhibitors with known effects on eukaryotic cell structures/processes allowed us to define in these genetically distinct bacterial genera unusual bacterial invasion mechanisms that uniquely require microtubules but not microfilaments. Campylobacterjejuni strain 81-176 uptake into 407 intestinal ceUls and Citrobacter entry into T24 bladder ceUls was blocked by microtubule depolymerization and inhibitors of coated-pit formation but not by microrflament depolymerization. Inhibitors of endosome acidification had no significant impact on intracellular survival of Campylobacterjejuni or Citrobacterfreundii, but monensin markedly reduced Citrobacter uptake. Epithelial cel invasion by both of these bacterial genera was dependent upon de novo bacterial protein synthesis but not upon de novo eukaryotic cel protein synthesis. In contrast to the T24 cel line-specific, strict microtubule-dependent uptake, Citrobacter entry into other cell lines was inhibited by both microtubule-and microfilament-depolymerization, suggesting that these bacteria encode two separate pathways for uptake (i, microtubule-dependent; ii, microfflament-dependent) that are cel line-specific and are recognized perhaps depending on the presence and abundance of appropriate eukaryotic receptors.

The reported isolation of nanobacteria from human kidney stones raises the intriguing possibility that these microorganisms are etiological agents of pathological extraskeletal calcification [Kajander, E. O. & Ç iftçioglu, N. (1998) Proc. Natl. Acad. Sci. USA 95, 8274 -8279]. Nanobacteria were previously isolated from FBS after prolonged incubation in DMEM. These bacteria initiated biomineralization of the culture medium and were identified in calcified particles and biofilms by nucleic acid stains, 16S rDNA sequencing, electron microscopy, and the demonstration of a transferable biomineralization activity. We have now identified putative nanobacteria, not only from FBS, but also from human saliva and dental plaque after the incubation of 0.45-m membrane-filtered samples in DMEM. Although biomineralization in our ''cultures'' was transferable to fresh DMEM, molecular examination of decalcified biofilms failed to detect nucleic acid or protein that would be expected from growth of a living entity. In addition, biomineralization was not inhibited by sodium azide. Furthermore, the 16S rDNA sequences previously ascribed to Nanobacterium sanguineum and Nanobacterium sp. were found to be indistinguishable from those of an environmental microorganism, Phyllobacterium mysinacearum, that has been previously detected as a contaminant in PCR. Thus, these data do not provide plausible support for the existence of a previously undiscovered bacterial genus. Instead, we provide evidence that biomineralization previously attributed to nanobacteria may be initiated by nonliving macromolecules and transferred on ''subculture'' by self-propagating microcrystalline apatite.