Primarily, three operons, hmsHFRS, hmsT and hmsP, are responsible for the development of a Yersinia pestis biofilm, which is essential for blockage-dependent transmission of plague from fleas to mammals. Here, using specific antibodies, a polymeric beta-1,6-N-acetyl-d-glucosamine-like polysaccharide was detected in the extracellular matrix of hmsHFRS-dependent Y. pestis biofilm. The production of this exopolysaccharide (EPS) was controlled by diguanylate cyclase HmsT and EAL domain phosphodiesterase HmsP, acting as positive and negative regulators respectively. Cellular compartmentalization of soluble segments of Hms inner membrane proteins, including the putative glycosyltransferase domain of HmsR, the diguanylate cyclase/GGDEF domain of HmsT and the phosphodiesterase/EAL domain of HmsP, was determined by a combination of topology prediction algorithms and construction of C-terminal translational fusions with beta-galactosidase and alkaline phosphatase. Multiple interactions of Hms inner membrane proteins were detected using bacterial cAMP based two-hybrid system. Biochemical analyses confirmed some of these protein-protein interactions. Our results indicate that synthesis and regulation of the Y. pestis biofilm EPS occurs in the cytoplasm by a proposed Hms enzymatic complex.

In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice.Adherence of bacterial pathogens to host cells is an early step in the infectious process and allows exploitation of host cell signaling pathways or cell entry (4). Yersinia pseudotuberculosis and Yersinia enterocolitica, both of which are food-borne pathogens, express two adhesins, YadA and InvA, that support bacterial docking at the mucosal surface and provide the intimate contact needed for functioning of the Ysc type III secretion system (12,14,21). Yersinia pestis lacks functional YadA and InvA (37), yet this pathogen adheres tightly to epithelial cells and macrophages and invades these cells as avidly as the enteropathogenic yersiniae (8, 44). One adhesin/invasin unique to Y. pestis is the surface aspartyl protease Pla; however, absence of Pla did not eliminate all invasiveness for epithelial cells and resulted in little reduction in adherence, indicating that additional adhesins/invasins must be present (8).Inspection of the genome sequences available for Y. pestis has revealed the presence of open reading frames that encode putative structural analogs of YadA (19). YadA and the structurally analogous Moraxella UspAs belong to a class of nonfimbrial adhesins called oligomeric coiled-coil adhesins (Oca) (19). The YadA molecule consists of five major domains: head, neck, stalk, coil-coil segment, and membrane anchor. Crystallography of the collagen-binding head domain of YadA resolved at 1.55 Å showed a stable trimeric locknut structure that is required for collagen binding (33, 34). More recently, the structure of the C-terminal membrane anchor, which forms a ␤-barrel, was resolved at 3.8 Å, and this anchor was shown to contain a helical part at its N terminus (51). Model studies have proposed a pore assembly scheme in which a 12-strand ␤-barrel is assembled by trimerization (41) of the four transmembrane ␤-strands, forming an opening through which the N-terminal head, neck, stalk, and coiled helical domains of the three monomer chains exit to the cell surface (23). The Oca family of proteins is now viewed as a subset of autotransporters, the type Vc or trimeric autotransporters (7,18). The Nterminal domains are not cleaved, which is commonly true for "conve...

Although Yersinia pestis epidemic biovars and Yersinia pseudotuberculosis are recently diverged, highly related species, they cause different diseases via disparate transmission routes. Since iron transport systems are important for iron acquisition from hosts and for survival in the environment, we have analyzed potential iron transport systems encoded by epidemic and non-epidemic or endemic strains of Y. pestis as well as two virulent Y. pseudotuberculosis strains. Computational biology analysis of these genomes showed a high degree of identity/similarity among 16 proven or possible iron/heme transporters identified. Of these, 7 systems were essentially the same in all seven genomes analyzed. The remaining 9 loci had 2-6 genetic variations among these genomes. Two untested, potential siderophore-dependent systems appear intact in Y. pseudotuberculosis but are disrupted or absent in all the endemic Y. pestis strains as well as the epidemic strains from the antiqua and mediaevalis biovars. Only one of these two loci are obviously disrupted in Y. pestis CO92 (epidemic orientalis biovar). Experimental studies failed to identify a role for hemin uptake systems in the virulence of pneumonic plague and suggest that Y. pestis CO92 does not make a siderophore other than Ybt.

Yersinia pestis biofilm formation causes massive adsorption of haemin or Congo red in vitro as well as colonization and eventual blockage of the flea proventriculus in vivo. This blockage allows effective transmission of plague from some fleas, like the oriental rat flea, to mammals. Four Hms proteins, HmsH, HmsF, HmsR and HmsS, are essential for biofilm formation, with HmsT and HmsP acting as positive and negative regulators, respectively. HmsH has a β-barrel structure with a large periplasmic domain while HmsF possesses polysaccharide deacetylase and COG1649 domains. HmsR is a putative glycosyltransferase while HmsS has no recognized domains. In this study, specific amino acids within conserved domains or within regions of high similarity in HmsH, HmsF, HmsR and HmsS proteins were selected for site-directed mutagenesis. Some but not all of the substitutions in HmsS and within the periplasmic domain of HmsH were critical for protein function. Substitutions within the glycosyltransferase domain of HmsR and the deacetylase domain of HmsF abolished biofilm formation in Y. pestis. Surprisingly, substitution of highly conserved residues within COG1649 did not affect HmsF function.

Yersinia pestis genomes contain genes homologous to the aerobactin receptor (iutA) and biosynthetic genes (iucABCD) as well as the ferric hydroxamate uptake system (fhuCDB) of Escherichia coli. However, iucA is disrupted by a frameshift mutation. An E. coli strain carrying the cloned Y. pestis aerobactin region was unable to produce aerobactin, but could use the siderophore as an iron source. Repair of the frameshift mutation in iucA did not allow aerobactin production in E. coli or Y. pestis. In contrast, a Y. pestis strain with a plasmid encoding the iucABCD-iutA genes from Shigella flexneri or pColV-K30 did produce and secrete the siderophore. In addition, Yersinia pseudotuberculosis PB1, which encodes the iucABCD-iutA locus without the Y. pestis-specific frameshift mutation, also failed to produce aerobactin. The Y. pestis fhuCDB operon, encoding an ABC transporter for a range of hydroxamate siderophores, was able to complement a strain of E. coli with a transposon insertion in fhuC, allowing utilization of aerobactin and ferrichrome. Y. pestis KIM6, a strain deficient in the production of the siderophore yersiniabactin, was able to use both the ferrichrome and the aerobactin siderophores as a source of iron. Mutations in iutA or the fhu operon abolished the ability of KIM6 to use aerobactin. Mutations in the fhu operon, but not in iutA, affected the ability of KIM6 to use ferrichrome. This demonstrates that Y. pestis uses both ferrichrome and aerobactin, but has lost the ability to synthesize aerobactin.

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RTX cytotoxins play an important role in virulence of numerous gram-negative pathogens. Unexpectedly, however, we show here that the RTX proteins of Neisseria meningitidis are dispensable for virulence in the infant rat model of infection.

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