SummaryThe YopM virulence protein of Yersinia pestis has been described as binding human ␣-thrombin and inhibiting thrombin-induced platelet aggregation in vitro. However, recent studies have shown that a YopM-CyaA fusion protein could be targeted vectorially into eukaryotic cells through the Yersinia type III secretion system. In this study, our objective was to characterize YopM's fate in more detail. We followed YopM in the culture medium and inside infected HeLa cells. We confirmed that the native YopM is targeted into HeLa cells, where it is insensitive to exogenous trypsin. The bacteria must be surface located to target YopM, and YopB and YopD are necessary, whereas the LcrE protein (called also YopN) makes this process more efficient. Immunofluorescence localization revealed that YopM, in contrast to YopE, is not only targeted to the cytoplasm but also trafficks to the cell's nucleus by means of a vesicle-associated pathway that is strongly inhibited by brefeldin A, perturbed by monensin or bafilomycin A1 and dependent upon microtubules (decreased by colchicine and nocodazole). These findings revealed a novel interaction of Yersinia pestis with its eukaryotic host.
The causative agent of plague, Yersinia pestis, is regarded as being noninvasive for epithelial cells and lacks the major adhesins and invasins of its enteropathogenic relatives Yersinia enterocolitica and Yersinia pseudotuberculosis. However, there are studies indicating that Y. pestis invades and causes systemic infection from ingestive and aerogenic routes of infection. Accordingly, we developed a gentamicin protection assay and reexamined invasiveness of Y. pestis for HeLa cells. By optimizing this assay, we discovered that Y. pestis is highly invasive. Several factors, including the presence of fetal bovine serum, the configuration of the tissue culture plate, the temperature at which the bacteria are grown, and the presence of the plasminogen activator protease Pla-encoding plasmid pPCP1, were found to influence invasiveness strongly. Suboptimal combinations of these factors may have contributed to negative findings by previous studies attempting to demonstrate invasion by Y. pestis. Invasion of HeLa cells was strongly inhibited by cytochalasin D and modestly inhibited by colchicine, indicating strong and modest respective requirements for microfilaments and microtubules. We found no significant effect of the iron status of yersiniae or of the pigmentation locus on invasion and likewise no significant effect of the Yops regulon. However, an unidentified thermally induced property (possibly the Y. pestis-specific capsular protein Caf1) did inhibit invasiveness significantly, and the plasmid pPCP1, unique to Y. pestis, was essential for highly efficient invasion. pPCP1 encodes an invasion-promoting factor and not just an adhesin, because Y. pestis lacking this plasmid still adhered to HeLa cells. These studies have enlarged our picture of Y. pestis biology and revealed the importance of properties that are unique to Y. pestis.
The low-Ca2' response is a multicomponent virulence regulon of the human-pathogenic yersiniae in which 12 known virulence genes are coordinately regulated in response to environmental cues of temperature, Ca2+, and nucleotides such as ATP. Yersinial growth also is regulated, with full growth yield being permitted at 37°C only if Ca2+ or a nucleotide is present. In this study, we constructed and characterized a mutant Yersinia pestis specifically defective in the gene encoding the V antigen, one of the virulence genes of the low-Ca2+ response.An in-frame internal deletion-insertion mutation was made by removing bases 51 through 645 of IcrV and inserting 61 new bases. The altered lcrV was introduced into the low-Ca2' response plasmid in Y. pestis by allelic exchange, and the resulting mutant was characterized for its two-dimensional protein profiles, growth, expression of an operon fusion to another low-Ca2' response virulence operon, and virulence in mice. The mutant had lost its Ca2' and nucleotide requirement for growth, showed diminished expression of Ca2+-and nucleotide-regulated virulence genes, and was avirulent in mice. The mutation could be complemented with respect to the growth property by supplying native V antigen operon sequences in trans in high copy number (on pBR322). Partial complementation of the growth defect and almost complete complementation of the virulence defect were seen with a lower-copy-number complementing replicon (a pACYC184 derivative). The data are consistent with the interpretation that V antigen is bifunctional, with a role in regulating growth and expression of low-Ca2' response virulence genes in addition to its putative role as a secreted virulence protein.The human-pathogenic yersiniae have a multicomponent virulence property called the low-Ca2" response (Lcr phenotype) (8), which contains a set of regulatory genes and 12 identified, coordinately regulated virulence genes. The virulence gene products include a set of 11 surface proteins called Yops, which can be shed into the medium, and a secreted protein called the V antigen (11,35,36,43). The regulatory genes modulate expression of the virulence genes in response to the environmental inputs of temperature, Ca2+, and nucleotides such as ATP (19). The low-Ca2+ response is encoded by a ca. 75-kb plasmid (3, 17, 36), the one in Yersinia pestis KIM being called pCD1 (19).A curious manifestation of the low-Ca2+ response is the requirement of millimolar Ca2+ or ca. 10 to 20 mM nucleotide for the bacteria to show full growth yield at 37°C (20,49
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...
Yersinia pestis, the etiologic agent of plague, secretes a set of environmentally regulated, plasmid pCD1-encoded virulence proteins termed Yops and V antigen (LcrV) by a type III secretion mechanism (Ysc). LcrV is a multifunctional protein that has been shown to act at the level of secretion control by binding the Ysc inner-gate protein LcrG and to modulate the host immune response by altering cytokine production. LcrV also is essential for the unidirectional targeting of Yops to the cytosol of infected eukaryotic cells. In this study, we constructed an in-frame deletion withinlcrG (ΔlcrG3) to further analyze the requirement of LcrV in Yop targeting. We confirmed the essentiality of LcrV and found that LcrG may have a facilitative role, perhaps by promoting efficient secretion of LcrV. We also constructed mutants oflcrV expressing LcrV truncated at the N or C terminus. Both the N and C termini of LcrV were required for the secretion of LcrV into the medium and targeting of Yops. LcrV was detected in punctate zones on the surface of fixed Y. pestis by laser-scanning confocal microscopy, and this localization required a functional Ysc. However, the truncated LcrV proteins were not found on the bacterial surface. Finally, we tested the ability of LcrV-specific Fab antibody fragments or full-length antibody to interfere with Yop targeting and found no interference, even though this antibody protects mice against plague. These results indicate that LcrV may function in Yop targeting at the extracellular surface of yersiniae and that the protective efficacy of LcrV-specific antibodies can be manifested without blocking Yop targeting.
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