Abstract
Pathogenic Yersinia species employ type III machines to transport virulence factors across the bacterial envelope. Some substrates for the type III machinery are secreted into the extracellular medium, whereas others are targeted into the cytosol of host cells. We found that during infection of tissue culture cells, yersiniae secrete small amounts of LcrV into the extracellular medium. Knockout mutations of lcrV abolish Yersinia targeting and reduce expression of the lcrGVHyopBD operon. In contrast, a block in LcrV secretion does not affect targeting, but results in premature expression and secretion of Yop proteins into the extracellular medium. LcrV-mediated activation of the type III pathway is thought to occur by sequestration of the regulatory factor LcrG, presumably via the formation of LcrV.LcrG complexes. These results suggest that intrabacterial LcrV regulates the expression and targeting of Yop proteins during Yersinia infection, whereas secreted LcrV is required to ensure specificity of Yop injection into eukaryotic cells.
Highlights
Pathogenic Yersinia species colonize the lymphoid tissues of their infected hosts and require the type III machinery to escape phagocytic killing by immune cells [1, 2]
Straley and co-workers [31, 38] examined the fate of LcrV during Yersinia pestis infection and reported that LcrV may be surface-exposed as well as injected into the cytosol of HeLa cells. These investigators observed no inhibition of type III targeting following the addition of LcrV antiserum to infected tissue culture cells [38]. lcrV mutant yersiniae have been reported to be defective in the type III secretion of YopB and YopD [30]
Small amounts of LcrV were found in the extracellular medium; the majority of LcrV sedimented with yersiniae after digitonin extraction of HeLa cells (Fig. 1A and Table I)
Summary
Bacterial Strains and Plasmids—Y. enterocolitica strain W22703 (wild type) has been previously described [40]. Cultures were centrifuged at 10,500 ϫ g for 15 min, and the supernatant was separated from the cell pellet. Proteins in both fractions were precipitated with trichloroacetic acid, washed with acetone, and suspended in sample buffer. The cell pellet was suspended in 20 ml of buffer E (50 mM Tris-HCl, 20% sucrose, and 1 mM dithiothreitol, pH 7.0), and bacteria were broken by two passages through a French pressure cell at 14,000 p.s.i. Unbroken cells and debris were removed by centrifugation at 32,500 ϫ g for 15 min. Coverslips were washed with PBS and incubated with DMEM prior to infection with Y. enterocolitica strains W22703/pDA37 (wild type, expressing plasmid-encoded Npt (neomycin phosphotransferase)), CT1/pDA37 (lcrVϪ, expressing plasmid-encoded Npt), CT1/pVL49 (expressing plasmid-encoded wild-type LcrV), and CT1/pVL47 (expressing plasmid-encoded GST-LcrV) for 3 h at an multiplicity of infection of 20.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
