LcrV of Yersinia pestis enters infected eukaryotic cells by a virulence plasmid-independent mechanism

Yersinia pestis is the causative agent of bubonic plague and possesses a se t of plasmid-encoded, secretable virulence proteins termed LcrV and Yops wh ich are essential for survival in mammalian hosts. Yops and LcrV are secret ed by a type III mechanism (YSc), and Yops are unidirectionally targeted in to the cytosol of associated eukaryotic cells in a tissue culture infection model. LcrV is required for Yops targeting, and recent findings have revea led that it can localize to the bacterial surface; however, its fate in thi s infection model has not been investigated in detail. In this study, we co mpared the localization of LcrV to that of the targeted proteins YopE and Y opM by immunoblot analysis of fractions of Yersinia-infected HeLa cultures or by laser-scanning confocal microscopy of infected monolayers. Both LcrV and YopE were secreted by contact-activated, extracellularly localized yers iniae and were targeted to the HeLa cell cytosol, Although a significant am ount of LcrV partitioned to the culture medium (unlike YopE), this extracel lular pool of LcrV was not the source of the LcrV that entered HeLa cells. Unlike targeting of YopE and YopM, targeting of LcrV occurred in the absenc e of a functional Ysc apparatus and other virulence plasmid (pCD1)-expresse d proteins. However, the Ysc is necessary for LcrV to be released into the medium, and our recent work has shown that localization of LcrV on the bact erial surface requires the Ysc. These results indicate that two mechanisms exist for the secretion of LcrV by Y. pestis, both of which are activated b y contact with eukaryotic cells. LcrV secreted by the Ysc reaches the bacte rial surface and the surrounding medium, whereas the second is a novel, Ysc -independent pathway which results in localization of LcrV in the cytosol o f infected cells but not the surrounding medium.