B-215. The Intracellular Trafficking of Yersinia pestis Virulence (V) Antigen during Macrophage Infection by the TTSS-Independent Mechanism

T. L. DiMezzo, G. T. Ruthel, S. L. Welkos;
USAMRIID, Frederick, MD.

Yersinia pestis, the causative agent of plague, has several essential Yersinia virulence factors, encoded by the 70-kB, low-calcium response plasmid (pLcr), including the Yersinia outer proteins (Yops), and virulence antigen (V). Besides being highly immunogenic, V stimulates the expression of anti-inflammatory cytokines such as interleukin-10 that inhibit the host leukocyte response. It is also required for the expression and secretion of the cytotoxic Yops and is a structural component of the injection apparatus involved in the type three secretion system (TTSS)-mediated translocation of the Yops into host target cells. V is the only pLcr-encoded protein secreted extracellularly in large amounts, and it can also enter cells by a novel TTSS-independent mechanism. However, V’s activities inside the host cells are not known. To investigate the intracellular functions of V and their role in the pathogenesis of plague, murine J774.A1 macrophages (MQs) were infected with a pigmentation-negative, pPst plasmid-cured mutant derivative of Y. pestis strain CO92; Y. pseudotuberculosis strains YpIII pIB19, which has an in-frame deletion mutation of the plasmid pLcr-encoded lcrV gene; strain Y.ptb pIB19, which had been transformed with a recombinant V expression plasmid; and YpIII IB604, which has an in-frame deletion in the yopB gene, required for injectisome formation. Immunofluorescence; confocal, and electron microscopy; density gradient centrifugation coupled with immunobloting; and flow cytometry experiments monitored co-localization and determined the intracellular pathway of V. Also, small-molecule inhibitors of TTSS were incubated with theY. ptb and Y. pestis strains to ensure that our findings were associated with the TTSS-independent entry of V into MQs. The results indicated an apparent interaction of V with: (1) endosomal proteins after infection for 10 - 45 min; (2) lysosomal marker(s) after 1 and 2 h of infection; (3) mitochondrial antigens after 3 h of infection; and (4) Golgi proteins after 4 h of infection. Further research is being pursued to characterize the specific host protein-V interaction(s) involved.