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Infect. Immun. doi:10.1128/IAI.00183-08
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Type III secretion decreases bacterial and host survival following phagocytosis of Yersinia by macrophages

Center for Infectious Diseases and Department of Molecular Genetics and Microbiology, SUNY Stony Brook, Stony Brook NY 11794-5222; Immunoregulation Unit, Autoimmunity Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892

^* To whom correspondence should be addressed. Email: yzhang{at}ms.cc.sunysb.edu.

	Abstract

Yersinia pseudotuberculosis uses a plasmid (pYV)-encoded type III secretion system (T3SS) to translocate a set of effectors called Yops into infected host cells. YopJ functions to induce apoptosis and YopT, YopE, and YopH act to antagonize phagocytosis in macrophages. Because Yops do not completely block phagocytosis, and Y. pseudotuberculosis can replicate in macrophages, it is important to determine if the T3SS modulates host responses to intracellular bacteria. Isogenic pYV-cured, pYV⁺ wild-type or yop mutant Y. pseudotuberculosis strains were allowed to infect bone-marrow derived murine macrophages at a low multiplicity of infection under conditions in which survival of extracellular bacteria was prevented. Phagocytosis, intracellular survival of the bacteria and apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20 min infection, allowing study of the macrophage response to internalized pYV⁺ Y. pseudotuberculosis. Interestingly, macrophages restricted survival of pYV⁺ but not pYV-cured or yopB Y. pseudotuberculosis within phagosomes: only a small fraction of pYV⁺ bacteria internalized replicated by 24 h. In addition, 20% of macrophages infected with wild-type pYV⁺ Y. pseudotuberculosis died of apoptosis after 20 h. Analysis of yop mutants expressing catalytically-inactive effectors revealed that YopJ was important for apoptosis, while a role for YopE, YopH and YopT in modulating macrophage responses to intracellular bacteria could not be identified. Apoptosis was reduced in TLR-4 deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV⁺ Y. pseudotuberculosis with chloramphicol reduced apoptosis, indicating that the de-novo bacterial protein synthesis was necessary for cell death. Our finding that the presence of a functional T3SS impacts survival of both bacterium and host following phagocytosis of Y. pseudotuberculosis suggests new roles for the T3SS in Yersinia pathogenesis.