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Infection and Immunity, September 2008, p. 4299-4310, Vol. 76, No. 9
0019-9567/08/$08.00+0     doi:10.1128/IAI.00183-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Type III Secretion Decreases Bacterial and Host Survival following Phagocytosis of Yersinia pseudotuberculosis by Macrophages

Yue Zhang,^1* James Murtha,¹ Margaret A. Roberts,^2, Richard M. Siegel,² and James B. Bliska¹

Center for Infectious Diseases and Department of Molecular Genetics and Microbiology, SUNY Stony Brook, Stony Brook, New York 11794-5222,¹ Immunoregulation Unit, Autoimmunity Branch, National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, Maryland 20892²

Received 11 February 2008/ Returned for modification 26 March 2008/ Accepted 23 June 2008

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, and yop mutant Y. pseudotuberculosis strains were allowed to infect bone marrow-derived murine macrophages at a low multiplicity of infection under conditions in which the survival of extracellular bacteria was prevented. Phagocytosis, the intracellular survival of the bacteria, and the apoptosis of the infected macrophages were analyzed. Forty percent of cell-associated wild-type bacteria were intracellular after a 20-min infection, allowing the 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 the 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 Toll-like receptor 4-deficient macrophages, indicating that cell death required signaling through this receptor. Treatment of macrophages harboring intracellular pYV⁺ Y. pseudotuberculosis with chloramphenicol 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 the survival of both bacterium and host following phagocytosis of Y. pseudotuberculosis suggests new roles for the T3SS in Yersinia pathogenesis.

Published ahead of print on 30 June 2008.

Editor: J. N. Weiser

Present address: School of Medicine, Virginia Commonwealth University, 1101 E. Marshall Street, Richmond, VA 23298-0565.