Igor E. Brodsky

Numerous microbial pathogens modulate or interfere with cell death pathways in cultured cells. However, the precise role of host cell death during in vivo infection remains poorly understood. Macrophages infected by pathogenic species of Yersinia typically undergo an apoptotic cell death. This is due to the activity of a Type III secreted effector protein, designated YopJ in Y. pseudotuberculosis and Y. pestis, and YopP in the closely related Y. enterocolitica. It has recently been reported that Y. enterocolitica YopP shows intrinsically greater capacity for being secreted than Y. pestis YopJ, and that this correlates with enhanced cytotoxicity observed for high virulence serotypes of Y. enterocolitica. The enzymatic activity and secretory capacity of YopP from different Y. enterocolitica serotypes have been shown to be variable. However, the underlying basis for differential secretion of YopJ/YopP, and whether reduced secretion of YopJ by Y. pestis plays a role in pathogenesis during in vivo infection, is not currently known. It has also been reported that similar to macrophages, Y. enterocolitica infection of dendritic cells leads to YopP-dependent cell death. We demonstrate here that in contrast to Y. enterocolitica, Y. pseudotuberculosis infection of bone marrow–derived dendritic cells does not lead to increased cell death. However, death of Y. pseudotuberculosis–infected dendritic cells is enhanced by ectopic expression of YopP in place of YopJ. We further show that polymorphisms at the N-terminus of the YopP/YopJ proteins are responsible for their differential secretion, translocation, and consequent cytotoxicity. Mutation of two amino acids in YopJ markedly enhanced both translocation and cytotoxicity. Surprisingly, expression of YopP or a hypersecreted mutant of YopJ in Y. pseudotuberculosis resulted in its attenuation in oral mouse infection. Complete absence of YopJ also resulted in attenuation of virulence, in accordance with previous observations. These findings suggest that control of cytotoxicity is an important virulence property for Y. pseudotuberculosis, and that intermediate levels of YopJ-mediated cytotoxicity are necessary for maximal systemic virulence of this bacterial pathogen. The ability of bacterial pathogens to modulate death of infected host cells is an important virulence determinant. For pathogenic members of the genus Yersinia, the type III secreted effector protein YopJ/YopP is required for Yersinia-induced macrophage death. The YopJ protein is expressed by Y. pseudotuberculosis, while the ninety-four percent identical YopP protein is expressed by Y. enterocolitica. Y. enterocolitica infection also triggers YopP-dependent killing of dendritic cells, which are critical antigen presenting cells of the immune system. We demonstrate that in contrast to macrophages, dendritic cells are resistant to Y. pseudotuberculosis-mediated cytotoxicity. However, Y. pseudotuberculosis expressing YopP in place of YopJ was highly cytotoxic toward dendritic cells. This difference in cytotoxicity was attributable to a difference in the delivery of YopJ and YopP into mammalian cells. Furthermore, mutation of two amino acids at the N-terminus of YopJ enhanced its delivery and cytotoxicity. Remarkably, we found that enhancing the cytotoxicity of Y. pseudotuberculosis by expression of YopP led to its attenuation in a mouse model of Yersinia infection. This indicates that optimal virulence for a given pathogen requires careful regulation of virulence properties and highlights the potential evolutionary tradeoffs between cellular cytotoxicity and in vivo virulence.

Summary: Salmonella enterica serovar Typhimurium (S. typhimurium) infects a wide variety of mammalian hosts and in rodents causes a typhoid‐like systemic disease involving replication of bacteria inside macrophages within reticuloendothelial tissues. Previous studies demonstrated that the mig‐14 and virK genes of Salmonella enterica are important in bacterial resistance to anti‐microbial peptides and are necessary for continued replication of S. typhimurium in the liver and spleen of susceptible mice after orogastric inoculation. In this work we report that inflammatory signalling via interferon‐gamma (IFN‐γ) is crucial to controlling replication of mig‐14 mutant bacteria within the liver and spleen of mice after oral infection. Using a Salmonella persistence model recently developed in our laboratory, we further demonstrate that mig‐14 contributes to long‐term persistence of Salmonella in the spleen and mesenteric lymph nodes of chronically infected mice. Both mig‐14 and virK contribute to the survival of Salmonella in macrophages treated with IFN‐γ and are necessary for resistance to cathelin‐related anti‐microbial peptide (CRAMP), an anti‐microbial peptide expressed at high levels in activated mouse macrophages. We also show that both Mig‐14 and VirK inhibit the binding of CRAMP to Salmonella, and demonstrate that Mig‐14 is an inner membrane‐associated protein. We further demonstrate by transmission electron microscopy that the primary locus of CRAMP activity appears to be intracytoplasmic, rather than at the outer membrane, suggesting that Mig‐14 may prevent the penetration of the inner membrane by CRAMP. Together, these data indicate an important role for mig‐14 in anti‐microbial peptide resistance in vivo, and show that this resistance is important to the survival of Salmonella in systemic sites during both acute and persistent infection.