CD8+ T Cells Restrict Yersinia pseudotuberculosis Infection: Bypass of Anti-Phagocytosis by Targeting Antigen-Presenting Cells

All Yersinia species target and bind to phagocytic cells, but uptake and destruction of bacteria are prevented by injection of anti-phagocytic Yop proteins into the host cell. Here we provide evidence that CD8+ T cells, which canonically eliminate intracellular pathogens, are important for restricting Yersinia, even though bacteria are primarily found in an extracellular locale during the course of disease. In a model of infection with attenuated Y. pseudotuberculosis, mice deficient for CD8+ T cells were more susceptible to infection than immunocompetent mice. Although exposure to attenuated Y. pseudotuberculosis generated TH1-type antibody responses and conferred protection against challenge with fully virulent bacteria, depletion of CD8+ T cells during challenge severely compromised protective immunity. Strikingly, mice lacking the T cell effector molecule perforin also succumbed to Y. pseudotuberculosis infection. Given that the function of perforin is to kill antigen-presenting cells, we reasoned that cell death marks bacteria-associated host cells for internalization by neighboring phagocytes, thus allowing ingestion and clearance of the attached bacteria. Supportive of this model, cytolytic T cell killing of Y. pseudotuberculosis–associated host cells results in engulfment by neighboring phagocytes of both bacteria and target cells, bypassing anti-phagocytosis. Our findings are consistent with a novel function for cell-mediated immune responses protecting against extracellular pathogens like Yersinia: perforin and CD8+ T cells are critical for hosts to overcome the anti-phagocytic action of Yops.Molecular and Cellular Biolog

RfaH Suppresses Small RNA MicA Inhibition of fimB Expression in Escherichia coli K-12

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σE , and hence MicA, in the absence of RfaH. Additional work con- firms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467–479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression

Yersinia Has a Tropism for B and T Cell Zones of Lymph Nodes That Is Independent of the Type III Secretion System

Pathogenic Yersinia have a pronounced tropism for lymphatic tissues and harbor a virulence plasmid that encodes a type III secretion system, pTTSS, that transports Yops into host cells. Yops are critical virulence factors that prevent phagocytosis by macrophages and neutrophils and Yersinia mutants lacking one or more Yops are defective for survival in lymphatic tissues, liver, and gastrointestinal tract. However, here we demonstrate that Y. pseudotuberculosis (Yptb) mutants lacking the pTTSS survived as well as or better than wild-type (WT) Yptb in the mesenteric lymph nodes (MLN). Infection with pTTSS mutants caused lymphadenitis with little necrosis, whereas infection with WT Yptb provoked lymphadenitis with multiple necrotic suppurative foci. Gentamicin protection assays and microscopic examination of the MLN revealed that pTTSS mutants resided extracellularly adjacent to B and T lymphocytes in the cortex and paracortex. WT Yptb was found extracellularly adjacent to neutrophils and macrophages in necrotic areas and adjacent to B and T lymphocytes in less-inflamed areas. To determine whether lymphocytes protected pTTSS mutants from phagocytic cells, Rag1(−/−) mice were infected with pTTSS mutants or WT Yptb. pTTSS mutants but not WT, were impaired for survival in MLN of Rag1(−/−) mice, suggesting that lymphocyte-rich regions constitute a protective niche for pTTSS mutants. Finally, we show that invasin and the chromosomally encoded TTSS were not required for Yptb survival in MLN. In summary, chromosomally encoded factors are sufficient for Yptb replication in the cortex and paracortex of MLN; the pTTSS enables Yersinia to survive within phagocyte-rich areas of lymph nodes, and spread to other tissues

Yersinia pseudotuberculosis disseminates directly from a replicating bacterial pool in the intestine

Dissemination of Yersinia pseudotuberculosis within mice after oral inoculation was analyzed. Y. pseudotuberculosis translocated to organs such as the liver and spleen shortly after oral inoculation, but was quickly cleared. In contrast, a second temporally distinct bacterial translocation event resulted in successful hepatosplenic replication of the bacteria. Replicating pools of bacteria could be established in these organs in mouse mutants that lacked Peyer's patches. These animals frequently had sterile mesenteric lymph nodes, a finding consistent with translocation taking place independently of regional lymph node colonization. In further contradiction to accepted models for dissemination of enteropathogens, clonal analysis revealed that bacteria causing disease in the spleen and liver of C57BL/6J mice were derived from populations located outside the intestinal lymph nodes. Replication of bacteria in the intestine before translocation appeared critical for dissemination, as transient selective suppression by streptomycin of bacterial growth in the intestine delayed dissemination of Y. pseudotuberculosis. These results collectively indicate that hepatosplenic colonization appears intimately connected with the ability of Y. pseudotuberculosis to successfully establish replication in the intestinal lumen and does not result from ordered spread leading from the intestine to regional lymph nodes before dissemination

Identification of genes required for soil survival in Burkholderia thailandensis by transposon-directed insertion site sequencing.

Transposon-directed insertion site sequencing was used to identify genes required by Burkholderia thailandensis to survive in plant/soil microcosms. A total of 1,153 genetic loci fulfilled the criteria as being likely to encode survival characteristics. Of these, 203 (17.6 %) were associated with uptake and transport systems; 463 loci (40.1 %) coded for enzymatic properties, 99 of these (21.4 %) had reduction/oxidation functions; 117 (10.1 %) were gene regulation or sensory loci; 61 (5.3 %) encoded structural proteins found in the cell envelope or with enzymatic activities related to it, distinct from these, 46 (4.0 %) were involved in chemotaxis and flagellum, or pilus synthesis; 39 (3.4 %) were transposase enzymes or were bacteriophage-derived; and 30 (2.6 %) were involved in the production of antibiotics or siderophores. Two hundred and twenty genes (19.1 %) encoded hypothetical proteins or those of unknown function. Given the importance of motility and pilus formation in microcosm persistence the nature of the colonization of the rhizosphere was examined by confocal microscopy. Wild type B. thailandensis expressing red fluorescent protein was inoculated into microcosms. Even though the roots had been washed, the bacteria were still present but they were motile with no attachment having taken place, perhaps being retained in a biofilm

Outer membrane protein folding from an energy landscape perspective

The cell envelope is essential for the survival of Gram-negative bacteria. This specialised membrane is densely packed with outer membrane proteins (OMPs), which perform a variety of functions. How OMPs fold into this crowded environment remains an open question. Here, we review current knowledge about OFMP folding mechanisms in vitro and discuss how the need to fold to a stable native state has shaped their folding energy landscapes. We also highlight the role of chaperones and the β-barrel assembly machinery (BAM) in assisting OMP folding in vivo and discuss proposed mechanisms by which this fascinating machinery may catalyse OMP folding