Description: Francisella tularensis , the bacterial cause of tularemia, infects the liver and replicates in hepatocytes in vivo and in vitro . However, the factors that govern adaptation of F. tularensis to the intrahepatocytic niche have not been identified. Using cDNA microarrays, we determined the transcriptional profile of the live vaccine strain (LVS) of F. tularensis grown in the FL83B murine hepatocytic cell line compared to that of F. tularensis cultured in broth. The fslC gene of the fsl operon was the most highly upregulated. Deletion of fslC eliminated the ability of the LVS to produce siderophore, which is involved in uptake of ferric iron, but it did not impair its growth in hepatocytes, A549 epithelial cells, or macrophages. Therefore, we sought an alternative means by which F. tularensis might obtain iron. Deletion of feoB , which encodes a putative ferrous iron transporter, retarded replication of the LVS in iron-restricted media, reduced its growth in hepatocytic and epithelial cells, and impaired its acquisition of iron. Survival of mice infected intradermally with a lethal dose of the LVS was slightly improved by deletion of fslC but was not altered by loss of feoB . However, the feoB mutant showed diminished ability to colonize the lungs, liver, and spleen of mice that received sublethal inocula. Thus, FeoB represents a previously unidentified mechanism for uptake of iron by F. tularensis . Moreover, failure to produce a mutant strain lacking both feoB and fslC suggests that FeoB and the proteins of the fsl operon are the only major means by which F. tularensis acquires iron.

Description: Immature myeloid cells in bone marrow are a heterogeneous population of cells that, under normal conditions, provide tissues with protective cell types such as granulocytes and macrophages. Under certain pathological conditions, myeloid cell homeostasis is altered and immature forms of these cells appear in tissues. Murine immature myeloid cells that express CD11b and Ly6C or Ly6G (two isoforms of Gr-1) have been associated with immunosuppression in cancer (in the form of myeloid-derived suppressor cells) and, more recently, infection. Here, we found that CD11b + Ly6C hi Ly6G – and CD11b + Ly6C int Ly6G + cells accumulated and persisted in tissues of mice infected with Salmonella enterica serovar Typhimurium ( S . Typhimurium). Recruitment of CD11b + Ly6C hi Ly6G – but not CD11b + Ly6C int Ly6G + cells from bone marrow into infected tissues depended on chemokine receptor CCR2. The CD11b + Ly6C hi Ly6G – cells exhibited a mononuclear morphology, whereas the CD11b + Ly6C int Ly6G + cells exhibited a polymorphonuclear or band-shaped nuclear morphology. The CD11b + Ly6C hi Ly6G – cells differentiated into macrophage-like cells following ex vivo culture and could present antigen to T cells in vitro . However, significant proliferation of T cells was observed only when the ability of the CD11b + Ly6C hi Ly6G – cells to produce nitric oxide was blocked. CD11b + Ly6C hi Ly6G – cells recruited in response to S . Typhimurium infection could also present antigen to T cells in vivo , but increasing their numbers by adoptive transfer did not cause a corresponding increase in T cell response. Thus, CD11b + Ly6C hi Ly6G – immature myeloid cells recruited in response to S . Typhimurium infection exhibit protective and immunosuppressive properties that may influence the outcome of infection.

Description: Francisella tularensis is a facultative intracellular, Gram-negative pathogen and the causative agent of tularemia. We previously identified TolC as a virulence factor of the F. tularensis live vaccine strain (LVS) and demonstrated that a tolC mutant exhibits increased cytotoxicity toward host cells and elicits increased proinflammatory responses compared to those of the wild-type (WT) strain. TolC is the outer membrane channel component used by the type I secretion pathway to export toxins and other bacterial virulence factors. Here, we show that the LVS delays activation of the intrinsic apoptotic pathway in a TolC-dependent manner, both during infection of primary macrophages and during organ colonization in mice. The TolC-dependent delay in host cell death is required for F. tularensis to preserve its intracellular replicative niche. We demonstrate that TolC-mediated inhibition of apoptosis is an active process and not due to defects in the structural integrity of the tolC mutant. These findings support a model wherein the immunomodulatory capacity of F. tularensis relies, at least in part, on TolC-secreted effectors. Finally, mice vaccinated with the tolC LVS are protected from lethal challenge and clear challenge doses faster than WT-vaccinated mice, demonstrating that the altered host responses to primary infection with the tolC mutant led to altered adaptive immune responses. Taken together, our data demonstrate that TolC is required for temporal modulation of host cell death during infection by F. tularensis and highlight how shifts in the magnitude and timing of host innate immune responses may lead to dramatic changes in the outcome of infection.

Description: YopE is a virulence factor that is secreted into host cells infected by Yersinia species. The YopE C-terminal domain has GTPase-activating protein (GAP) activity. The YopE N-terminal domain contains an epitope that is an immunodominant CD8 + T cell antigen during primary infection of C57BL/6 mice with Yersinia pseudotuberculosis . The characteristics of the CD8 + T cells generated in response to the epitope, which comprises YopE amino acid residues 69 to 77 (YopE 69–77 ), and the features of YopE that are important for antigenicity during primary infection, are unknown. Following intravenous infection of naïve C57BL/6 mice with a yopE GAP mutant (the R144A mutant), flow cytometry analysis of splenocytes by tetramer and intracellular cytokine staining over a time course showed that YopE 69–77 -specific CD8 + T cells producing gamma interferon (IFN-) and tumor necrosis factor alpha (TNF-α) were generated by day 7, with a peak at day 14. In addition, ~80% of YopE 69–77 -specific CD8 + T cells were positive for KLRG1, a memory phenotype marker, at day 21. To determine if residues that regulate YopE activity by ubiquitination or membrane localization affect the antigenicity of YopE 69–77 , mice were infected with a yopE ubiquitination or membrane localization mutant (the R62K or L55N I59N L63N mutant, respectively). These mutants elicited YopE 69–77 -specific CD8 + T cells producing IFN- and TNF-α with kinetics and magnitudes similar to those of the parental R144A strain, indicating that primary infection primes effector CD8 + T cells independently of the ubiquitination or membrane localization of YopE. Additionally, at day 7, there was an unexpected positive correlation between the numbers of YopE 69–77 -specific CD8 + T cells and CD11b + cells, but not between the numbers of YopE 69–77 -specific CD8 + T cells and bacterial cells, in spleens, suggesting that the innate immune response contributes to the immunodominance of YopE 69–77 .

Description: Recent studies have linked accumulation of the Gr-1 + CD11b + cell phenotype with functional immunosuppression in diverse pathological conditions, including bacterial and parasitic infections and cancer. Gr-1 + CD11b + cells were the largest population of cells present in the spleens of mice infected with sublethal doses of the Francisella tularensis live vaccine strain (LVS). In contrast, the number of T cells present in the spleens of these mice did not increase during early infection. There was a significant delay in the kinetics of accumulation of Gr-1 + CD11b + cells in the spleens of B-cell-deficient mice, indicating that B cells play a role in recruitment and maintenance of this population in the spleens of mice infected with F. tularensis . The splenic Gr-1 + CD11b + cells in tularemia were a heterogeneous population that could be further subdivided into monocytic (mononuclear) and granulocytic (polymorphonuclear) cells using the Ly6C and Ly6G markers and differentiated into antigen-presenting cells following ex vivo culture. Monocytic, CD11b + Ly6C hi Ly6G – cells but not granulocytic, CD11b + Ly6C int Ly6G + cells purified from the spleens of mice infected with F. tularensis suppressed polyclonal T-cell proliferation via a nitric oxide-dependent pathway. Although the monocytic, CD11b + Ly6C hi Ly6G – cells were able to suppress the proliferation of T cells, the large presence of Gr-1 + CD11b + cells in mice that survived F. tularensis infection also suggests a potential role for these cells in the protective host response to tularemia.

Description: Pathogenic Yersinia species modulate host immune responses through the activity of a plasmid-encoded type III secretion system and its associated effector proteins. One effector, YopM, is a leucine-rich-repeat-containing protein that is important for virulence in murine models of Yersinia infection. Although the mechanism by which YopM promotes virulence is unknown, we previously demonstrated that YopM was required for the induction of high levels of the immunosuppressive cytokine interleukin-10 (IL-10) in sera of C57BL/6J mice infected with Yersinia pseudotuberculosis . To determine if IL-10 production is important for the virulence function of YopM, C57BL/6J or congenic IL-10 –/– mice were infected intravenously with wild-type or yopM mutant Y. pseudotuberculosis strains. Analysis of cytokine levels in serum and bacterial colonization in the spleen and liver showed that YopM is required for IL-10 induction in C57BL/6J mice infected with either the IP32953 or the 32777 strain of Y. pseudotuberculosis , demonstrating that the phenotype is conserved in the species. In single-strain infections, the ability of the 32777 yopM mutant to colonize the liver was significantly increased by the delivery of exogenous IL-10 to C57BL/6J mice. In mixed infections, the competitive advantage of a yopM + 32777 strain over an isogenic yopM mutant to colonize spleen and liver, as observed for C57BL/6J mice, was significantly reduced in IL-10 –/– animals. Thus, by experimentally controlling IL-10 levels in a mouse infection model, we obtained evidence that the induction of this cytokine is an important mechanism by which YopM contributes to Y. pseudotuberculosis virulence.