Akt and SHIP Modulate Francisella Escape from the Phagosome and Induction of the Fas-Mediated Death Pathway

Francisella tularensis infects macrophages and escapes phago-lysosomal fusion to replicate within the host cytosol, resulting in host cell apoptosis. Here we show that the Fas-mediated death pathway is activated in infected cells and correlates with escape of the bacterium from the phagosome and the bacterial burden. Our studies also demonstrate that constitutive activation of Akt, or deletion of SHIP, promotes phago-lysosomal fusion and limits bacterial burden in the host cytosol, and the subsequent induction of Fas expression and cell death. Finally, we show that phagosomal escape/intracellular bacterial burden regulate activation of the transcription factors sp1/sp3, leading to Fas expression and cell death. These data identify for the first time host cell signaling pathways that regulate the phagosomal escape of Francisella, leading to the induction of Fas and subsequent host cell death

Macrophage Pro-Inflammatory Response to Francisella novicida Infection Is Regulated by SHIP

Francisella tularensis, a Gram-negative facultative intracellular pathogen infecting principally macrophages and monocytes, is the etiological agent of tularemia. Macrophage responses to F. tularensis infection include the production of pro-inflammatory cytokines such as interleukin (IL)-12, which is critical for immunity against infection. Molecular mechanisms regulating production of these inflammatory mediators are poorly understood. Herein we report that the SH2 domain-containing inositol phosphatase (SHIP) is phosphorylated upon infection of primary murine macrophages with the genetically related F. novicida, and negatively regulates F. novicida–induced cytokine production. Analyses of the molecular details revealed that in addition to activating the MAP kinases, F. novicida infection also activated the phosphatidylinositol 3-kinase (PI3K)/Akt pathway in these cells. Interestingly, SHIP-deficient macrophages displayed enhanced Akt activation upon F. novicida infection, suggesting elevated PI3K-dependent activation pathways in absence of SHIP. Inhibition of PI3K/Akt resulted in suppression of F. novicida–induced cytokine production through the inhibition of NFκB. Consistently, macrophages lacking SHIP displayed enhanced NFκB-driven gene transcription, whereas overexpression of SHIP led to decreased NFκB activation. Thus, we propose that SHIP negatively regulates F. novicida–induced inflammatory cytokine response by antagonizing the PI3K/Akt pathway and suppressing NFκB-mediated gene transcription. A detailed analysis of phosphoinositide signaling may provide valuable clues for better understanding the pathogenesis of tularemia

Targeting CBLB as a potential therapeutic approach for disseminated candidiasis

We thank J.M. Penninger (University of Toronto) for providing Cblb−/− mice, Y. Iwakura (Tokyo University of Science) for providing Clec4n−/− mice, S. Lipkowitz (National Cancer Institute, US National Institutes of Health) for providing Cblb constructs, X. Lin (MD Anderson Cancer Center) for providing the antibody to mouse dectin-3 and Card9−/− bone marrow cells, P.R. Sundstrom (Dartmouth University) for providing the C. albicans cap1 mutant, and L.D. Chaves (University at Buffalo) for flow cytometric analysis of myeloid cells in the kidneys. We also thank A. Lovett-Racke (Ohio State University) for her advice on in vivo Cblb-knockdown experiments. This work was supported by the US National Institutes of Health (grants R01 AI090901, R01 AI123253, and R21 AI117547; all to J.Z.), the American Heart Association (AHA Great Rivers Associate Grant-in-Aid grant 16GRNT26990004; J.Z.), a start-up fund from the Ohio State University College of Medicine (J.Z.), and the Wellcome Trust (G.D.B.).Peer reviewedPostprin

Correction: Fine Tuning Inflammation at the Front Door: Macrophage Complement Receptor 3-mediates Phagocytosis and Immune Suppression for Francisella tularensis.

Correction: Fine Tuning Inflammation at the Front Door: Macrophage Complement Receptor 3-mediates Phagocytosis and Immune Suppression for <i>Francisella tularensis</i

Type A Francisella tularensis acid phosphatases contribute to pathogenesis.

Different Francisella spp. produce five or six predicted acid phosphatases (AcpA, AcpB, AcpC, AcpD, HapA and HapB). The genes encoding the histidine acid phosphatases (hapA, hapB) and acpD of F. tularensis subsp. Schu S4 strain are truncated or disrupted. However, deletion of HapA (FTT1064) in F. tularensis Schu S4 resulted in a 33% reduction in acid phosphatase activity and loss of the four functional acid phosphatases in F. tularensis Schu S4 (ΔABCH) resulted in a>99% reduction in acid phosphatase activity compared to the wild type strain. All single, double and triple mutants tested, demonstrated a moderate decrease in mouse virulence and survival and growth within human and murine phagocytes, whereas the ΔABCH mutant showed >3.5-fold decrease in intramacrophage survival and 100% attenuation of virulence in mouse. While the Schu S4 ΔABCH strain was attenuated in the mouse model, it showed only limited protection against wild type challenge. F. tularensis Schu S4 failed to stimulate reactive oxygen species production in phagocytes, whereas infection by the ΔABCH strain stimulated 5- and 56-fold increase in reactive oxygen species production in neutrophils and human monocyte-derived macrophages, respectively. The ΔABCH mutant but not the wild type strain strongly co-localized with p47 (phox) and replicated in macrophages isolated from p47 (phox) knockout mice. Thus, F. tularensis Schu S4 acid phosphatases, including the truncated HapA, play a major role in intramacrophage survival and virulence of this human pathogen

Mannose Receptor-Dependent Delay in Phagosome Maturation by Mycobacterium avium Glycopeptidolipids▿

The ability of pathogenic mycobacteria to block phagosome-lysosome fusion is critical for its pathogenesis. The molecules expressed by mycobacteria that inhibit phagosome maturation and the mechanism of this inhibition have been extensively studied. Recent work has indicated that mannosylated lipoarabinomannan (ManLAM) isolated from Mycobacterium tuberculosis can function to delay phagosome-lysosome fusion and that this delay requires the interaction of ManLAM with the mannose receptor (MR). However, the molecules expressed by other pathogenic mycobacteria that function to inhibit phagosome maturation have not been well described. In the present study, we show that phagosomes containing silica beads coated with glycopeptidolipids (GPLs), a major surface component of Mycobacterium avium, showed limited acidification and delayed recruitment of late endosomal/lysosomal markers compared to those of phosphatidylcholine-coated beads. The carbohydrate component of the GPLs was required, as beads coated only with the lipopeptide core failed to delay phagosome-lysosome fusion. Moreover, the ability of GPLs to delay phagosome maturation was dependent on the macrophage expression of the MR. Using CHO cells expressing the MR, we confirmed that the GPLs bind this receptor. Finally, human monocyte-derived macrophages knocked down for MR expression showed increased M. avium phagosome-lysosome fusion relative to control cells. Together, the data indicate that GPLs can function to delay phagosome-lysosome fusion and suggest that GPLs, like ManLAM, work through the MR to mediate this activity