Homeostatic maintenance of pathogen-containing vacuoles requires TBK1-dependent regulation of aquaporin-1

Membranes are an integral component of many cellular functions and serve as a barrier to keep pathogenic bacteria from entering the nutrient-rich host cytosol. TANK-binding-kinase-1 (TBK1), a kinase of the IκB kinase family, is required for maintaining integrity of pathogen-containing vacuoles (PCV) upon bacterial invasion of host cells. Here we investigate how vacuolar integrity is maintained during bacterial infection, even in the presence of bacterial membrane damaging agents. We found that Aquaporin-1 (AQP1), a water channel that regulates swelling of secretory vesicles, associated with PCV. AQP1 levels were elevated in TBK1-deficient cells, and overexpression of AQP1 in wild-type cells led to PCV destabilization, similar to that observed in tbk1 −/ − cells. Inhibition of physiological levels of AQP1 in multiple cell types also led to increased instability of PCV, demonstrating a need for tightly regulated AQP1 function to maintain vacuole homeostasis during bacterial infection. AQP1-dependent modulation of PCV was triggered by bacterially induced membrane damage and ion flux. These results highlight the contribution of water channels to promoting PCV membrane integrity, and reveal an unexpected role for TBK1 and AQP1 in restricting bacterial pathogens to the vacuolar compartment.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72109/1/j.1462-5822.2008.01199.x.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/72109/2/CMI_1199_sm_Figs_S1-S3_and_Table_S1.pd

Caspase-2 mediates a Brucella abortus RB51-induced hybrid cell death having features of apoptosis and pyroptosis

Programmed cell death (PCD) can play a crucial role in tuning the immune response to microbial infection. Although PCD can occur in different forms, all are mediated by a family of proteases called caspases. Caspase-2 is the most conserved caspase, however, its function in cell death is ill-defined. Previously we demonstrated that live attenuated cattle vaccine strain Brucella abortus RB51 induces caspase-2-mediated and caspase-1-independent PCD of infected macrophages. We also discovered that rough attenuated B. suis strain VTRS1 induces a caspase-2-mediated and caspase-1-independent proinflammatory cell death in infected macrophages, which was tentatively coined “caspase-2-mediated pyroptosis”. However, the mechanism of caspase-2-mediated cell death pathway remained unclear. In this study, we found that caspase-2 mediated proinflammatory cell death of RB51-infected macrophages and regulated many genes in different PCD pathways. We show that the activation of proapoptotic caspases-3 and -8 was dependent upon caspase-2. Caspase-2 regulated mitochondrial cytochrome c release and TNFα production, both of which are known to activate caspase-3 and caspase-8, respectively. In addition to TNFα, RB51-induced caspase-1 and IL-1β production was also driven by caspase-2-mediated mitochondrial dysfunction. Interestingly, pore formation, a phenomenon commonly associated with caspase-1-mediated pyroptosis, occurred; however, unlike its role in S. typhimurium-induced pyroptosis, pore formation did not contribute to RB51-induced proinflammatory cell death. Our data suggest that caspase-2 acts as an initiator caspase that mediates a novel RB51-induced hybrid cell death that simulates but differs from typical non-proinflammatory apoptosis and caspase-1-mediated proinflammatory pyroptosis. The initiator role of the caspase-2-mediated cell death was also conserved in cellular stress-induced cell death of macrophages treated with etoposide, naphthalene, or anti-Fas. Caspase-2 also regulated caspase-3 and -8 activation, as well as cell death in macrophages treated with each of the three reagents. Taken together, our data has demonstrated that caspase-2 can play an important role in mediating a proinflammatory response and a hybrid cell death that demonstrates features of both apoptosis and pyroptosis

XIAP Regulates Cytosol-Specific Innate Immunity to Listeria Infection

The inhibitor of apoptosis protein (IAP) family has been implicated in immune regulation, but the mechanisms by which IAP proteins contribute to immunity are incompletely understood. We show here that X-linked IAP (XIAP) is required for innate immune control of Listeria monocytogenes infection. Mice deficient in XIAP had a higher bacterial burden 48 h after infection than wild-type littermates, and exhibited substantially decreased survival. XIAP enhanced NF-κB activation upon L. monocytogenes infection of activated macrophages, and prolonged phosphorylation of Jun N-terminal kinase (JNK) specifically in response to cytosolic bacteria. Additionally, XIAP promoted maximal production of pro-inflammatory cytokines upon bacterial infection in vitro or in vivo, or in response to combined treatment with NOD2 and TLR2 ligands. Together, our data suggest that XIAP regulates innate immune responses to L. monocytogenes infection by potentiating synergy between Toll-like receptors (TLRs) and Nod-like receptors (NLRs) through activation of JNK- and NF-κB–dependent signaling

A LysM and SH3-Domain Containing Region of the Listeria monocytogenes p60 Protein Stimulates Accessory Cells to Promote Activation of Host NK Cells

Listeria monocytogenes (Lm) infection induces rapid and robust activation of host natural killer (NK) cells. Here we define a region of the abundantly secreted Lm endopeptidase, p60, that potently but indirectly stimulates NK cell activation in vitro and in vivo. Lm expression of p60 resulted in increased IFNγ production by naïve NK cells co-cultured with treated dendritic cells (DCs). Moreover, recombinant p60 protein stimulated activation of naive NK cells when co-cultured with TLR or cytokine primed DCs in the absence of Lm. Intact p60 protein weakly digested bacterial peptidoglycan (PGN), but neither muropeptide recognition by RIP2 nor the catalytic activity of p60 was required for NK cell activation. Rather, the immune stimulating activity mapped to an N-terminal region of p60, termed L1S. Treatment of DCs with a recombinant L1S polypeptide stimulated them to activate naïve NK cells in a cell culture model. Further, L1S treatment activated NK cells in vivo and increased host resistance to infection with Francisella tularensis live vaccine strain (LVS). These studies demonstrate an immune stimulating function for a bacterial LysM domain-containing polypeptide and suggest that recombinant versions of L1S or other p60 derivatives can be used to promote NK cell activation in therapeutic contexts

Branched-Chain Fatty Acids Promote Listeria monocytogenes Intracellular Infection and Virulence ▿

Anteiso-branched-chain fatty acids (BCFA) represent the dominant group of membrane fatty acids and have been established as crucial determinants in resistance against environmental stresses in Listeria monocytogenes, a facultative intracellular pathogen. Here, we investigate the role of anteiso-BCFA in L. monocytogenes virulence by using mutants deficient in branched-chain alpha-keto acid dehydrogenase (BKD), an enzyme complex involved in the synthesis of BCFA. In tissue culture models of infection, anteiso-BCFA contributed to intracellular growth and survival in macrophages and significantly enhanced plaque formation upon prolonged infection in L2 fibroblasts. The intracellular defects observed could be attributed partially to insufficient listeriolysin O (LLO) production, indicating a requirement for anteiso-BCFA in regulating virulence factor production. In a murine model of infection, the BKD-deficient mutant was highly attenuated, further emphasizing the importance of BKD-mediated metabolism in L. monocytogenes virulence. This study demonstrates an underappreciated role for BCFA in bacterial pathogenesis, which may provide insight into the development and application of antimicrobial agents

TBK1 protects vacuolar integrity during intracellular bacterial infection.

TANK-binding kinase-1 (TBK1) is an integral component of Type I interferon induction by microbial infection. The importance of TBK1 and Type I interferon in antiviral immunity is well established, but the function of TBK1 in bacterial infection is unclear. Upon infection of murine embryonic fibroblasts with Salmonella enterica serovar Typhimurium (Salmonella), more extensive bacterial proliferation was observed in tbk1(-/-) than tbk1(+/+) cells. TBK1 kinase activity was required for restriction of bacterial infection, but interferon regulatory factor-3 or Type I interferon did not contribute to this TBK1-dependent function. In tbk1(-/-)cells, Salmonella, enteropathogenic Escherichia coli, and Streptococcus pyogenes escaped from vacuoles into the cytosol where increased replication occurred, which suggests that TBK1 regulates the integrity of pathogen-containing vacuoles. Knockdown of tbk1 in macrophages and epithelial cells also resulted in increased bacterial localization in the cytosol, indicating that the role of TBK1 in maintaining vacuolar integrity is relevant in different cell types. Taken together, these data demonstrate a requirement for TBK1 in control of bacterial infection distinct from its established role in antiviral immunity

In the Absence of TBK1, Bacteria Enter the Host Cytosol

<div><p>(A) <i>Tbk1^+/+</i> and <i>tbk1^−/−</i> MEFs were infected with <i>Salmonella</i>-GFP (green). Infected cells were fixed at 2 h p.i., stained with anti-LAMP-1 antibody followed by a TRITC-labeled secondary antibody (red), and analyzed by confocal immunofluorescence microscopy. Percent colocalization was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with LAMP-1. The experiment shown is representative of three independent experiments.</p><p>(B) MEFs were infected for 1 h with the <i>Salmonella</i>-GFP (green), fixed at 4 h p.i., and stained with an anti-ubiquitin monoclonal antibody followed by a TRITC-labeled secondary antibody (red). The percent of ubiquitin-associated bacteria was determined by counting the number of bacteria out of 150 bacteria per experiment that colocalized with ubiquitin as observed by confocal microscopy. The experiment shown is representative of three independent experiments.</p><p>(C) Transmission electron microscopy was performed on MEFs infected for 1 h with <i>Salmonella,</i> and images acquired at 64,000× magnification<i>.</i> White arrowheads point to bacterial membranes, black arrowheads point to host vacuolar membranes, and arrows point to bacteria no longer completely surrounded by vacuolar membranes.</p><p>(D) MEFs were incubated with ¹²⁵I-EGF and the rate of endocytic uptake (cell associated), degradation (TCA soluble), and accumulation of undegraded product (TCA insoluble) was determined at 0.5, 1, 2, and 3 h after endocytosis. The data represent the mean of four independent experiments.</p></div