Listeria monocytogenes exploits efferocytosis to promote cell-to-cell spread

Efferocytosis, the process by which dying/dead cells are removed by phagocytosis, plays an important role in development, tissue homeostasis and innate immunity1. Efferocytosis is mediated, in part, by receptors that bind to exofacial phosphatidylserine (PS) on cells or cellular debris after loss of plasma membrane asymmetry. Here we show that a bacterial pathogen, Listeria monocytogenes (Lm), can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells using the pore-forming toxin Listeriolysin O (LLO) and two phospholipases C2. Expression of the cell surface protein ActA allows Lm to activate host actin regulatory factors and undergo actin-based motility in the cytosol, eventually leading to formation of actin-rich protrusions at the cell surface. We show that protrusion formation is associated with plasma membrane damage due to LLO’s pore-forming activity. LLO also promotes the release of bacteria-containing protrusions from the host cell, generating membrane-derived vesicles with exofacial PS. The PS-binding receptor TIM-4 contributes to efficient cell-to-cell spread by Lm in macrophages in vitro and growth of these bacteria is impaired in TIM-4−/− mice. Thus, Lm promotes its dissemination in a host by exploiting efferocytosis. Our study suggests that PS-targeted therapeutics may be useful in the fight against infections by Lm and other bacteria that utilize similar strategies of cell-to-cell spread during infection

Multiple host kinases contribute to Akt activation during Salmonella infection.

SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3-5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3-5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host

Activation of antibacterial autophagy by NADPH oxidases

Autophagy plays an important role in immunity to microbial pathogens. The autophagy system can target bacteria in phagosomes, promoting phagosome maturation and preventing pathogen escape into the cytosol. Recently, Toll-like receptor (TLR) signaling from phagosomes was found to initiate their targeting by the autophagy system, but the mechanism by which TLR signaling activates autophagy is unclear. Here we show that autophagy targeting of phagosomes is not exclusive to those containing TLR ligands. Engagement of either TLRs or the Fcγ receptors (FcγRs) during phagocytosis induced recruitment of the autophagy protein LC3 to phagosomes with similar kinetics. Both receptors are known to activate the NOX2 NADPH oxidase, which plays a central role in microbial killing by phagocytes through the generation of reactive oxygen species (ROS). We found that NOX2-generated ROS are necessary for LC3 recruitment to phagosomes. Antibacterial autophagy in human epithelial cells, which do not express NOX2, was also dependent on ROS generation. These data reveal a coupling of oxidative and nonoxidative killing activities of the NOX2 NADPH oxidase in phagocytes through autophagy. Furthermore, our results suggest a general role for members of the NOX family in regulating autophagy

Class II PI3-Kinase beta isoform (PI3K-C2β) plays a role in SopB-mediated Akt activation.

<p>(A) HeLa cells were treated with control, PI3K-C2A, PI3K-C2B or PI3K-C2G siRNA for 48 h. Cells were then infected with wild type <i>S</i><i>. Typhimurium</i> for 30 min. As controls, cells were uninfected or incubated with 100 ng/mL EGF for 5 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD induced by wild type <i>S</i><i>. Typhimurium</i> or EGF for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown.</p

IPMK plays a role in SopB-mediated Akt activation.

<p>(A) HeLa cells were treated with control or IPMK siRNA for 48 h. Cells were infected with wild type <i>S</i><i>. Typhimurium</i> for 30 min. As a control, cells were uninfected. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Wild type (ES10) or IPMK knockout (ES2 and ES5) embryonic stem cells were infected with wild type or ΔSopB mutant <i>S</i><i>. Typhimurium</i> for 30 min. As a control, cells were uninfected. Where indicated, cells were treated with 100 µM LY294002 for 30 min. prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (C) Western blot results of ES10 and ES5 from B were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD for three separate experiments, calculated as outlined in the Materials and Methods. The p-values from one-way ANOVA analysis are shown.</p

SopB-mediated Akt activation at <i>Salmonella</i> invasion ruffles is PI(3,4) P₂/PI(3–5) P₃-dependent.

<p>(A) HeLa cells were infected with wild type or Δ<i>sopB</i> mutant <i>S</i><i>. Typhimurium</i> and fixed at 30 min p.i. Cells were examined by epifluorescence microscopy after co-staining for activated Akt with a phospho-specific (Ser473) antibody, actin with fluorescently-labeled phalloidin and bacteria with DAPI. Insets are enlarged from boxed areas. (B) HeLa cells were transiently transfected with GFP-PH-Akt for 16 h prior to <i>Salmonella</i> infection. Cells were infected and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin and bacteria with a polyclonal antibody to <i>S</i><i>. Typhimurium</i>. Insets are enlarged from boxed areas. (C) HeLa cells were transiently transfected with GFP or PTEN-A4-YFP for 16 h prior to <i>Salmonella</i> infection. Cells were infected with wild type <i>S</i><i>. Typhimurium</i> and fixed as in A. Cells were co-stained for actin with fluorescently-labeled phalloidin, Akt with a phospho-specific (Ser473) antibody and bacteria with a polyclonal antibody to <i>S</i><i>. Typhimurium</i>. Insets are enlarged from boxed areas. Size bars, 10 µm. (D) The percentage of phospho-Akt⁺<i>Salmonella</i> invasion ruffles from C was quantified (n ≥ 50). Averages ± SD for three separate experiments are shown. Asterisk indicates that the percent value is significantly different from the control (P < 0.001) as determined by one-way ANOVA analysis. (E) HeLa cells were transiently transfected with GFP-Akt and PTEN-A4-YFP or GFP for 16 h prior to infection. Cells were uninfected, infected as in A, or incubated with 100 ng/mL EGF for 5 min. Where indicated, cells were treated with 100 µM LY294002 for 30 min prior to infection. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. GFP antibodies were used to ensure equal Akt transfection.</p

SopB-mediated Akt activation in HeLa cells is only partially sensitive to PI3-Kinase inhibitor LY294002.

<p>(A) HeLa cells were treated with different concentrations of LY294002 (1 µM to 100 µM) for 30 min. Cells were then infected with wild type <i>S</i><i>. Typhimurium</i> for 30 min or incubated with 100 ng/mL EGF for 5 min. As controls, cells were either uninfected or infected with Δ<i>sopB</i> mutant <i>S</i><i>. Typhimurium</i> for 30 min. Akt activation was determined by immunoblotting the cell lysates with a phospho-specific anti-Ser473 Akt antibody. Pan-Akt antibodies were used to ensure equal protein loading. (B) Western blot results from A were analyzed by estimating the intensities of protein bands with the ImageJ software. Shown on the graph are the relative and normalized expression levels of phospho-Akt ± SD induced by wild type <i>S</i><i>. Typhimurium</i> or EGF for three separate experiments, calculated as outlined in the Materials and Methods. Asterisks indicate that the percent value is significantly different from the control (P < 0.05).</p

Salmonella exploits host Rho GTPase signalling pathways through the phosphatase activity of SopB

12 p.-7 fig.Salmonella uses Type 3 secretion systems (T3SSs) to deliver virulence factors, called effectors, into host cells during infection. The T3SS effectors promote invasion into host cells and the generation of a replicative niche. SopB is a T3SS effector that plays an important role in Salmonella pathogenesis through its lipid phosphatase activity. Here, we show that SopB mediates the recruitment of Rho GTPases (RhoB, RhoD, RhoH, and RhoJ) to bacterial invasion sites. RhoJ contributes to Salmonella invasion, and RhoB and RhoH play an important role in Akt activation. R‐Ras1 also contributes to SopB‐dependent Akt activation by promoting the localised production of PI(3,4)P2/PI(3,4,5)P3. Our studies reveal new signalling factors involved in SopB‐dependent Salmonella infection.This work was supported by an operating grant from the Canadian Institutes of Health Research (FDN 154329).Peer reviewe