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The LC3 recruitment mechanism is separate from Atg9L1-dependent membrane formation in the autophagic response against Salmonella.

ABSTRACT: Salmonella develops into resident bacteria in epithelial cells, and the autophagic machinery (Atg) is thought to play an important role in this process. In this paper, we show that an autophagosome-like double-membrane structure surrounds the Salmonella still residing within the Salmonella-containing vacuole (SCV). This double membrane is defective in Atg9L1- and FAK family-interacting protein of 200 kDa (FIP200)-deficient cells. Atg9L1 and FIP200 are important for autophagy-specific recruitment of the phosphatidylinositol 3-kinase (PI3K) complex. However, in the absence of Atg9L1, FIP200, and the PI3K complex, LC3 and its E3-like enzyme, the Atg16L complex, are still recruited to Salmonella. We propose that the LC3 system is recruited through a mechanism that is independent of isolation membrane generation.

SUBMITTER: Kageyama S

PROVIDER: S-EPMC3128531 | biostudies-other | 2011 Jul

REPOSITORIES: biostudies-other

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The LC3 recruitment mechanism is separate from Atg9L1-dependent membrane formation in the autophagic response against Salmonella.

Kageyama Shun S Omori Hiroko H Saitoh Tatsuya T Sone Takefumi T Guan Jun-Lin JL Akira Shizuo S Imamoto Fumio F Noda Takeshi T Yoshimori Tamotsu T

Molecular biology of the cell 20110427 13

Salmonella develops into resident bacteria in epithelial cells, and the autophagic machinery (Atg) is thought to play an important role in this process. In this paper, we show that an autophagosome-like double-membrane structure surrounds the Salmonella still residing within the Salmonella-containing vacuole (SCV). This double membrane is defective in Atg9L1- and FAK family-interacting protein of 200 kDa (FIP200)-deficient cells. Atg9L1 and FIP200 are important for autophagy-specific recruitment ...[more]

PMID: 21525242

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Project description:Intracellular pathogens such as Salmonella depend on their molecular virulence factors to evade host defense responses like autophagy. Using a zebrafish systemic infection model, we have previously shown that phagocytes, predominantly macrophages, target Salmonella Typhimurium by an autophagy-related pathway known as Lc3-associated phagocytosis (LAP), which is dependent on the host protein Rubicon. Here, we explore the influence of Salmonella virulence factors on pathogenicity in the zebrafish model and induction of LAP as a defense response. We investigated five mutant strains that all could trigger GFP-Lc3 recruitment as puncta or rings around single bacteria or bacterial clusters, in a Rubicon-dependent manner. We found that S. Typhimurium strains carrying mutations in PhoP or PurA, responsible for adaptation to the intracellular environment and efficient metabolism of purines, respectively, are attenuated in the zebrafish model. However, both strains show increased virulence when LAP is inhibited by knockdown of Rubicon. Mutations in type III secretion systems 1 and 2, SipB and SsrB, which are important for invading and replicating in non-phagocytic cells, did not affect the ability to establish successful infection in the zebrafish model. This observation is in line with our previous characterization of this infection model revealing that macrophages actively phagocytose the majority of S. Typhimurium. In contrast to SipB mutants, SsrB mutants were unable to become more virulent in Rubicon-deficient hosts, suggesting that type III system 2 effectors are important for intracellular replication of Salmonella in the absence of LAP. Finally, we found that mutation of FlhD, required for production of flagella, renders S. Typhimurium hypervirulent both in wild type zebrafish embryos and in Rubicon-deficient hosts. FlhD mutation also led to lower levels of GFP-Lc3 recruitment compared with the wild type strain, indicating that recognition of flagellin by the host innate immune system promotes the LAP response. Together, our results provide new evidence that the Rubicon-dependent LAP process is an important defense mechanism against S. Typhimurium.

Project description:UnlabelledOuter membrane vesicles (OMV) are proposed to mediate multiple functions during pathogenesis and symbiosis. However, the mechanisms responsible for OMV formation remain poorly understood. It has been shown in eukaryotic membranes that lipids with an inverted-cone shape favor the formation of positive membrane curvatures. Based on these studies, we formulated the hypothesis that lipid A deacylation might impose shape modifications that result in the curvature of the outer membrane (OM) and subsequent OMV formation. We tested the effect of lipid A remodeling on OMV biogenesis employing Salmonella enterica serovar Typhimurium as a model organism. Expression of the lipid A deacylase PagL resulted in increased vesiculation, without inducing an envelope stress response. Mass spectrometry analysis revealed profound differences in the patterns of lipid A in OM and OMV, with accumulation of deacylated lipid A forms exclusively in OMV. OMV biogenesis by intracellular bacteria upon macrophage infection was drastically reduced in a pagL mutant strain. We propose a novel mechanism for OMV biogenesis requiring lipid A deacylation in the context of a multifactorial process that involves the orchestrated remodeling of the outer membrane.ImportanceThe role of lipid remodeling in vesiculation is well documented in eukaryotes. Similarly, bacteria produce membrane-derived vesicles; however, the molecular mechanisms underlying their production are yet to be determined. In this work, we investigated the role of outer membrane remodeling in OMV biogenesis in S Typhimurium. We showed that the expression of the lipid A deacylase PagL results in overvesiculation with deacylated lipid A accumulation exclusively in OMV. An S Typhimurium ΔpagL strain showed a significant reduction in intracellular OMV secretion relative to the wild-type strain. Our results suggest a novel mechanism for OMV biogenesis that involves outer membrane remodeling through lipid A modification. Understanding how OMV are produced by bacteria is important to advance our understanding of the host-pathogen interactions.

Dataset Information

The LC3 recruitment mechanism is separate from Atg9L1-dependent membrane formation in the autophagic response against Salmonella.

Publications

The LC3 recruitment mechanism is separate from Atg9L1-dependent membrane formation in the autophagic response against Salmonella.

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