An ATG16L1-dependent pathway promotes plasma membrane repair and limits Listeria monocytogenes cell-to-cell spread.

Plasma membrane integrity is essential for the viability of eukaryotic cells. In response to bacterial pore-forming toxins, disrupted regions of the membrane are rapidly repaired. However, the pathways that mediate plasma membrane repair are unclear. Here we show that autophagy-related (ATG) protein ATG16L1 and its binding partners ATG5 and ATG12 are required for plasma membrane repair through a pathway independent of macroautophagy. ATG16L1 is required for lysosome fusion with the plasma membrane and blebbing responses that promote membrane repair. ATG16L1 deficiency causes accumulation of cholesterol in lysosomes that contributes to defective membrane repair. Cell-to-cell spread by Listeria monocytogenes requires membrane damage by the bacterial toxin listeriolysin O, which is restricted by ATG16L1-dependent membrane repair. Cells harbouring the ATG16L1 T300A allele associated with inflammatory bowel disease were also found to accumulate cholesterol and be defective in repair, linking a common inflammatory disease to plasma membrane integrity. Thus, plasma membrane repair could be an important therapeutic target for the treatment of bacterial infections and inflammatory disorders.

Listeriolysin O: from bazooka to Swiss army knife.

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen. Infections in humans can lead to listeriosis, a systemic disease with a high mortality rate. One important mechanism of Lm dissemination involves cell-to-cell spread after bacteria have entered the cytosol of host cells. Listeriolysin O (LLO; encoded by the hly gene) is a virulence factor present in Lm that plays a central role in the cell-to-cell spread process. LLO is a member of the cholesterol-dependent cytolysin (CDC) family of toxins that were initially thought to promote disease largely by inducing cell death and tissue destruction-essentially acting like a 'bazooka'. This view was supported by structural studies showing CDCs can form large pores in membranes. However, it is now appreciated that LLO has many subtle activities during Lm infection of host cells, and many of these likely do not involve large pores, but rather small membrane perforations. It is also appreciated that membrane repair pathways of host cells play a major role in limiting membrane damage by LLO and other toxins. LLO is now thought to represent a 'Swiss army knife', a versatile tool that allows Lm to induce many membrane alterations and cellular responses that promote bacterial dissemination during infection.This article is part of the themed issue 'Membrane pores: from structure and assembly, to medicine and technology'.

Type I interferon promotes cell-to-cell spread of Listeria monocytogenes.

Type I interferons (IFNs) play a critical role in antiviral immune responses, but can be deleterious to the host during some bacterial infections. Listeria monocytogenes (Lm) induces a type I IFN response by activating cytosolic antiviral surveillance pathways. This is beneficial to the bacteria as mice lacking the type I IFN receptor (IFNAR1-/- ) are resistant to systemic infection by Lm. The mechanisms by which type I IFNs promote Lm infection are unclear. Here, we show that IFNAR1 is required for dissemination of Lm within infection foci in livers of infected mice and for efficient cell-to-cell spread in vitro in macrophages. IFNAR1 promotes ActA polarization and actin-based motility in the cytosol of host cells. Our studies suggest type I IFNs directly impact the intracellular life cycle of Lm and provide new insight into the mechanisms used by bacterial pathogens to exploit the type I IFN response.

Characterization of DalS, an ATP-binding cassette transporter for D-alanine, and its role in pathogenesis in Salmonella enterica.

Expansion into new host niches requires bacterial pathogens to adapt to changes in nutrient availability and to evade an arsenal of host defenses. Horizontal acquisition of Salmonella Pathogenicity Island (SPI)-2 permitted the expansion of Salmonella enterica serovar Typhimurium into the intracellular environment of host cells by allowing it to deliver bacterial effector proteins across the phagosome membrane. This is facilitated by the SsrA-SsrB two-component regulatory system and a type III secretion system encoded within SPI-2. SPI-2 acquisition was followed by evolution of existing regulatory DNA, creating an expanded SsrB regulon involved in intracellular fitness and host infection. Here, we identified an SsrB-regulated operon comprising an ABC transporter in Salmonella. Biochemical and structural studies determined that the periplasmic solute-binding component, STM1633/DalS, transports D-alanine and that DalS is required for intracellular survival of the bacteria and for fitness in an animal host. This work exemplifies the role of nutrient exchange at the host-pathogen interface as a critical determinant of disease outcome.

Transcriptional priming of Salmonella Pathogenicity Island-2 precedes cellular invasion.

Invasive salmonellosis caused by Salmonella enterica involves an enteric stage of infection where the bacteria colonize mucosal epithelial cells, followed by systemic infection with intracellular replication in immune cells. The type III secretion system encoded in Salmonella Pathogenicity Island (SPI)-2 is essential for intracellular replication and the regulators governing high-level expression of SPI-2 genes within the macrophage phagosome and in inducing media thought to mimic this environment have been well characterized. However, low-level expression of SPI-2 genes is detectable in media thought to mimic the extracellular environment suggesting that additional regulatory pathways are involved in SPI-2 gene expression prior to cellular invasion. The regulators involved in this activity are not known and the extracellular transcriptional activity of the entire SPI-2 island in vivo has not been studied. We show that low-level, SsrB-independent promoter activity for the ssrA-ssrB two-component regulatory system and the ssaG structural operon encoded in SPI-2 is dependent on transcriptional input by OmpR and Fis under non-inducing conditions. Monitoring the activity of all SPI-2 promoters in real-time following oral infection of mice revealed invasion-independent transcriptional activity of the SPI2 T3SS in the lumen of the gut, which we suggest is a priming activity with functional relevance for the subsequent intracellular host-pathogen interaction.

Expression and secretion hierarchy in the nonflagellar type III secretion system.

Type III secretion systems that deliver bacterial proteins into eukaryotic cells are the basis for both symbiotic and pathogenic relationships between many Gram-negative bacteria and their hosts. Exploration of the structure, function and assembly of this secretion system has greatly enhanced our knowledge of bacterial ecology in the context of infectious disease and has spawned new avenues in anti-infective research with a view towards inhibiting virulence functions. We outline advances in understanding type III secretion system function with specific focus on how assembly is hierarchically coordinated at the level of expression and how the type III secretion system mediates transitions in substrate specificity.

RpoE fine tunes expression of a subset of SsrB-regulated virulence factors in Salmonella enterica serovar Typhimurium.

BACKGROUND: The survival of Salmonella enterica within the intracellular host niche requires highly co-ordinated expression of virulence effectors predominantly regulated by the SsrAB two-component regulatory system. S. enterica serovar Typhimurium mutants lacking the ssrAB genes are avirulent in mice, highlighting the importance of this regulatory system in vivo. Mutants lacking the gene encoding the alternative sigma factor sigmaE (rpoE) are also highly attenuated for intracellular survival, pointing to a potential connection with the SsrAB regulatory system. RESULTS: In this study we demonstrate that RpoE is involved in fine-tuning the expression of a subset of SsrB-regulated genes found in the Salmonella pathogenicity island-2 (SPI-2) genetic locus that encodes a horizontally acquired type III secretion system, and unlinked genes integrated into this regulon that are required for virulence in host animals. CONCLUSION: These data point to a potential connection between the virulence phenotype of strains lacking ssrB and rpoE, and highlight new transcriptional regulation that might be essential for appropriate temporal and spatial control of the virulence-associated type III secretion system during host infection.

Pathogenic adaptation of intracellular bacteria by rewiring a cis-regulatory input function.

The acquisition of DNA by horizontal gene transfer enables bacteria to adapt to previously unexploited ecological niches. Although horizontal gene transfer and mutation of protein-coding sequences are well-recognized forms of pathogen evolution, the evolutionary significance of cis-regulatory mutations in creating phenotypic diversity through altered transcriptional outputs is not known. We show the significance of regulatory mutation for pathogen evolution by mapping and then rewiring a cis-regulatory module controlling a gene required for murine typhoid. Acquisition of a binding site for the Salmonella pathogenicity island-2 regulator, SsrB, enabled the srfN gene, ancestral to the Salmonella genus, to play a role in pathoadaptation of S. typhimurium to a host animal. We identified the evolved cis-regulatory module and quantified the fitness gain that this regulatory output accrues for the bacterium using competitive infections of host animals. Our findings highlight a mechanism of pathogen evolution involving regulatory mutation that is selected because of the fitness advantage the new regulatory output provides the incipient clones.

Salmonella-containing vacuoles display centrifugal movement associated with cell-to-cell transfer in epithelial cells.

Intracellular Salmonella enterica serovar Typhimurium (serovar Typhimurium) occupies a Salmonella-containing vacuole (SCV) where bacterial effector proteins are secreted into the host cell using type III secretion systems (T3SS). Cytoskeletal motor proteins and T3SS-delivered effector proteins facilitate SCV positioning to juxtanuclear positions where bacterial replication occurs. Here, we show that this characteristic SCV positioning is not maintained by all SCVs during infection of HeLa cells. Notably, juxtanuclear SCV localization that occurs by 8 to 14 h postinfection is followed by significant centrifugal displacement of a subset of SCVs toward the host cell periphery by 24 h postinfection. This novel phenotype requires bacterial protein synthesis, a functional Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS, intact microtubules, and kinesin-1 motor protein. Bacteria lacking PipB2, a kinesin-recruiting T3SS effector, did not exhibit centrifugal displacement and remained at juxtanuclear positions throughout 24 h of infection. While levels of the SPI-2 effectors PipB2 and SifA increased during 24 h postinfection, a corresponding decrease in levels of the SPI-1 T3SS effectors SipA and SopB, both known to mediate juxtanuclear SCV positioning, was observed. A fluorescence-based assay indicated that wild-type serovar Typhimurium transferred from infected to uninfected epithelial cells while strains deficient in SPI-2 T3SS secretion or PipB2 did not. Our results reveal a novel SCV phenotype implicated in the cell-to-cell spread of serovar Typhimurium during infection.