Suzanne E. Osborne

SseL Is a Salmonella-Specific Translocated Effector Integrated into the SsrB-Controlled Salmonella Pathogenicity Island 2 Type III Secretion System

ABSTRACT Bacterial pathogens use horizontal gene transfer to acquire virulence factors that influence host colonization, alter virulence traits, and ultimately shape the outcome of disease following infection. One hallmark of the host-pathogen interaction is the prokaryotic type III secretion system that translocates virulence factors into host cells during infection. Salmonella enterica possesses two type III secretion systems that are utilized during host colonization and intracellular replication. Salmonella pathogenicity island 2 (SPI2) is a genomic island containing approximately 30 contiguous genes required to assemble a functional secretion system including the two-component regulatory system called SsrA-SsrB that positively regulates transcription of the secretion apparatus. We used transcriptional profiling with DNA microarrays to search for genes that coregulate with the SPI2 type III secretion machinery in an SsrB-dependent manner. Here we report the identification of a Salmonella-specific translocated effector called SseL that is required for full virulence during murine typhoid-like disease. Analysis of infected macrophages using fluorescence-activated cell sorting revealed that sseL is induced inside cells and requires SsrB for expression. SseL is retained predominantly in the cytoplasm of infected cells following translocation by the type III system encoded in SPI2. Animal infection experiments with sseL mutant bacteria indicate that integration of SseL into the SsrB response regulatory system contributes to systemic virulence of this pathogen.

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

Efferocytosis, the process by which dying or dead cells are removed by phagocytosis, has an important role in development, tissue homeostasis and innate immunity. 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, can exploit efferocytosis to promote cell-to-cell spread during infection. These bacteria can escape the phagosome in host cells by using the pore-forming toxin listeriolysin O (LLO) and two phospholipase C enzymes. Expression of the cell surface protein ActA allows L. monocytogenes 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. Here 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 (encoded by the Timd4 gene) contributes to efficient cell-to-cell spread by L. monocytogenes in macrophages in vitro and growth of these bacteria is impaired in Timd4−/− mice. Thus, L. monocytogenes promotes its dissemination in a host by exploiting efferocytosis. Our results indicate that PS-targeted therapeutics may be useful in the fight against infections by L. monocytogenes and other bacteria that use similar strategies of cell-to-cell spread during 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.

Thermosensing coordinates a cis-regulatory module for transcriptional activation of the intracellular virulence system in Salmonella enterica serovar Typhimurium.

The expression of bacterial virulence genes is tightly controlled by the convergence of multiple extracellular signals. As a zoonotic pathogen, virulence gene regulation in Salmonella enterica serovar Typhimurium must be responsive to multiple cues from the general environment as well as from multiple niches within animal and human hosts. Previous work has identified combined magnesium and phosphate limitation as an environmental cue that activates genes required for intracellular virulence. One unanswered question is how virulence genes that are expressed within the host are inhibited in non-host environments that satisfy the phosphate and magnesium limitation cues. We report here that thermosensing is the major mechanism controlling incongruous activation of the intracellular virulence phenotype. Bacteria grown at 30 °C or lower were unable to activate the intracellular type III secretion system even under strong inducing signals such as synthetic medium, contact with macrophages, and exposure to the murine gut. Thermoregulation was fully recapitulated in a Salmonella bongori strain engineered to contain the intracellular virulence genes of S. enterica sv. Typhimurium, suggesting that orthologous thermoregulators were available. Accordingly, virulence gene repression at the nonpermissive temperature required Hha and H-NS, two nucleoid-like proteins involved in virulence gene control. The use of combined environmental cues to control transcriptional “logic gates” allows for transcriptional selectivity of virulence genes that would otherwise be superfluous if activated in the non-host environment. Thus, thermosensing by Salmonella provides integrated control of host niche-specific virulence factors.

Salmonella-Containing Vacuoles Display Centrifugal Movement Associated with Cell-to-Cell Transfer in Epithelial Cells

ABSTRACT 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.

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.

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

BackgroundThe 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 σE (rpoE) are also highly attenuated for intracellular survival, pointing to a potential connection with the SsrAB regulatory system.ResultsIn 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.ConclusionThese 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.

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

Background: Bacterial pathogens must acquire nutrients for survival during host infection. Results: DalSTUV is an ABC transporter for d-alanine and contributes to virulence in vivo. Conclusion: Nutrient exchange during the host-pathogen interaction can direct disease outcome. Significance: This is the first report of an ABC transporter for d-alanine. 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.

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.

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’.