Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

The mammalian immune system is constantly challenged by signals from both pathogenic and non-pathogenic microbes. Many of these non-pathogenic microbes have pathogenic potential if the immune system is compromised. The importance of type I interferons (IFNs) in orchestrating innate immune responses to pathogenic microbes has become clear in recent years. However, the control of opportunistic pathogens-and especially intracellular bacteria-by type I IFNs remains less appreciated. In this study, we use the opportunistic, Gram-negative bacterial pathogen Burkholderia cenocepacia (Bc) to show that type I IFNs are capable of limiting bacterial replication in macrophages, preventing illness in immunocompetent mice. Sustained type I IFN signaling through cytosolic receptors allows for increased expression of autophagy and linear ubiquitination mediators, which slows bacterial replication. Transcriptomic analyses and in vivo studies also show that LPS stimulation does not replicate the conditions of intracellular Gram-negative bacterial infection as it pertains to type I IFN stimulation or signaling. This study highlights the importance of type I IFNs in protection against opportunistic pathogens through innate immunity, without the need for damaging inflammatory responses.

Correction: Type I IFNs facilitate innate immune control of the opportunistic bacteria Burkholderia cenocepacia in the macrophage cytosol.

[This corrects the article DOI: 10.1371/journal.ppat.1009395.].

A role for the Salmonella Type III Secretion System 1 in bacterial adaptation to the cytosol of epithelial cells.

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that invades the intestinal epithelium. Following invasion of epithelial cells, Salmonella survives and replicates within two distinct intracellular niches. While all of the bacteria are initially taken up into a membrane bound vacuole, the Salmonella-containing vacuole or SCV, a significant proportion of them promptly escape into the cytosol. Cytosolic Salmonella replicates more rapidly compared to the vacuolar population, although the reasons for this are not well understood. SipA, a multi-function effector protein, has been shown to affect intracellular replication and is secreted by cytosolic Salmonella via the invasion-associated Type III Secretion System 1 (T3SS1). Here, we have used a multipronged microscopy approach to show that SipA does not affect bacterial replication rates per se, but rather mediates intra-cytosolic survival and/or initiation of replication following bacterial egress from the SCV. Altogether, our findings reveal an important role for SipA in the early survival of cytosolic Salmonella.

A second wave of Salmonella T3SS1 activity prolongs the lifespan of infected epithelial cells.

Type III secretion system 1 (T3SS1) is used by the enteropathogen Salmonella enterica serovar Typhimurium to establish infection in the gut. Effector proteins translocated by this system across the plasma membrane facilitate invasion of intestinal epithelial cells. One such effector, the inositol phosphatase SopB, contributes to invasion and mediates activation of the pro-survival kinase Akt. Following internalization, some bacteria escape from the Salmonella-containing vacuole into the cytosol and there is evidence suggesting that T3SS1 is expressed in this subpopulation. Here, we investigated the post-invasion role of T3SS1, using SopB as a model effector. In cultured epithelial cells, SopB-dependent Akt phosphorylation was observed at two distinct stages of infection: during and immediately after invasion, and later during peak cytosolic replication. Single cell analysis revealed that cytosolic Salmonella deliver SopB via T3SS1. Although intracellular replication was unaffected in a SopB deletion mutant, cells infected with ΔsopB demonstrated a lack of Akt phosphorylation, earlier time to death, and increased lysis. When SopB expression was induced specifically in cytosolic Salmonella, these effects were restored to levels observed in WT infected cells, indicating that the second wave of SopB protects this infected population against cell death via Akt activation. Thus, T3SS1 has two, temporally distinct roles during epithelial cell colonization. Additionally, we found that delivery of SopB by cytosolic bacteria was translocon-independent, in contrast to canonical effector translocation across eukaryotic membranes, which requires formation of a translocon pore. This mechanism was also observed for another T3SS1 effector, SipA. These findings reveal the functional and mechanistic adaptability of a T3SS that can be harnessed in different microenvironments.

Salmonella Meningitis Associated with Monocyte Infiltration in Mice.

In the current study, we examined the ability of Salmonella enterica serovar Typhimurium to infect the central nervous system and cause meningitis following the natural route of infection in mice. C57BL/6J mice are extremely susceptible to systemic infection by Salmonella Typhimurium because of loss-of-function mutations in Nramp1 (SLC11A1), a phagosomal membrane protein that controls iron export from vacuoles and inhibits Salmonella growth in macrophages. Therefore, we assessed the ability of Salmonella to disseminate to the central nervous system (CNS) after oral infection in C57BL/6J mice expressing either wild-type (resistant) or mutant (susceptible) alleles of Nramp1. In both strains, oral infection resulted in focal meningitis and ventriculitis with recruitment of inflammatory monocytes to the CNS. In susceptible Nramp1-/- mice, there was a direct correlation between bacteremia and the number of bacteria in the brain, which was not observed in resistant Nramp1+/+ mice. A small percentage of Nramp1+/+ mice developed severe ataxia, which was associated with high bacterial loads in the CNS as well as clear histopathology of necrotizing vasculitis and hemorrhage in the brain. Thus, Nramp1 is not essential for Salmonella entry into the CNS or neuroinflammation, but may influence the mechanisms of CNS entry as well as the severity of meningitis.

Genetic Determinants of Salmonella enterica Serovar Typhimurium Proliferation in the Cytosol of Epithelial Cells.

Intestinal epithelial cells provide an important colonization niche for Salmonella enterica serovar Typhimurium during gastrointestinal infections. In infected epithelial cells, a subpopulation of S Typhimurium bacteria damage their internalization vacuole, leading to escape from the Salmonella-containing vacuole (SCV) and extensive proliferation in the cytosol. Little is known about the bacterial determinants of nascent SCV lysis and subsequent survival and replication of Salmonella in the cytosol. To pinpoint S Typhimurium virulence factors responsible for these steps in the intracellular infectious cycle, we screened a S Typhimurium multigene deletion library in Caco-2 C2Bbe1 and HeLa epithelial cells for mutants that had an altered proportion of cytosolic bacteria compared to the wild type. We used a gentamicin protection assay in combination with a chloroquine resistance assay to quantify total and cytosolic bacteria, respectively, for each strain. Mutants of three S Typhimurium genes, STM1461 (ydgT), STM2829 (recA), and STM3952 (corA), had reduced cytosolic proliferation compared to wild-type bacteria, and one gene, STM2120 (asmA), displayed increased cytosolic replication. None of the mutants were affected for lysis of the nascent SCV or vacuolar replication in epithelial cells, indicating that these genes are specifically required for survival and proliferation of S Typhimurium in the epithelial cell cytosol. These are the first genes identified to contribute to this step of the S Typhimurium infectious cycle.

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages.

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages.

Bacterial colonization of host cells in the absence of cholesterol.

Reports implicating important roles for cholesterol and cholesterol-rich lipid rafts in host-pathogen interactions have largely employed sterol sequestering agents and biosynthesis inhibitors. Because the pleiotropic effects of these compounds can complicate experimental interpretation, we developed a new model system to investigate cholesterol requirements in pathogen infection utilizing DHCR24(-/-) mouse embryonic fibroblasts (MEFs). DHCR24(-/-) MEFs lack the Δ24 sterol reductase required for the final enzymatic step in cholesterol biosynthesis, and consequently accumulate desmosterol into cellular membranes. Defective lipid raft function by DHCR24(-/-) MEFs adapted to growth in cholesterol-free medium was confirmed by showing deficient uptake of cholera-toxin B and impaired signaling by epidermal growth factor. Infection in the absence of cholesterol was then investigated for three intracellular bacterial pathogens: Coxiella burnetii, Salmonella enterica serovar Typhimurium, and Chlamydia trachomatis. Invasion by S. Typhimurium and C. trachomatis was unaltered in DHCR24(-/-) MEFs. In contrast, C. burnetii entry was significantly decreased in -cholesterol MEFs, and also in +cholesterol MEFs when lipid raft-associated α(V)ß(3) integrin was blocked, suggesting a role for lipid rafts in C. burnetii uptake. Once internalized, all three pathogens established their respective vacuolar niches and replicated normally. However, the C. burnetii-occupied vacuole within DHCR24(-/-) MEFs lacked the CD63-positive material and multilamellar membranes typical of vacuoles formed in wild type cells, indicating cholesterol functions in trafficking of multivesicular bodies to the pathogen vacuole. These data demonstrate that cholesterol is not essential for invasion and intracellular replication by S. Typhimurium and C. trachomatis, but plays a role in C. burnetii-host cell interactions.

The Annexin A2/p11 complex is required for efficient invasion of Salmonella Typhimurium in epithelial cells.

The facultative intracellular pathogen, Salmonella enterica, triggers its own uptake into non-phagocytic epithelial cells. Invasion is dependent on a type 3 secretion system (T3SS), which delivers a cohort of effector proteins across the plasma membrane where they induce dynamic actin-driven ruffling of the membrane and ultimately, internalization of the bacteria into a modified phagosome. In eukaryotic cells, the calcium- and phospholipid-binding protein Annexin A2 (AnxA2) functions as a platform for actin remodelling in the vicinity of dynamic cellular membranes. AnxA2 is mostly found in a stable heterotetramer, with p11, which can interact with other proteins such as the giant phosphoprotein AHNAK. We show here that AnxA2, p11 and AHNAK are required for T3SS-mediated Salmonella invasion of cultured epithelial cells and that the T3SS effector SopB is required for recruitment of AnxA2 and AHNAK to Salmonella invasion sites. Altogether this work shows that, in addition to targeting Rho-family GTPases, Salmonella can intersect the host cell actin pathway via AnxA2.

Exploitation of the ubiquitin system by invading bacteria.

A variety of bacterial intracellular pathogens target the host cell ubiquitin system during invasion, a process that involves transient but fundamental changes in the actin cytoskeleton and plasma membrane. These changes are induced by bacterial proteins, which can be surface associated, secreted or injected directly into the host cell. Here, the invasion strategies of two extensively studied intracellular bacteria, Salmonella enterica serovar Typhimurium and Listeria monocytogenes, are used to illustrate some of the diverse ways by which bacterial pathogens intersect the host cell ubiquitin pathway.

Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia.

Salmonella enterica is an intracellular bacterial pathogen that resides and proliferates within a membrane-bound vacuole in epithelial cells of the gut and gallbladder. Although essential to disease, how Salmonella escapes from its intracellular niche and spreads to secondary cells within the same host, or to a new host, is not known. Here, we demonstrate that a subpopulation of Salmonella hyperreplicating in the cytosol of epithelial cells serves as a reservoir for dissemination. These bacteria are transcriptionally distinct from intravacuolar Salmonella. They are induced for the invasion-associated type III secretion system and possess flagella; hence, they are primed for invasion. Epithelial cells laden with these cytosolic bacteria are extruded out of the monolayer, releasing invasion-primed and -competent Salmonella into the lumen. This extrusion mechanism is morphologically similar to the process of cell shedding required for turnover of the intestinal epithelium. In contrast to the homeostatic mechanism, however, bacterial-induced extrusion is accompanied by an inflammatory cell death characterized by caspase-1 activation and the apical release of IL-18, an important cytokine regulator of gut inflammation. Although epithelial extrusion is obviously beneficial to Salmonella for completion of its life cycle, it also provides a mechanistic explanation for the mucosal inflammation that is triggered during Salmonella infection of the gastrointestinal and biliary tracts.

IL-27 induces an IFN-like signature in murine macrophages which in turn modulate colonic epithelium.

Mucosal delivery of IL-27 has been shown to have a therapeutic benefit in murine models of inflammatory bowel disease (IBD). The IL-27 effect was associated with phosphorylated STAT1 (pSTAT1), a product of IL27 receptor signaling, in bowel tissue. To determine whether IL-27 acted directly on colonic epithelium, murine colonoids and primary intact colonic crypts were shown to be unresponsive to IL-27 in vitro and to lack detectable IL-27 receptors. On the other hand, macrophages, which are present in inflamed colon tissue, were responsive to IL-27 in vitro. IL-27 induced pSTAT1 in macrophages, the transcriptome indicated an IFN-like signature, and supernatants induced pSTAT1 in colonoids. IL-27 induced anti-viral activity in macrophages and MHC Class II induction. We conclude that the effects of mucosal delivery of IL-27 in murine IBD are in part based on the known effects of IL27 inducing immunosuppression of T cells mediated by IL-10. We also conclude that IL-27 has potent effects on macrophages in inflamed colon tissue, generating mediators that in turn act on colonic epithelium.

Coiled-coil domains enhance the membrane association of Salmonella type III effectors.

Coiled-coil domains in eukaryotic and prokaryotic proteins contribute to diverse structural and regulatory functions. Here we have used in silico analysis to predict which proteins in the proteome of the enteric pathogen, Salmonella enterica serovar Typhimurium, harbour coiled-coil domains. We found that coiled-coil domains are especially prevalent in virulence-associated proteins, including type III effectors. Using SopB as a model coiled-coil domain type III effector, we have investigated the role of this motif in various aspects of effector function including chaperone binding, secretion and translocation, protein stability, localization and biological activity. Compared with wild-type SopB, SopB coiled-coil mutants were unstable, both inside bacteria and after translocation into host cells. In addition, the putative coiled-coil domain was required for the efficient membrane association of SopB in host cells. Since many other Salmonella effectors were predicted to contain coiled-coil domains, we also investigated the role of this motif in their intracellular targeting in mammalian cells. Mutation of the predicted coiled-coil domains in PipB2, SseJ and SopD2 also eliminated their membrane localization in mammalian cells. These findings suggest that coiled-coil domains represent a common membrane-targeting determinant for Salmonella type III effectors.

Protocol for RNA fluorescence in situ hybridization in mouse meningeal whole mounts.

The multilayered meninges surrounding the brain and spinal cord harbor distinct immune cell populations with prominent roles in health and diseases. Here we present an optimized protocol for RNA fluorescence in situ hybridization (RNA FISH) in meningeal whole mounts, allowing the visualization of gene expression. We also describe the combination of this protocol with immunohistochemistry for simultaneous visualization of mRNA and proteins. This protocol can be used for assessing spatial gene expression within the meninges.

HilD-mediated transcriptional cross-talk between SPI-1 and SPI-2.

The acquisition of new genetic traits by horizontal gene transfer and their incorporation into preexisting regulatory networks have been essential events in the evolution of bacterial pathogens. An example of successful assimilation of virulence traits is Salmonella enterica, which acquired, at distinct evolutionary times, Salmonella pathogenicity island 1 (SPI-1), required for efficient invasion of the intestinal epithelium and intestinal disease, and SPI-2, essential for Salmonella replication and survival within macrophages and the progression of a systemic infection. A positive regulatory cascade mainly composed of HilD, HilA, and InvF, encoded in SPI-1, controls the expression of SPI-1 genes, whereas the two-component regulatory system SsrA/B, encoded in SPI-2, controls expression of SPI-2 genes. In this study, we report a previously undescribed transcriptional cross-talk between SPI-1 and SPI-2, where the SPI-1-encoded regulator HilD is essential for the activation of both the SPI-1 and SPI-2 regulons but at different times during the stationary phase of growth in Luria-Bertani medium. Our data indicate that HilD counteracts the H-NS-mediated repression exerted on the OmpR-dependent activation of the ssrAB operon by specifically interacting with its regulatory region. In contrast, HilD is not required for SPI-2 regulon expression under the in vitro growth conditions that are thought to resemble the intracellular environment. Our results suggest that two independent SPI-2 activation pathways evolved to take advantage of the SPI-2-encoded information at different niches and, in consequence, in response to different growth conditions.

Cytosolic replication in epithelial cells fuels intestinal expansion and chronic fecal shedding of Salmonella Typhimurium.

Persistent and intermittent fecal shedding, hallmarks of Salmonella infections, are important for fecal-oral transmission. In the intestine, Salmonella enterica serovar Typhimurium (STm) actively invades intestinal epithelial cells (IECs) and survives in the Salmonella-containing vacuole (SCV) and the cell cytosol. Cytosolic STm replicate rapidly, express invasion factors, and induce extrusion of infected epithelial cells into the intestinal lumen. Here, we engineered STm that self-destruct in the cytosol (STmCytoKill), but replicates normally in the SCV, to examine the role of cytosolic STm in infection. Intestinal expansion and fecal shedding of STmCytoKill are impaired in mouse models of infection. We propose a model whereby repeated rounds of invasion, cytosolic replication, and release of invasive STm from extruded IECs fuels the high luminal density required for fecal shedding.

Regulatory protein HilD stimulates Salmonella Typhimurium invasiveness by promoting smooth swimming via the methyl-accepting chemotaxis protein McpC.

In the enteric pathogen Salmonella enterica serovar Typhimurium, invasion and motility are coordinated by the master regulator HilD, which induces expression of the type III secretion system 1 (T3SS1) and motility genes. Methyl-accepting chemotaxis proteins (MCPs) detect specific ligands and control the direction of the flagellar motor, promoting tumbling and changes in direction (if a repellent is detected) or smooth swimming (in the presence of an attractant). Here, we show that HilD induces smooth swimming by upregulating an uncharacterized MCP (McpC), and this is important for invasion of epithelial cells. Remarkably, in vitro assays show that McpC can suppress tumbling and increase smooth swimming in the absence of exogenous ligands. Expression of mcpC is repressed by the universal regulator H-NS, which can be displaced by HilD. Our results highlight the importance of smooth swimming for Salmonella Typhimurium invasiveness and indicate that McpC can act via a ligand-independent mechanism when incorporated into the chemotactic receptor array.

Global mapping of Salmonella enterica-host protein-protein interactions during infection.

Intracellular bacterial pathogens inject effector proteins to hijack host cellular processes and promote their survival and proliferation. To systematically map effector-host protein-protein interactions (PPIs) during infection, we generated a library of 32 Salmonella enterica serovar Typhimurium (STm) strains expressing chromosomally encoded affinity-tagged effectors and quantified PPIs in macrophages and epithelial cells. We identified 446 effector-host PPIs, 25 of which were previously described, and validated 13 by reciprocal co-immunoprecipitation. While effectors converged on the same host cellular processes, most had multiple targets, which often differed between cell types. We demonstrate that SseJ, SseL, and SifA modulate cholesterol accumulation at the Salmonella-containing vacuole (SCV) partially via the cholesterol transporter Niemann-Pick C1 protein. PipB recruits the organelle contact site protein PDZD8 to the SCV, and SteC promotes actin bundling by phosphorylating formin-like proteins. This study provides a method for probing host-pathogen PPIs during infection and a resource for interrogating STm effector mechanisms.

Dynamic behavior of Salmonella-induced membrane tubules in epithelial cells.

Salmonella Typhimurium is a facultative intracellular pathogen that causes acute gastroenteritis in man. Intracellular Salmonella survive and replicate within a modified phagosome known as the Salmonella-containing vacuole (SCV). The onset of intracellular replication is accompanied by the appearance of membrane tubules, called Salmonella-induced filaments (Sifs), extending from the SCV. Sifs are enriched in late endosomal/lysosomal membrane proteins such as lysosome-associated membrane protein 1, but their formation and ability to interact with endosomal compartments are not characterized. In this study, we use live cell imaging techniques to define the dynamics of Sif formation in infected epithelial cells. At early time-points, Sifs are simple tubules extending from the surface of SCVs. These tubules are highly dynamic and exhibit bidirectional, microtubule-dependent movement. At the distal ends of individual Sif tubules, furthest from the SCV, a distinct 'leader' domain was often observed. At later times, Sifs develop into highly complex tubular networks that extend throughout the cell and appear less dynamic than nascent Sifs; however, individual tubules continue to display bidirectional dynamics. Sifs can acquire endocytic content by fusion, indicating a sustained interaction with the endocytic pathway. Together, these results show that these Salmonella-induced tubules form a highly dynamic network that involves both microtubule-dependent motility and interactions with endosomal compartments.

Induction of Salmonella pathogenicity island 1 under different growth conditions can affect Salmonella-host cell interactions in vitro.

Salmonella invade non-phagocytic cells by inducing massive actin rearrangements, resulting in membrane ruffle formation and phagocytosis of the bacteria. This process is mediated by a cohort of effector proteins translocated into the host cell by type III secretion system 1, which is encoded by genes in the Salmonella pathogenicity island (SPI) 1 regulon. This network is precisely regulated and must be induced outside of host cells. In vitro invasive Salmonella are prepared by growth in synthetic media although the details vary. Here, we show that culture conditions affect the frequency, and therefore invasion efficiency, of SPI1-induced bacteria and also can affect the ability of Salmonella to adapt to its intracellular niche following invasion. Aerobically grown late-exponential-phase bacteria were more invasive and this was associated with a greater frequency of SPI1-induced, motile bacteria, as revealed by single-cell analysis of gene expression. Culture conditions also affected the ability of Salmonella to adapt to the intracellular environment, since they caused marked differences in intracellular replication. These findings show that induction of SPI1 under different pre-invasion growth conditions can affect the ability of Salmonella to interact with eukaryotic host cells.