Summary
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.
Summary
The intracellular pathogen, Salmonella enterica, translocates type III effectors across its vacuolar membrane into host cells. Herein we describe a new Salmonella effector, PipB2, which has ...sequence similarity to another type III effector, PipB. In phagocytic cells, PipB2 localizes to the Salmonella‐containing vacuole (SCV) and tubular extensions from the SCV, Salmonella‐induced filaments (Sifs). We used the specific targeting of PipB2 in macrophages to characterize Sifs in phagocytic cells for the first time. In epithelial cells, PipB2 has a unique localization pattern, localizing to SCVs and Sifs and additionally to vesicles at the periphery of infected cells. We further show that the N‐terminal 225‐amino‐acid residues of PipB2 are sufficient for type III translocation and association with SCVs and Sifs, but not peripheral vesicles. Subcellular fractionation demonstrated that both PipB and PipB2 associate with host cell membranes and resist extraction by high salt, high pH and to a significant extent, non‐ionic detergent. Furthermore, PipB and PipB2 are enriched in detergent‐resistant microdomains (DRMs), also known as lipid rafts, present on membranes of SCVs and Sifs. The enrichment of Salmonella effectors in DRMs on these intracellular membranes probably permits specific interactions with host cell molecules that are concentrated in these signalling platforms.
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.
Summary
Type III secretion systems (TTSS) are used by Gram‐negative pathogens to translocate proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium
(S. Typhimurium) has two of ...these specialized systems, which are encoded on
separate Salmonellapathogenicity islands (SPI‐1 and
SPI‐2) and translocate unique sets of effectors. The specific roles of these systems
in Salmonella pathogenesis remain undefined, although SPI‐1 is required for bacterial
invasion of epithelial cells and SPI‐2 for survival/replication in phagocytic cells.
However, because SPI‐1 TTSS mutants are invasion‐incompetent, the role of this TTSS
in post‐invasion processes has not been investigated. In this study, we have used
two distinct methods to internalize a non‐invasive SPI‐1 TTSS mutant (invA)
into cultured epithelial cells: (i) co‐internalization with wild‐type S. Typhimurium
(SPI‐1‐dependent) and (ii) complementation with the Yersinia pseudotuberculosis
invasin (inv) gene (SPI‐1‐independent). In both cases, internalized invA
mutants were unable to replicate intracellularly, indicating that SPI‐1 effectors
are essential for this process and cannot be complemented by wild‐type bacteria in
the same cell. Analysis of the biogenesis of SCVs showed that vacuoles containing
mutant bacteria displayed abnormal maturation that was dependent on the mechanism
of entry. Manipulation of Salmonella‐containing vacuole (SCV) biogenesis by
pharmacologically perturbing membrane trafficking in the host cell increased intracellular
replication of wild‐type but not mutant S. Typhimurium This demonstrates a previously unknown role for SPI‐1 in vacuole biogenesis and intracellular survival in non‐phagocytic cells.
Invasion of epithelial cells by Salmonella enterica is mediated by bacterial “effector” proteins that are delivered into the host cell by a type III secretion system. Although primarily known for ...their roles in actin rearrangements and membrane ruffling, translocated effectors also affect host cell processes that are not directly associated with invasion. Here, we show that SopB/SigD, an effector with phosphoinositide phosphatase activity, has anti-apoptotic activity in Salmonella-infected epithelial cells. Salmonella induced the sustained activation of Akt/protein kinase B, a pro-survival kinase, in a SopB-dependent manner. Failure to activate Akt resulted in increased levels of apoptosis after infection with a sopB deletion mutant (ΔsopB). Furthermore, cells infected with wild type bacteria, but not the ΔsopB strain, were protected from camptothecin-induced cleavage of caspase-3 and subsequent apoptosis. The anti-apoptotic activity of SopB was dependent on its phosphatase activity, because a catalytically inactive mutant was unable to protect cells from the effects of camptothecin. Finally, small interfering RNA was used to demonstrate the essential role of Akt in SopB-mediated protection against apoptosis. These results provide new insights into the mechanisms of apoptosis and highlight how bacterial effectors can intercept signaling pathways to manipulate host responses.
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.
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•A protocol for the isolation of mouse meninges and preparation of dried whole mounts•Optimization of whole mount in situ hybridization for assessing spatial gene expression•RNA FISH protocol can be combined with immunohistochemistry•Allows simultaneous visualization of gene expression and protein localization
Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
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.
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.
Summary
The ability of Salmonella enterica to invade and replicate within host cells depends on two type III secretion systems (TTSSs) encoded on pathogenicity islands 1 and 2 (SPI1 and SPI2). The ...current paradigm holds that these systems translocate two classes of effectors that operate sequentially and independently. In essence, the SPI1 TTSS mediates early events (i.e. invasion) whereas the SPI2 TTSS mediates post‐invasion processes (i.e. replication, vacuole maturation). Contrary to this model, we have found in infected macrophages that a SPI1 effector, SopB/SigD, increased inducible nitric oxide synthase levels and nitric oxide production, host cell process previously known only to be a target of the SPI2 TTSS. Furthermore, SopB protein and message persist many hours after invasion. Our findings reveal an unanticipated potential for dialogue between the SPI1 and SPI2 TTSS and the host cell response.
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