Structural and Biochemical Characterization of SrcA, a Multi-Cargo Type III Secretion Chaperone in Salmonella Required for Pathogenic Association with a Host

Many Gram-negative bacteria colonize and exploit host niches using a protein apparatus called a type III secretion system (T3SS) that translocates bacterial effector proteins into host cells where their functions are essential for pathogenesis. A suite of T3SS-associated chaperone proteins bind cargo in the bacterial cytosol, establishing protein interaction networks needed for effector translocation into host cells. In Salmonella enterica serovar Typhimurium, a T3SS encoded in a large genomic island (SPI-2) is required for intracellular infection, but the chaperone complement required for effector translocation by this system is not known. Using a reverse genetics approach, we identified a multi-cargo secretion chaperone that is functionally integrated with the SPI-2-encoded T3SS and required for systemic infection in mice. Crystallographic analysis of SrcA at a resolution of 2.5 Å revealed a dimer similar to the CesT chaperone from enteropathogenic E. coli but lacking a 17-amino acid extension at the carboxyl terminus. Further biochemical and quantitative proteomics data revealed three protein interactions with SrcA, including two effector cargos (SseL and PipB2) and the type III-associated ATPase, SsaN, that increases the efficiency of effector translocation. Using competitive infections in mice we show that SrcA increases bacterial fitness during host infection, highlighting the in vivo importance of effector chaperones for the SPI-2 T3SS

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

Identification of the Regulatory Logic Controlling Salmonella Pathoadaptation by the SsrA-SsrB Two-Component System

Sequence data from the past decade has laid bare the significance of horizontal gene transfer in creating genetic diversity in the bacterial world. Regulatory evolution, in which non-coding DNA is mutated to create new regulatory nodes, also contributes to this diversity to allow niche adaptation and the evolution of pathogenesis. To survive in the host environment, Salmonella enterica uses a type III secretion system and effector proteins, which are activated by the SsrA-SsrB two-component system in response to the host environment. To better understand the phenomenon of regulatory evolution in S. enterica, we defined the SsrB regulon and asked how this transcription factor interacts with the cis-regulatory region of target genes. Using ChIP-on-chip, cDNA hybridization, and comparative genomics analyses, we describe the SsrB-dependent regulon of ancestral and horizontally acquired genes. Further, we used a genetic screen and computational analyses integrating experimental data from S. enterica and sequence data from an orthologous regulatory system in the insect endosymbiont, Sodalis glossinidius, to identify the conserved yet flexible palindrome sequence that defines DNA recognition by SsrB. Mutational analysis of a representative promoter validated this palindrome as the minimal architecture needed for regulatory input by SsrB. These data provide a high-resolution map of a regulatory network and the underlying logic enabling pathogen adaptation to a host

GogB Is an Anti-Inflammatory Effector that Limits Tissue Damage during Salmonella Infection through Interaction with Human FBXO22 and Skp1

Bacterial pathogens often manipulate host immune pathways to establish acute and chronic infection. Many Gram-negative bacteria do this by secreting effector proteins through a type III secretion system that alter the host response to the pathogen. In this study, we determined that the phage-encoded GogB effector protein in Salmonella targets the host SCF E3 type ubiquitin ligase through an interaction with Skp1 and the human F-box only 22 (FBXO22) protein. Domain mapping and functional knockdown studies indicated that GogB-containing bacteria inhibited IκB degradation and NFκB activation in macrophages, which required Skp1 and a eukaryotic-like F-box motif in the C-terminal domain of GogB. GogB-deficient Salmonella were unable to limit NFκB activation, which lead to increased proinflammatory responses in infected mice accompanied by extensive tissue damage and enhanced colonization in the gut during long-term chronic infections. We conclude that GogB is an anti-inflammatory effector that helps regulate inflammation-enhanced colonization by limiting tissue damage during infection

Coordinated Regulation of Virulence during Systemic Infection of Salmonella enterica Serovar Typhimurium

To cause a systemic infection, Salmonella must respond to many environmental cues during mouse infection and express specific subsets of genes in a temporal and spatial manner, but the regulatory pathways are poorly established. To unravel how micro-environmental signals are processed and integrated into coordinated action, we constructed in-frame non-polar deletions of 83 regulators inferred to play a role in Salmonella enteriditis Typhimurium (STM) virulence and tested them in three virulence assays (intraperitoneal [i.p.], and intragastric [i.g.] infection in BALB/c mice, and persistence in 129X1/SvJ mice). Overall, 35 regulators were identified whose absence attenuated virulence in at least one assay, and of those, 14 regulators were required for systemic mouse infection, the most stringent virulence assay. As a first step towards understanding the interplay between a pathogen and its host from a systems biology standpoint, we focused on these 14 genes. Transcriptional profiles were obtained for deletions of each of these 14 regulators grown under four different environmental conditions. These results, as well as publicly available transcriptional profiles, were analyzed using both network inference and cluster analysis algorithms. The analysis predicts a regulatory network in which all 14 regulators control the same set of genes necessary for Salmonella to cause systemic infection. We tested the regulatory model by expressing a subset of the regulators in trans and monitoring transcription of 7 known virulence factors located within Salmonella pathogenicity island 2 (SPI-2). These experiments validated the regulatory model and showed that the response regulator SsrB and the MarR type regulator, SlyA, are the terminal regulators in a cascade that integrates multiple signals. Furthermore, experiments to demonstrate epistatic relationships showed that SsrB can replace SlyA and, in some cases, SlyA can replace SsrB for expression of SPI-2 encoded virulence factors

Physiotherapy alone or in combination with corticosteroid injection for acute lateral epicondylitis in general practice: A protocol for a randomised, placebo-controlled study

<p>Abstract</p> <p>Background</p> <p>Lateral epicondylitis is a painful condition responsible for loss of function and sick leave for long periods of time. In many countries, the treatment guidelines recommend a wait-and-see policy, reflecting that no conclusions on the best treatment can be drawn from the available research, published studies and meta-analyses.</p> <p>Methods/Design</p> <p>Randomized double blind controlled clinical trial in a primary care setting. While earlier trials have either compared corticosteroid injections to physical therapy or to naproxen orally, we will compare the clinical effect of physiotherapy alone or physiotherapy combined with corticosteroid injection in the initial treatment of acute tennis elbow. Patients seeing their general practitioner with lateral elbow pain of recent onset will be randomised to one of three interventions: 1: physiotherapy, corticosteroid injection and naproxen or 2: physiotherapy, placebo injection and naproxen or 3: wait and see treatment with naproxen alone. Treatment and assessments are done by two different doctors, and the contents of the injection is unknown to both the treating doctor and patient. The primary outcome measure is the patient's evaluation of improvement after 6, 12, 26 and 52 weeks. Secondary outcome measures are pain, function and severity of main complaint, pain-free grip strength, maximal grip strength, pressure-pain threshold, the patient's satisfaction with the treatment and duration of sick leave.</p> <p>Conclusion</p> <p>This article describes a randomized, double blind, controlled clinical trial with a one year follow up to investigate the effects of adding steroid injections to physiotherapy in acute lateral epicondylitis.</p> <p>Trial Registration</p> <p>ClinicalTrials.gov Identifier: NCT00826462</p

Chlamydia trachomatis Co-opts the FGF2 Signaling Pathway to Enhance Infection

The molecular details of Chlamydia trachomatis binding, entry, and spread are incompletely understood, but heparan sulfate proteoglycans (HSPGs) play a role in the initial binding steps. As cell surface HSPGs facilitate the interactions of many growth factors with their receptors, we investigated the role of HSPG-dependent growth factors in C. trachomatis infection. Here, we report a novel finding that Fibroblast Growth Factor 2 (FGF2) is necessary and sufficient to enhance C. trachomatis binding to host cells in an HSPG-dependent manner. FGF2 binds directly to elementary bodies (EBs) where it may function as a bridging molecule to facilitate interactions of EBs with the FGF receptor (FGFR) on the cell surface. Upon EB binding, FGFR is activated locally and contributes to bacterial uptake into non-phagocytic cells. We further show that C. trachomatis infection stimulates fgf2 transcription and enhances production and release of FGF2 through a pathway that requires bacterial protein synthesis and activation of the Erk1/2 signaling pathway but that is independent of FGFR activation. Intracellular replication of the bacteria results in host proteosome-mediated degradation of the high molecular weight (HMW) isoforms of FGF2 and increased amounts of the low molecular weight (LMW) isoforms, which are released upon host cell death. Finally, we demonstrate the in vivo relevance of these findings by showing that conditioned medium from C. trachomatis infected cells is enriched for LMW FGF2, accounting for its ability to enhance C. trachomatis infectivity in additional rounds of infection. Together, these results demonstrate that C. trachomatis utilizes multiple mechanisms to co-opt the host cell FGF2 pathway to enhance bacterial infection and spread

Choice of Bacterial Growth Medium Alters the Transcriptome and Phenotype of Salmonella enterica Serovar Typhimurium

The type of bacterial culture medium is an important consideration during design of any experimental protocol. The aim of this study was to understand the impact of medium choice on bacterial gene expression and physiology by comparing the transcriptome of Salmonella enterica SL1344 after growth in the widely used LB broth or the rationally designed MOPS minimal medium. Transcriptomics showed that after growth in MOPS minimal media, compared to LB, there was increased expression of 42 genes involved in amino acid synthesis and 23 genes coding for ABC transporters. Seven flagellar genes had decreased expression after growth in MOPS minimal medium and this correlated with a decreased motility. In both MOPS minimal medium and MEM expression of genes from SPI-2 was increased and the adhesion of S. Typhimurium to intestinal epithelial cells was higher compared to the levels after growth in LB. However, SL1344 invasion was not significantly altered by growth in either MOPs minimal media or MEM. Expression of SPI-2 was also measured using chromosomal GFP reporter fusions followed by flow cytometry which showed, for the first time, that the reduction in SPI-2 transcript after growth in different media related to a reduction in the proportion of the bacterial population expressing SPI-2. These data highlight the profound differences in the global transcriptome after in vitro growth in different media and show that choice of medium should be considered carefully during experimental design, particularly when virulence related phenotypes are being measured

A Pivotal Role of Vitamin B9 in the Maintenance of Regulatory T Cells In Vitro and In Vivo

Dietary factors regulate immunological function, but the underlying mechanisms remain elusive. Here we show that vitamin B9 is a survival factor for regulatory T (Treg) cells expressing high levels of vitamin B9 receptor (folate receptor 4). In vitamin B9-reduced condition in vitro, Treg cells could be differentiated from naïve T cells but failed to survive. The impaired survival of Treg cells was associated with decreased expression of anti-apoptotic Bcl2 and independent of IL-2. In vivo depletion of dietary vitamin B9 resulted in the reduction of Treg cells in the small intestine, a site for the absorption of dietary vitamin B9. These findings provide a new link between diet and the immune system, which could maintain the immunological homeostasis in the intestine