Type III Secretion Research Articles

Genomic characterization of an extensively drug-resistant chicken-borne Salmonella Indiana isolate carrying an IncHI2-IncHI2A plasmid

The recent emergence of extensively drug-resistant (XDR) foodborne bacteria is posing a huge threat to food safety and public health. In this study, we reported the genome features of an XDR Salmonella Indiana isolate (SJTUF87912v2) recovered from a chicken carcass in 2012 in Guangdong, China. This isolate was resistant to 10 antimicrobial classes including penicillins, β-lactam combination agents, cephems, aminoglycosides, folate pathway antagonists, fosfomycins, macrolides, phenicols, quinolones, and tetracyclines, and thus, it should be considered as an XDR isolate. A prominent feature of its genome was that it possessed 28 genomic islands (GIs) encoding proteins for phage, type IV secretion, DNA repair, and biofilm formation, as well as 5 Salmonellapathogenicity islands (SPIs) including C63PI, SPI-2, SPI-3, SPI-4 and SPI-5. Type Ⅲ secretion system (TTSS) genes (hilACD, iacP, iagB, invABCEFGHIJ, orgABC, prgHIJK, sicAP, sipD, spaOPQRS and sprB) were located on SPI-1 and TTSS genes (ssaCDEGHIJKLMNOPQRSTUV, sscAB, sseABCDE and ssrAB) were located on SPI-2. A variety of 27 acquired antimicrobial resistance genes (ARGs) was located on the IncHI2-IncHI2A-type plasmid p87912 with 236, 217 bp in size, including blaCTX-M-65, fosA3, mphA, qepA and rmtB interspersed with different insertion sequences and transposons (IS26, IS903, IS6100, ISCR3 and ΔTn2). The co-transfer of blaCTX-M-65, fosA3, mphA, rmtB, and qepA genes and corresponding antibiotic resistance profiles driven by plasmid p87912 occurred. Taken together, these results revealed the genomic characterization of an XDR chicken isolates in S. Indiana, and that the co-existence of numbers of ARGs on plasmid played an important role in XDR profile in S. Indiana isolates.

Interfering with Autophagy: The Opposing Strategies Deployed by Legionella pneumophila and Coxiella burnetii Effector Proteins.

Autophagy is a fundamental and highly conserved eukaryotic process, responsible for maintaining cellular homeostasis and releasing nutrients during times of starvation. An increasingly important function of autophagy is its role in the cell autonomous immune response; a process known as xenophagy. Intracellular pathogens are engulfed by autophagosomes and targeted to lysosomes to eliminate the threat to the host cell. To counteract this, many intracellular bacterial pathogens have developed unique approaches to overcome, evade, or co-opt host autophagy to facilitate a successful infection. The intracellular bacteria Legionella pneumophila and Coxiella burnetii are able to avoid destruction by the cell, causing Legionnaires’ disease and Q fever, respectively. Despite being related and employing homologous Dot/Icm type 4 secretion systems (T4SS) to translocate effector proteins into the host cell, these pathogens have developed their own unique intracellular niches. L. pneumophila evades the host endocytic pathway and instead forms an ER-derived vacuole, while C. burnetii requires delivery to mature, acidified endosomes which it remodels into a large, replicative vacuole. Throughout infection, L. pneumophila effectors act at multiple points to inhibit recognition by xenophagy receptors and disrupt host autophagy, ensuring it avoids fusion with destructive lysosomes. In contrast, C. burnetii employs its effector cohort to control autophagy, hypothesized to facilitate the delivery of nutrients and membrane to support the growing vacuole and replicating bacteria. In this review we explore the effector proteins that these two organisms utilize to modulate the host autophagy pathway in order to survive and replicate. By better understanding how these pathogens manipulate this highly conserved pathway, we can not only develop better treatments for these important human diseases, but also better understand and control autophagy in the context of human health and disease.

Virulence potential of Aeromonas hydrophila isolated from apparently healthy freshwater food fish

Aeromonas hydrophila is considered an important pathogen of fish causing substantial economic loss in aquaculture worldwide. The objective of the current study was to assess the virulence potential of A. hydrophila strains isolated from gills of apparently healthy fish using phenotypic assays, PCR based genotypic tests, and in-vivo pathogenicity assays in Indian catfish, Clarias magur. For this, motile aeromonads (n = 50) were isolated from gills of apparently healthy freshwater food fish (n = 50) collected from domestic fish market. Gill swabs were incubated for primary enrichment in alkaline peptone water supplemented with cephalothin (10 mg/mL) for 12 hours followed by plating on Starch Ampicillin Agar. A single big yellow colony was selected from each sample, purified, and presumptively identified as Aeromonas with the help of Aerokey-II. Out of the fifty isolates of Aeromonas, five isolates of diverse origin (9C, 10G1, 7C, 10C and 4P) were confirmed to be A. hydrophila by automated bacterial identification system VITEK 2 with confidence interval of 90–98%. In the phenotypic assays, strains 9C and 10G1 showed high serum resistance, swimming and swarming activity and low biofilm producing capabilities, which were indicative of putative virulence. Both the strains belonged to genotype aah+act+ alt+ascV+eno+lip+ast+ on the basis of detection of virulence genes by PCR, i.e. extracellular haemolysin (aah), cytolytic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), type-III secretion system (ascV), enolase (eno), lipase (lip) and heat-stable cytotonic enterotoxin (ast). In-vivo pathogenicity assays confirmed that both the strains were pathogenic to magur fish. The LD50 for 9C strain was 6.81 × 104 CFU/mL and 7.62 × 105 CFU/mL for 10G1strain. In conclusion, our phenotypic and genotypic findings showed that A. hydrophila isolated from apparently healthy fish harbour number of important virulence genes/factors and could have important implications in triggering disease in farmed fish under stress.

Enhanced autophagy promotes the clearance of Pseudomonas aeruginosa in diabetic rats with wounds.

BackgroundTo investigate the effects of the Pseudomonas aeruginosa (P. aeruginosa) type 3 secretion system (T3SS) on diabetic wound healing and autophagy-associated proteins.MethodsP. aeruginosa and P. aeruginosa without T3SS were used to infect back wounds in 36 rats (18 normal and 18 diabetic). Followed infection with P. aeruginosa, another 36 rats (18 normal and 18 diabetic) with back wounds were treated with autophagy inducer rapamycin or gentamicin (positive control). Wound healing, colony count, HE and Masson staining were recorded. Western blot and immunofluorescent were used to determine the expression of the autophagy markers (LC3, beclin-1 and p62) in wound tissues.ResultsThe number of P. aeruginosa colonies in infected wounds began to decrease on day 3 in normal rats and on day 7 in diabetic rats. The decrease was more apparent in P. aeruginosa without T3SS. The expression of LC3-II/LC3-I and beclin-1 gradually increased, and p62 gradually decreased in the wounds in of all groups; however, the changes were more dramatic in normal rats compared with diabetic rats on day 14. Rapamycin increased LC3-II/LC3-I and beclin-1 expression, and decreased p62 expression, gentamicin had no effect on their expression of autophagy markers.ConclusionsT3SS of P. aeruginosa inhibited the entire autophagy process in wounds. Thus inducing autophagy could enhance the clearance of P. aeruginosa in diabetic wounds and is expected to become a new method of anti-P. aeruginosa infection.

Early Intracellular Trafficking and Subsequent Activity of Programmed Cell Death in Channel Catfish Macrophages Infected with Edwardsiella ictaluri.

The development of Edwardsiella-containing-vacuoles (ECV) and the ability of Edwardsiella ictaluri to survive and replicate within macrophages suggests a unique process relative to normal phagosomal/lysosomal maturation and programed cell death. Developing ECV showed that endosomal membrane markers Rab5, EEA1, and Rab7 were all detected in both the wild type (WT) and an E. ictaluri type-3 secretion system (T3SS) mutant, 65ST. Co-localization with Lamp1, however, was significantly lower in the WT. The host cell endoplasmic reticulum marker, calnexin, co-localized to 65ST ECV significantly more than WT ECV, while Golgi vesicle marker, giantin, was recruited to WT ECV significantly more than 65ST. The autophagosomal marker LC3 was significantly lower in WT than in 65ST and Western blotting demonstrated significantly greater induction of the membrane localized, lipidated form, LC3-II, in 65ST ECV than in WT ECV. Activity of the apoptosis initiator caspase-8 increased post-infection in 65ST and was significantly lower in WT-infected cells. Executioner caspase-3/7 activity also increased significantly in 65ST-infected cells compared to WT-infected cells. Repression of apoptosis was further demonstrated with flow cytometry using Alexa Fluor 647-labeled Annexin V and propidium iodide. Results indicate that WT ECV fused with early and late endosomes but that phagosomal/lysosomal fusion did not occur. Additionally, WT-infected cells recruited Golgi vesicles for vacuolar size increase and bacterial growth material, and both autophagy and apoptosis were repressed in the WT. This activity was all based on the function of the E. ictaluri T3SS.

A bacterial F-box effector suppresses SAR immunity through mediating the proteasomal degradation of OsTrxh2 in rice.

Plant bacterial pathogens usually cause diseases by secreting and translocating numerous virulence effectors into host cells and suppressing various host immunity pathways. It has been demonstrated that the extensive ubiquitin systems of host cells are frequently interfered with or hijacked by numerous pathogenic bacteria, through various strategies. Some type-III secretion system (T3SS) effectors of plant pathogens have been demonstrated to impersonate the F-box protein (FBP) component of the SKP1/CUL1/F-box (SCF) E3 ubiquitin system for their own benefit. Although numerous putative eukaryotic-like F-box effectors have been screened for different bacterial pathogens by bioinformatics analyses, the targets of most F-box effectors in host immune systems remain unknown. Here, we show that XopI, a putative F-box effector of African Xoo (Xanthomonas oryzae pv. oryzae) strain BAI3, strongly inhibits the host's OsNPR1-dependent resistance to Xoo. The xopI knockout mutant displays lower virulence in Oryza sativa (rice) than BAI3. Mechanistically, we identify a thioredoxin protein, OsTrxh2, as an XopI-interacting protein in rice. Although OsTrxh2 positively regulates rice immunity by catalyzing the dissociation of OsNPR1 into monomers in rice, the XopI effector serves as an F-box adapter to form an OSK1-XopI-OsTrxh2 interaction complex, and further disrupts OsNPR1-mediated resistance through proteasomal degradation of OsTrxh2. Our results indicate that XopI targets OsTrxh2 and further represses OsNPR1-dependent signaling, thereby subverting systemic acquired resistance (SAR) immunity in rice.

SopB- and SifA-dependent shaping of the Salmonella-containing vacuole proteome in the social amoeba Dictyostelium discoideum.

The ability of Salmonella to survive and replicate within mammalian host cells involves the generation of a membranous compartment known as the Salmonella-containing vacuole (SCV). Salmonella employs a number of effector proteins that are injected into host cells for SCV formation using its type-3 secretion systems encoded in SPI-1 and SPI-2 (T3SS-1 and T3SS-2, respectively). Recently, we reported that S. Typhimurium requires T3SS-1 and T3SS-2 to survive in the model amoeba Dictyostelium discoideum. Despite these findings, the involved effector proteins have not been identified yet. Therefore, we evaluated the role of two major S. Typhimurium effectors SopB and SifA during D. discoideum intracellular niche formation. First, we established that S. Typhimurium resides in a vacuolar compartment within D. discoideum. Next, we isolated SCVs from amoebae infected with wild type or the ΔsopB and ΔsifA mutant strains of S. Typhimurium, and we characterised the composition of this compartment by quantitative proteomics. This comparative analysis suggests that S. Typhimurium requires SopB and SifA to modify the SCV proteome in order to generate a suitable intracellular niche in D. discoideum. Accordingly, we observed that SopB and SifA are needed for intracellular survival of S. Typhimurium in this organism. Thus, our results provide insight into the mechanisms employed by Salmonella to survive intracellularly in phagocytic amoebae.

The Aeromonas salmonicida plasmidome: a model of modular evolution and genetic diversity.

High-throughput genomic sequencing has helped to reveal the plasmidome of Aeromonas salmonicida. This literature review provides an overview of A. salmonicida's rich plasmidome by presenting all the plasmids identified so far, addressing their biological importance and the functional links between them. The plasmids of A. salmonicida, especially those bearing antibiotic resistance genes, can provide clues about interactions of this species with other pathogens (animals and humans), as is the case for pRAS3-3432 and Chlamydia suis or pSN254b and Salmonella enterica. In addition to antibiotic resistance, plasmids play an important role in the virulence of A.salmonicida, particularly for the subspecies salmonicida and the plasmid pAsa5, which carries genes for the type-three secretion system, a virulence factor essential for the bacterium. The A.salmonicida plasmidome also has many cryptic plasmids with no known biological function, but which can be used for the acquisition of new genetic elements. Striking examples are pAsa7 and pAsaXII that provide, respectively, resistance to chloramphenicol and formaldehyde and are derivatives of cryptic pAsa2.

Keeping in Touch with Type-III Secretion System Effectors: Mass Spectrometry-Based Proteomics to Study Effector-Host Protein-Protein Interactions.

Manipulation of host cellular processes by translocated bacterial effectors is key to the success of bacterial pathogens and some symbionts. Therefore, a comprehensive understanding of effectors is of critical importance to understand infection biology. It has become increasingly clear that the identification of host protein targets contributes invaluable knowledge to the characterization of effector function during pathogenesis. Recent advances in mapping protein–protein interaction networks by means of mass spectrometry-based interactomics have enabled the identification of host targets at large-scale. In this review, we highlight mass spectrometry-driven proteomics strategies and recent advances to elucidate type-III secretion system effector–host protein–protein interactions. Furthermore, we highlight approaches for defining spatial and temporal effector–host interactions, and discuss possible avenues for studying natively delivered effectors in the context of infection. Overall, the knowledge gained when unravelling effector complexation with host factors will provide novel opportunities to control infectious disease outcomes.

Sodium Butyrate Reduces Salmonella Enteritidis Infection of Chicken Enterocytes and Expression of Inflammatory Host Genes in vitro

Salmonella Enteritidis (SE) is a facultative intracellular pathogen that colonizes the chicken gut leading to contamination of carcasses during processing. A reduction in intestinal colonization by SE could result in reduced carcass contamination thereby reducing the risk of illnesses in humans. Short chain fatty acids such as butyrate are microbial metabolites produced in the gut that exert various beneficial effects. However, its effect on SE colonization is not well known. The present study investigated the effect of sub-inhibitory concentrations (SICs) of sodium butyrate on the adhesion and invasion of SE in primary chicken enterocytes and chicken macrophages. In addition, the effect of sodium butyrate on the expression of SE virulence genes and selected inflammatory genes in chicken macrophages challenged with SE were investigated. Based on the growth curve analysis, the two SICs of sodium butyrate that did not reduce SE growth were 22 and 45 mM, respectively. The SICs of sodium butyrate did not affect the viability and proliferation of chicken enterocytes and macrophage cells. The SICs of sodium butyrate reduced SE adhesion by ∼1.7 and 1.8 Log CFU/mL, respectively. The SE invasion was reduced by ∼2 and 2.93 Log CFU/mL, respectively in chicken enterocytes (P < 0.05). Sodium butyrate did not significantly affect the adhesion of SE to chicken macrophages. However, 45 mM sodium butyrate reduced invasion by ∼1.7 Log CFU/mL as compared to control (P < 0.05). Exposure to sodium butyrate did not change the expression of SE genes associated with motility (flgG, prot6E), invasion (invH), type 3 secretion system (sipB, pipB), survival in macrophages (spvB, mgtC), cell wall and membrane integrity (tatA), efflux pump regulator (mrr1) and global virulence regulation (lrp) (P > 0.05). However, a few genes contributing to type-3 secretion system (ssaV, sipA), adherence (sopB), macrophage survival (sodC) and oxidative stress (rpoS) were upregulated by at least twofold. The expression of inflammatory genes (Il1β, Il8, and Mmp9) that are triggered by SE for host colonization was significantly downregulated (at least 25-fold) by sodium butyrate as compared to SE (P < 0.05). The results suggest that sodium butyrate has an anti-inflammatory potential to reduce SE colonization in chickens.

Enterococcal PrgA Extends Far Outside the Cell and Provides Surface Exclusion to Protect against Unwanted Conjugation

Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via type 4 secretion systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a second copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggests that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.

Comprehensive Host Cell-Based Screening Assays for Identification of Anti-Virulence Drugs Targeting Pseudomonas aeruginosa and Salmonella Typhimurium.

The prevalence of bacterial pathogens being resistant to antibiotic treatment is increasing worldwide, leading to a severe global health challenge. Simultaneously, the development and approval of new antibiotics stagnated in the past decades, leading to an urgent need for novel approaches to avoid the spread of untreatable bacterial infections in the future. We developed a highly comprehensive screening platform based on quantification of pathogen driven host-cell death to detect new anti-virulence drugs targeting Pseudomonas aeruginosa (Pa) and Salmonella enterica serovar Typhimurium (ST), both known for their emerging antibiotic resistance. By screening over 10,000 small molecules we could identify several substances showing promising effects on Pa and ST pathogenicity in our in vitro infection model. Importantly, we could detect compounds potently inhibiting bacteria induced killing of host cells and one novel comipound with impact on the function of the type 3 secretion system (T3SS) of ST. Thus, we provide proof of concept data of rapid and feasible medium- to high-throughput drug screening assays targeting virulence mechanisms of two major Gram-negative pathogens.

Targeted Delivery of the Mitochondrial Target Domain of Noxa to Tumor Tissue via Synthetic Secretion System in E. coli.

Targeted delivery of drugs is a key aspect of the successful treatment of serious conditions such as tumors. In the pursuit of accurate delivery with high specificity and low size limit for peptide drugs, a synthetic type 3 secretion system (T3SS) has been repurposed from a native genetic system encoded in Salmonella pathogenicity island-1 (SPI-1) with no virulence effectors. Here, we tested the potential of synthetic T3SS as drug delivery machinery for peptide-based drugs owing to its modular nature. First, the genetic system for synthetic T3SS was introduced into non-native host E. coli, which was chosen for its lack of Salmonella-driven virulence factors. Next, the mitochondrial targeting domain (MTD) of Noxa was tested as a cargo protein with anti-tumor activity. To this end, the gene encoding MTD was engineered for secretion through synthetic T3SS, thereby resulting in the tagged MTD at the N-terminus. When E. coli carrying synthetic T3SS and MTD on plasmids was administered into tumor-bearing mice, MTD with a secretion tag at the N-terminus was clearly detected in the tumor tissue after induction. Also, the tumor growth and mortality of tumor-bearing animals were mitigated by the cytotoxic activity of the delivered. Thus, this work potentiates the use of biotherapeutic bacteria for the treatment of tumors by implanting a dedicated delivery system.

The Type VI secretion system in commensal Neisseria spp.

A type 6 secretion system (T6SS) was recently identified in the genome sequence data of an isolate sourced from a throat swab of a volunteer that is believed to be N. subflava. The T6SS is one of the most recently discovered bacterial secretion systems and this is the first time it has been reported in Neisseriaceae. Since this discovery, genome sequence analyses for a number of other commensal Neisseria spp. has identified that in fact, two distinct T6SS types exist across Neisseriaceae. These two types are clearly defined and are different to one another in both their core gene sequences and organisation. The two systems also differ in the number of VgrG proteins required for toxic effector protein delivery, as well as type of effector associated with them. The predicted VgrG/effector combinations identified in our isolate are not common to all members of the same species and further analysis has identified a wide range of diversity in these components between different strains of the same species. The data provides possible evidence that T6SS positive commensal Neisseria spp. can acquire new VgrG/effector combinations through competitor killing. A number of putative effectors have so far been identified within the genome of our original isolate, including hydrolases, phospholipases, and nucleases. Whilst these are predicted to be antibacterial effectors, the conditions under which the T6SS system is activated, as well as demonstration of the function of the effectors still needs to be investigated experimentally.

Hepatic Stellate Cells and Hepatocytes as Liver Antigen-Presenting Cells during B. abortus Infection.

In Brucellosis, the role of hepatic stellate cells (HSCs) in the induction of liver fibrosis has been elucidated recently. Here, we study how the infection modulates the antigen-presenting capacity of LX-2 cells. Brucella abortus infection induces the upregulation of class II transactivator protein (CIITA) with concomitant MHC-I and -II expression in LX-2 cells in a manner that is independent from the expression of the type 4 secretion system (T4SS). In concordance, B. abortus infection increases the phagocytic ability of LX-2 cells and induces MHC-II-restricted antigen processing and presentation. In view of the ability of B. abortus-infected LX-2 cells to produce monocyte-attracting factors, we tested the capacity of culture supernatants from B. abortus-infected monocytes on MHC-I and –II expression in LX-2 cells. Culture supernatants from B. abortus-infected monocytes do not induce MHC-I and -II expression. However, these supernatants inhibit MHC-II expression induced by IFN-γ in an IL-10 dependent mechanism. Since hepatocytes constitute the most abundant epithelial cell in the liver, experiments were conducted to determine the contribution of these cells in antigen presentation in the context of B. abortus infection. Our results indicated that B. abortus-infected hepatocytes have an increased MHC-I expression, but MHC-II levels remain at basal levels. Overall, B. abortus infection induces MHC-I and -II expression in LX-2 cells, increasing the antigen presentation. Nevertheless, this response could be modulated by resident or infiltrating monocytes/macrophages.

Bactericidal Effect of Entomopathogenic Bacterium Pseudomonas entomophila Against Xanthomonas citri Reduces Citrus Canker Disease Severity.

The bacterium Pseudomonas entomophila has been recognized as an exceptional species within the Pseudomonas genus, capable of naturally infecting and killing insects from at least three different orders. P. entomophila ingestion leads to irreversible gut damage resulting from a global blockage of translation, which impairs both immune and tissue repair systems in the insect intestine. In this study we isolated a P. entomophila bacterial strain from soil samples which displayed a strong activity against Xanthomonas citri subsp, citri (Xcc), the etiological agent of citrus canker disease. The antagonism potential of isolated bacteria against Xcc and its ability to reduce citrus canker severity was assessed both ex planta and in planta. Our findings show that pathogenicity assays in Citrus x limonia by pressure infiltration and spray with a mixture of P. entomophila and Xcc leaded to a significant reduction in the number of canker lesions in high susceptible citrus leaves, at 21 days post-infection. To the best of our knowledge this is the first report of antibacterial activity of P. entomophila against a phytopathogenic bacterium. Collective action of P. entomophila factors such as diketopiperazine production and the type 6 secretion system (T6SS) may be involved in this type of biological control of citrus canker. The results suggest that the P. entomophila strain could be a promising biocontrol agent acting directly against Xcc.

The Gli1-Snail axis contributes to Salmonella Typhimurium-induced disruption of intercellular junctions of intestinal epithelial cells.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a facultative intracellular pathogen that damages gastrointestinal tissue and causes severe diarrhoea. The mechanisms by which Salmonella disrupts epithelial barrier and increases the paracellular permeability are incompletely understood. Our present study aims to determine the role of Gli1, a transcription factor activated in the sonic hedgehog (Shh) pathway, in decreasing the levels of apical junction proteins in a Salmonella-infected human colonic epithelial cancer cell line, Caco-2, and in the intestinal tissue of Salmonella-infected mice. Here, we report that S. Typhimurium increased the mRNA and protein levels of Gli1 and Snail, a downstream transcription factor that plays an important role in the epithelial-to-mesenchymal transition (EMT). S. Typhimurium also decreased the levels of E-cadherin and three tight junction proteins (ZO-1, claudin-1, and occludin). Gli1 siRNA and GANT61, a Gli1-specific inhibitor, blocked S. Typhimurium-induced Snail expression, restored the levels of E-cadherin and tight junction proteins, and prevented S. Typhimurium-increased paracellular permeability. Further study showed that Gli1 was cross-activated by the MAP and PI-3 kinase pathways. S. Typhimurium devoid of sopB, an effector of the Type 3 secretion system (T3SS) responsible for AKT activation, was unable to induce Snail expression and to decrease the expression of apical junction proteins. Our study uncovered a novel role of Gli1 in mediating the Salmonella-induced disruption of the intestinal epithelial barrier.

Staying out or Going in? The Interplay between Type 3 and Type 5 Secretion Systems in Adhesion and Invasion of Enterobacterial Pathogens

Enteric pathogens rely on a variety of toxins, adhesins and other virulence factors to cause infections. Some of the best studied pathogens belong to the Enterobacterales order; these include enteropathogenic and enterohemorrhagic Escherichia coli, Shigella spp., and the enteropathogenic Yersiniae. The pathogenesis of these organisms involves two different secretion systems, a type 3 secretion system (T3SS) and type 5 secretion systems (T5SSs). The T3SS forms a syringe-like structure spanning both bacterial membranes and the host cell plasma membrane that translocates toxic effector proteins into the cytoplasm of the host cell. T5SSs are also known as autotransporters, and they export part of their own polypeptide to the bacterial cell surface where it exerts its function, such as adhesion to host cell receptors. During infection with these enteropathogens, the T3SS and T5SS act in concert to bring about rearrangements of the host cell cytoskeleton, either to invade the cell, confer intracellular motility, evade phagocytosis or produce novel structures to shelter the bacteria. Thus, in these bacteria, not only the T3SS effectors but also T5SS proteins could be considered “cytoskeletoxins” that bring about profound alterations in host cell cytoskeletal dynamics and lead to pathogenic outcomes.

L-Arginine sensing regulates virulence gene expression and disease progression in enteric pathogens

Microbiota, host and dietary metabolites/signals compose the rich gut chemical environment, which profoundly impacts virulence of enteric pathogens. Enterohemorrhagic Escherichia coli (EHEC) engages a syringe-like machinery named type-III secretion system (T3SS) to inject effectors within host cells that lead to intestinal colonization and disease. We previously conducted a high-throughput screen to identify metabolic pathways that affect T3SS expression. Here we show that in the presence of arginine, the arginine sensor ArgR, identified through this screen, directly activates expression of the genes encoding the T3SS. Exogenously added arginine induces EHEC virulence gene expression in vitro. Congruently, a mutant deficient in arginine transport (ΔartP) had decreased virulence gene expression. ArgR also augments murine disease caused by Citrobacter rodentium, which is a murine pathogen extensively employed as a surrogate animal model for EHEC. The source of arginine sensed by C. rodentium is not dietary. At the peak of C. rodentium infection, increased arginine concentration in the colon correlated with down-regulation of the host SLC7A2 transporter. This increase in the concentration of colonic arginine promotes virulence gene expression in C. rodentium Arginine is an important modulator of the host immune response to pathogens. Here we add that arginine also directly impacts bacterial virulence. These findings suggest that a delicate balance between host and pathogen responses to arginine occur during disease progression.

Yersinia pseudotuberculosisYopH targets SKAP2-dependent and independent signaling pathways to block neutrophil antimicrobial mechanisms during infection.

Yersinia suppress neutrophil responses by using a type 3 secretion system (T3SS) to inject 6–7 Yersinia effector proteins (Yops) effectors into their cytoplasm. YopH is a tyrosine phosphatase that causes dephosphorylation of the adaptor protein SKAP2, among other targets in neutrophils. SKAP2 functions in reactive oxygen species (ROS) production, phagocytosis, and integrin-mediated migration by neutrophils. Here we identify essential neutrophil functions targeted by YopH, and investigate how the interaction between YopH and SKAP2 influence Yersinia pseudotuberculosis (Yptb) survival in tissues. The growth defect of a ΔyopH mutant was restored in mice defective in the NADPH oxidase complex, demonstrating that YopH is critical for protecting Yptb from ROS during infection. The growth of a ΔyopH mutant was partially restored in Skap2-deficient (Skap2KO) mice compared to wild-type (WT) mice, while induction of neutropenia further enhanced the growth of the ΔyopH mutant in both WT and Skap2KO mice. YopH inhibited both ROS production and degranulation triggered via integrin receptor, G-protein coupled receptor (GPCR), and Fcγ receptor (FcγR) stimulation. SKAP2 was required for integrin receptor and GPCR-mediated ROS production, but dispensable for degranulation under all conditions tested. YopH blocked SKAP2-independent FcγR-stimulated phosphorylation of the proximal signaling proteins Syk, SLP-76, and PLCγ2, and the more distal signaling protein ERK1/2, while only ERK1/2 phosphorylation was dependent on SKAP2 following integrin receptor activation. These findings reveal that YopH prevents activation of both SKAP2-dependent and -independent neutrophilic defenses, uncouple integrin- and GPCR-dependent ROS production from FcγR responses based on their SKAP2 dependency, and show that SKAP2 is not required for degranulation.