YopK regulates the Yersinia pestis type III secretion system from within host cells

The pathogenic Yersinia species share a conserved type III secretion system, which delivers cytotoxic effectors known as Yops into target mammalian cells. In all three species, YopK (also called YopQ) plays an important role in regulating this process. In cell culture infections, yopK mutants inject higher levels of Yops, leading to increase cytotoxicity; however, in vivo the same mutants are highly attenuated. In this work, we investigate the mechanism behind this paradox. Using a β-lactamase reporter assay to directly measure the effect of YopK on translocation, we demonstrated that YopK controls the rate of Yop injection. Furthermore, we find that YopK cannot regulate effector Yop translocation from within the bacterial cytosol. YopE is also injected into host cells and was previously shown to contribute to regulation of the injectisome. In this work we show that YopK and YopE work at different steps to regulate Yop injection, with YopK functioning independently of YopE. Finally, by expressing YopK within tissue culture cells, we confirm that YopK regulates translocation from inside the host cell, and we show that cells pre-loaded with YopK are resistant to Yop injection. These results suggest a novel role for YopK in controlling the Yersinia type III secretion system.     

Interaction of Salmonella with host cells through the centisome 63 type III secretion system

Salmonella enterica engages host cells in a complex two-way biochemical interaction that results in a variety of responses from both the bacteria and the host cell. Central to this interaction is the function of a type III protein secretion system that delivers effector proteins into the host cell. During the past year we have seen major advances in our knowledge of both the bacterial determinants and the host-signal transduction pathways involved in these interactions. A coherent picture of the mechanisms by which Salmonella engages the host cell is now beginning to emerge.     

The regulatory network of Vibrio parahaemolyticus type VI secretion system 1

Type VI secretion systems (T6SSs) are widespread, tightly regulated, protein delivery apparatuses used by Gram-negative bacteria to outcompete their neighbours. The pathogen, Vibrio parahaemolyticus, encodes two T6SSs. These T6SSs are differentially regulated by external conditions. T6SS1, an antibacterial system predominantly found in pathogenic isolates, requires warm marine-like conditions and surface sensing for activation. The regulatory network that governs this activation is not well understood. In this work, we devised a screening methodology that allows us to easily monitor the outcome of bacterial competitions and thus to identify mutants that are defective in T6SS1-mediated bacterial killing. The methodology, termed Ba cterial Co mpetition F luorescence (BaCoF), relies on detection of a fluorescent signal as an indicator of the survival and growth of a T6SS-sensitive, GFP-expressing prey that has been co-cultured with mutants derived from a T6SS⁺ attacker of interest. Using BaCoF, we screened a random transposon insertion mutant library and identified genes required for V. parahaemolyticus T6SS1 activation, among them TfoY and Tmk. We used epistasis experiments to determine the relationships between the newly identified components and other regulators that were previously described. Thus, we present here a detailed biological understanding of the T6SS1 regulatory network.     

The cost of virulence: retarded growth of Salmonella Typhimurium cells expressing type III secretion system 1

Virulence factors generally enhance a pathogen's fitness and thereby foster transmission. However, most studies of pathogen fitness have been performed by averaging the phenotypes over large populations. Here, we have analyzed the fitness costs of virulence factor expression by Salmonella enterica subspecies I serovar Typhimurium in simple culture experiments. The type III secretion system ttss-1, a cardinal virulence factor for eliciting Salmonella diarrhea, is expressed by just a fraction of the S. Typhimurium population, yielding a mixture of cells that either express ttss-1 (TTSS-1(+) phenotype) or not (TTSS-1(-) phenotype). Here, we studied in vitro the TTSS-1(+) phenotype at the single cell level using fluorescent protein reporters. The regulator hilA controlled the fraction of TTSS-1+ individuals and their ttss-1 expression level. Strikingly, cells of the TTSS-1(+) phenotype grew slower than cells of the TTSS-1(-) phenotype. The growth retardation was at least partially attributable to the expression of TTSS-1 effector and/or translocon proteins. In spite of this growth penalty, the TTSS-1(+) subpopulation increased from <10% to approx. 60% during the late logarithmic growth phase of an LB batch culture. This was attributable to an increasing initiation rate of ttss-1 expression, in response to environmental cues accumulating during this growth phase, as shown by experimental data and mathematical modeling. Finally, hilA and hilD mutants, which form only fast-growing TTSS-1(-) cells, outcompeted wild type S. Typhimurium in mixed cultures. Our data demonstrated that virulence factor expression imposes a growth penalty in a non-host environment. This raises important questions about compensating mechanisms during host infection which ensure successful propagation of the genotype.     

Horizontal gene transfer of a genetic island encoding a type III secretion system distributed in Vibrio cholerae

Twelve Vibrio cholerae isolates with genes for a type III secretion system (T3SS) were detected among 110 environmental and 14 clinical isolates. T3SS‐related genes were distributed among the various serogroups and pulsed‐field gel electrophoresis of NotI‐digested genomes showed genetic diversity in these strains. However, the restriction fragment length polymorphism profiles of the T3SS‐related genes had similar patterns. Additionally, naturally competent T3SS‐negative V. cholerae incorporated the ca. 47 kb gene cluster of T3SS, which had been integrated into a site on the chromosome by recombination. Therefore, it is suggested that horizontal gene transfer of T3SS‐related genes occurs among V. cholerae in natural ecosystems.     

Binding to type I collagen is essential for the infectivity of Vibrio parahaemolyticus to host cells

Vibrio parahaemolyticus is a globally present marine bacterium that often leads to acute gastroenteritis. Two type III secretion systems (T3SSs), T3SS1 and T3SS2, are important for host infection. Type I collagen is a component of the extracellular matrix and is abundant in the small intestine. However, whether type I collagen serves as the cellular receptor for V. parahaemolyticus infection of host cells remains enigmatic. In this study, we discovered that type I collagen is not only important for the attachment of V. parahaemolyticus to host cells but is also involved in T3SS1‐dependent cytotoxicity. In addition, 2 virulence factors, MAM7 and VpadF enable V. parahaemolyticus to interact with type I collagen and mediate T3SS2‐dependent host cell invasion. Type I collagen, the collagen receptor α1 integrin, and its downstream factor phosphatidylinositol 3‐kinase (PI3K) are responsible for V. parahaemolyticus invasion of host cells. Further biochemical studies revealed that VpadF mainly relies on the C‐terminal region for type I collagen binding and MAM7 relies on mce domains to bind to type I collagen. As MAM7 and/or VpadF homologues are widely distributed in the genus Vibrio, we propose that Vibrios have evolved a unique strategy to infect host cells by binding to type I collagen.     

Identification and characterization of a type III secretion-associated chaperone in the type III secretion system 1 of Vibrio parahaemolyticus

Vibrio parahaemolyticus causes human gastroenteritis. Genomic sequencing of this organism has revealed that it has two sets of type III secretion systems, T3SS1 and T3SS2, both of which are important for its pathogenicity. However, the mechanism of protein secretion via T3SSs is unknown. A characteristic of many effectors is that they require specific chaperones for efficient delivery via T3SSs; however, no chaperone has been experimentally identified in the T3SSs of V. parahaemolyticus . In this study, we identified candidate T3SS1-associated chaperones from genomic sequence data and examined their roles in effector secretion/translocation and binding to their cognate substrates. From these experiments, we concluded that there is a T3S-associated chaperone, VecA, for a cytotoxic T3SS1-dependent effector, VepA. Further analysis using pulldown and secretion assays characterized the chaperone-binding domain encompassing the first 30–100 amino acids and an amino terminal secretion signal encompassing the first 5–20 amino acids on VepA. These findings will provide a strategy to clarify how the T3SS1 of V. parahaemolyticus secretes its specific effectors.     

Induction of cytotoxic T lymphocytes specific for a syngeneic tumor expressing the E6 oncoprotein of human papillomavirus type 16.

Human papillomavirus (HPV) type 16 has been implicated in the etiology of cervical carcinomas, but it is unknown whether HPV-specific immunity can function in controlling the growth of HPV-associated carcinomas. We previously demonstrated that CD8+ T lymphocytes can inhibit the in vivo outgrowth of murine tumor cells transfected with the HPV-16 E7 gene and have now established a murine model to study the CTL responses to the E6 oncoprotein of HPV-16. Immunization of C3H/HeN mice with syngeneic fibroblasts expressing a transfected HPV-16 E6 gene induced regression of transplanted tumors expressing this gene. Populations of CTL isolated from the spleens of mice whose E6+ tumors had regressed were shown to specifically lyse E6+ target cells. The cytolytic activity was mediated by CD8+ CTL in a MHC restricted pattern. These data and our previous findings with transfected tumor cells expressing the E7 gene, support the conclusion that tumor cells associated with HPV-16 can be inhibited by CTL specific for molecules encoded by the HPV-16 E6 and E7 genes.     

Global impact of Salmonella type III secretion effector SteA on host cells

Salmonella enterica is a Gram-negative bacterium that causes gastroenteritis, bacteremia and typhoid fever in several animal species including humans. Its virulence is greatly dependent on two type III secretion systems, encoded in pathogenicity islands 1 and 2. These systems translocate proteins called effectors into eukaryotic host cell. Effectors interfere with host signal transduction pathways to allow the internalization of pathogens and their survival and proliferation inside vacuoles. SteA is one of the few Salmonella effectors that are substrates of both type III secretion systems. Here, we used gene arrays and bioinformatics analysis to study the genetic response of human epithelial cells to SteA. We found that constitutive synthesis of SteA in HeLa cells leads to induction of genes related to extracellular matrix organization and regulation of cell proliferation and serine/threonine kinase signaling pathways. SteA also causes repression of genes related to immune processes and regulation of purine nucleotide synthesis and pathway-restricted SMAD protein phosphorylation. In addition, a cell biology approach revealed that epithelial cells expressing steA show altered cell morphology, and decreased cytotoxicity, cell–cell adhesion and migration.     

Modulation of host immune responses, induction of apoptosis and inhibition of NF-kappaB activation by the Bordetella type III secretion system

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild-type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long-term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti- Bordetella antibodies upon infection compared with wild-type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID-beige mice. These observations suggest that type III-secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF-κB into large complexes in the cytoplasm. NF-κB aggregation was dependent on type III secretion and aggregated NF-κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF-κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.     

Efficient secretion of small proteins in mammalian cells relies on Sec62-dependent posttranslational translocation

Mammalian cells secrete a large number of small proteins, but their mode of translocation into the endoplasmic reticulum is not fully understood. Cotranslational translocation was expected to be inefficient due to the small time window for signal sequence recognition by the signal recognition particle (SRP). Impairing the SRP pathway and reducing cellular levels of the translocon component Sec62 by RNA interference, we found an alternate, Sec62-dependent translocation path in mammalian cells required for the efficient translocation of small proteins with N-terminal signal sequences. The Sec62-dependent translocation occurs posttranslationally via the Sec61 translocon and requires ATP. We classified preproteins into three groups: 1) those that comprise ≤100 amino acids are strongly dependent on Sec62 for efficient translocation; 2) those in the size range of 120-160 amino acids use the SRP pathway, albeit inefficiently, and therefore rely on Sec62 for efficient translocation; and 3) those larger than 160 amino acids depend on the SRP pathway to preserve a transient translocation competence independent of Sec62. Thus, unlike in yeast, the Sec62-dependent translocation pathway in mammalian cells serves mainly as a fail-safe mechanism to ensure efficient secretion of small proteins and provides cells with an opportunity to regulate secretion of small proteins independent of the SRP pathway.     

Purification of essentially RNA free plasmid DNA using a modified Escherichia coli host strain expressing ribonuclease A

Regulatory agencies have stringent requirements for the large-scale production of biotherapeutics. One of the difficulties associated with the manufacture of plasmid DNA for gene therapy is the removal of the host cell-related impurity RNA following cell lysis. We have constructed a modified Escherichia coli JM107 plasmid host (JMRNaseA), containing a bovine pancreatic ribonuclease (RNaseA) expression cassette, integrated into the host chromosome at the dif locus. The expressed RNaseA is translocated to the periplasm of the cell, and is released during primary plasmid extraction by alkaline lysis. The RNaseA protein is stable throughout incubation at high pH ( approximately 12-12.5), and subsequently acts to hydrolyse host cell RNA present in the neutralised solution following alkaline lysis. Results with this strain harbouring pUC18, and a 2.4 kb pUC18DeltalacO, show that sufficient levels of ribonuclease (RNase) activity are produced to hydrolyse the bulk of the host RNA. This provides a suitable methodology for the removal of RNA, whilst avoiding the addition of exogenous animal sourced RNase and its associated regulatory requirements.     

Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells

Background  

Chlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (Innate Pharmaceuticals AB) were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness.     

Structural and functional studies of EpsC, a crucial component of the type 2 secretion system from Vibrio cholerae

The type 2 secretion system (T2SS) occurring in Gram-negative bacteria is composed of 12-15 different proteins which form large assemblies spanning two membranes and secreting several virulence factors in folded state across the outer membrane. The T2SS component EpsC of Vibrio cholerae plays an important role in this machinery. While anchored in the inner membrane, by far the largest part of EpsC is periplasmic, containing a so-called homology region (HR) domain and a PDZ domain. Here we report studies on the structure and function of both periplasmic domains of EpsC. The crystal structures of two variants of the PDZ domain of EpsC from V. cholerae were determined at better than 2 A resolution. Compared to the short variant, the longer variant contains an additional N-terminal helix, and reveals a significant difference in the position of helix alphaB with respect to the beta-sheet. Both our structures show that the PDZ domain of EpsC adopts a more open form than in previously reported structures of other PDZ domains. Most interestingly, in the crystals of the short EpsC-PDZ domain the peptide binding groove interacts with an alpha-helix from a neighboring subunit burying approximately 921 A2 solvent accessible surface. This makes it possible that the PDZ domain of this bacterial protein binds proteins in a manner which is altogether different from that seen in any other PDZ domain so far. We also determined that the HR domain of EpsC is primarily responsible for the interaction with the secretin EpsD, while the PDZ is not, or much less, so. This new finding, together with studies of others, leads to the suggestion that the PDZ domain of EpsC may interact with exoproteins to be secreted while the HR domain plays a key role in linking the inner-membrane sub-complex of the T2SS in V. cholerae to the outer membrane secretin.     

Genetic analysis of anti-amoebae and anti-bacterial activities of the type VI secretion system in Vibrio cholerae

A type VI secretion system (T6SS) was recently shown to be required for full virulence of Vibrio cholerae O37 serogroup strain V52. In this study, we systematically mutagenized each individual gene in T6SS locus and characterized their functions based on expression and secretion of the hemolysin co-regulated protein (Hcp), virulence towards amoebae of Dictyostelium discoideum and killing of Escherichia coli bacterial cells. We group the 17 proteins characterized in the T6SS locus into four categories: twelve (VipA, VipB, VCA0109-VCA0115, ClpV, VCA0119, and VasK) are essential for Hcp secretion and bacterial virulence, and thus likely function as structural components of the apparatus; two (VasH and VCA0122) are regulators that are required for T6SS gene expression and virulence; another two, VCA0121 and valine-glycine repeat protein G 3 (VgrG-3), are not essential for Hcp expression, secretion or bacterial virulence, and their functions are unknown; the last group is represented by VCA0118, which is not required for Hcp expression or secretion but still plays a role in both amoebae and bacterial killing and may therefore be an effector protein. We also showed that the clpV gene product is required for Dictyostelium virulence but is less important for killing E. coli. In addition, one vgrG gene (vgrG-2) outside of the T6SS gene cluster was required for bacterial killing but another (vgrG-1) was not. However, a bacterial killing defect was observed when vgrG-1 and vgrG-3 were both deleted. Several genes encoded in the same putative operon as vgrG-1 and vgrG-2 also contribute to virulence toward Dictyostelium but have a smaller effect on bacterial killing. Our results provide new insights into the functional requirements of V. cholerae's T6SS in the context of secretion as well as killing of bacterial and eukaryotic phagocytic cells.     

VopB1 and VopD1 are essential for translocation of type III secretion system 1 effectors of Vibrio parahaemolyticus

Vibrio parahaemolyticus is a pathogenic Vibrio species that causes food-borne acute gastroenteritis, often related to the consumption of raw or undercooked seafood. Vibrio parahaemolyticus has 2 type III secretion systems (T3SS1 and T3SS2). Here, we demonstrate that VP1657 (VopB1) and VP1656 (VopD1), which share sequence similarity with Pseudomonas genes popB (38%) and popD (36%), respectively, are essential for translocation of T3SS1 effectors into host cells. A VP1680CyaA fusion reporter system was constructed to observe effector translocation. Using this reporter assay we showed that the VopB1 and VopD1 deletion strains were unable to translocate VP1680 to host cell but that the secretion of VP1680 into the culture medium was not affected. VopB1 or VopD1 deletion strains did not enhance cytotoxicity and failed to activate mitogen-activated protein kinases and secretion of interleukin-8, which depend on VP1680. Thus, we conclude that VopB1 and VopD1 are essential components of the translocon. To target VopB1 and VopD1 may have therapeutic potential for the treatment or prevention in V.?parahaemolyticus infection.