Search

Phosphatidylcholine (PC) is a typical eukaryotic phospholipid absent from most prokaryotes. Thus, its presence in some intracellular bacteria is intriguing as it may constitute host mimicry. The role of PC in Brucella abortus was examined by generating mutants in pcs (BApcs) and pmtA (BApmtA), which encode key enzymes of the two bacterial PC biosynthetic routes, the choline and methyl-transferase pathways. In rich medium, BApcs and the double mutant BApcspmtA but not BApmtA displayed reduced growth, increased phosphatidylethanolamine and no PC, showing that Pcs is essential for PC synthesis under these conditions. In minimal medium, the parental strain, BApcs and BApmtA showed reduced but significant amounts of PC suggesting that PmtA may also be functional. Probing with phage Tb, antibiotics, polycations and serum demonstrated that all mutants had altered envelopes. In macrophages, BApcs and BApcspmtA showed reduced ability to evade fusion with lysosomes and establish a replication niche. In mice, BApcs showed attenuation only at early times after infection, BApmtA at later stages and BApcspmtA throughout. The results suggest that Pcs and PmtA have complementary roles in vivo related to nutrient availability and that PC and the membrane properties that depend on this typical eukaryotic phospholipid are essential for Brucella virulence.

Proteases are powerful enzymes, which cleave peptide bonds, leading most of the time to irreversible fragmentation or degradation of their substrates. Therefore they control many critical cell fate decisions in eukaryotes. Bacterial pathogens exploit this power and deliver protease effectors through specialised secretion systems into host cells. Research over the past years revealed that the functions of protease effectors during infection are diverse, reflecting the lifestyles and adaptations to specific hosts; however, only a small number of peptidase families seem to have given rise to most of these protease virulence factors by the evolution of different substrate‐binding specificities, intracellular activation and subcellular targeting mechanisms. Here, we review our current knowledge about the enzymology and function of protease effectors, which Gram‐negative bacterial pathogens translocate via type III and IV secretion systems to irreversibly manipulate host processes. We highlight emerging concepts such as signalling by protease cleavage products and effector‐triggered immunity, which host cells employ to detect and defend themselves against a protease attack. Take Away: Proteases irreversibly cleave proteins to control critical cell fate decisions.Gram‐negative bacteria use type III and IV secretion systems to inject effectors.Protease effectors are integral weapons for the manipulation of host processes.Effectors evolved from few peptidase families to target diverse substrates.Effector‐triggered immunity upon proteolytic attack emerges as host defence. [ABSTRACT FROM AUTHOR]

The strategies by which intracellular pathogenic bacteria manipulate innate immunity to establish chronicity are poorly understood. Here, we show that Brucella abortus outer membrane protein Omp25 specifically binds the immune cell receptor SLAMF1 in vitro. The Omp25‐dependent engagement of SLAMF1 by B. abortus limits NF‐κB translocation in dendritic cells (DCs) with no impact on Brucella intracellular trafficking and replication. This in turn decreases pro‐inflammatory cytokine secretion and impairs DC activation. The Omp25‐SLAMF1 axis also dampens the immune response without affecting bacterial replication in vivo during the acute phase of Brucella infection in a mouse model. In contrast, at the chronic stage of infection, the Omp25/SLAMF1 engagement is essential for Brucella persistence. Interaction of a specific bacterial protein with an immune cell receptor expressed on the DC surface at the acute stage of infection is thus a powerful mechanism to support microbe settling in its replicative niche and progression to chronicity. [ABSTRACT FROM AUTHOR]

Cyclic β-1,2-D-glucans (CβG) are natural bionanopolymers present in the periplasmic space of many Proteobacteria. These molecules are sugar rings made of 17 to 25 Dglucose units linked exclusively by β-1,2-glycosidic bonds. CβG are important for environmental sensing and osmoadaptation in bacteria, but most importantly, they play key roles in complex host-cell interactions such as symbiosis, pathogenesis, and immunomodulation. In the last years, the identification and characterisation of the enzymes involved in the synthesis of CβG allowed to know in detail the steps necessary for the formation of these sugar rings. Due to its peculiar structure, CβG can complex large hydrophobic molecules, a feature possibly related to its function in the interaction with the host. The capabilities of the CβG to function as molecular boxes and to solubilise hydrophobic compounds are attractive for application in the development of drugs, in food industry, nanotechnology, and chemistry. More importantly, its excellent immunomodulatory properties led to the proposal of CβG as a new class of adjuvants for vaccine development. [ABSTRACT FROM AUTHOR]

Flagella are nanofibers that drive bacterial movement. The filaments are generally composed of thousands of tightly packed flagellin subunits with a terminal cap protein, named FliD. Here, we report that the FliD protein of the bacterial pathogen Campylobacter jejuni binds to host cells. Live-cell imaging and confocal microscopy showed initial contact of the bacteria with epithelial cells via the flagella tip. Recombinant FliD protein bound to the surface of intestinal epithelial cells in a dose-dependent fashion. Search for the FliD binding site on the host cell using cells with defined glycosylation defects indicated glycosaminoglycans as a putative target. Heparinase treatment of wild type cells and an excess of soluble heparin abolished FliD binding. Binding assays showed direct and specific binding of FliD to heparin. Addition of an excess of purified FliD or heparin reduced the attachment of viable C. jejuni to the host cells. The host cell binding domain of FliD was mapped to the central region of the protein. Overall, our results indicate that the C. jejuni flagellar tip protein FliD acts as an attachment factor that interacts with cell surface heparan sulfate glycosaminoglycan receptors. [ABSTRACT FROM AUTHOR]

The current paradigm suggests that Y ersinia enterocolitica ( Y e) adheres to host cells via the outer membrane proteins Yersinia adhesin A ( YadA) or invasin ( Inv) to facilitate injection of Yops by the type III secretion system. In this process Inv binds directly to β1 integrins of host cells while YadA may bind indirectly via extracellular matrix proteins to β1 integrins. Here we challenged this paradigm and investigated the requirements for Yop injection. We demonstrate that Inv- but not YadA-mediated adhesion depends on β1 integrin binding and activation, and that tight adhesion is a prerequisite for Yop injection. By means of novel transgenic cell lines, sh RNA approaches and RGD peptides, we found that YadA, in contrast to Inv, may use a broad host cell receptor repertoire for host cell adhesion. In the absence of β1 integrins, YadA mediates Yop injection by interaction with α V integrins in cooperation with yet unknown cofactors expressed by epithelial cells, but not fibroblasts. Electron microscopic and flow chamber studies revealed that a defined intimate contact area between Y e and host cells resulting in adhesion forces resisting shear stress is required for Yop injection. Thus, the indirect binding of YadA to a broad extracellular matrix ( ECM) binding host cell receptor repertoire of different cell types makes YadA a versatile tool to ensure Yop injection. In conclusion, given the differential expression of the outer membrane proteins Inv and YadA in the course of Y e infection and differential expression of integrins by various host cell populations, the data demonstrate that Y e is flexibly armed to accomplish Yop injection in different host cell types, a central event in its immune evasion strategy. [ABSTRACT FROM AUTHOR]

Yersinia species display a tropism for lymphoid tissues during infection, and the bacteria select innate immune cells for delivery of cytotoxic effectors by the type III secretion system. Yet, the mechanism for target cell selection remains a mystery. Here we investigate the interaction of Y ersinia pestis with murine splenocytes to identify factors that participate in the targeting process. We find that interactions with primary immune cells rely on multiple factors. First, the bacterial adhesin Ail is required for efficient targeting of neutrophils in vivo. However, Ail does not appear to directly mediate binding to a specific cell type. Instead, we find that host serum factors direct Y . pestis to specific innate immune cells, particularly neutrophils. Importantly, specificity towards neutrophils was increased in the absence of bacterial adhesins because of reduced targeting of other cell types, but this phenotype was only visible in the presence of mouse serum. Addition of antibodies against complement receptor 3 and CD14 blocked target cell selection, suggesting that a combination of host factors participate in steering bacteria towards neutrophils during plague infection. [ABSTRACT FROM AUTHOR]

A Y ersinia pseudotuberculosis ( Yptb ) murine model of lung infection was previously developed using the serotype III IP2666 NdeI strain, which robustly colonized lungs but only sporadically disseminated to the spleen and liver. We demonstrate here that a serotype Ib Yptb strain, IP32953, colonizes the lungs at higher levels and disseminates more efficiently to the spleen and liver compared with IP2666 NdeI. The role of adhesins was investigated during IP32953 lung infection by constructing isogenic Δ ail , Δ inv , Δ psaE and Δ yadA mutants. An IP32953Δ ailΔ yadA mutant initially colonized but failed to persist in the lungs and disseminate to the spleen and liver. Yptb expressing these adhesins selectively bound to and targeted neutrophils for translocation of Yops. This selective targeting was critical for virulence because persistence of the Δ ailΔ yadA mutant was restored following intranasal infection of neutropenic mice. Furthermore, Ail and YadA prevented killing by complement-mediated mechanisms during dissemination to and/or growth in the spleen and liver, but not in the lungs. Combined, these results demonstratethat Ail and YadA are critical, redundant virulence factors during lung infection, because they thwart neutrophils by directing Yop-translocation specifically into these cells. [ABSTRACT FROM AUTHOR]

Many Gram-negative pathogens utilize type 3 secretion systems ( T3 SSs) for a successful infection. The T3 SS is a large macromolecular complex which spans both bacterial membranes and delivers effector proteins into the host cell. The infection requires spatiotemporal control of diverse sets of secreted effectors and various mechanisms have evolved to regulate T3 SS in response to external stimuli. This review will describe mechanisms that may control type 3 secretion, revealing a multi-step regulatory strategy. We then propose an updated model of T3 SS that illustrates different stages of secretion and integrates the most recent structural and functional data. [ABSTRACT FROM AUTHOR]

Dendritic cells ( DCs) serve as the primers of adaptive immunity, which is indispensable for the control of the majority of infections. Interestingly, some pathogenic intracellular bacteria can subvert DC function and gain the advantage of an ineffective host immune reaction. This scenario appears to be the case particularly with so-called stealth pathogens, which are the causative agents of several under-diagnosed chronic diseases. However, there is no consensus how less explored stealth bacteria like Coxiella, Brucella and Francisella cross-talk with DCs. Therefore, the aim of this review was to explore the issue and to summarize the current knowledge regarding the interaction of above mentioned pathogens with DCs as crucial hosts from an infection strategy view. Evidence indicates that infected DCs are not sufficiently activated, do not undergo maturation and do not produce expected proinflammatory cytokines. In some cases, the infected DCs even display immunosuppressive behaviour that may be directly linked to the induction of tolerogenicity favouring pathogen survival and persistence. [ABSTRACT FROM AUTHOR]

Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopDI262P and YopDK267P poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD- YopB translocon. Mutants YopDA263P and YopDA270P had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells. [ABSTRACT FROM AUTHOR]

Summary NadA ( N eisseria meningitidis adhesin A), a meningococcal surface protein, mediates adhesion to and invasion of human cells, an activity in which host membrane proteins have been implicated. While investigating these host factors in human epithelial cells by affinity chromatography, we discovered an unanticipated interaction of NadA with heat shock protein (Hsp) 90, a molecular chaperone. The specific in vitro interaction of recombinant soluble NadA and Hsp90 was confirmed by co-immunoprecipitations, dot and far-Western blot. Intriguingly, ADP, but not ATP, was required for this association, and the Hsp90 inhibitor 17-AAG promoted complex formation. Hsp90 binding to an Escherichia coli strain used as carrier to express surface exposed NadA confirmed these results in live bacteria. We also examined RNA interference, plasmid-driven overexpression, addition of exogenous rHsp90 and 17-AAG inhibition in human epithelial cells to further elucidate the involvement of Hsp90 in NadA-mediated adhesion and invasion. Together, these data suggest an inverse correlation between the amount of host Hsp90 and the NadA adhesive/invasive phenotype. Confocal microscopy also demonstrated that meningococci interact with cellular Hsp90, a completely novel finding. Altogether our results show that variation of host Hsp90 expression or activity interferes with adhesive and invasive events driven by NadA. [ABSTRACT FROM AUTHOR]

The pathogenic bacteria Yersinia spp. contain a virulence plasmid that encodes a type III secretion system and effectors. During infection, four of the effectors target the actin cytoskeleton, crippling the phagocytic machinery in the infected cell. The remaining two effectors dampen the innate immune response by targeting important signalling pathways. Although the biochemical activity for each of these effectors is known, the mechanisms involved in their ordered secretion and delivery remain elusive. [ABSTRACT FROM AUTHOR]

Bacteria from the genus Yersinia deliver a number of effectors into host cells via type III secretion (T3S). Injected Yop effectors interfere and prevent pro-inflammatory warning signals by hijacking the host's intracellular machinery. While macrophages infected by wild-type Yersinia enterocolitica did not release mature IL-1β, macrophages infected by Y. enterocolitica deprived of all effectors released mature IL-1β. Surprisingly, macrophages infected by Y. enterocolitica deficient for secretion of all T3S proteins, including effectors and translocators, did not release mature IL-1β. Using different genetic constructs, we show that insertion of T3S translocation pores trigger activation of caspase-1, maturation of proIL-1β and release of mature IL-1β, which occurs independently of cell osmotic lysis. These data show that T3S translocation is intrinsically a pro-inflammatory phenomenon. However, in the case of Yersinia, this effect is neutralized by the action of effectors. [ABSTRACT FROM AUTHOR]

Yersinia enterocolitica (Ye) targets mouse dendritic cells (DCs) and inhibits their ability to trigger T cell activation. Here we have investigated whether Ye might interfere with antigen presentation in DCs. Infection of DCs with the Ye wild-type strain reduced OVA uptake by DCs as demonstrated by flow cytometry and confocal laser scan microscopy. In contrast, DCs infected with Yersinia outer protein P (YopP)-deficient mutant strain rapidly internalized OVA. Furthermore, transfection of DCs with YopP, but not with a cysteine protease deficient YopP-C172A mutant, reduced uptake of OVA. This finding suggests that YopP, a virulence factor of Ye, inhibits OVA uptake by DCs. By the use of MAPK inhibitors we provide evidence that YopP mediates reduction of OVA uptake by its ability to block MAPK signalling pathways in host cells. Using transferrin (Tf) as specific marker for clathrin-mediated endocytosis (CME) and lucifer yellow (LY) as specific marker for macropinocytosis (MP) we could show that YopP inhibits CME, whereas other Yops inhibit MP. In keeping with these data, activation and proliferation of OVA-specific T cells was reduced when DCs were treated with MAPK inhibitors. Together, our data demonstrate that (i) MAPK play an important role in antigen uptake by CME in DCs, and (ii) that YopP inhibits thispathway of antigen uptake in DCs, which might contribute to evasion of adaptive immunity. [ABSTRACT FROM AUTHOR]

Pathogenic Yersinia species share a type III secretion system that translocates Yop effector proteins into host cells to counteract signalling responses during infection. Two of these effectors, YopE and YopT, downregulate Rho GTPases by different mechanisms. Here, we investigate whether YopT and YopE are functionally redundant by dissecting the contribution of these two effectors to the pathogenesis of Yersinia pseudotuberculosis in a mouse infection and tissue culture model. Four days after oral infection, a YopE+T- strain and a YopE+T+ strain colonized spleens of mice at similar levels, suggesting that YopT is not required for virulence. In contrast, spleen colonization by a YopE-T- strain was significantly reduced. A YopE-T+ strain colonized spleen at levels comparable to those of the YopE+T- strain, arguing that YopT can promote virulence in the absence of YopE. Infection of HeLa cells with a YopE-T- strain expressing either YopE or YopT showed that YopE had a stronger antiphagocytic activity than YopT. Expression of YopE strongly inhibited activation of JNK, ERK and NFκB, and prevented production of IL-8; whereas YopT moderately inhibited these responses. On the other hand, pore formation was inhibited equally by YopE or YopT. In conclusion, YopE is a potent inhibitor of infection-induced signalling cascades, and YopT can only partially compensate for the loss of YopE. [ABSTRACT FROM AUTHOR]

YopE of Yersinia pseudotuberculosis inactivates three members of the small RhoGTPase family (RhoA, Rac1 and Cdc42) in vitro and mutation of a critical arginine abolishes both in vitro GTPase-activating protein (GAP) activity and cytotoxicity towards HeLa cells, and renders the pathogen avirulent in a mouse model. To understand the functional role of YopE, in vivo studies of the GAP activity in infected eukaryotic cells were conducted. Wild-type YopE inactivated Rac1 as early as 5 min after infection whereas RhoA was downregulated about 30 min after infection. No effect of YopE was found on the activation state of Cdc42 in Yersinia-infected cells. Single-amino-acid substitution mutants of YopE revealed two different phenotypes: (i) mutants with significantly lowered in vivo GAP activity towards RhoA and Rac1 displaying full virulence in mice, and (ii) avirulent mutants with wild-type in vivo GAP activity towards RhoA and Rac1. Our results show that Cdc42 is not an in vivo target for YopE and that YopE interacts preferentially with Rac1, and to a lesser extent with RhoA, during in vivo conditions. Surprisingly, we present results suggesting that these interactions are not a prerequisite to establish infection in mice. Finally, we show that avirulent yopE mutants translocate YopE in about sixfold higher amount compared with wild type. This raises the question whether YopE’s primary function is to sense the level of translocation rather than being directly involved in downregulation of the host defence. [ABSTRACT FROM AUTHOR]

Secretion by the type III pathway of Gram-negative microbes transports polypeptides into the extracellular medium or into the cytoplasm of host cells during infection. In pathogenic Yersinia spp., type III machines recognize 14 different Yop protein substrates via discrete signals genetically encoded in 7–15 codons at the 5′ portion of yop genes. Although the signals necessary and sufficient for substrate recognition of Yop proteins have been mapped, a clear mechanism on how proteins are recognized by the machinery and then initiated into the transport pathway has not yet emerged. As synonymous substitutions, mutations that alter mRNA sequence but not codon specificity, affect the function of some secretion signals, recent work with several different microbes tested the hypothesis of an RNA-encoded secretion signal for polypeptides that travel the type III pathway. This review summarizes experimental observations and mechanistic models for substrate recognition in this field. [ABSTRACT FROM AUTHOR]

Photorhabdusis an entomopathogenic bacterium belonging to the Enterobacteriaceae. The genome of the TT01 strain ofPhotorhabdus luminescenswas recently sequenced and a large number of toxin-encoding genes were found. Genomic analysis predicted the presence on the chromosome of genes encoding a type three secretion system (TTSS), the main role of which is the delivery of effector proteins directly into eukaryotic host cells. We report here the functional characterization of the TTSS. The locus identified encodes the secretion/translocation apparatus, gene expression regulators and an effector protein– LopT– homologous to theYersiniacysteine protease cytotoxin YopT. Heterologous expression inYersiniademonstrated that LopT was translocated into mammal cells in an active form, as shown by the appearance of a form of the RhoA GTPase with modified electrophoretic mobility.In vitrostudy showed that recombinant LopT was able to release RhoA and Rac from human and insect cell membrane.In vivoassays of infection of the cutwormSpodoptera littoralisand the locustLocusta migratoriawith a TT01 strain carrying a translational fusion of thelopTgene with thegfpreporter gene revealed that thelopTgene was switched on only at sites of cellular defence reactions, such as nodulation, in insects. TTSS-mutant did not induce nodule formation and underwent phagocytosis by insect macrophage cells, suggesting that the LopT effector plays an essential role in preventing phagocytosis and indicating an unexpected link between TTSS expression and the nodule reaction in insects. [ABSTRACT FROM AUTHOR]

The YadA protein ofYersinia pseudotuberculosispromotes tight adhesion and invasion into mammalian cells throughβ1-integrins. In this work, we demonstrate that YadA also triggers the production of interleukin-8 (IL-8) in host cells and we identify intracellular signal transduction mechanisms involved in YadA-initiated cell invasion and/or IL-8 synthesis. Tyrosine protein kinases, including the focal adhesion kinase (FAK) and c-Src, as well as the small GTPase Ras, were shown to play a significant role in both YadA-promoted cell processes. YadA-mediated cell contact led to autophosphorylation of FAK at position Tyr397 and induced GTP-loading of Ras. Furthermore, IL-8 production and invasion induced by YadA were strongly reduced in FAK- and c-Src-deficient cells and in cells overexpressing dominant interfering forms of FAK, c-Src or Ras. We also demonstrate that YadA activates the Ras-dependent Raf–MEK1/2–ERK1/2 pathway and mitogen-activated protein kinases (MAPKs) p38 and JNK. Moreover, inhibition of ERK1/2 by pharmacological agents or overexpression of dominant negative FAK, c-Src or Ras abrogated IL-8 release, whereas invasion remained unaffected. In contrast, actin polymerization and phosphatidylinositol 3-kinase (PI3K) activity is essential for YadA-promoted cell entry, but not for cytokine secretion. We conclude that YadA triggers FAK–Src complex formation and subsequent Ras activation, which leads to the stimulation of MAPKs-dependent IL-8 production or to PI3K-dependent invasion. [ABSTRACT FROM AUTHOR]

Type III secretion systems (TTSSs) are essential mediators of the interaction of many Gram-negative bacteria with human, animal or plant hosts. Extensive sequence and functional similarities exist between components of TTSS from bacteria as diverse as animal and plant pathogens. Recent crystal structure determinations of TTSS proteins reveal extensive structural homologies and novel structural motifs and provide a basis on which protein interaction networks start to be drawn within the TTSSs, that are consistent with and help rationalize genetic and biochemical data. Such studies, along with electron microscopy, also established common architectural design and function among the TTSSs of plant and mammalian pathogens, as well as between the TTSS injectisome and the flagellum. Recent comparative genomic analysis, bioinformatic genome mining and genome-wide functional screening have revealed an unsuspected number of newly discovered effectors, especially in plant pathogens and uncovered a wider distribution of TTSS in pathogenic, symbiotic and commensal bacteria. Functional proteomics and analysis further reveals common themes in TTSS effector functions across phylogenetic host and pathogen boundaries. Based on advances in TTSS biology, new diagnostics, crop protection and drug development applications, as well as new cell biology research tools are beginning to emerge. [ABSTRACT FROM AUTHOR]

Epithelial cells express genes whose products signal the presence of pathogenic microorganisms to the immune system. Pathogenicity factors of enteric bacteria modulate host cell gene expression. Using microarray technology we have profiled epithelial cell gene expression upon interaction with Yersinia enterocolitica. Yersinia enterocolitica wild-type and isogenic mutant strains were used to identify host genes modulated by invasin protein (Inv), which is involved in enteroinvasion, and Yersinia outer protein P (YopP) which inhibits innate immune responses. Among 22 283 probesets (14 239 unique genes), we found 193 probesets (165 genes) to be regulated by Yersinia infection. The majority of these genes were induced by Inv, whose recognition leads to expression of NF-κB-regulated factors such as cytokines and adhesion molecules. Yersinia virulence plasmid (pYV)-encoded factors counter regulated Inv-induced gene expression. Thus, YopP repressed Inv-induced NF-κB regulated genes at 2 h post infection whereas other pYV-encoded factors repressed host cell genes at 4 and 8 h post infection. Chromosomally encoded factors of Yersinia, other than Inv, induced expression of genes known to be induced by TGF-β receptor signalling. These genes were also repressed by pYV-encoded factors. Only a few host genes were exclusively induced by pYV-encoded factors. We hypothesize that some of these genes may contribute to pYV-mediated silencing of host cells. In conclusion, the data demonstrates that epithelial cells express a limited number of genes upon interaction with enteric Yersinia. Both Inv and YopP appear to modulate gene expression in order to subvert epithelial cell functions involved in innate immunity. [ABSTRACT FROM AUTHOR]

Summary Type III secretion-dependent translocation of Yop (Yersinia o uter p roteins) effector proteins into host cells is an essential virulence mechanism common to the pathogenic Yersinia species. One unique feature of this mechanism is the polarized secretion of Yops, i.e. Yops are only secreted at the site of contact with the host cell and not to the surrounding medium. In vitro , secretion occurs in Ca2+ -depleted media, a condition believed to somehow mimic cell contact. Three proteins, YopN, LcrG and TyeA have been suggested to control secretion and mutating any of these genes results in constitutive secretion. In addition, in Y. enterocolitica TyeA has been implied to be specifically required for delivery of a subset of Yop effectors into infected cells. In this work we have investigated the role of TyeA in secretion and translocation of Yop effectors by Y. pseudotuberculosis . An in frame deletion mutant of tyeA was found to be temperature-sensitive for growth and this phenotype correlated to a lowered expression of the negative regulatory element LcrQ. In medium containing Ca2+ , Yop expression was somewhat elevated compared to the wild-type strain and low levels of Yop secretion was also seen. Somewhat surprisingly, expression and secretion of Yops was lower than for the wild-type strain when the tyeA mutant was grown in Ca2+ -depleted medium. Translocation of YopE, YopH, YopJ and YopM into infected HeLa cells was significantly lower in comparison with the isogenic wild-type strain and Yop proteins could also be recovered in the tissue culture medium. This indicated that the tyeA mutant had lost the ability to translocate Yop proteins by a polarized mechanism. In order to exclude that the defect in translocation seen in the tyeA mutant was a result of lowered expression/secretion of Yops, a double lcrQ/tyeA mutant was constructed. This strain was de-repressed for Yop expression and secretion but was still impaired for... [ABSTRACT FROM AUTHOR]

Summary Animals have an immune system to fight off challenges from both viruses and bacteria. The first line of defence is innate immunity, which is composed of cells that engulf pathogens as well as cells that release potent signalling molecules to activate an inflammatory response and the adaptive immune system. Pathogenic bacteria have evolved a set of weapons, or effectors, to ensure survival in the host. Yersinia spp. use a type III secretion system to translocate these effector proteins, called Yops, into the host. This report outlines how Yops thwart the signalling machinery of the host immune system. [ABSTRACT FROM AUTHOR]

Examines the role of Rho kinase in the disturbance of endothelial barrier function by bacterial toxins and atherogenic mediators. Composition of atherogenic agonists and bacterial toxins in the endothelial barrier; Inactivation of the bacterial toxins; Investigation of the signalling networks in endothelium.

Yersinia spp. inject effector proteins (Yersinia outer proteins, Yops) into target cells via a type III secretion apparatus. The effector YopE was recently shown to possess GAP activity towards the Rho GTPases RhoA, Rac and CDC42 in vitro. To investigate the intracellular, 'in vivo' targets of YopE we generated a Yersinia enterocolitica strain [WA(pYLCR+E)] that injects 'life-like' amounts of YopE as only effector. Primary human umbilical vein endothelial cells (HUVEC) were infected with WA(pYLCR+E) and were then stimulated with: (i) bradykinin to induce actin microspikes followed by ruffles as an assay for CDC42 activity followed by CDC42 stimulated Rac activity; (ii) sphingosine-1-phosphate to form ruffles by direct Rac activation; or (iii) thrombin to generate actin stress fibres through Rho activation. In WA(pYLCR+E)-infected HUVEC microspike formation stimulated with bradykinin remained intact but the subsequent development of ruffles was abolished. Furthermore, ruffle formation after stimulation with sphingosine-1-phosphate or thrombin induced production of stress fibres was unaltered in the infected cells. These data suggest that YopE is able to inhibit Rac- but not Rho- or CDC42-regulated actin structures and, more specifically, that YopE is capable of blocking CDC42Hs dependent Rac activation but not direct Rac activation in HUVEC. This provides evidence for a considerable specificity of YopE towards selective Rac-mediated signalling pathways in primary target cells of Yersinia. [ABSTRACT FROM AUTHOR]

Microbe–macrophage interactions play a central role in the pathogenesis of many infections. Several bacterial pathogens induce apoptosis specifically in macrophages, but the mechanisms by which it occurs differ, and the resulting pathology can take different courses. Macrophage death caused by Shigella flexneri and Salmonella spp. has been shown to result in the release of pro-inflammatory cytokines. Conversely, Yersinia spp. induce apoptosis by suppressing the signalling pathways that lead to the production of tumour necrosis factor (TNF)-α, a cytokine essential for the control of this infection. It is likely that there are a variety of reasons why macrophages are particularly susceptible to pathogen-induced apoptosis. One reason may be the expression of surface receptors that recognize highly conserved bacterial components, such as lipopolysaccharide (LPS) and bacterial lipoproteins (BLPs). These receptors have recently been shown to activate pro-apoptotic signalling pathways. The roles of macrophage apoptosis in different disease processes are discussed. [ABSTRACT FROM AUTHOR]

Yersinia enterocolitica cross the intestinal epithelium via translocation through M cells, which are located in the follicle-associated epithelium (FAE) of Peyer's patches (PP). To investigate the molecular basis of this process, studies were performed using a recently developed in vitro model, in which the enterocyte-like cell line Caco-2 and PP lymphocytes are co-cultured in order to establish FAE-like structures including M cells. Here, we demonstrate that Y. enterocolitica does not adhere significantly to the apical membrane of differentiated enterocyte-like Caco-2 cells that express binding sites for Ulex europaeus agglutinin (UEA)-1. In contrast, Y. enterocolitica adhered to, and was internalized by, cells that lacked UEA-1 binding sites and displayed a disorganized brush border. These cells were considered to be converted to M-like cells. Further analysis revealed that part of these cells expressed β1 integrins at their apical surface and, as revealed by comparison of wild-type and mutant strains, interacted with invasin of Y. enterocolitica. Consistently, anti-β1 integrin antibodies significantly inhibited internalization of inv-expressing yersiniae. Experiments with Yersinia mutant strains deficient in YadA or Yop secretion revealed that these virulence factors play a minor role in this process. After internalization, yersiniae were transported within LAMP-1-negative vacuoles to, and released at, the basal surface. Internalization and transport of yersiniae was inhibited by cytochalasin D, suggesting that F-actin assembly is required for this process. These results provide direct evidence that expression of β1 integrins at the apical surface of M cells enables interaction with the invasin of Y. enterocolitica, and thereby initiates internalization and translocation of bacteria. [ABSTRACT FROM AUTHOR]