Edwardsiella tarda EscE (Orf13 Protein) Is a Type III Secretion System-Secreted Protein That Is Required for the Injection of Effectors,Secretion of Translocators,and Pathogenesis in Fish

The type III secretion system (T3SS) of Edwardsiella tarda is crucial for its intracellular survival and pathogenesis in fish. The orf13 gene (escE) of E. tarda is located 84 nucleotides (nt) upstream of esrC in the T3SS gene cluster. We found that EscE is secreted and translocated in a T3SS-dependent manner and that amino acids 2 to 15 in the N terminus were required for a completely functional T3SS in E. tarda. Deletion of escE abolished the secretion of T3SS translocators, as well as the secretion and translocation of T3SS effectors, but did not influence their intracellular protein levels in E. tarda. Complementation of the escE mutant with a secretion-incompetent EscE derivative restored the secretion of translocators and effectors. Interestingly, the effectors that were secreted and translocated were positively correlated with the EscE protein level in E. tarda. The escE mutant was attenuated in the blue gourami fish infection model, as its 50% lethal dose (LD₅₀) increased to 4 times that of the wild type. The survival rate of the escE mutant-strain-infected fish was 69%, which was much higher than that of the fish infected with the wild-type bacteria (6%). Overall, EscE represents a secreted T3SS regulator that controls effector injection and translocator secretion, thus contributing to E. tarda pathogenesis in fish. The homology of EscE within the T3SSs of other bacterial species suggests that the mechanism of secretion and translocation control used by E. tarda may be commonly used by other bacterial pathogens.     

Identification and Functional Characterization of the Novel Edwardsiella tarda Effector EseJ

Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and gastro- and extraintestinal infections in humans. The type III secretion system (T3SS) of E. tarda has been identified as a key virulence factor that contributes to pathogenesis in fish. However, little is known about the associated effectors translocated by this T3SS. In this study, by comparing the profile of secreted proteins of the wild-type PPD130/91 and its T3SS ATPase ΔesaN mutant, we identified a new effector by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. This effector consists of 1,359 amino acids, sharing high sequence similarity with Orf29/30 of E. tarda strain EIB202, and is renamed EseJ. The secretion and translocation of EseJ depend on the T3SS. A ΔeseJ mutant strain adheres to epithelioma papillosum of carp (EPC) cells 3 to 5 times more extensively than the wild-type strain does. EseJ inhibits bacterial adhesion to EPC cells from within bacterial cells. Importantly, the ΔeseJ mutant strain does not replicate efficiently in EPC cells and fails to replicate in J774A.1 macrophages. In infected J774A.1 macrophages, the ΔeseJ mutant elicits higher production of reactive oxygen species than wild-type E. tarda. The replication defect is consistent with the attenuation of the ΔeseJ mutant in the blue gourami fish model: the 50% lethal dose (LD₅₀) of the ΔeseJ mutant is 2.34 times greater than that of the wild type, and the ΔeseJ mutant is less competitive than the wild type in mixed infection. Thus, EseJ represents a novel effector that contributes to virulence by reducing bacterial adhesion to EPC cells and facilitating intracellular bacterial replication.     

EseG,an Effector of the Type III Secretion System of Edwardsiella tarda,Triggers Microtubule Destabilization

Edwardsiella tarda is a Gram-negative enteric pathogen that causes hemorrhagic septicemia in fish and both gastrointestinal and extraintestinal infections in humans. A type III secretion system (T3SS) was recently shown to contribute to pathogenesis, since deletions of various T3SS genes increased the 50% lethal dose (LD₅₀) by about 1 log unit in the blue gourami infection model. In this study, we report EseG as the first identified effector protein of T3SS. EseG shares partial homology with two Salmonella T3SS effectors (SseG and SseF) over a conserved domain (amino acid residues 142 to 192). The secretion of EseG is dependent on a functional T3SS and, in particular, requires the chaperone EscB. Experiments using TEM-1 β-lactamase as a fluorescence-based reporter showed that EseG was translocated into HeLa cells at 35°C. Fractionation of infected HeLa cells demonstrated that EseG was localized to the host membrane fraction after translocation. EseG is able to disassemble microtubule structures when overexpressed in mammalian cells. This phenotype may require a conserved motif of EseG (EseG_142-192), since truncated versions of EseG devoid of this motif lose their ability to cause microtubule destabilization. By demonstrating the function of EseG, our study contributes to the understanding of E. tarda pathogenesis. Moreover, the approach established in this study to identify type III effectors can be used to identify and characterize more type III and possible type VI effectors in Edwardsiella.Edwardsiella tarda is a Gram-negative bacterium associated with septicemia and fatal infections in many animals, including fish and humans (18, 36, 48). In humans, it causes gastrointestinal infections as well as extraintestinal infections such as myonecrosis, bacteremia, septic arthritis, and wound infections (18). Using a comparative proteomics approach, our group reported the presence of two secretion systems, namely, a type III secretion system (T3SS) and a type VI secretion system (T6SS), which are vital for E. tarda pathogenesis (44, 45, 46, 54).T3SSs are contact-dependent translocation systems that have been found in many Gram-negative bacteria of animals, commensals, and plant-symbiotic rhizobia (3, 31). They have many components, such as secretion and translocon apparatuses, effectors, regulators, and chaperones. The protein secretion machinery directs the secretion and translocation of many bacterial effectors into host cells. The concerted action of these effectors stimulates or interferes with host cellular processes, thereby dictating the terms of bacterium-host cell interactions (8). In Salmonella spp., more than 30 effectors are secreted by two different T3SSs. These effectors are involved in several diverse functions, such as forced entry into epithelial cells, intracellular replication inside vacuoles, and suppression of cellular immune responses (16, 29, 50). In Yersinia species, six Yop effectors have been identified, and they have been found to disrupt vital signaling cascades that are required for innate immunity (20, 41). T3SS effectors are generally less conserved than other components of T3SSs, and they perform unique functions adapted to a pathogen''s virulence strategy.The T3SS facilitates the survival of E. tarda in phagocytes and HEp-2 cells (19, 33, 34, 45) and contributes to virulence in vivo (45). The deletion of various single T3SS genes, such as escC, eseB, eseD, or escA, increased the 50% lethal dose (LD₅₀) by approximately 1 log unit in blue gourami (49, 55). EscC has been shown to act as the chaperone for EseB and EseD, whereas EscA is the chaperone for EseC (49, 55). EseB, EseC, and EseD, which are secreted in large quantities by E. tarda, are homologous to Salmonella sp. SseB, SseC, and SseD, respectively (45). The SseB, SseC, and SseD proteins are secreted by the T3SS of Salmonella pathogenicity island 2 (SPI-2) and predominantly assemble into complexes (SseBCD) that function as a translocon for effector proteins (32). EseB, EseC, and EseD can also form a protein complex (EseBCD) after secretion (55), suggesting that the EseBCD complex functions as a translocon component, rather than as T3SS effectors. To our knowledge, no T3SS effectors have been identified in E. tarda yet.The E. tarda T3SS has almost a full set of genes that are homologous to those of SPI-2 (45). A bioinformatics analysis of E. tarda T3SS proteins revealed that EseG bears some similarity to two Salmonella effectors, SseG and SseF. SseG is an effector of SPI-2 that targets intracellular Salmonella to the Golgi network, where the close association of bacterial cells allows Salmonella to multiply efficiently (40). SseF, another SPI-2 effector, is involved in positioning and maintaining the Salmonella-containing vacuoles (SCV) in a juxtanuclear position in infected epithelial cells (1). The translocated Salmonella effector proteins SseF and SseG interact with each other and are required for Salmonella to establish an intracellular replication niche (12).In this study, we used a modified TEM-1 β-lactamase system (TEM) to show that EseG is an E. tarda T3SS effector that is localized in the host membrane fraction after translocation. A functional study showed that EseG interacts with α-tubulin and destabilizes the microtubule through a conserved domain. The ΔeseG mutant showed slightly reduced virulence in blue gourami.     

Conserved type III secretion system exerts important roles in Chlamydia trachomatis

Upon infection, Chlamydiae alter host cellular functions in a variety of ways. Chlamydial infection prevents host cell apoptosis, induces re-organization of the actin cytoskeleton and alters host cellular signaling mechanisms. Chlamydia is among the many pathogenic Gram-negative bacteria that employ the type III secretion system (T3SS) to overcome host defenses and exploit available resources. T3SS are used by many Gram-negative bacterial pathogens to manipulate eukaryotic host cells through the delivery of effector proteins into their cytosol and membranes. T3SS is an evolutionarily refined, virulence determinant of Gram-negative bacteria where more than 20 proteins form an apparatus, generally termed injectisome, to achieve the vectorial secretion and translocation of anti-host effector proteins. This review describes challenges and recent advances that have revealed how Chlamydia trachomatis utilizes diversification to produce a conserved T3SS that exerts an important role in Chlamydia trachomatis.     

Bacterial adhesion and host cell factors leading to effector protein injection by type III secretion system

Type III secretion systems (T3SS) play a crucial role for virulence in many Gram-negative bacteria. After tight bacterial contact to host cells, the T3SS injects effector proteins into the host cells, which leads to cell invasion, tissue destruction and/or immune evasion. Over the last decade several attempts were made to characterize the host-cell interactions which precede and determine effector protein injection during infection. The development of the TEM-β-lactamase reporter was an important breakthrough to achieve this goal. By this means it was demonstrated that during infection with many Gram-negative pathogens such as Salmonella, Pseudomonas or Yersinia the main targets of T3SS are leukocytes of the myeloid lineage such as neutrophils, macrophages or dendritic cells. This is due to the recruitment of these cells to the site of infection, but also due to the specific interplay between bacterial and host cells. Comprehensive studies on Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis effector translocation show that adhesins such as Invasin (Inv), Yersinia adhesin A (YadA) and attachment and invasion locus (Ail) are critical for effector translocation. Here, mainly the complex interaction of YadA and Ail with various host cell receptor repertoires on leukocytes and the modulatory effects of serum factors direct effector translocation predominantly towards myeloid cells. The current understanding suggests that mostly protein based interactions between bacteria and host determine host cell specific effector translocation during Yersinia infection. However, for Shigella dysenteriae infection it was shown that glycan-glycan interactions can also play a critical role for the adhesion preceding effector translocation. In addition, the Shigella infection model revealed that the activation status of cells is a further criterium directing effector translocation into a distinct cell population. In this review the current understanding of the complex and species-specific interaction between bacteria and host cells leading to type III secretion is discussed.     

Edwardsiella tarda Ivy,a Lysozyme Inhibitor That Blocks the Lytic Effect of Lysozyme and Facilitates Host Infection in a Manner That Is Dependent on the Conserved Cysteine Residue

Edwardsiella tarda is a Gram-negative bacterial pathogen with a broad host range that includes fish and humans. In this study, we examined the activity and function of the lysozyme inhibitor Ivy (named Ivy_Et) identified in the pathogenic E. tarda strain TX01. Ivy_Et possesses the Ivy signature motif CKPHDC in the form of ⁸²CQPHNC⁸⁷ and contains several highly conserved residues, including a tryptophan (W55). For the purpose of virulence analysis, an isogenic TX01 mutant, TXivy, was created. TXivy bears an in-frame deletion of the ivy_Et gene. A live infection study in a turbot (Scophthalmus maximus) model showed that, compared to TX01, TXivy exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, an impaired ability to replicate in host macrophages, and decreased resistance against the bactericidal effect of host serum. To facilitate functional analysis, recombinant Ivy_Et (rIvy) and three mutant proteins, i.e., rIvyW55A, rIvyC82S, and rIvyH85D, which bear Ala, Ser, and Asp substitutions at W55, C82, and H85, respectively, were prepared. In vitro studies showed that rIvy, rIvyW55A, and rIvyH85D were able to block the lytic effect of lysozyme on a Gram-positive bacterium, whereas rIvyC82S could not do so. Likewise, rIvy, but not rIvyC82S, inhibited the serum-facilitated killing effect of lysozyme on E. tarda. In vivo analysis showed that rIvy, but not rIvyC82S, restored the lost pathogenicity of TXivy and enhanced the infectivity of TX01. Together these results indicate that Ivy_Et is a lysozyme inhibitor and a virulence factor that depends on the conserved C82 for biological activity.     

Large antibiotic-resistance plasmid of Edwardsiella tarda contributes to virulence in fish

Edwardsiella tarda, an enteric gram negative bacterium, infects a wide range of fish and causes a systemic fish disease called edwardsiellosis. E. tarda CK41, isolated from Japanese flounder diagnosed with edwardsiellosis, has exhibited a high degree of resistance to multiple antibiotics, including kanamycin, tetracycline, streptomycin, among others. As the bacterial antibiotic-resistance genes are usually contained in plasmids, we hypothesized that E. tarda CK41 may harbor one or more plasmids for antibiotic resistance. We showed the existence of plasmids in E. tarda CK41, and the size of the plasmid, designated as pCK41, was estimated to be approximately 70 kb. Escherichia coli DH5α transformed by the pCK41 plasmid exhibited an antibiotic-resistance phenotype against kanamycin (30 μg/mL), tetracycline (30 μg/mL), and streptomycin (10 μg/mL), indicating the existence of at least 3 antibiotic-resistance genes in pCK41. Through a procedure for pCK41 plasmid curing, a plasmid-cured strain, designated as E. tarda CK108, was identified, which was unable to grow in the presence of either kanamycin or tetracycline. As virulence-associated genes are occasionally encoded in bacterial plasmids, we examined the virulence of E. tarda CK108 in Japanese flounder. The virulence of plasmid-cured E. tarda CK108 was lower (survival rate 80%) than that of CK41 (20%), indicating the existence of virulence-associated genes in pCK41. The strain also appeared to be attenuated in both goldfish and zebrafish pathogenesis models. To analyze genes for antibiotic resistance and virulence in pCK41, the entire nucleotide sequences of pCK41 were determined (GenBank accession number: HQ332785). A total of 84 open reading frames (ORFs) were annotated. The pCK41 plasmid consists of potential virulence genes, transposases, plasmid maintenance genes, antibiotic-resistance genes (including kanamycin, tetracycline, and streptomycin), conjugal transfer genes, and unknown ORFs. These results suggest that pCK41 is a virulence plasmid of substantial importance in the E. tarda pathogenesis to fish.     

Essential role of the type III secretion system effector NleB in colonization of mice by Citrobacter rodentium

Attaching and effacing (A/E) pathogens are a significant cause of gastrointestinal illness in humans and animals. All A/E pathogens carry a large pathogenicity island, termed the locus for enterocyte effacement (LEE), which encodes a type III secretion system that translocates several effector proteins into host cells. To identify novel virulence determinants in A/E pathogens, we performed a signature-tagged mutagenesis screen in C57BL/6 mice by using the mouse A/E pathogen Citrobacter rodentium. Five hundred seventy-six derivatives of C. rodentium were tested in pools of 12 mutants. One attenuated mutant carried a transposon insertion in nleB, which encodes a putative effector of the LEE-encoded type III secretion system (T3SS). nleB is present in a genomic pathogenicity island that also encodes another putative effector, NleE, immediately downstream. Using translational fusions with β-lactamase (TEM-1), we showed that both NleB and NleE were translocated into host cells by the LEE-encoded T3SS of enteropathogenic Escherichia coli. In addition, deletion of the gene encoding NleB in C. rodentium resulted in reduced colonization of mice in single infections and reduced colonic hyperplasia. In contrast, the deletion of other non-LEE-encoded effector genes in C. rodentium, nleC, nleD, or nleE, had no effect on host colonization or disease. These results suggest that nleB encodes an important virulence determinant of A/E pathogens.     

Evaluation of a Yersinia pestis mutant impaired in a thermoregulated type VI-like secretion system in flea,macrophage and murine models

Type VI secretion systems (T6SSs) have been identified recently in several Gram-negative organisms and have been shown to be associated with virulence in some bacterial pathogens. A T6SS of Yersinia pestis CO92 (locus YPO0499–YPO0516) was deleted followed by investigation of the phenotype of this mutation. We observed that this T6SS locus of Y. pestis was preferentially expressed at 26 °C in comparison to 37 °C suggesting a possible role in the flea cycle. However, we found that the deletion of T6SS locus YPO0499–YPO0516 in Y. pestis CO92 had no effect on the ability of this strain to infect the oriental rat flea, Xenopsylla cheopis. Nevertheless, this mutant displayed increased intracellular numbers in macrophage-like J774.A1 cells after 20 h post-infection for bacterial cells pre-grown at 26 °C indicating that expression of this T6SS locus limited intracellular replication in macrophages. In addition, deletion of the YPO0499–YPO0516 locus reduced the uptake by macrophages of the Y. pestis mutant pre-grown at 37 °C, suggesting that this T6SS locus has phagocytosis-promoting activity. Further study of the virulence of the T6SS mutant in murine bubonic and inhalation plague models revealed no attenuation in comparison with the parental CO92 strain.     

Coxiella burnetii Type IV Secretion-Dependent Recruitment of Macrophage Autophagosomes

Coxiella burnetii is an intracellular Gram-negative bacterium that causes human Q fever, a flu-like disease that can progress to chronic, life-threatening endocarditis. In humans, C. burnetii infects alveolar macrophages and promotes phagosomal fusion with autophagosomes and lysosomes, establishing a unique parasitophorous vacuole (PV) in which to replicate. The pathogen uses a Dot/Icm type IV secretion system (T4SS) to deliver effector proteins to the host cytoplasm, where they alter cellular processes to benefit the pathogen. The T4SS is required for PV expansion and prevention of apoptosis, but little else is known about the role of the system during intracellular growth. Recent reports suggest that C. burnetii actively recruits autophagosomes to the PV to deliver nutrients to the pathogen and provide membrane for the expanding vacuole. In this study, we examined the role of the T4SS in mediating PV interactions with autophagosomes. We found that the autophagy-related proteins LC3 and p62 localized to wild-type PV but not to T4SS mutant organism-containing phagosomes in human macrophage-like cells, primary human alveolar macrophages, and Chinese hamster ovary cells. However, while lipidated LC3 levels were elevated regardless of T4SS activity, no p62 turnover was observed during C. burnetii growth in macrophages, suggesting that the pathogen recruits preformed autophagosomes. When the T4SS was activated 24 h after infection, autophagosome recruitment ensued, indicating that autophagosome interactions are dispensable for initial PV maturation to a phagolysosome-like compartment but are involved in vacuole expansion. Together, these results demonstrate that C. burnetii actively directs PV-autophagosome interactions by using the Dot/Icm T4SS.     

Involvement of an Skp-Like Protein,PGN_0300, in the Type IX Secretion System of Porphyromonas gingivalis

The oral Gram-negative anaerobic bacterium Porphyromonas gingivalis is an important pathogen involved in chronic periodontitis. Among its virulence factors, the major extracellular proteinases, Arg-gingipain and Lys-gingipain, are of interest given their abilities to degrade host proteins and process other virulence factors. Gingipains possess C-terminal domains (CTDs) and are translocated to the cell surface or into the extracellular milieu by the type IX secretion system (T9SS). Gingipains contribute to the colonial pigmentation of the bacterium on blood agar. In this study, Omp17, the PGN_0300 gene product, was found in the outer membrane fraction. A mutant lacking Omp17 did not show pigmentation on blood agar and showed reduced proteolytic activity of the gingipains. CTD-containing proteins were released from bacterial cells without cleavage of the CTDs in the omp17 mutant. Although synthesis of the anionic polysaccharide (A-LPS) was not affected in the omp17 mutant, the processing of and A-LPS modification of CTD-containing proteins was defective. PorU, a C-terminal signal peptidase that cleaves the CTDs of other CTD-containing proteins, was not detected in any membrane fraction of the omp17 mutant, suggesting that the defective maturation of CTD-containing proteins by impairment of Omp17 is partly due to loss of function of PorU. In the mouse subcutaneous infection experiment, the omp17 mutant was less virulent than the wild type. These results suggested that Omp17 is involved in P. gingivalis virulence.     

Flagellin-dependent and -independent inflammatory responses following infection by enteropathogenic Escherichia coli and Citrobacter rodentium

Enteropathogenic Escherichia coli (EPEC) and the murine pathogen Citrobacter rodentium belong to the attaching and effacing (A/E) family of bacterial pathogens. These noninvasive bacteria infect intestinal enterocytes using a type 3 secretion system (T3SS), leading to diarrheal disease and intestinal inflammation. While flagellin, the secreted product of the EPEC fliC gene, causes the release of interleukin 8 (IL-8) from epithelial cells, it is unclear whether A/E bacteria also trigger epithelial inflammatory responses that are FliC independent. The aims of this study were to characterize the FliC dependence or independence of epithelial inflammatory responses to direct infection by EPEC or C. rodentium. Following infection of Caco-2 intestinal epithelial cells by wild-type and DeltafliC EPEC, a rapid activation of several proinflammatory genes, including those encoding IL-8, monocyte chemoattractant protein 1, macrophage inflammatory protein 3alpha (MIP3alpha), and beta-defensin 2, occurred in a FliC-dependent manner. These responses were accompanied by mitogen-activated protein kinase activation, as well as the Toll-like receptor 5 (TLR5)-dependent activation of NF-kappaB. At later infection time points, a subset of these proinflammatory genes (IL-8 and MIP3alpha) was also induced in cells infected with DeltafliC EPEC. The nonmotile A/E pathogen C. rodentium also triggered similar innate responses through a TLR5-independent but partially NF-kappaB-dependent mechanism. Moreover, the EPEC FliC-independent responses were increased in the absence of the locus of enterocyte effacement-encoded T3SS, suggesting that translocated bacterial effectors suppress rather than cause the FliC-independent inflammatory response. Thus, we demonstrate that infection of intestinal epithelial cells by A/E pathogens can trigger an array of proinflammatory responses from epithelial cells through both FliC-dependent and -independent pathways, expanding our understanding of the innate epithelial response to infection by these pathogens.     

Legionella pneumophila Effector LpdA Is a Palmitoylated Phospholipase D Virulence Factor

Legionella pneumophila is a bacterial pathogen that thrives in alveolar macrophages, causing a severe pneumonia. The virulence of L. pneumophila depends on its Dot/Icm type IV secretion system (T4SS), which delivers more than 300 effector proteins into the host, where they rewire cellular signaling to establish a replication-permissive niche, the Legionella-containing vacuole (LCV). Biogenesis of the LCV requires substantial redirection of vesicle trafficking and remodeling of intracellular membranes. In order to achieve this, several T4SS effectors target regulators of membrane trafficking, while others resemble lipases. Here, we characterized LpdA, a phospholipase D effector, which was previously proposed to modulate the lipid composition of the LCV. We found that ectopically expressed LpdA was targeted to the plasma membrane and Rab4- and Rab14-containing vesicles. Subcellular targeting of LpdA required a C-terminal motif, which is posttranslationally modified by S-palmitoylation. Substrate specificity assays showed that LpdA hydrolyzed phosphatidylinositol, -inositol-3- and -4-phosphate, and phosphatidylglycerol to phosphatidic acid (PA) in vitro. In HeLa cells, LpdA generated PA at vesicles and the plasma membrane. Imaging of different phosphatidylinositol phosphate (PIP) and organelle markers revealed that while LpdA did not impact on membrane association of various PIP probes, it triggered fragmentation of the Golgi apparatus. Importantly, although LpdA is translocated inefficiently into cultured cells, an L. pneumophila ΔlpdA mutant displayed reduced replication in murine lungs, suggesting that it is a virulence factor contributing to L. pneumophila infection in vivo.     

The Type VI Secretion System Encoded in Salmonella Pathogenicity Island 19 Is Required for Salmonella enterica Serotype Gallinarum Survival within Infected Macrophages

Salmonella enterica serotype Gallinarum is the causative agent of fowl typhoid, a disease characterized by high morbidity and mortality that causes major economic losses in poultry production. We have reported that S. Gallinarum harbors a type VI secretion system (T6SS) encoded in Salmonella pathogenicity island 19 (SPI-19) that is required for efficient colonization of chicks. In the present study, we aimed to characterize the SPI-19 T6SS functionality and to investigate the mechanisms behind the phenotypes previously observed in vivo. Expression analyses revealed that SPI-19 T6SS core components are expressed and produced under in vitro bacterial growth conditions. However, secretion of the structural/secreted components Hcp1, Hcp2, and VgrG to the culture medium could not be determined, suggesting that additional signals are required for T6SS-dependent secretion of these proteins. In vitro bacterial competition assays failed to demonstrate a role for SPI-19 T6SS in interbacterial killing. In contrast, cell culture experiments with murine and avian macrophages (RAW264.7 and HD11, respectively) revealed production of a green fluorescent protein-tagged version of VgrG soon after Salmonella uptake. Furthermore, infection of RAW264.7 and HD11 macrophages with deletion mutants of SPI-19 or strains with genes encoding specific T6SS core components (clpV and vgrG) revealed that SPI-19 T6SS contributes to S. Gallinarum survival within macrophages at 20 h postuptake. SPI-19 T6SS function was not linked to Salmonella-induced cytotoxicity or cell death of infected macrophages, as has been described for other T6SS. Our data indicate that SPI-19 T6SS corresponds to a novel tool used by Salmonella to survive within host cells.     

TLR5 and Ipaf: dual sensors of bacterial flagellin in the innate immune system

The innate immune system precisely modulates the intensity of immune activation in response to infection. Flagellin is a microbe-associated molecular pattern that is present on both pathogenic and nonpathogenic bacteria. Macrophages and dendritic cells are able to determine the virulence of flagellated bacteria by sensing whether flagellin remains outside the mammalian cell, or if it gains access to the cytosol. Extracellular flagellin is detected by TLR5, which induces expression of proinflammatory cytokines, while flagellin within the cytosol of macrophages is detected through the Nod-like receptor (NLR) Ipaf, which activates caspase-1. In macrophages infected with Salmonella typhimurium or Legionella pneumophila, Ipaf becomes activated in response to flagellin that appears to be delivered to the cytosol via specific virulence factor transport systems (the SPI1 type III secretion system (T3SS) and the Dot/Icm type IV secretion system (T4SS), respectively). Thus, TLR5 responds more generally to flagellated bacteria, while Ipaf responds to bacteria that express both flagellin and virulence factors.     

The Secreted Effector Protein EspZ Is Essential for Virulence of Rabbit Enteropathogenic Escherichia coli

Attaching and effacing (A/E) pathogens adhere intimately to intestinal enterocytes and efface brush border microvilli. A key virulence strategy of A/E pathogens is the type III secretion system (T3SS)-mediated delivery of effector proteins into host cells. The secreted protein EspZ is postulated to promote enterocyte survival by regulating the T3SS and/or by modulating epithelial signaling pathways. To explore the role of EspZ in A/E pathogen virulence, we generated an isogenic espZ deletion strain (ΔespZ) and corresponding cis-complemented derivatives of rabbit enteropathogenic Escherichia coli and compared their abilities to regulate the T3SS and influence host cell survival in vitro. For virulence studies, rabbits infected with these strains were monitored for bacterial colonization, clinical signs, and intestinal tissue alterations. Consistent with data from previous reports, espZ-transfected epithelial cells were refractory to infection-dependent effector translocation. Also, the ΔespZ strain induced greater host cell death than did the parent and complemented strains. In rabbit infections, fecal ΔespZ strain levels were 10-fold lower than those of the parent strain at 1 day postinfection, while the complemented strain was recovered at intermediate levels. In contrast to the parent and complemented mutants, ΔespZ mutant fecal carriage progressively decreased on subsequent days. ΔespZ mutant-infected animals gained weight steadily over the infection period, failed to show characteristic disease symptoms, and displayed minimal infection-induced histological alterations. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining of intestinal sections revealed increased epithelial cell apoptosis on day 1 after infection with the ΔespZ strain compared to animals infected with the parent or complemented strains. Thus, EspZ-dependent host cell cytoprotection likely prevents epithelial cell death and sloughing and thereby promotes bacterial colonization.     

ExsB Is Required for Correct Assembly of the Pseudomonas aeruginosa Type III Secretion Apparatus in the Bacterial Membrane and Full Virulence In Vivo

Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa. Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon.