Novel T3SS effector EseK in Edwardsiella piscicida is chaperoned by EscH and EscS to express virulence

Bacterium usually utilises type III secretion systems (T3SS) to deliver effectors directly into host cells with the aids of chaperones. Hence, it is very important to identify bacterial T3SS effectors and chaperones for better understanding of host–pathogen interactions. Edwardsiella piscicida is an invasive enteric bacterium, which infects a wide range of hosts from fish to human. Given E. piscicida encodes a functional T3SS to promote infection, very few T3SS effectors and chaperones have been identified in this bacterium so far. Here, we reported that EseK is a new T3SS effector protein translocated by E. piscicida. Bioinformatic analysis indicated that escH and escS encode two putative class I T3SS chaperones. Further investigation indicated that EscH and EscS can enhance the secretion and translocation of EseK. EscH directly binds EseK through undetermined binding domains, whereas EscS binds EseK via its N‐terminal α‐helix. We also found that EseK has an N‐terminal chaperone‐binding domain, which binds EscH and EscS to form a ternary complex. Zebrafish infection experiments showed that EseK and its chaperones EscH and EscS are necessary for bacterial colonisation in zebrafish. This work identified a new T3SS effector, EseK, and its two T3SS chaperones, EscH and EscS, in E. piscicida, which enriches our knowledge of bacterial T3SS effector–chaperone interaction and contributes to our understanding of bacterial pathogenesis.     

Edwardsiella piscicida type III protein EseJ suppresses apoptosis through down regulating type 1 fimbriae,which stimulate the cleavage of caspase‐8

The type III secretion system effector EseJ plays a regulatory role inside bacteria. It suppresses the adherence of Edwardsiella piscicida (E. piscicida) to host epithelial cells by down regulating type 1 fimbriae. In this study, we observed that more macrophages infected with ΔeseJ strain of E. piscicida detached as compared with those infected with the wild‐type (WT) strain. Terminal deoxynucleotidyl transferase dUTP nick‐end labelling (TUNEL) staining and cleaved caspase‐3 examination revealed that the detachment is due to increased apoptosis, suggesting that EseJ suppresses macrophage apoptosis. However, apoptosis inhibition by EseJ is not relative to a type III secretion system (T3SS) and is not related to EseJ's translocation. Since EseJ negatively regulates type 1 fimbriae, murine J774A.1 cells were infected with ΔeseJΔfimA or ΔeseJΔfimH strains. It was demonstrated that ΔeseJ stimulates macrophage apoptosis through type 1 fimbriae. Moreover, we found that infecting J774A.1 cells with the ΔeseJ strain increased levels of cleaved caspase‐8, caspase‐9, and caspase‐3, demonstrating that EseJ inhibits apoptosis through either an extrinsic or a combination of extrinsic and intrinsic pathways. Pre‐treatment of macrophages with caspase‐8 inhibitor prior to infection with the ΔeseJ strain decreased the levels of cleaved caspase‐8, caspase‐9, and caspase‐3, indicating that the ΔeseJ strain stimulates apoptosis, mainly through an extrinsic pathway by up regulating type 1 fimbriae. Zebrafish larvae or blue gourami fish infected with the ΔeseJ strain consistently exhibited higher apoptosis than those infected with the E. piscicida WT strain or ΔeseJΔfimA strain. Taken together, we revealed that the T3SS protein EseJ of E. piscicida inhibits host apoptosis, mainly through an extrinsic pathway by down regulating type 1 fimbriae.     

Involvement of PorK,a component of the type IX secretion system,in Prevotella melaninogenica pathogenicity

Prevotella melaninogenica is a gram‐negative anaerobic commensal bacterium that resides in the human oral cavity and is isolated as a pathogen of suppurative diseases both inside and outside the mouth. However, little is known about the pathogenic factors of P. melaninogenica. The periodontal pathogens Porphyromonas gingivalis and Tanerella forsythia secrete virulence factors such as protease and bacterial cell surface proteins via a type IX secretion system (T9SS) that are involved in pathogenicity. P. melaninogenica also possesses all known orthologs of T9SS. In this study, a P. melaninogenica GAI 07411 mutant deficient in the orthologue of the T9SS‐encoding gene, porK, was constructed. Hemagglutination and biofilm formation were decreased in the porK mutant. Furthermore, following growth on skim milk‐containing medium, the diameters of the halos surrounding the porK mutant were smaller than those of the wild‐type strain, suggesting a decrease in secretion of proteases outside the bacterium. To investigate this in detail, culture supernatants of wild‐type and porK mutant strains were purified and compared by two‐dimensional electrophoresis. In the mutant strain, fewer spots were detected, indicating fewer secreted proteins. In infection experiments, the mortality rate of mice inoculated with the porK mutant strain was significantly lower than in the wild‐type strain. These results suggest that P. melaninogenica secretes potent virulence factors via the T9SS that contribute to its pathogenic ability.

     

Role of distinct type‐IV‐secretion systems and secreted effector sets in host adaptation by pathogenic Bartonella species

The α‐proteobacterial genus Bartonella comprises a large number of facultative intracellular pathogens that share a common lifestyle hallmarked by hemotrophic infection and arthropod transmission. Speciation in the four deep‐branching lineages (L1–L4) occurred by host adaptation facilitating the establishment of long lasting bacteraemia in specific mammalian reservoir host(s). Two distinct type‐IV‐secretion systems (T4SSs) acquired horizontally by different Bartonella lineages mediate essential host interactions during infection and represent key innovations for host adaptation. The Trw‐T4SS confined to the species‐rich L4 mediates host‐specific erythrocyte infection and likely has functionally replaced flagella as ancestral virulence factors implicated in erythrocyte colonisation by bartonellae of the other lineages. The VirB/VirD4‐T4SS translocates Bartonella effector proteins (Bep) into various host cell types to modulate diverse cellular and innate immune functions involved in systemic spreading of bacteria following intradermal inoculation. Independent acquisition of the virB/virD4/bep locus by L1, L3, and L4 was likely driven by arthropod vectors associated with intradermal inoculation of bacteria rather than facilitating direct access to blood. Subsequently, adaptation to colonise specific niches in the new host has shaped the evolution of complex species‐specific Bep repertoires. This diversification of the virulence factor repertoire of Bartonella spp. represents a remarkable example for parallel evolution of host adaptation.     

The cell death response to enteropathogenic Escherichia coli infection

Given the critical roles of inflammation and programmed cell death in fighting infection, it is not surprising that many bacterial pathogens have evolved strategies to inactivate these defences. The causative agent of infant diarrhoea, enteropathogenic Escherichia coli (EPEC), is an extracellular, intestinal pathogen that blocks both inflammation and programmed cell death. EPEC attaches to enterocytes, remains in the gut lumen and utilizes a type III secretion system (T3SS) to inject multiple virulence effector proteins directly into the infected cell, many of which subvert host antimicrobial processes through the disruption of signalling pathways. Recently, T3SS effector proteins from EPEC have been identified that inhibit death receptor‐induced apoptosis. Here we review the mechanisms used by EPEC T3SS effectors to manipulate apoptosis and promote host cell survival and discuss the role of these activities during infection.     

Interfacial amino acids support Spa47 oligomerization and shigella type three secretion system activation

Like many Gram-negative pathogens, Shigella rely on a type three secretion system (T3SS) for injection of effector proteins directly into eukaryotic host cells to initiate and sustain infection. Protein secretion through the needle-like type three secretion apparatus (T3SA) requires ATP hydrolysis by the T3SS ATPase Spa47, making it a likely target for in vivo regulation of T3SS activity and an attractive target for small molecule therapeutics against shigellosis. Here, we developed a model of an activated Spa47 homo-hexamer, identifying two distinct regions at each protomer interface that we hypothesized to provide intermolecular interactions supporting Spa47 oligomerization and enzymatic activation. Mutational analysis and a series of high-resolution crystal structures confirm the importance of these residues, as many of the engineered mutants are unable to form oligomers and efficiently hydrolyze ATP in vitro. Furthermore, in vivo evaluation of Shigella virulence phenotype uncovered a strong correlation between T3SS effector protein secretion, host cell membrane disruption, and cellular invasion by the tested mutant strains, suggesting that perturbation of the identified interfacial residues/interactions influences Spa47 activity through preventing oligomer formation, which in turn regulates Shigella virulence. The most impactful mutations are observed within the conserved Site 2 interface where the native residues support oligomerization and likely contribute to a complex hydrogen bonding network that organizes the active site and supports catalysis. The critical reliance on these conserved residues suggests that aspects of T3SS regulation may also be conserved, providing promise for the development of a cross-species therapeutic that broadly targets T3SS ATPase oligomerization and activation.     

Chromobacterium pathogenicity island 1 type III secretion system is a major virulence determinant for Chromobacterium violaceum‐induced cell death in hepatocytes

Chromobacterium violaceum is a Gram‐negative bacterium that causes fatal septicaemia in humans and animals. C. violaceum ATCC 12472 possesses genes associated with two distinct type III secretion systems (T3SSs). One of these systems is encoded by Chromobacterium pathogenicity islands 1 and 1a (Cpi‐1/‐1a), another is encoded by Chromobacterium pathogenicity island 2 (Cpi‐2). Here we show that C. violaceum causes fulminant hepatitis in a mouse infection model, and Cpi‐1/‐1a‐encoded T3SS is required for its virulence. In addition, using C. violaceum strains with defined mutations in the genes that encode the Cpi‐1/‐1a or Cpi‐2 locus in combination with cultured mammalian cell lines, we found that C. violaceum is able to induce cytotoxicity in a Cpi‐1/‐1a‐dependent manner. Characterization of Chromobacterium‐induced cytotoxicity revealed that cell lysis by C. violaceum infection involves the formation of pore structures on the host cell membrane, as demonstrated by protection by cytotoxicity in the presence of osmoprotectants. Finally, we demonstrated that CipB, a Cpi‐1/‐1a effector, is implicated in translocator‐mediated pore formation and the ability of CipB to form a pore is essential for Chromobacterium‐induced cytotoxicity. These results strongly suggest that Cpi‐1/‐1a‐encoded T3SS is a virulence determinant that causes fatal infection by the induction of cell death in hepatocytes.     

Identification of a regulatory cascade controlling Type III Secretion System 3 gene expression in Burkholderia pseudomallei

A major and critical virulence determinant of many Gram‐negative bacterial pathogens is the Type III Secretion Systems (T3SS). T3SS3 in Burkholderia pseudomallei is critical for bacterial virulence in mammalian infection models but its regulation is unknown. B. pseudomallei is the causative agent of melioidosis, a potentially fatal disease endemic in Southeast Asia and northern Australia. While screening for bacterial transposon mutants with a defective T3SS function, we discovered a TetR family regulator (bspR) responsible for the control of T3SS3 gene expression. The bspR mutant exhibited significant virulence attenuation in mice. BspR acts through BprP, a novel transmembrane regulator located adjacent to the currently delineated T3SS3 region. BprP in turn regulates the expression of structural and secretion components of T3SS3 and the AraC family regulator bsaN. BsaN and BicA likely form a complex to regulate the expression of T3SS3 effectors and other regulators which in turn affect the expression of Type VI Secretion Systems (T6SS). The complete delineation of the bspR initiated T3SS regulatory cascade not only contributes to the understanding of B. pseudomallei pathogenesis but also provides an important example of how bacterial pathogens could co‐opt and integrate various regulatory motifs to form a new regulatory network adapted for its own purposes.     

Syntaxin 3 SPI-2 dependent crosstalk facilitates the division of Salmonella containing vacuole

Intracellular membrane fusion is mediated by membrane-bridging complexes of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SNARE proteins are one of the key players in vesicular transport. Several reports shed light on intracellular bacteria modulating host SNARE machinery to establish infection successfully. The critical SNAREs in macrophages responsible for phagosome maturation are Syntaxin 3 (STX3) and Syntaxin 4 (STX4). Reports also suggest that Salmonella actively modulates its vacuole membrane composition to escape lysosomal fusion. Salmonella containing vacuole (SCV) harbours recycling endosomal SNARE Syntaxin 12 (STX12). However, the role of host SNAREs in SCV biogenesis and pathogenesis remains unclear. Upon knockdown of STX3, we observed a reduction in bacterial proliferation, which is concomitantly restored upon the overexpression of STX3. Live-cell imaging of Salmonella-infected cells showed that STX3 localises to the SCV membranes and thus might help in the fusion of SCV with intracellular vesicles to acquire membrane for its division. We also found the interaction STX3-SCV was abrogated when we infected with SPI-2 encoded Type 3 secretion system (T3SS) apparatus mutant (STM ∆ssaV) but not with SPI-1 encoded T3SS apparatus mutant (STM ∆invC). These observations were also consistent in the mice model of Salmonella infection. Together, these results shed light on the effector molecules secreted through T3SS encoded by SPI-2, possibly involved in interaction with host SNARE STX3, which is essential to maintain the division of Salmonella in SCV and help to maintain a single bacterium per vacuole.     

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.     

Roles of mitogen-activated protein kinase pathways during <Emphasis Type="Italic">Escherichia coli-</Emphasis>induced apoptosis in U937 cells

Escherichia coli (E. coli) infections play an important and growing role in the clinic. In the present study, we investigated the involvement of members of the mitogen-activated protein kinase (MAPK) superfamily, including extracellular signal-regulated kinase (ERK), c-Jun NH₂-terminal kinase (JNK) and p38 MAPK, and caspase-3 and 9 activity in E. coli-induced apoptosis in human U937 cells. We found that E. coli induces apoptosis in U937 cell lines in a dose- and time-dependent manner, p38 MAPK and JNK were activated after 10 min of infection with E. coli. In contrast, ERK1/2 was down-regulated in a time-dependent manner. The levels of total (phosphorylation state-independent) p38 MAPK, JNK and ERK1/2 did not change in E. coli-infected U937 cells at all times examined. Moreover, exposure of U937 cells to E. coli led to caspase-3 and 9 activity. For the evaluation of the role of MAPKs, PD98059, SB203580 and SP600125 were used as MAPKs inhibitors for ERK1/2, p38 MAPK and JNK. Inhibition of ERK1/2 with PD98059 caused further enhancement in apoptosis and caspase-3 and 9 activity, while a selective p38 MAPK inhibitor, SB203580 and JNK inhibitor, SP600125 significantly inhibited E. coli-induced apoptosis and caspase-3 and 9 activity in U937 cells. The results were further confirmed by the observation that the caspase inhibitors Z-DEVD-FMK and Z-LEHD-FMK blocked E. coli-induced U937 apoptosis. Taken together, we have shown that E. coli increase p38 MAPK and JNK and decrease ERK1/2 phosphorylation and increase caspase-3 and 9 activity in U937 cells.     

The noncanonical type III secretion system of Xanthomonas translucens pv. graminis is essential for forage grass infection

Xanthomonas translucens pv. graminis (Xtg) is a gammaproteobacterium that causes bacterial wilt on a wide range of forage grasses. To gain insight into the host–pathogen interaction and to identify the virulence factors of Xtg, we compared a draft genome sequence of one isolate (Xtg29) with other Xanthomonas spp. with sequenced genomes. The type III secretion system (T3SS) encoding a protein transport system for type III effector (T3E) proteins represents one of the most important virulence factors of Xanthomonas spp. In contrast with other Xanthomonas spp. assigned to clade 1 on the basis of phylogenetic analyses, we identified an hrp (hypersensitive response and pathogenicity) gene cluster encoding T3SS components and a representative set of 35 genes encoding putative T3Es in the genome of Xtg29. The T3SS was shown to be divergent from the hrp gene clusters of other sequenced Xanthomonas spp. Xtg mutants deficient in T3SS regulating and structural genes were constructed to clarify the role of the T3SS in forage grass colonization. Italian ryegrass infection with these mutants led to significantly reduced symptoms (P < 0.05) relative to plants infected with the wild‐type strain. This showed that the T3SS is required for symptom evocation. In planta multiplication of the T3SS mutants was not impaired significantly relative to the wild‐type, indicating that the T3SS is not required for survival until 14 days post‐infection. This study represents the first major step to understanding the bacterial colonization strategies deployed by Xtg and may assist in the identification of resistance (R) genes in forage grasses.     

A revised model for the role of GacS/GacA in regulating type III secretion by Pseudomonas syringae pv. tomato DC3000

GacS/GacA is a conserved two-component system that functions as a master regulator of virulence-associated traits in many bacterial pathogens, including Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA-regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogen Pseudomonas aeruginosa, GacS/GacA negatively regulates the expression of type III secretion system (T3SS)-encoding genes. However, in the plant pathogenic bacterium Pseudomonas syringae, strain-to-strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re-evaluated the function of GacA in P. syringae pv. tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence inside Arabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface-inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulating P. syringae virulence.     

Type VI secretion system MIX‐effectors carry both antibacterial and anti‐eukaryotic activities

Most type VI secretion systems (T6SSs) described to date are protein delivery apparatuses that mediate bactericidal activities. Several T6SSs were also reported to mediate virulence activities, although only few anti‐eukaryotic effectors have been described. Here, we identify three T6SSs in the marine bacterium Vibrio proteolyticus and show that T6SS1 mediates bactericidal activities under warm marine‐like conditions. Using comparative proteomics, we find nine potential T6SS1 effectors, five of which belong to the polymorphic MIX‐effector class. Remarkably, in addition to six predicted bactericidal effectors, the T6SS1 secretome includes three putative anti‐eukaryotic effectors. One of these is a MIX‐effector containing a cytotoxic necrotizing factor 1 domain. We demonstrate that T6SS1 can use this MIX‐effector to target phagocytic cells, resulting in morphological changes and actin cytoskeleton rearrangements. In conclusion, the V. proteolyticus T6SS1, a system homologous to one found in pathogenic vibrios, uses a suite of polymorphic effectors that target both bacteria and eukaryotic neighbors.     

The Vibrio parahaemolyticus effector VopC mediates Cdc42‐dependent invasion of cultured cells but is not required for pathogenicity in an animal model of infection

Vibrio parahaemolyticus is a Gram‐negative marine bacterium that causes acute gastroenteritis in humans. The virulence of V. parahaemolyticus is dependent upon a type III secretion system (T3SS2). One effector for T3SS2, VopC, is a homologue of the catalytic domain of cytotoxic necrotizing factor (CNF), and was recently reported to be a Rho family GTPase activator and to be linked to internalization of V. parahaemolyticus by non‐phagocytic cultured cells. Here, we provide direct evidence that VopC deamidates Rac1 and CDC42, but not RhoA, in vivo. Our results alsosuggest that VopC, through its activation of Rac1, contributes to formation of actin stress fibres in infected cells. Invasion of host cells, which occurs at a low frequency, does not seem linked to Rac1 activation, but instead appears to require CDC42. Finally, using an infant rabbit model of V. parahaemolyticus infection, we show that the virulence of V. parahaemolyticus is not dependent upon VopC‐mediated invasion. Genetic inactivation of VopC did not impair intestinal colonization nor reduce signs of disease, including fluid accumulation, diarrhoea and tissue destruction. Thus, although VopC can promote host cell invasion, such internalization is not a critical step of the disease process, consistent with the traditional view of V. parahaemolyticus as an extracellular pathogen.