Type-three Secretion Research Articles

Pseudomonas aeruginosa type three-secretion system as a target for development of antivirulence drugs

Pseudomonas aeruginosa type three-secretion system as a target for development of antivirulence drugs

Development of a multiplex PCR assay for the detection of major virulence genes in Vibrio cholerae including non-O1 and non-O139 serogroups

Vibrio cholerae strains producing cholera toxin (CT) and toxin co-regulated pilus (TCP) and belonging to O1 and O139 serogroups are responsible for cholera. However, non-CT producing V. cholerae from non-O1/non-O139 serogroups have been increasingly isolated from diarrheal stools and extra-intestinal infections. In this study, we have developed a multiplex PCR for the simultaneous detection of heat-stable enterotoxin (stn), type three-secretion system (vopF), and cholix toxin (chxA), along with CT (ctx) in V. cholerae strains. As other species from genus Vibrio carries homologous virulence genes, V. cholerae specific ompW was also included to differentiate V. cholerae from other vibrios. This assay was 100% specific and sensitive, and could detect homologous virulence genes like ctxA in V. mimicus and vopF in V. parahaemolyticus. This multiplex PCR assay, which can detect four major virulence genes in V. cholerae, is novel and important for epidemiologic and environmental surveillance of pathogenic V. cholerae.

Was the Chlamydial Adaptative Strategy to Tryptophan Starvation an Early Determinant of Plastid Endosymbiosis?

Chlamydiales were recently proposed to have sheltered the future cyanobacterial ancestor of plastids in a common inclusion. The intracellular pathogens are thought to have donated those critical transporters that triggered the efflux of photosynthetic carbon and the consequent onset of symbiosis. Chlamydiales are also suspected to have encoded glycogen metabolism TTS (Type Three Secretion) effectors responsible for photosynthetic carbon assimilation in the eukaryotic cytosol. We now review the reasons underlying other chlamydial lateral gene transfers evidenced in the descendants of plastid endosymbiosis. In particular we show that half of the genes encoding enzymes of tryptophan synthesis in Archaeplastida are of chlamydial origin. Tryptophan concentration is an essential cue triggering two alternative modes of replication in Chlamydiales. In addition, sophisticated tryptophan starvation mechanisms are known to act as antibacterial defenses in animal hosts. We propose that Chlamydiales have donated their tryptophan operon to the emerging plastid to ensure increased synthesis of tryptophan by the plastid ancestor. This would have allowed massive expression of the tryptophan rich chlamydial transporters responsible for symbiosis. It would also have allowed possible export of this valuable amino-acid in the inclusion of the tryptophan hungry pathogens. Free-living single cell cyanobacteria are devoid of proteins able to transport this amino-acid. We therefore investigated the phylogeny of the Tyr/Trp transporters homologous to E. coli TyrP/Mre and found yet another LGT from Chlamydiales to Archaeplastida thereby considerably strengthening our proposal.

DspA/E Contributes to Apoplastic Accumulation of ROS in Non-host A. thaliana

The bacterium Erwinia amylovora is responsible for the fire blight disease of Maleae, which provokes necrotic symptoms on aerial parts. The pathogenicity of this bacterium in hosts relies on its type three-secretion system (T3SS), a molecular syringe that allows the bacterium to inject effectors into the plant cell. E. amylovora-triggered disease in host plants is associated with the T3SS-dependent production of reactive oxygen species (ROS), although ROS are generally associated with resistance in other pathosystems. We showed previously that E. amylovora can multiply transiently in the non-host plant Arabidopsis thaliana and that a T3SS-dependent production of intracellular ROS occurs during this interaction. In the present work we characterize the localization and source of hydrogen peroxide accumulation following E. amylovora infection. Transmission electron microscope (TEM) analysis of infected tissues showed that hydrogen peroxide accumulation occurs in the cytosol, plastids, peroxisomes, and mitochondria as well as in the apoplast. Furthermore, TEM analysis showed that an E. amylovora dspA/E-deficient strain does not induce hydrogen peroxide accumulation in the apoplast. Consistently, a transgenic line expressing DspA/E accumulated ROS in the apoplast. The NADPH oxidase-deficient rbohD mutant showed a very strong reduction in hydrogen peroxide accumulation in response to E. amylovora inoculation. However, we did not find an increase in bacterial titers of E. amylovora in the rbohD mutant and the rbohD mutation did not suppress the toxicity of DspA/E when introgressed into a DspA/E-expressing transgenic line. Co-inoculation of E. amylovora with cycloheximide (CHX), which we found previously to suppress callose deposition and allow strong multiplication of E. amylovora in A. thaliana leaves, led to a strong reduction of apoplastic ROS accumulation but did not affect intracellular ROS. Our data strongly suggest that apoplastic ROS accumulation is one layer of the non-host defense response triggered by the type three effector (T3E) DspA/E, together with callose deposition.

Identification of putative TAL effector targets of the citrus canker pathogens shows functional convergence underlying disease development and defense response

BackgroundTranscriptional activator-like (TAL) effectors, formerly known as the AvrBs3/PthA protein family, are DNA-binding effectors broadly found in Xanthomonas spp. that transactivate host genes upon injection via the bacterial type three-secretion system. Biologically relevant targets of TAL effectors, i.e. host genes whose induction is vital to establish a compatible interaction, have been reported for xanthomonads that colonize rice and pepper; however, citrus genes modulated by the TAL effectors PthA“s” and PthC“s” of the citrus canker bacteria Xanthomonas citri (Xc) and Xanthomonas aurantifolii pathotype C (XaC), respectively, are poorly characterized. Of particular interest, XaC causes canker disease in its host lemon (Citrus aurantifolia), but triggers a defense response in sweet orange.ResultsBased on, 1) the TAL effector-DNA binding code, 2) gene expression data of Xc and XaC-infiltrated sweet orange leaves, and 3) citrus hypocotyls transformed with PthA2, PthA4 or PthC1, we have identified a collection of Citrus sinensis genes potentially targeted by Xc and XaC TAL effectors. Our results suggest that similar with other strains of Xanthomonas TAL effectors, PthA2 and PthA4, and PthC1 to some extent, functionally converge. In particular, towards induction of genes involved in the auxin and gibberellin synthesis and response, cell division, and defense response. We also present evidence indicating that the TAL effectors act as transcriptional repressors and that the best scoring predicted DNA targets of PthA“s” and PthC“s” in citrus promoters predominantly overlap with or localize near to TATA boxes of core promoters, supporting the idea that TAL effectors interact with the host basal transcriptional machinery to recruit the RNA pol II and start transcription.ConclusionsThe identification of PthA“s” and PthC“s” targets, such as the LOB (LATERAL ORGAN BOUNDARY) and CCNBS genes that we report here, is key for the understanding of the canker symptoms development during host susceptibility, or the defenses of sweet orange against the canker bacteria. We have narrowed down candidate targets to a few, which pointed out the host metabolic pathways explored by the pathogens.

The lipopolysaccharide modification regulator PmrA limitsSalmonellavirulence by repressing the type three-secretion system Spi/Ssa

The regulatory protein PmrA controls expression of lipopolysaccharide (LPS) modification genes in Salmonella enterica serovar Typhimurium, the etiologic agent of human gastroenteritis and murine typhoid fever. PmrA-dependent LPS modifications confer resistance to serum, Fe(3+), and several antimicrobial peptides, suggesting that the pmrA gene is required for Salmonella virulence. We now report that, surprisingly, a pmrA null mutant is actually hypervirulent when inoculated i.p. into C3H/HeN mice. We establish that the PmrA protein binds to the promoter and represses transcription of ssrB, a virulence regulatory gene required for expression of the Spi/Ssa type three-secretion system inside macrophages. The pmrA mutant displayed heightened expression of SsrB-dependent genes and faster Spi/Ssa-dependent macrophage killing than wild-type Salmonella. A mutation in the ssrB promoter that abolished repression by the PmrA protein rendered Salmonella as hypervirulent as the pmrA null mutant. The antivirulence function of the PmrA protein may limit the acute phase of Salmonella infection, thereby enhancing pathogen persistence in host tissues.

The orchestrators of Shigella flexneri infection

Shigella flexneri is a pathogenic bacterium with an intriguing set of effectors which collectively orchestrate a truly successful mode of infection. The main effector contributing to the process of entry is IpgB1 associated with Spa15, a type three secretion (TTS) chaperone. Once secreted, IpgB1 skilfully hijacks the RhoG-ELMO-Dock180 pathway to stimulate membrane ruffling resulting in S. flexneri entry into the host. Escape from autophagy is the role of the IcsB effector which is associated with another TTS chaperone, IpgA. The secreted IcsB is able to mask VirG from detection by the autophagy protein and prevents destruction of the bacterium upon entry. Colonization of the host cell is mainly down to a late secreted effector, IpaH9.8, which enters the nucleus and inhibits the role of U2AF35 in splicing activity. IpaH9.8 C-terminal domain which has structural flexibility and ubiquitin ligase activity binds to U2AF35 and down-regulates pro-inflammatory responses by the host cell, allowing S. flexneri to colonize. Intracellular and intercellular movements of S. flexneri and thus the ability for the bacterium to spread to neighbouring cells is orchestrated by the surface-exposed protein VirG which interacts with vinculin and N-WASP and promotes actin polymerization.

Expression of the Bradyrhizobium japonicum Type III Secretion System in Legume Nodules and Analysis of the Associated tts box Promoter

In Bradyrhizobium japonicum, as in some other rhizobia, symbiotic efficiency is influenced by a type III secretion system (T3SS). Most genes encoding the transport machinery and secreted proteins are preceded by a conserved 30-bp motif, the type-three secretion (tts) box. In this study, we found that regions downstream of 34 tts boxes are transcribed. For nopB, nopL, and gunA2, the transcriptional start sites were found to be 12, 11, and 10 bp downstream of their tts boxes, respectively. The deletion of this motif or modification of two or more conserved residues strongly reduced expression of nopB. This indicates that the tts box is an essential promoter element. Data obtained with lacZ reporter gene fusions of five genes preceded by a tts box (gunA2, nopB, rhcV, nopL, and blr1806) revealed that they are expressed in 4-week-old nodules of Macroptilium atropurpureum. These data suggest that the T3SS is active in mature nitrogen-fixing nodules. The two-component response regulator TtsI is required for the expression of rhcV, nopL, and blr1806 in bacteroids. Staining of inoculated roots showed that nopB is also expressed in early infection stages.