Exploration of chlamydial type III secretion system reconstitution in Escherichia coli

BACKGROUND: Type III secretion system is a virulent factor for many pathogens, and is thought to play multiple roles in the development cycle and pathogenesis of chlamydia, an important human pathogen. However, due to the obligate intracellular parasitical nature of chlamydiae and a lack of convenient genetic methodology for the organisms, very limited approaches are available to study the chlamydial type III secretion system. In this study, we explored the reconstitution of a chlamydial type III secretion in Escherichia coli. RESULTS: We successfully cloned all 6 genomic DNA clusters of the chlamydial type III secretion system into three bacterial plasmids. 5 of the 6 clusters were found to direct mRNA synthesis from their own promoters in Escherichia coli transformed with the three plasmids. Cluster 5 failed to express mRNA using its own promoters. However, fusion of cluster 5 to cluster 6 resulted in the expression of cluster 5 mRNA. Although only two of the type III secretion system proteins were detected transformed E. coli due to limited antibody availability, type III secretion system-like structures were detected in ultrathin sections in a small proportion of transformed E. coli. CONCLUSIONS: We have successfully generated E. coli expressing all genes of the chlamydial type III secretion system. This serves as a foundation for optimal expression and assembly of the recombinant chlamydial type III secretion system, which may be extremely useful for the characterization of the chlamydial type III secretion system and for studying its role in chlamydial pathogenicity

Expression and quorum sensing regulation of type III secretion system genes of <i>Vibrio harveyi</i> during infection of gnotobiotic brine shrimp

Type III secretion systems enable pathogens to inject their virulence factors directly into the cytoplasm of the host cells. The type III secretion system of Vibrio harveyi, a major pathogen of aquatic organisms and a model species in quorum sensing studies, is repressed by the quorum sensing master regulator LuxR. In this study, we found that during infection of gnotobiotic brine shrimp larvae, the expression levels of three type III secretion operons in V. harveyi increased within the first 12h after challenge and decreased again thereafter. The in vivo expression levels were highest in a mutant with a quorum sensing system that is locked in low cell density configuration (minimal LuxR levels) and lowest in a mutant with a quorum sensing system that is locked in the high cell density configuration (maximal LuxR levels), which is consistent with repression of type III secretion by LuxR. Remarkably, in vivo expression levels of the type III secretion system genes were much (> 1000 fold) higher than the in vitro expression levels, indicating that (currently unknown) host factors significantly induce the type III secretion system. Given the fact that type III secretion is energy-consuming, repression by the quorum sensing master regulators might be a mechanism to save energy under conditions where it does not provide an advantage to the cells

Genomics and transcriptomics of Xanthomonas campestris species challenge the concept of core type III effectome

The bacterial species Xanthomonas campestris infects a wide range of Brassicaceae. Specific pathovars of this species cause black rot (pv. campestris), bacterial blight of stock (pv. incanae) or bacterial leaf spot (pv. raphani). In this study, we extended the genomic coverage of the species by sequencing and annotating the genomes of strains from pathovar incanae (CFBP 1606R and CFBP 2527R), pathovar raphani (CFBP 5828R) and a pathovar formerly named barbareae (CFBP 5825R). While comparative analyses identified a large core ORFeome at the species level, the core type III effectome was limited to only three putative type III effectors (XopP, XopF1 and XopAL1). In Xanthomonas, these effector proteins are injected inside the plant cells by the type III secretion system and contribute collectively to virulence. A deep and strand-specific RNA sequencing strategy was adopted in order to experimentally refine genome annotation for strain CFBP 5828R. This approach also allowed the experimental definition of novel ORFs and non-coding RNA transcripts. Using a constitutively active allele of hrpG, a master regulator of the type III secretion system, a HrpG-dependent regulon of 141 genes co-regulated with the type III secretion system was identified. Importantly, all these genes but seven are positively regulated by HrpG and 56 of those encode components of the Hrp type III secretion system and putative effector proteins. This dataset is an important resource to mine for novel type III effector proteins as well as for bacterial genes which could contribute to pathogenicity of X. campestris

Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface

BACKGROUND: Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein. RESULTS: In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking. CONCLUSION: Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53

Yersiniae Virulence Factors: Type III Secretion System

Several Gram-negative pathogenic bacteria have evolved a complex protein secretion system termed the Type Three Secretion System (TTSS) to deliver bacterial effector proteins into host-cells that then modulate host-cellular functions. These bacterial devices are evolutionarily related to the flagellar apparatus. Although the TTSSs are substantially conserved among different species, the effector molecules they deliver are species-unique. There exist three human pathogenic Yersiniae. Yersinia enterocolitica and Yersinia pseudotuberculosis cause self-limiting gastro-enteric diseases and infect mesenteric lymph nodes, while Yersinia pestis is transmitted by fleas and can be aerosolized, causing the lethal disease known as plague (also known as Black Death). The TTSS is composed of over 20 proteins making up the injectisome (inserted directly into the host-cell), in addition to translocator, regulator, and modulator proteins, as well as chaperones for several effector proteins. Today, plague is still a health concern due to the ability of Y. pestis to be aerosolized. No effective vaccines are currently available to the public. However, research is being implemented to create a vaccine that can be widely used. The purpose of this paper is to update the state of the Yersiniae TTSSs by providing a review of recently published primary articles

Patterns of expression and translocation of the ubiquitin ligase SlrP in Salmonella enterica serovar typhimurium.

SlrP is an E3 ubiquitin ligase that can be translocated into eukaryotic host cells by the two type III secretion systems that are expressed by Salmonella enterica serovar Typhimurium and are encoded in Salmonella pathogenicity islands 1 (SPI1) and 2 (SPI2). Expression of slrP and translocation of its product were examined using lac, 3×FLAG, and cyaA′ translational fusions. Although slrP was expressed in different media, optimal expression was found under conditions that imitate the intravacuolar environment and promote synthesis of the SPI2-encoded type III secretion system. Translocation into mammalian cells took place through the SPI1- or the SPI2-encoded type III secretion system, depending on specific host cell type and timing. A search for genetic factors involved in controlling the expression of slrP unveiled LeuO, Lon, and the two-component system PhoQ/PhoP as novel regulators of slrP. Our experiments suggest that LeuO and Lon act through HilD under SPI1-inducing conditions, whereas PhoP directly interacts with the slrP promoter to activate transcription under SPI2 inducing conditions

SrfJ, a salmonella type III secretion system effector regulated by PhoP, RcsB, and IolR.

Virulence-related type III secretion systems are present in many Gram-negative bacterial pathogens. These complex devices translocate proteins, called effectors, from the bacterium into the eukaryotic host cell. Here, we identify the product of srfJ, a Salmonella enterica serovar Typhimurium gene regulated by SsrB, as a new substrate of the type III secretion system encoded by Salmonella pathogenicity island 2. The N-terminal 20-amino-acid segment of SrfJ was recognized as a functional secretion and translocation signal specific for this system. Transcription of srfJ was positively regulated by the PhoP/PhoQ system in an SsrBdependent manner and was negatively regulated by the Rcs system in an SsrB-independent manner. A screen for regulators of an srfJ-lacZ transcriptional fusion using the T-POP transposon identified IolR, the regulator of genes involved in myo-inositol utilization, as an srfJ repressor. Our results suggest that SrfJ is synthesized both inside the host, in response to intracellular conditions, and outside the host, in myo-inositol-rich environments

YopN and TyeA Hydrophobic Contacts Required for Regulating Ysc-Yop Type III Secretion Activity by Yersinia pseudotuberculosis

Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W 279 functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopN W 279 G mutant lost all ability to bind TyeA. The TyeA residue F 8 was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity

Comparative Analysis of the Type III Secretion System Effector Repertoires of Pseudomonas savastanoi Pathovars Pathogenic on Woody Hosts

Comunicación de tipo pósterThe species Pseudomonas savastanoi, a member of the Pseudomonas syringae complex, includes four pathovars causing knots or excrescences in woody hosts: P. savastanoi pv. savastanoi (Psv), pv. fraxini (Psf), pv. nerii (Psn) and pv. retacarpa (Psr), comprising isolates from olive, ash, oleander and broom plants, respectively. Pathogenicity of P. savastanoi is dependent, among other factors, on the type III secretion system (T3SS) and its effector (T3E) repertoire. Furthermore, a putative role in the interaction with woody hosts has been suggested for several of these T3E. The recent availability of the genome sequences of several P. savastanoi strains isolated from different hosts has facilitated bioinformatics predictions of their T3SS genes and T3E pools, the study of their distribution in other strains of the P. syringae complex isolated from woody hosts and the functional analysis of several of these secreted proteins. As previously reported for Psv, Psn and Psf, here we show that pathogenicity of Psr ICMP16945, is also dependent on the T3SS. Psv strains NCPPB 3335, ICMP4352 and PseNe107 share a core set of at least 22 T3E, 18 of which are also encoded in Psn ICMP16943, Psf ICMP7711 and Psr ICMP16945. However, these three strains encode truncated versions of 1-2 of these 18 T3E and, Psr ICMP16945 contains three pathovarspecific T3E. Our results also show that several T3E, including HopAO1, are phylogenetically clustered across the P. syringae complex according to the woody/herbaceous nature of their host of isolation, suggesting host specialization of these effectors in this complex.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tec

A Salmonella virulence factor activates the NOD1/NOD2 signaling pathway.

The invasion-associated type III secretion system (T3SS-1) of Salmonella enterica serotype Typhimurium (S. Typhimurium) activates the transcription factor NF-κB in tissue culture cells and induces inflammatory responses in animal models through unknown mechanisms. Here we show that bacterial delivery or ectopic expression of SipA, a T3SS-1-translocated protein, led to the activation of the NOD1/NOD2 signaling pathway and consequent RIP2-mediated induction of NF-κB-dependent inflammatory responses. SipA-mediated activation of NOD1/NOD2 signaling was independent of bacterial invasion in vitro but required an intact T3SS-1. In the mouse colitis model, SipA triggered mucosal inflammation in wild-type mice but not in NOD1/NOD2-deficient mice. These findings implicate SipA-driven activation of the NOD1/NOD2 signaling pathway as a mechanism by which the T3SS-1 induces inflammatory responses in vitro and in vivo