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

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

An indirect enzyme-linked immunosorbent assay for rapid and quantitative assessment of Type III virulence phenotypes of Pseudomonas aeruginosa isolates

BACKGROUND: The presence of a Type III secretion system in clinical isolates of Pseudomonas aeruginosa is associated with severe disease and poor outcomes in infections caused by this pathogen. We describe an indirect enzyme-linked immunosorbent assay that rapidly and quantitatively detects two exotoxins, ExoU and ExoT, and two structural components, PopD and PcrV, of the P. aeruginosa Type III secretion system after in-vitro growth in a calcium-free minimal medium. METHODS: We used this assay to characterize the Type III secretion phenotype of 74 clinical isolates of P. aeruginosa. Findings were compared with results of standard immunoblotting and correlated with Type III secretion-dependent virulence of isolates toward cultured epithelial cells. RESULTS: Results of the ELISA assay were concordant with immunoblot detection of the secreted antigens for 73 of 74 isolates. The Type III secretion phenotype assessed by this immunoassay predicted bacterial virulence toward epithelial cells in vitro for all but five of the clinical isolates. CONCLUSION: The availability of an ELISA assay for rapid detection of Type III secreted virulence factors will facilitate large clinical studies to examine whether the Type III secretion phenotype of a P. aeruginosa isolate predicts the course of clinical disease in a patient and should be taken into account in determining optimal treatment strategies for infected patients

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

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

Inhibition Of The Ysc Type III Secretion System Of Yersinia Pestis By Compound D And Interaction Of Type III Secretion System Needle Proteins With Host Receptors

The type III secretion system of gram-negative bacterial pathogens is a major virulence factor and functions to modulate host immune responses. Immune modulation occurs in many ways, including direct injection of effector proteins or indirect methods such as the detection of bacterial components by host immune receptors. Knowledge of these immune modulations allows for development of treatment options in an ever-increasing antibiotic-resistance climate. The studies presented here explore both areas of immune modulation. We identify Compound D as a potent inhibitor of the type III secretion system of Yersinia pestis. Through evaluation of effector secretion by bacteria grown in the presence of Compound D, we establish that inhibition of secretion occurs through translocon protein YopD and is also affected by LcrQ and YopD\u27s chaperone, LcrH. Type III Secretion inhibition by Compound D also requires a secretion active state of the type III secretion system as determined by analysis of strains that constitutively secrete effectors. The other study focuses on host recognition of bacterial proteins, specifically the needle protein of type III secretion systems. Via utilization of cells that secrete a measurable signal protein when NF-κB or AP-1 is activated, we show that needle proteins from Yersinia pestis, Salmonella enterica serovar Typhimurium, and Shigella flexneri are capable of activating cells through Toll-like receptors 2 and 4. This interaction appears to be modulated by the N-terminus, that is reported to reside on the outside of the fully formed needle structure, exposed to host receptors. Activation of NF-κB/AP-1 correlates with production of TNF-α in response to needle proteins

Construction and Characteristics of a Recombinant Single- Chain Antibody Fragment against Bacterial Type III Secretion

Pseudomonas aeruginosa, a Gram-negative pathogen, causes life-threatening infections. Lung injury and the development of sepsis depend largely on expression of the virulence genes associated with the type III secretion system of this bacterium. The type III secretion system functions as a molecular syringe to deliver type III secretory toxins directly into the cytosol of eukaryotic cells and also acts to inhibit innate immune mechanisms, thereby preventing bacterial clearance. Antibodies against PcrV, the cap structure in the translocational needle of type III secretory apparatus of P. aeruginosa, block toxin translocation of the type III secretion system. We have been investigating the therapeutic use of a recombinant anti-PcrV single-chain antibody. In this chapter, as a preliminary step toward an antibody-based immunotherapy against bacterial infections, we summarize our experience of constructing a recombinant single-chain antibody (called scFv166), in which the heavy (VH) and light chain (VL) variable regions of the anti-PcrV monoclonal IgG are joined by a flexible peptide linker. The practical methodologies used to make recombinant scFv166 against a bacterial protein component are described in detail

Identification and biochemical characterization of the CHLAMYDIA TRACHOMATIS type III secretion chaperone, SLC1, and its role in the translocation of the invasion-associated effector TARP.

Chlamydia trachomatis is an obligate intracellular pathogen that utilizes a type III secretion system to enter mammalian cells and establish an intracellular niche. TARP, the translocated actin recruitment protein, is a chlamydial invasion protein known to be type III secreted by the metabolically inert elementary body upon docking to the mammalian cell surface. Because immediate secretion of TARP into host cells is necessary for entry, I hypothesized that a chlamydial chaperone binds to TARP and facilitates its translocation through the type III secretion apparatus. Most effector-binding type III secretion chaperones are small (14-18 kDa), have an acidic pI, and share a specific secondary structure of alternating alpha-helices (a) and beta-sheets (~). Typically, type III secretion chaperones dimerize and interact with their effectors as a complex of two molecules of chaperone to one effector molecule. Only 3 Chlamydia trachomatis proteins have been identified in EB\u27 s that are predicted to be putative chlamydial type III effector chaperones. These are CT043, CT663, and CT088, which I have designated as SIc 1, SIc2, and Scc 1, respectively. These chaperones were tested for their interaction with the N-terminal 200 amino acids of TARP (HIS6 TARpl-200) by co immunoprecipitation. HIS6 TARpl-200 interacted specifically with SlcI, but not Scc1 or Slc2. This interaction was enhanced by coexpression of the recombinant proteins. To confirm this interaction and rule out the possibility of Slc 1 heterodimerization enhancing the interaction with TARP, I employed a 2-hybrid system to test for TARP: chaperone and chaperone:chaperone interactions. I confirmed the specific interaction between Cya18- TARpl-200 and Cya25-Slc 1. I was also able to detect SIc1 interaction with itself as well as confirm a few other previously described chaperone-chaperone interactions. Analysis by crosslinking and gel filtration chromatography indicated that Slc 1 forms a stable dimer in solution. Complexes of the Slci chaperone dimer with TARP in a 2:1 stoichiometry were detected following purification from co-expressing bacteria, but not following addition of singly purified species. Expression ofbeta-Iactamase fused to TARl-200 by the heterologous system Yersinia enterocolitica allowed for secretion of TARP into type-III inducing media (low calcium). Furthermore I was able to detect SIc I-dependent translocation of T ARP into HeLa cells via the heterologous type III secretion system of Y enterocolitica, and also by the SPI-2 system of Salmonella enterica serovar typhimurium

Identification of Chromosomal Genes in Yersinia pestis that Influence Type III Secretion and Delivery of Yops into Target Cells

Pathogenic Yersinia species possess a type III secretion system, which is required for the delivery of effector Yop proteins into target cells during infection. Genes encoding the type III secretion machinery, its substrates, and several regulatory proteins all reside on a 70-Kb virulence plasmid. Genes encoded in the chromosome of yersiniae are thought to play important roles in bacterial perception of host environments and in the coordinated activation of the type III secretion pathway. Here, we investigate the contribution of chromosomal genes to the complex regulatory process controlling type III secretion in Yersinia pestis. Using transposon mutagenesis, we identified five chromosomal genes required for expression or secretion of Yops in laboratory media. Four out of the five chromosomal mutants were defective to various extents at injecting Yops into tissue culture cells. Interestingly, we found one mutant that was not able to secrete in vitro but was fully competent for injecting Yops into host cells, suggesting independent mechanisms for activation of the secretion apparatus. When tested in a mouse model of plague disease, three mutants were avirulent, whereas two strains were severely attenuated. Together these results demonstrate the importance of Y. pestis chromosomal genes in the proper function of type III secretion and in the pathogenesis of plague