The <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis> type three secretion chaperone SycO is integrated into the Yop regulatory network and binds to the Yop secretion protein YscM1

Background  

Pathogenic yersiniae (Y. pestis, Y. pseudotuberculosis, Y. enterocolitica) share a virulence plasmid encoding a type three secretion system (T3SS). This T3SS comprises more than 40 constituents. Among these are the transport substrates called Yops (Yersinia outer proteins), the specific Yop chaperones (Sycs), and the Ysc (Yop secretion) proteins which form the transport machinery. The effectors YopO and YopP are encoded on an operon together with SycO, the chaperone of YopO. The characterization of SycO is the focus of this study.     

Small molecule inhibitors of the Yersinia type III secretion system impair the development of Chlamydia after entry into host cells

Background  

Chlamydiae are obligate intracellular pathogens that possess a type III secretion system to deliver proteins into the host cell during infection. Small molecule inhibitors of type III secretion in Yersinia, termed INPs (Innate Pharmaceuticals AB) were reported to strongly inhibit Chlamydia growth in epithelial cells. In this study we have analyzed the effect of these drugs on bacterial invasiveness.     

Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation

All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94 Å resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.     

LcrG secretion is not required for blocking of Yops secretion in <Emphasis Type="Italic">Yersinia pestis</Emphasis>

Background  

LcrG, a negative regulator of the Yersinia type III secretion apparatus has been shown to be primarily a cytoplasmic protein, but is secreted at least in Y. pestis. LcrG secretion has not been functionally analyzed and the relevance of LcrG secretion on LcrG function is unknown.     

Rtt107/Esc4 binds silent chromatin and DNA repair proteins using different BRCT motifs

Background  

By screening a plasmid library for proteins that could cause silencing when targeted to the HMR locus in Saccharomyces cerevisiae, we previously reported the identification of Rtt107/Esc4 based on its ability to establish silent chromatin. In this study we aimed to determine the mechanism of Rtt107/Esc4 targeted silencing and also learn more about its biological functions.     

Bioinformatics analysis of the locus for enterocyte effacement provides novel insights into type-III secretion

Background  

Like many other pathogens, enterohaemorrhagic and enteropathogenic strains of Escherichia coli employ a type-III secretion system to translocate bacterial effector proteins into host cells, where they then disrupt a range of cellular functions. This system is encoded by the locus for enterocyte effacement. Many of the genes within this locus have been assigned names and functions through homology with the better characterised Ysc-Yop system from Yersinia spp. However, the functions and homologies of many LEE genes remain obscure.     

<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> vesicle-mediated protein transport pathways

Background  

Protein secretion is a universal cellular process involving vesicles which bud and fuse between organelles to bring proteins to their final destination. Vesicle budding is mediated by protein coats; vesicle targeting and fusion depend on Rab GTPase, tethering factors and SNARE complexes. The Génolevures II sequencing project made available entire genome sequences of four hemiascomycetous yeasts, Yarrowia lipolytica, Debaryomyces hansenii, Kluyveromyces lactis and Candida glabrata. Y. lipolytica is a dimorphic yeast and has good capacities to secrete proteins. The translocation of nascent protein through the endoplasmic reticulum membrane was well studied in Y. lipolytica and is largely co-translational as in the mammalian protein secretion pathway.     

Systematic analysis of the regulation of type three secreted effectors in <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Typhimurium

Background  

The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions.     

Yersinia outer protein YopE affects the actin cytoskeleton in Dictyostelium discoideum through targeting of multiple Rho family GTPases

Background  

All human pathogenic Yersinia species share a virulence-associated type III secretion system that translocates Yersinia effector proteins into host cells to counteract infection-induced signaling responses and prevent phagocytosis. Dictyostelium discoideum has been recently used to study the effects of bacterial virulence factors produced by internalized pathogens. In this study we explored the potential of Dictyostelium as model organism for analyzing the effects of ectopically expressed Yersinia outer proteins (Yops).     

The Arabidopsis <Emphasis Type="Italic">AtRaptor</Emphasis> genes are essential for post-embryonic plant growth

Background  

Flowering plant development is wholly reliant on growth from meristems, which contain totipotent cells that give rise to all post-embryonic organs in the plant. Plants are uniquely able to alter their development throughout their lifespan through the generation of new organs in response to external signals. To identify genes that regulate meristem-based growth, we considered homologues of Raptor proteins, which regulate cell growth in response to nutrients in yeast and metazoans as part of a signaling complex with the target of rapamycin (TOR) kinase.     

Acid stress damage of DNA is prevented by Dps binding in <Emphasis Type="Italic">Escherichia coli</Emphasis>O157:H7

Background  

Acid tolerance in Escherichia coli O157:H7 contributes to persistence in its bovine host and is thought to promote passage through the gastric barrier of humans. Dps (DNA-binding protein in starved cells) mutants of E. coli have reduced acid tolerance when compared to the parent strain although the role of Dps in acid tolerance is unclear. This study investigated the mechanism by which Dps contributes to acid tolerance in E. coli O157:H7.     

The <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> Type III Secretion Systems manipulate host cell MAPK for critical steps in pathogenesis

Background  

Vibrio parahaemolyticus is a food-borne pathogen causing inflammation of the gastrointestinal epithelium. Pathogenic strains of this bacterium possess two Type III Secretion Systems (TTSS) that deliver effector proteins into host cells. In order to better understand human host cell responses to V. parahaemolyticus, the modulation of Mitogen Activated Protein Kinase (MAPK) activation in epithelial cells by an O3:K6 clinical isolate, RIMD2210633, was investigated. The importance of MAPK activation for the ability of the bacterium to be cytotoxic and to induce secretion of Interleukin-8 (IL-8) was determined.     

Susceptibility of four inbred mouse strains to a low-pathogenic isolate of <Emphasis Type="Italic">Yersinia enterocolitica</Emphasis>

EUMORPHIA (European Union Mouse Research for Public Health and Industrial Application) is a research program involved in developing new approaches in phenotyping, mutagenesis, and informatics to improve characterization of mouse models for understanding human physiology and disease. Secondary screen experiments include the development of assays to identify mice with altered susceptibility or resistance to infections. In this context we developed a new model and established a standard operating procedure for the experimental infection of mice with Yersinia (Y.) enterocolitica. In contrast with previous studies that dealt with high-pathogenic Y. enterocolitica, we used the low-pathogenic Y. enterocolitica strain E40 to analyze differences in the immune response of four strains of inbred mice (BALB/c, C3H/HeN, 129P2, C57BL/6) after oral infection. The determination of colony-forming units in Peyer’s patches and histologic analysis supported the observations that BALB/c are less able to ameliorate the infection within 21 days. The immune defense of C57BL/6 mice against Yersinia was the most effective resulting in a nearly complete elimination of bacteria after 21 days. C3H/HeN and 129P2 were intermediate. Analysis of serum immunoglobulins (Ig) by Luminex showed a significant increase of IgG2b levels 21 days after infection in all four inbred strains. The other immunoglobulins remained nearly constant. Our infection model discriminates between the efficiency of an infection at an early time point (3 days) and immunity at a later time point (21 days). It is furthermore an appropriate model to characterize genetic differences in resistance and immunity of inbred and mutant mouse lines.     

P80, the HinT interacting membrane protein,is a secreted antigen of <Emphasis Type="Italic">Mycoplasma hominis</Emphasis>

Background  

Mycoplasmas are cell wall-less bacteria which encode a minimal set of proteins. In Mycoplasma hominis, the genes encoding the surface-localized membrane complex P60/P80 are in an operon with a gene encoding a cytoplasmic, nucleotide-binding protein with a characteristic Histidine triad motif (HinT). HinT is found in both procaryotes and eukaryotes and known to hydrolyze adenosine nucleotides in eukaryotes. Immuno-precipitation and BIACore analysis revealed an interaction between HinT and the P80 domain of the membrane complex. As the membrane anchored P80 carries an N-terminal uncleaved signal peptide we have proposed that the N-terminus extends into the cytoplasm and interacts with the cytosolic HinT.     

Rapid identification and typing of <Emphasis Type="Italic">Yersinia pestis</Emphasis> and other <Emphasis Type="Italic">Yersinia</Emphasis> species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

Background  

Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species.     

Modulation of Rho GTPases by type III secretion system translocated effectors of Yersinia

Pathogenic species of the bacterial genus Yersinia subdue the immune system to proliferate and spread within the host organism. For this purpose yersiniae employ a type III secretion apparatus which governs injection of six effector proteins (Yersinia outer proteins; Yops) into host cells. Yops control various regulatory and signalling proteins in a unique and highly specific manner. YopE, YopT, and YpkA/YopO modulate the activity of Rho GTP-binding proteins, whereas YopH dephosphorylates phospho-tyrosine residues in focal adhesion proteins. Furthermore, YopP/YopJ and YopM affect cell survival/apoptosis and cell proliferation, respectively. In this review the focus will be on the biochemistry and cellular effects of YopT, YopE, YopO/YpkA, and YopH.     

Direct and negative regulation of the sycO-ypkA-ypoJ operon by cyclic AMP receptor protein (CRP) in Yersinia pestis

Background  

Pathogenic yersiniae, including Y. pestis, share a type III secretion system (T3SS) that is composed of a secretion machinery, a set of translocation proteins, a control system, and six Yop effector proteins including YpkA and YopJ. The cyclic AMP receptor protein (CRP), a global regulator, was recently found to regulate the laterally acquired genes (pla and pst) in Y. pestis. The regulation of T3SS components by CRP is unknown.     

Effects of site-directed mutagenesis of <Emphasis Type="Italic">mglA</Emphasis> on motility and swarming of <Emphasis Type="Italic">Myxococcus xanthus</Emphasis>

Background  

The mglA gene from the bacterium Myxococcus xanthus encodes a 22kDa protein related to the Ras superfamily of monomeric GTPases. MglA is required for the normal function of A-motility (adventurous), S-motility (social), fruiting body morphogenesis, and sporulation. MglA and its homologs differ from all eukaryotic and other prokaryotic GTPases because they have a threonine (Thr78) in place of the highly conserved aspartate residue of the consensus PM3 (phosphate-magnesium binding) region. To identify residues critical for MglA function or potential protein interactions, and explore the function of Thr78, the phenotypes of 18 mglA mutants were characterized.     

Quantification of the physiochemical constraints on the export of spider silk proteins by <Emphasis Type="Italic">Salmonella</Emphasis> type III secretion

Background  

The type III secretion system (T3SS) is a molecular machine in gram negative bacteria that exports proteins through both membranes to the extracellular environment. It has been previously demonstrated that the T3SS encoded in Salmonella Pathogenicity Island 1 (SPI-1) can be harnessed to export recombinant proteins. Here, we demonstrate the secretion of a variety of unfolded spider silk proteins and use these data to quantify the constraints of this system with respect to the export of recombinant protein.