PubMed Health⌕ Search

Biomedical subjects

Junkal Garmendia

Publications and source records attributed to Junkal Garmendia.

14 recordsLinked to original sources
PubMed2006

TccP2 of O157:H7 and non-O157 enterohemorrhagic Escherichia coli (EHEC): challenging the dogma of EHEC-induced actin polymerization.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 and enteropathogenic E. coli (EPEC) trigger actin polymerization at the site of bacterial adhesion by inducing different signaling pathways. Actin assembly by EPEC requires tyrosine phosphorylation of Tir, which subsequently binds the host adaptor protein Nck. In contrast, Tir(EHEC O157) is not tyrosine phosphorylated and instead of Nck utilizes the bacterially encoded Tir-cytoskeleton coupling protein (TccP)/EspF(U), which mimics the function of Nck. tccP is carried on prophage CP-933U/Sp14 (TccP). Typical isolates of EHEC O157:H7 harbor a pseudo-tccP gene that is carried on prophage CP-933 M/Sp4 (tccP2). Here we report that atypical, beta-glucuronidase-positive and sorbitol-fermenting, strains of EHEC O157 harbor intact tccP and tccP2 genes, both of which are secreted by the LEE-encoded type III secretion system. Non-O157 EHEC strains, including O26, O103, O111, and O145, are typically tccP negative and translocate a Tir protein that encompasses an Nck binding site. Unexpectedly, we found that most clinical non-O157 EHEC isolates carry a functional tccP2 gene that encodes a secreted protein that can complement an EHEC O157:H7 DeltatccP mutant. Using discriminatory, allele-specific PCR, we have demonstrated that over 90% of tccP2-positive non-O157 EHEC strains contain a Tir protein that can be tyrosine phosphorylated. These results suggest that the TccP pathway can be used by both O157 and non-O157 EHEC and that non-O157 EHEC can also trigger actin polymerization via the Nck pathway.

Actins↗
PubMed2006

Function and distribution of EspG2, a type III secretion system effector of enteropathogenic Escherichia coli.

The enteropathogenic Escherichia coli (EPEC) effector protein EspG, like the Shigella effector VirA, functions through disruption of the host cell microtubule network. Reports have differed as to whether the EspG homologue, EspG2, is also responsible for microtubule disruption. In this study we show that following translocation, EspG2 and VirA are localised under adherent bacteria and able to restore the microtubule disruption phenotype to an espG/espG2 double EPEC mutant. The espG/espG2 double mutant produced A/E lesions similar to wild-type EPEC on human intestinal in vitro organ cultures. Determining the distribution of espG and espG2 among clinical EPEC isolates revealed two different types of espG (espG alpha and espG beta) and espG2 (intact and pseudo genes), which were associated with specific EPEC serotypes and closely followed the EPEC lineage. This investigation has established a role for EspG2 in the disruption of the microtubule network and associated different espG and espG2 types with different groups of EPEC.

Caco-2 Cells↗
PubMed2006

Subversion of actin dynamics by EPEC and EHEC.

During the course of infection, enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC, respectively) subvert the host cell signalling machinery and hijack the actin cytoskeleton to tighten their interaction with the gut epithelium, while avoiding phagocytosis by professional phagocytes. Much progress has been made recently in our understanding of how EPEC and EHEC regulate the pathways leading to local activation of two regulators of actin cytoskeleton dynamics, the Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex. A recent highlight is the unravelling of functions for effector proteins (particularly Tir, TccP, Map and EspG/EspG2) that are injected into the host cell by a type III secretion system.

Actin-Related Protein 2-3 Complex↗
PubMed2006

Surveying biotransformations with à la carte genetic traps: translating dehydrochlorination of lindane (gamma-hexachlorocyclohexane) into lacZ-based phenotypes.

The ability of the product of a desired reaction to activate a bacterial transcriptional regulator was exploited to develop genetic traps that render the catalytic activity born by a DNA clone into a selectable/scorable phenotype. We established this strategy with a system to expose the activity of dehydrochlorinases acting upon gamma-hexachlorocyclohexane (gamma-HCH or lindane). To this end, the effector-binding protein, XylR, was evolved by gene shuffling plus mutagenic polymerase chain reaction to be optimally responsive to the major product of gamma-HCH dehydrochlorination, 1,2,4-trichlorobenzene (TCB). We then derived Escherichia coli strains that constitutively expressed the modified XylR variant (named XylR5) and had lacZ under control of the Pu promoter, which is activated by XylR. A robotic beta-galactosidase assay indicated that when the resulting strain was transformed with a linA+ clone (expressing a gamma-HCH dehydrochlorinase from Sphingomonas paucimobilis UT26), it had levels of beta-galactosidase that were dependent on the gamma-HCH concentration. This à la carte host thus translated the conversion of gamma-HCH to TCB into upregulation of lacZ. An alternate host additionally expressing LacY grew efficiently on lactose only when LacZ was upregulated in a fashion dependent on TCB or other effectors of XylR5. These results demonstrated the power of deriving a host for the genetic scrutiny, rather than enzymatic screening, of clones expressing a given catabolic enzyme.

Bacterial Proteins↗
PubMed2006

A novel category of enteropathogenic Escherichia coli simultaneously utilizes the Nck and TccP pathways to induce actin remodelling.

Enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) induce drastic reorganization of the microfilament cytoskeleton. EHEC and EPEC translocate Tir (translocated intimin receptor) which, once inserted into the host plasma membrane, binds the bacterial outer membrane adhesin intimin. Tir(EPEC) then becomes tyrosine phosphorylated facilitating the recruitment and site-specific binding of the eukaryotic adaptor Nck, which in turn binds and activates the Wiskott-Aldrich syndrome protein (N-WASP), leading to actin-related protein 2/3 (Arp2/3) complex-mediated actin polymerization. In contrast, Tir(EHEC) has no Nck binding site; instead, EHEC utilizes the translocated effector TccP (Tir-cytoskeleton coupling protein) to bind and activate N-WASP. Here we report a novel class of EPEC that translocates both TccP and Tir(EPEC)-like effector molecules. Consistent with these characteristics, we show that both the Tir-Nck and Tir:TccP actin remodelling pathways function simultaneously during infection, making this a novel and versatile EPEC category.

Actins↗
PubMed2006

Characterization of TccP-mediated N-WASP activation during enterohaemorrhagic Escherichia coli infection.

Subversion of the host cell cytoskeleton is the hallmark of enterohaemorrhagic Escherichia coli (EHEC) infection. EHEC translocates the trans-membrane receptor protein Tir (translocated intimin receptor), which links the extracellular bacterium to the eukaryotic cell actin cytoskeleton, triggering formation of actin-rich pedestals beneath adherent bacteria. Tir-mediated actin accretion by EHEC requires TccP (Tir cytoskeleton coupling protein), a recently discovered type III secretion system effector protein which, following translocation, binds and activates Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates the actin-related protein 2/3 complex leading to localized polymerization of actin. In this study, truncated N-WASP and TccP derivatives were generated and tested in in vitro actin polymerization and epithelial cell infection assays. The C-terminal amino acids 253-276 of the GTPase binding domain (GBD) of N-WASP were identified as essential, although not sufficient, for TccP:N-WASP protein:protein interaction, TccP-mediated N-WASP activation and induction of actin polymerization. TccP from EHEC O157:H7 strain EDL933 consists of a unique N-terminal domain and six proline-rich repeats. Progressive deletions within the N-terminus of TccP revealed that residues 1-21 are necessary and sufficient for its translocation, while amino acids 1-181, encompassing the N-terminal translocation signal and two proline-rich repeats, are sufficient for triggering actin polymerization in EHEC-infected epithelial cells and in in vitro actin polymerization assays. This study defines the modular domain structure of TccP and the molecular basis of TccP-mediated N-WASP activation and EHEC-induced remodelling of the host actin cytoskeleton.

Actins↗
PubMed2006

Role of intimin-tir interactions and the tir-cytoskeleton coupling protein in the colonization of calves and lambs by Escherichia coli O157:H7.

Intimin facilitates intestinal colonization by enterohemorrhagic Escherichia coli O157:H7; however, the importance of intimin binding to its translocated receptor (Tir) as opposed to cellular coreceptors is unknown. The intimin-Tir interaction is needed for optimal actin assembly under adherent bacteria in vitro, a process which requires the Tir-cytoskeleton coupling protein (TccP/EspF(U)) in E. coli O157:H7. Here we report that E. coli O157:H7 tir mutants are at least as attenuated as isogenic eae mutants in calves and lambs, implying that the role of intimin in the colonization of reservoir hosts can be explained largely by its binding to Tir. Mutation of tccP uncoupled actin assembly from the intimin-Tir-mediated adherence of E. coli O157:H7 in vitro but did not impair intestinal colonization in calves and lambs, implying that pedestal formation may not be necessary for persistence. However, an E. coli O157:H7 tccP mutant induced typical attaching and effacing lesions in a bovine ligated ileal loop model of infection, suggesting that TccP-independent mechanisms of actin assembly may operate in vivo.

Adhesins, Bacterial↗
PubMed2005

Operon structure and gene expression of the espJ--tccP locus of enterohaemorrhagic Escherichia coli O157:H7.

In this study, we investigated the genetic organisation and expression of the espJ--tccP locus of enterohaemorrhagic Escherichia coli (EHEC O157:H7), which encodes the type III secretion system effector proteins EspJ and TccP. Using flow cytometry and fluorescence microscopy, we found that espJ and tccP constitute an operon, whose expression in vitro is affected by the composition of the medium and environmental signals such as temperature, pH, osmolarity and O(2) pressure. Moreover, expression of the espJ--tccP operon is not regulated by Ler, a LEE-encoded transcriptional regulator, but by an as yet unidentified regulatory system(s).

Escherichia coli O157↗
PubMed2005

Enteropathogenic Escherichia coli type III effectors EspG and EspG2 disrupt the microtubule network of intestinal epithelial cells.

Enteropathogenic Escherichia coli infection of intestinal epithelial cells leads to localized depletion of the microtubule cytoskeleton, an effect that is dependent on delivery of type III translocated effector proteins EspG and Orf3 (designated EspG2) to the site of depletion. Microtubule depletion involved disruption rather than displacement of microtubules.

Actins↗
PubMed2005

Distribution of tccP in clinical enterohemorrhagic and enteropathogenic Escherichia coli isolates.

Enterohemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) are diarrheagenic pathogens that colonize the gut through the formation of attaching and effacing lesions, which depend on the translocation of effector proteins via a locus of enterocyte effacement-encoded type III secretion system. Recently, two effector proteins, EspJ and TccP, which are encoded by adjacent genes on prophage CP-933U in EHEC O157:H7, have been identified. TccP consists of a unique N-terminus region and several proline-rich domains. In this project we determined the distribution of tccP in O157:H7, in non-O157 EHEC, and in typical and atypical EPEC isolates. All the EHEC O157:H7 strains tested were tccP(+). Unexpectedly, tccP was also found in non-O157 EHEC, and in typical and atypical EPEC isolates, particularly in strains belonging to serogroups O26 (EHEC), O119 (typical EPEC), and O55 (atypical EPEC). We recorded some variation in the length of tccP, which reflects diversity in the number of the proline-rich repeats. These results show the existence of a class of "attaching and effacing" pathogens which express a combination of EPEC and EHEC virulence determinants.

Animals↗
PubMed2004

TccP is an enterohaemorrhagic Escherichia coli O157:H7 type III effector protein that couples Tir to the actin-cytoskeleton.

Subversion of host cell actin microfilaments is the hallmark of enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli infections. Both pathogens translocate the trans-membrane receptor protein-translocated intimin receptor (Tir), which links the extracellular bacterium to the cell cytoskeleton. While both converge on neural Wiskott-Aldrich syndrome protein (N-WASP), Tir-mediated actin accretion by EPEC and EHEC differ in that Tir(EPEC) requires both tyrosine phosphorylation and the host adaptor protein Nck, whereas Tir(EHEC) is not phosphorylated and utilizes an unidentified linker. Here we report the identification of Tir-cytoskeleton coupling protein (TccP), a novel EHEC effector that displays an Nck-like coupling activity following translocation into host cells. A tccP mutant did not affect Tir translocation and focusing but failed to recruit alpha-actinin, Arp3, N-WASP and actin to the site of bacterial adhesion. When expressed in EPEC, bacterial-derived TccP restored actin polymerization activity following infection of an Nck-deficient cell line. TccP has a similar biological activity on infected human intestinal explants ex vivo. Purified TccP activates N-WASP stimulating, in the presence of Arp2/3, actin polymerization in vitro. These results show that EHEC translocates both its own receptor (Tir) and an Nck-like protein (TccP) to facilitate actin polymerization.

Actins↗
PubMed2003

The roles of SsrA-SsrB and OmpR-EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system.

The type III secretion system (TTSS) encoded by Salmonella typhimurium pathogenicity island 2 (SPI-2) is expressed after bacterial entry into host cells. The SPI-2 TTSS secretes the translocon components SseBCD, which translocate across the vacuolar membrane a number of effector proteins whose action is required for intracellular bacterial replication. Several of these effectors, including SifA and SifB, are encoded outside SPI-2. The two-component regulatory system SsrA-SsrB, encoded within SPI-2, controls the expression of components of the SPI-2 TTSS apparatus as well as its translocated effectors. The expression of SsrA-B is in turn regulated by the OmpR-EnvZ two-component system, by direct binding of OmpR to the ssrAB promoter. Several environmental signals have been shown to induce in vitro expression of genes regulated by the SsrA-B or OmpR-EnvZ systems. In this work, immunoblotting and flow cytometry were used to analyse the roles of SsrA-B and OmpR-EnvZ in coupling different environmental signals to changes in expression of a SPI-2 TTSS translocon component (SseB) and two effector genes (sifA and sifB). Using single and double mutant strains the relative contribution of each regulatory system to the response generated by low osmolarity, acidic pH or the absence of Ca2+ was determined. SsrA-B was found to be essential for the induction of SPI-2 gene expression in response to each of these individual signals. OmpR-EnvZ was found to play a minor role in sensing these signals and to require a functional SsrA-B system to mediate their effect on SPI-2 TTSS gene expression.

Bacterial Proteins↗
PubMed2000

Visualization of DNA-protein intermediates during activation of the Pu promoter of the TOL plasmid of Pseudomonas putida.

The ATP-dependent multimerization process undergone by the sigma(54)-dependent activator XylR of the TOL plasmid pWW0 of Pseudomonas putida when bound to the upstream activating sequences (UAS) of the cognate PU: promoter was examined by transmission electron microscopy (TEM). To this end, supercoiled DNA templates were combined with increasing concentrations of the constitutive XylR variant XylRDeltaA, with or without ATP or its non-hydrolysable analogue ATPgammaS, and the resulting complexes were visualized by TEM. The different types of DNA-protein association were analysed and a statistical study of the frequency of the various forms was made. ATP appeared to establish an equilibrium between different molecular associations, as well as major changes in the physical shape of the DNA-protein complexes. The formation of higher nucleoprotein structures frequently bearing DNA bends became manifest. Such complexes often engaged otherwise separated UAS-containing plasmids, indicating that the ATP-driven multimer included XylR molecules recruited in trans. Whilst ATP caused the different types of XylR-DNA complex to occur at quite balanced frequencies, ATPgammaS appeared to displace the distribution predominantly towards the higher order forms. These data are compatible with the notion that each time ATP is hydrolysed the transcriptional activation complex is disassembled.

Adenosine Triphosphate↗