21 research outputs found

    Yersinia Controls Type III Effector Delivery into Host Cells by Modulating Rho Activity

    Get PDF
    Yersinia pseudotuberculosis binds to β1 integrin receptors, and uses the type III secretion proteins YopB and YopD to introduce pores and to translocate Yop effectors directly into host cells. Y. pseudotuberculosis lacking effectors that inhibit Rho GTPases, YopE and YopT, have high pore forming activity. Here, we present evidence that Y. pseudotuberculosis selectively modulates Rho activity to induce cellular changes that control pore formation and effector translocation. Inhibition of actin polymerization decreased pore formation and YopE translocation in HeLa cells infected with Y. pseudotuberculosis. Inactivation of Rho, Rac, and Cdc42 by treatment with Clostridium difficile toxin B inhibited pore formation and YopE translocation in infected HeLa cells. Expression of a dominant negative form of Rac did not reduce the uptake of membrane impermeable dyes in HeLa cells infected with a pore forming strain YopEHJT−. Similarly, the Rac inhibitor NSC23766 did not decrease pore formation or translocation, although it efficiently hindered Rac-dependent bacterial uptake. In contrast, C. botulinum C3 potently reduced pore formation and translocation, implicating Rho A, B, and/or C in the control of the Yop delivery. An invasin mutant (Y. pseudotuberculosis invD911E) that binds to β1 integrins, but inefficiently transduces signals through the receptors, was defective for YopE translocation. Interfering with the β1 integrin signaling pathway, by inhibiting Src kinase activity, negatively affected YopE translocation. Additionally, Y. pseudotuberculosis infection activated Rho by a mechanism that was dependent on YopB and on high affinity bacteria interaction with β1 integrin receptors. We propose that Rho activation, mediated by signals triggered by the YopB/YopD translocon and from engagement of β1 integrin receptors, stimulates actin polymerization and activates the translocation process, and that once the Yops are translocated, the action of YopE or YopT terminate delivery of Yops and prevents pore formation

    CS22, a novel human enterotoxigenic Escherichia coli adhesin, is related to CS15

    No full text
    Enterotoxigenic Escherichia coli (ETEC) expresses a broad spectrum of O:H antigens. Serogroup O20 is one of the most prevalent among the ETEC strains lacking any of the defined colonization factors (CFs), in Argentina. An O20:H− strain, ARG-3, adhered to Caco-2 cells and exhibited a thermoregulated 15.7-kDa protein band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An antiserum against this protein inhibited ARG-3 adhesion to Caco-2 cells and bound to very thin fibrilla-like structures on the bacterial surface. A 15.7-kDa protein-defective mutant failed to adhere to Caco-2 cells and lacked immunogold-labeled surface structures. The N-terminal amino acid sequence of the structural subunit showed 95% homology to that of CS15 of ETEC (former antigen 8786) and 65% homology with fimbria SEF14 of Salmonella enterica serovar Enteritidis. Nevertheless, the molecular size of ARG-3 adhesin was different from that of CS15, as revealed by SDS-PAGE and mass spectrometry. Both proteins are immunologically related, yet not identical, since an antiserum against the 15.7-kDa protein reacted solely with ARG-3 after absorption with bacteria bearing CS15. Moreover, only under low stringency conditions could DNA from strain ARG-3 be amplified by PCR using primers derived from the nfaA sequence of CS15. Thus, from the DNA sequence obtained from the ARG-3 PCR product, it could be deduced that the subunit protein differed in 30 residues from that of CS15. ARG-3 adhesin was found in 60% of the O20:H- CF-negative ETEC strains from Argentina; however, it appeared restricted to this serotype. We propose the designation CS22 for the herein identified nonfimbrial adhesin of human ETEC.Fil: Pichel, Mariana G. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Viboud, Gloria I. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina

    The Diverse Roles of the Global Transcriptional Regulator PhoP in the Lifecycle of Yersinia pestis

    No full text
    Yersinia pestis, the causative agent of plague, has a complex infectious cycle that alternates between mammalian hosts (rodents and humans) and insect vectors (fleas). Consequently, it must adapt to a wide range of host environments to achieve successful propagation. Y. pestis PhoP is a response regulator of the PhoP/PhoQ two-component signal transduction system that plays a critical role in the pathogen’s adaptation to hostile conditions. PhoP is activated in response to various host-associated stress signals detected by the sensor kinase PhoQ and mediates changes in global gene expression profiles that lead to cellular responses. Y. pestis PhoP is required for resistance to antimicrobial peptides, as well as growth under low Mg2+ and other stress conditions, and controls a number of metabolic pathways, including an alternate carbon catabolism. Loss of phoP function in Y. pestis causes severe defects in survival inside mammalian macrophages and neutrophils in vitro, and a mild attenuation in murine plague models in vivo, suggesting its role in pathogenesis. A Y. pestisphoP mutant also exhibits reduced ability to form biofilm and to block fleas in vivo, indicating that the gene is also important for establishing a transmissible infection in this vector. Additionally, phoP promotes the survival of Y. pestis inside the soil-dwelling amoeba Acanthamoeba castellanii, a potential reservoir while the pathogen is quiescent. In this review, we summarize our current knowledge on the mechanisms of PhoP-mediated gene regulation in Y. pestis and examine the significance of the roles played by the PhoP regulon at each stage of the Y. pestis life cycle

    Identification of a cluster of strains bearing a new adhesin among genetically diverse enterotoxigenic Escherichia coli isolates of serogroup O20

    No full text
    About one-third of the enterotoxigenic Escherichia coli isolates lack any of the known colonization factors. Among this group, those of serogroup O20 are the most frequently isolated in Argentina. By combining analysis of adhesion to Caco-2 cells, random amplified polymorphic DNA, and pulsed-field gel electrophoresis techniques, we were able to identify three sets of closely related strains with different binding properties. Further analysis of the most prevalent group revealed that all these isolates expressed the recently described adhesin CS22.Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Gutkind, Gabriel. Universidad de Buenos Aires. Cátedra de Microbiología; Argentina.Fil: Viboud, Gloria. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina

    Identification of a cluster of strains bearing a new adhesin among genetically diverse enterotoxigenic Escherichia coli isolates of serogroup O20

    No full text
    About one-third of the enterotoxigenic Escherichia coli isolates lack any of the known colonization factors. Among this group, those of serogroup O20 are the most frequently isolated in Argentina. By combining analysis of adhesion to Caco-2 cells, random amplified polymorphic DNA, and pulsed-field gel electrophoresis techniques, we were able to identify three sets of closely related strains with different binding properties. Further analysis of the most prevalent group revealed that all these isolates expressed the recently described adhesin CS22.Fil: Pichel, Mariana. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Gutkind, Gabriel. Universidad de Buenos Aires. Cátedra de Microbiología; Argentina.Fil: Viboud, Gloria. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina

    Prospective Cohort Study of Enterotoxigenic Escherichia coli Infections in Argentinean Children

    No full text
    In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P = 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P = 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P = 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P = 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P = 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P = 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea

    Prospective Cohort Study of Enterotoxigenic Escherichia coli Infections in Argentinean Children

    No full text
    Fil: Viboud, Gloria I. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Jouve, Mabel J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Vergara, Marta. Universidad Nacional de Misiones. Cátedra de Bacteriología; Argentina.Fil: Rivas, Marta. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Quiroga, Marina. Universidad Nacional de Misiones. Cátedra de Bacteriología; Argentina.Fil: Svennerholm, Ann-Mari. University of Göteborg. Department of Medical Microbiology and Immunology; Suiza.In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P 5 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P 5 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P 5 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P 5 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P 5 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P 5 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea

    Prospective Cohort Study of Enterotoxigenic Escherichia coli Infections in Argentinean Children

    No full text
    Fil: Viboud, Gloria I. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Jouve, Mabel J. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Binsztein, Norma. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Vergara, Marta. Universidad Nacional de Misiones. Cátedra de Bacteriología; Argentina.Fil: Rivas, Marta. ANLIS Dr.C.G.Malbrán. Instituto Nacional de Enfermedades Infecciosas. Departamento de Bacteriología; Argentina.Fil: Quiroga, Marina. Universidad Nacional de Misiones. Cátedra de Bacteriología; Argentina.Fil: Svennerholm, Ann-Mari. University of Göteborg. Department of Medical Microbiology and Immunology; Suiza.In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P 5 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P 5 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P 5 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P 5 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P 5 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P 5 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea

    Phenotype of the <i>Y</i>. <i>pseudotuberculosis</i> expressing <i>yopD</i> mutations in its native location.

    No full text
    <p>HeLa cells were infected with <i>yopEHJ</i>, <i>yopEHJD</i>, and <i>yopEHJD</i> expressing <i>yopD</i>I168T, G196R and A273T in their native location. <b>A.</b> Effector translocation was determined by infecting HeLa cells at a MOI of 100 for 2h. Cells were washed, lysed with 1% Triton X100, and soluble and insoluble fractions were separated by SDS-PAGE and analyzed by immunoblotting with rabbit anti-phospho-<i>GSK</i>-3β (Ser9). Monoclonal antibody against GAPDH and total <i>GSK</i>-3β (Ser9) antibody were used as a loading control for the soluble and insoluble fractions, respectively. Quantification of the signal intensities was performed using Odyssey imaging system software. Results are expressed as the ratio between total ph-ERK signal and GAPDH. <b>B.</b> Pore formation was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120471#pone.0120471.g004" target="_blank">Fig. 4</a> by analyzing the amount of LDH released from culture supernatants of infected cells. Results were normalized to <i>yopEHJ</i> (100%). Error bars represent the standard deviation of the mean values obtained from three duplicate experiments. * P<0.005 determined by t-test. <b>C.</b> YopD-dependent activation of proinflammatory signaling was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120471#pone.0120471.g005" target="_blank">Fig. 5</a>. MAPK activation was analyzed in cell lysates by immunoblotting using rabbit anti-phospho ERK. A monoclonal antibody against GAPDH was used as a loading control and quantification of the signal intensities was performed using Odyssey imaging system software. Results are expressed as the ratio between total ph-ERK signal and GAPDH.</p
    corecore