Silvia Giono-Cerezo

Cell vacuolation caused by Vibrio cholerae hemolysin.

ABSTRACT Non-O1 strains of Vibrio cholerae implicated in gastroenteritis and diarrhea generally lack virulence determinants such as cholera toxin that are characteristic of epidemic strains; the factors that contribute to their virulence are not understood. Here we report that at least one-third of diarrhea-associated nonepidemicV. cholerae strains from Mexico cause vacuolation of cultured Vero cells. Detailed analyses indicated that this vacuolation was related to that caused by aerolysin, a pore-forming toxin ofAeromonas; it involved primarily the endoplasmic reticulum at early times (∼1 to 4 h after exposure), and resulted in formation of large, acidic, endosome-like multivesicular vacuoles (probably autophagosomes) only at late times (∼16 h). In contrast to vacuolation caused by Helicobacter pylori VacA protein, that induced by V. cholerae was exacerbated by agents that block vacuolar proton pumping but not by endosome-targeted weak bases. It caused centripetal redistribution of endosomes, reflecting cytoplasmic alkalinization. The gene for V. choleraevacuolating activity was cloned and was found to correspond tohlyA, the structural gene for hemolysin. HlyA protein is a pore-forming toxin that causes ion leakage and, ultimately, eukaryotic cell lysis. Thus, a distinct form of cell vacuolation precedes cytolysis at low doses of hemolysin. We propose that this vacuolation, in itself, contributes to the virulence of V. choleraestrains, perhaps by perturbing intracellular membrane trafficking or ion exchange in target cells and thereby affecting local intestinal inflammatory or other defense responses.

Interaction between pathogenic bacteria and intrauterine leukocytes triggers alternative molecular signaling cascades leading to labor in women.

ABSTRACT Increased risk of preterm labor has been linked to cervicovaginal infection with Ureaplasma urealyticum and group B streptococci. Although various experimental models have been developed to study the role of amniochorion infection in preterm labor, they typically exclude the initial interaction between intrauterine leukocytes (recruited from decidual vessels into the avascular fetal membranes) and infecting bacteria. In this work, we ascertained whether inflammatory molecules secreted by bacterium-activated intrauterine leukocytes stimulate the amniochorion production of mediators involved in human labor. Using a two-step process beginning with placental circulating leukocytes as a proxy for intrauterine leukocytes, we found that coincubation of amniochorion explants with plasma from placental whole blood preincubated with group B streptococci resulted in a significant increase in tumor necrosis factor alpha (TNF-α) and matrix metalloproteinase 9 (MMP-9) levels in tissue. Extensive changes in the connective tissue arrangement and a decrease in collagen content demonstrated the degradation of the extracellular matrix following this treatment. In contrast, plasma from blood preconditioned with U. urealyticum induced a highly significant secretion of interleukin-1β (IL-1β) and prostaglandin E2 (PGE2) by the amniochorion without changes in the extracellular matrix organization or content. These data demonstrate that group B streptococci induce degradation of the amniochorion as a result of MMP-9 production, probably via TNF-α, whereas U. urealyticum stimulates the secretion of PGE2, probably via IL-1β, potentially stimulating myometrial contraction. Our study provides novel evidence that the immunological cells circulating within the uterine microenvironment respond differentially to an infectious agent, triggering alternative molecular signaling pathways leading to human labor.

vacA genotypes of Helicobacter pylori in the oral cavity and stomach of patients with chronic gastritis and gastric ulcer

INTRODUCTION Helicobacter pylori adheres to various components of the human saliva. Therefore, the objective of this research was to simultaneously detect H. pylori in saliva and in gastric biopsy, and to determine the agreement between the vacA genotypes in both saliva and gastric biopsy. MATERIALS AND METHODS A total of 162 patients with chronic gastritis and 34 with gastric ulcer were studied, and saliva and biopsy samples were collected from each patient. H. pylori DNA was detected by conventional PCR and nested PCR was used for vacA genotyping. RESULTS In 24% of the patients (47/196) H. pylori DNA was found in saliva and in biopsy; 52.5% (103/196) were saliva(negative)/biopsy(positive) and 6.6% (13/196) were saliva(positive)/biopsy(negative). In either or both H. pylori vacAs1m1 or s1m2 genotypes were detected in saliva in 41.5% of the patients with chronic gastritis. Forty-seven percent had >1 genotype, and the s1m1/s1m2 combination was found in 36% of them. H. pylori vacAs1m1 and s1m2 were also found in the saliva and biopsy of patients with gastric ulcer. The genotypes found in saliva and biopsy of the same patient had 51.1% agreement. In 27.6% of the 47 patients saliva(positive)/biopsy(positive) two genotypes were found in saliva, and one or both in the stomach. CONCLUSIONS The s1m1/s1m2 genotypes, alone or together, are found simultaneously in saliva and gastric biopsy of the same patient. These results suggest that H. pylori reaches the oral cavity by various ways, and that saliva can be the transmitting and re-infecting vector.

Preserved Ex Vivo Inflammatory Status in Decidual Cells from Women with Preterm Labor and Subclinical Intrauterine Infection

Objective To compare the inflammatory response preserved ex vivo by decidual cells isolated from women who experienced preterm labor with and without subclinical intrauterine infection. Methods Fetal membranes were obtained after cesarean section from 35 women who delivered before 37 weeks of gestation following spontaneous preterm labor, with no clinical evidence of intrauterine infection. Decidua was microbiologically tested and cultured. Concentrations of anti-inflammatory cytokines (IL-2, IL-4, IL-10), pro-inflammatory cytokines (IL-6, IL-8, IL-1β and TNF-α), and matrix metalloproteinases (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9) were measured in the supernatants using Bio-Plex, and prostaglandin E2 (PGE2) was measured by enzyme immunoassay. Results Subclinical infection was confirmed in 10 women (28.5%). Microorganisms isolated were Ureaplasma urealyticum (4), group B streptococci (3), Gardnerella vaginalis (1), and Escherichia coli (2). We found a significant increase of pro-inflammatory cytokines and a significant decrease of anti-inflammatory cytokines in supernatants from decidual cells obtained from women with preterm labor and subclinical intrauterine infection compared to women without infection. Secretion of MMP-1, MMP-8, MMP-9 and PGE2 was significantly higher in infected women. Secretion of IL-8 by decidual cells from infected women persisted upon repeated in vitro culture passages. Conclusions Almost 30% of idiopathic preterm labor cases were associated with subclinical intrauterine infection, and decidual cells isolated from these cases preserved an ex vivo inflammatory status after in vivo bacterial exposure.

Evolution of bacterial genes: Evidences of positive Darwinian selection and fixation of base substitutions in virulence genes of Helicobacter pylori

Gene diversity in Helicobacter pylori from different origins results in a phylogeographic differentiation, and this genetic variation among populations might be driven by random drift or by selective forces. However, only the selective forces would contribute to adaptation of the bacteria to the physiology and environment of its local host and to its association with gastroduodenal diseases. We studied evolutionary forces acting on variable regions of virulence genes cagA, babA and oipA, which present geographic differences among H. pylori strains from different human groups. Gene sequences in H. pylori strains from Asia, Europe and America were analysed using state of the art analytical methods like the Maximum Likelihood method. The rate and nature of polymorphisms in these virulence genes were also compared among populations using the AMOVA and McDonald-Kreitman tests. We found strong and significant positive selection acting on variable regions of cagA, babA and oipA. We found in cagA from Asian strains regions under positive selection, which localised in amino acid sites defining the Asian fingerprint for this gene and in sites with important biological activity. Different evolutionary forces are acting on the variable region of virulence genes; they partly explain the source of genetic diversity and the differences in risk for gastroduodenal diseases among different human populations.

Impact of cagPAI and T4SS on the Inflammatory Response of Human Neutrophils to Helicobacter pylori Infection

Helicobacter pylori contains a pathogenicity island, cagPAI, with genes homologous to components of the type IV secretion system (T4SS) of Agrobacterium tumefaciens. The T4SS components assemble a structure that transfers CagA protein and peptidoglycan into host epithelial cells, causing the increased release of interleukin 8 (IL8) from the cells. The Toll-like receptors on neutrophils recognize H. pylori, initiating signaling pathways that enhance the activation of NF-κB. However, the roles of cagPAI and T4SS in the inflammatory response of neutrophils are unknown. We evaluated the participation of cagPAI and T4SS in the response of human neutrophils to H. pylori infection. Neutrophils were isolated from the blood of healthy donors and infected with H. pylori cagPAI+, cagPAI–, and cagPAI mutant strains virB4 – and virD4 –. Whereas cagPAI+ strain 26695 induced the greatest IL8 production, a proinflammatory response, cagPAI– strain 8822 induced the greatest IL10 production, an anti-inflammatory response. In contrast, the virB4 – and virD4 – mutant strains produced significantly more of the two proinflammatory cytokines IL1β and tumor necrosis factor αthan the cagPAI+ strain 26695. We observed that H. pylori downregulated the expression of TLRs 2 and 5 but upregulated TLR9 expression in a cagPAI and T4SS-independent manner. These results show for the first time that the response of human neutrophils to H. pylori may vary from a pro-inflammatory to an anti-inflammatory response, depending on cagPAI and the integrity of T4SS.

Molecular Mechanisms Associated with Nosocomial Carbapenem-resistant Acinetobacter baumannii in Mexico

BACKGROUND AND AIMS Acinetobacter baumannii is an emerging pathogen worldwide that is most commonly associated with nosocomial infections and multi-drug resistance. In the present study we determined the mechanisms of carbapenem resistance and clonal diversity of A. baumannii nosocomial isolates in Hospital Civil de Guadalajara, Mexico. METHODS A total of 303 clinical isolates of A. baumannii identified during a period expanding from 2004-2011 were analyzed for carbapenem resistance using several microbiological and molecular methods. Clonal relatedness of these isolates was determined using pulsed-field gel electrophoresis. RESULTS Of the 303 isolates, 84% were resistant to meropenem, 71.3% to imipenem and 78.3% the resistant isolates were positive for metallo-β-lactamases as determined by the phenotypic assay. In addition, 49.6% of carbapenem-intermediate or -resistant isolates carried the blaOXA-72 gene and 1.2% carried the blaVIM-1 gene. Efflux pump phenotype was responsible for reduced susceptibility to meropenem in 14.5% and to imipenem in 31.6% of the resistant isolates, respectively in the presence of the efflux pump inhibitor, carbonyl cyanide 3-chlorophenylhydrazone. Strains representing different carbapenem-resistant patterns exhibited reduced expression of 22, 29, 33, and 43 kDa OMPs. Among the bacterial collection studied, 48 different clones were identified, two of which were predominant and persistently transmitted. CONCLUSIONS Carbapenemase production in combination with efflux pump expression, reduction in OMPs expression and the cross-transmission of clones appear to be major contributors to the high frequency of carbapenem-resistance observed in A. baumannii. To our knowledge, this is the first study to define the molecular mechanisms associated with carbapenem-resistance in A. baumannii in Mexico.

Helicobacter pylori: detection of iceA1 and iceA2 genes in the same strain in Mexican isolates.

BACKGROUND AND AIMS Helicobacter pylori iceA1 and iceA2 gene amplification is usually performed to identify mixed populations as both genes are apparently reportedly exclusive. However, some strains isolated from Mexico show both iceA genes. The aim of this study was to establish the frequency of these genes in Mexican isolates and genomic diversity of the H. pylori strains. METHODS One hundred thirty six biopsies were obtained from 68 patients (39 children and 29 adults). The presence of H. pylori was confirmed in 3/18 children and 6/19 adults by culture. There were 93 clinical strains isolated from nine patients. Additionally, we studied 37 strains from a strain collection isolated from 10 patients. The strains were genotyped and dual iceA genes were identified by polymerase chain reaction (PCR) and amplicons were sequenced. In addition, an enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) assay was performed as fingerprinting method. RESULTS The genotypification of the H. pylori isolates indicated that all strains were vacA+; 86% babA2+, 86% cagA+, 82% vacA s1m1+, 19% iceA1+, 9% iceA2+, and 72% of them carried both iceA1 and iceA2 genes. The ERIC-PCR profiling revealed that the strains clustered in eight genetic groups depending on the presence of iceA1, iceA2 or both. A basic local multiple alignment analysis of the nucleotide sequences revealed that the iceA1 and iceA2 genes exhibited no relevant similarity. CONCLUSION The results here showed the presence of triple-positive strains (babA, cagA, vacA) of H. pylori and strains carrying simultaneously both iceA1 and iceA2 genes.

Receptores tipo Toll, patogénesis y respuesta inmune a Helicobacter pylori

Helicobacter pylori colonize the gastric epithelial, most infected people are asymptomatic, 10 to 20% develop atrophic gastritis, peptic ulcer and less than 3% gastric cancer. These diseases are determined by the relationship between virulence factors of bacteria, host factors such as, genetic predisposition, and immune response. The innate immune response mainly represented by Toll-like receptors and Nod-like receptors that recognize their specific ligands, activate transcription factors as NF-kB, AP-1, CREB-1, inducing production of inflammatory cytokines such as IL -8, IL-12, IL-6, IL-1β, IL-18, TNF-α and IL-10. Chronic inflammation promotes gastric morphological changes, prevents apoptosis and allows angiogenesis generating neoplasic lesions and cancer. The aim of this review is to analyze the mechanisms proposed to date of the innate and adaptative immune response involved in H. pylori infection; remarking the mechanisms related in the elimination or persistence.

Effects of the plasmid-encoded toxin of enteroaggregative Escherichia coli on focal adhesion complexes

Enteroaggregative Escherichia coli (EAEC) is an emerging diarrheal pathogen. Many EAEC strains produce the plasmid-encoded toxin (Pet), which exerts cytotoxic effects on human intestinal tissue. Pet-intoxicated HEp-2 cells exhibit rounding and detachment from the substratum, accompanied by loss of F-actin stress fibers and condensation of the spectrin-containing membrane cytoskeleton. Although studies suggest that Pet directly cleaves spectrin, it is not known whether this is the essential mode of action of the toxin. In addition, the effects of Pet on cytoskeletal elements other than actin and spectrin have not been reported. Here, we demonstrate by immunofluorescence that upon Pet intoxication, HEp-2 and HT29 cells lose focal adhesion complexes (FAC), a process that includes the redistribution of focal adhesion kinase (FAK), α-actinin, paxillin, vinculin, F-actin, and spectrin itself. This redistribution was coupled with the depletion of phosphotyrosine labeling at FACs. Immunoblotting and immunoprecipitation experiments revealed that FAK was tyrosine dephosphorylated, before the redistribution of FAK and spectrin. Moreover, phosphatase inhibition blocked cell retraction, suggesting that tyrosine dephosphorylation is an event that precedes FAK cleavage. Finally, we show that in vitro tyrosine-dephosphorylated FAK was susceptible to Pet cleavage. These data suggest that mechanisms other than spectrin redistribution occur during Pet intoxication.