Newly Isolated Animal Pathogen Corynebacterium silvaticum Is Cytotoxic to Human Epithelial Cells.

Corynebacterium silvaticum is a newly identified animal pathogen of forest animals such as roe deer and wild boars. The species is closely related to the emerging human pathogen Corynebacterium ulcerans and the widely distributed animal pathogen Corynebacterium pseudotuberculosis. In this study, Corynebacterium silvaticum strain W25 was characterized with respect to its interaction with human cell lines. Microscopy, measurement of transepithelial electric resistance and cytotoxicity assays revealed detrimental effects of C. silvaticum to different human epithelial cell lines and to an invertebrate animal model, Galleria mellonella larvae, comparable to diphtheria toxin-secreting C. ulcerans. Furthermore, the results obtained may indicate a considerable zoonotic potential of this newly identified species.

Effects of Escherichia coli subtilase cytotoxin and Shiga toxin 2 on primary cultures of human renal tubular epithelial cells.

Shiga toxin (Stx)-producing Escherichia coli (STEC) cause post-diarrhea Hemolytic Uremic Syndrome (HUS), which is the most common cause of acute renal failure in children in many parts of the world. Several non-O157 STEC strains also produce Subtilase cytotoxin (SubAB) that may contribute to HUS pathogenesis. The aim of the present work was to examine the cytotoxic effects of SubAB on primary cultures of human cortical renal tubular epithelial cells (HRTEC) and compare its effects with those produced by Shiga toxin type 2 (Stx2), in order to evaluate their contribution to renal injury in HUS. For this purpose, cell viability, proliferation rate, and apoptosis were assayed on HRTEC incubated with SubAB and/or Stx2 toxins. SubAB significantly reduced cell viability and cell proliferation rate, as well as stimulating cell apoptosis in HRTEC cultures in a time dependent manner. However, HRTEC cultures were significantly more sensitive to the cytotoxic effects of Stx2 than those produced by SubAB. No synergism was observed when HRTEC were co-incubated with both SubAB and Stx2. When HRTEC were incubated with the inactive SubAA272B toxin, results were similar to those in untreated control cells. Similar stimulation of apoptosis was observed in Vero cells incubated with SubAB or/and Stx2, compared to HRTEC. In conclusion, primary cultures of HRTEC are significantly sensitive to the cytotoxic effects of SubAB, although, in a lesser extent compared to Stx2.

Peptide pheromone plantaricin a produced by Lactobacillus plantarum permeabilizes liver and kidney cells.

Certain antimicrobial peptides from multicellular animals kill a variety of tumor cells at concentrations not affecting normal eukaryotic cells. Recently, it was reported that also plantaricin A (PlnA), which is a peptide pheromone with strain-specific antibacterial activity produced by Lactobacillus plantarum, permeabilizes cancerous rat pituitary cells (GH(4) cells), whereas normal rat anterior pituitary cells are resistant to the peptide. To examine whether the preferential permeabilization of cancerous cells is a general feature of PlnA, we studied its effect on primary cultures of cells from rat liver (hepatocytes, endothelial, and Kupffer cells) and rat kidney cortex, as well as two epithelial cell lines of primate kidney origin (Vero cells from green monkey and human Caki-2 cells). The Vero cell line is derived from normal cells, whereas the Caki-2 cell line is derived from a cancerous tumor. The membrane effects were studied by patch clamp recordings and microfluorometric (fura-2) monitoring of the cytosolic concentrations of Ca(2+) ([Ca(2+)](i)) and fluorophore. In all the tested cell types except Kupffer cells, exposure to 10-100 microM PlnA induced a nearly instant permeabilization of the membrane, indicated by the following criteria: increased membrane conductance, membrane depolarization, increased [Ca(2+)](i), and diffusional loss of fluorophore from the cytosol. At a concentration of 5 microM, PlnA had no effect on any of the cell types. The Kupffer cells were permeabilized by 500 microM PlnA. We conclude that the permeabilizing effect of PlnA is not restricted to cancerous cells.

Culture supernatants from V. cholerae O1 ElTor strains isolated from different geographic areas induce cell vacuolation and cytotoxicity / Cepas de V. cholerae O1 biotipo ElTor aisladas de diferente origen geográfico inducen vacuolización celular y citotoxicidad

OBJECTIVE: To investigate whether the HlyA-induced vacuolating effect is produced by V. cholerae O1 ElTor strains isolated from different geographic origins, including Mexico. MATERIAL AND METHODS: Supernatant-induced haemolysis, vacuolating activity and cytotoxicity in Vero cells were recorded. PCR, RFLP analysis and molecular cloning were performed. RESULTS: All ElTor strains analyzed induced cellular vacuolation. Ribotype 2 strains isolates from the U.S. gulf coast yielded the highest titer of vacuolating activity. Eight of nine strains were haemolytic, while all strains were PCR positive for the hlyA gene. We cloned the hlyA gene from two ElTor strains, a toxigenic (2514-88, ctxAB+) and a non-toxigenic Mexican strain (CM 91-3, ctxAB-). Supernatant from those recombinant E. coli strains induced haemolysis, cell vacuolation and cytotoxicity. RFLP-PCR analysis revealed similarities in the hlyA gene from all strains tested. CONCLUSION: The HlyA-induced vacuolating effect is a widespread phenotype of epidemic V. cholerae O1 ElTor strains.

OBJETIVO: Analizar el efecto vacuolizante de cepas de V. cholerae O1 ElTor aisladas de diferente origen geográfico, incluyendo México. MATERIAL Y MÉTODOS: Se realizaron pruebas de hemolisis, vacuolización y citotoxicidad en células Vero, así como PCR, análisis por RFLP y clonación molecular. RESULTADOS: Todas las cepas indujeron el efecto vacuolizante. Las cepas del ribotipo 2, aisladas de las costas del Golfo en Estados Unidos, presentaron títulos altos de vacuolización. El gen hlyA fue amplificado en las nueve cepas mediante PCR, aunque sólo ocho fueron hemolíticas. Se clonó el gen hlyA de una cepa toxigénica (2514-88, ctxAB+) y de una cepa no toxigénica aislada en México (CM 91-3, ctxAB-). El sobrenadante de las clonas recombinantes indujo hemólisis, efecto vacuolizante y citotoxicidad. El RFLP mostró alta similitud del gen hlyA de las cepas estudiadas. CONCLUSIÓN: El efecto vacuolizante es un fenotipo ampliamente distribuido en cepas epidémicas de V. cholerae O1 biotipo ElTor.

Rickettsial outer-membrane protein B (rOmpB) mediates bacterial invasion through Ku70 in an actin, c-Cbl, clathrin and caveolin 2-dependent manner.

Rickettsia conorii, an obligate intracellular tick-borne pathogen and the causative agent of Mediterranean spotted fever, binds to and invades non-phagocytic mammalian cells. Previous work identified Ku70 as a mammalian receptor involved in the invasion process and identified the rickettsial autotransporter protein, rOmpB, as a ligand; however, little is known about the role of Ku70-rOmpB interactions in the bacterial invasion process. Using an Escherichia coli heterologous expression system, we show here that rOmpB mediates attachment to mammalian cells and entry in a Ku70-dependent process. A purified recombinant peptide corresponding to the rOmpB passenger domain interacts with Ku70 and serves as a competitive inhibitor of adherence. We observe that rOmpB-mediated infection culminates in actin recruitment at the bacterial foci, and that this entry process relies in part on actin polymerization likely imparted through protein tyrosine kinase and phosphoinositide 3-kinase-dependent activities and microtubule stability. Small-interfering RNA studies targeting components of the endocytic pathway reveal that entry by rOmpB is dependent on c-Cbl, clathrin and caveolin-2. Together, these results illustrate that rOmpB is sufficient to mediate Ku70-dependent invasion of mammalian cells and that clathrin- and caveolin-dependent endocytic events likely contribute to the internalization process.

Serogroups and virulence genotypes of Escherichia coli isolated from patients with sepsis

Sixty strains of Escherichia coli, isolated by hemoculture, from septicemic Brazilian patients were evaluated to determine their serogroup and invasivity to Vero cells. All 60 patients died within 2 days of hospitalization. Furthermore, the molecular study of the following extraintestinal pathogenic E. coli-associated virulence factor (VF) genes was performed by PCR: i) adhesins: type 1 fimbria (fimH), S fimbria (sfaD/E), P fimbria (papC and papG alleles) and afimbrial adhesin (afaB/C); ii) capsule K1/K5 (kpsMTII); iii) siderophores: aerobactin (iucD), yersiniabactin (fyuA) and salmochelin (iroN); iv) toxins hemolysin (hlyA), necrotizing cytotoxic factor type 1 (cnf1) and secreted autotransporter toxin (sat); v) miscellaneous: brain microvascular endothelial cells invasion (ibeA), serum resistance (traT), colicin V (cvaC) and specific uropathogenic protein (usp). Our results showed that isolates are able to invade Vero cells (96.6 percent), differing from previous research on uropathogenic E. coli (UPEC). The O serogroups associated with UPEC were prevalent in 60 percent of strains vs 11.7 percent of other serogroups. The PCR results showed a conserved virulence subgroup profile and a prevalence above 75 percent for fimH, fyuA, kpsMTII and iucD, and between 35-65 percent for papC, papG, sat, iroN, usp and traT. The evasion from the immunological system of the host and also iron uptake are essential for the survival of extraintestinal pathogenic E. coli strains. Interestingly, among our isolates, a low prevalence of VF genes appeared. Therefore, the present study contributes to the identification of a bacterial profile for sepsis-associated E. coli.

Effect of Gb3 in lipid rafts in resistance to Shiga-like toxin of mutant Vero cells.

Shiga-like toxin 1 (Stx1), produced by enterohemorrhagic Escherichia coli, binds to its receptor, globotriaosylceramide (Gb3), on target cell membranes, as a prerequisite for inducing host cell intoxication. To examine further toxin-receptor interactions, we established an Stx1-resistant clone of Vero cells by chemical mutagenesis. The mutant cells, expressed Gb3, but did not bind Stx1. These mutant cells were larger and had more Gb3 per cell than wild-type cells. Gb3 from both wild-type and mutant Vero cells was recovered in lipid rafts, isolated from cell lysates as detergent resistant membranes (DRMs); the DRMs derived from mutant cells had a lower density of Gb3 than did those from wild-type cells. Stx1 did not bind to the DRMs of mutant cells, both by ELISA and surface plasmon resonance. However, Stx1 bound to Gb3 separated by thin-layer chromatograms from the DRMs of mutant cells. The results indicate that not only presence of Gb3 but also Gb3 density on lipid rafts were important for Stx binding.

Interactions of Paracoccidioides brasiliensis with host cells: recent advances.

Host-fungal interactions are inherently complex and dynamic. In order to identify new microbial targets and develop more effective antifungal therapies, it is important to understand the cellular and molecular mechanisms of disease. Paracoccidioidomycosis provokes a variety of clinical symptoms, and Paracoccidioides brasiliensis can reach many tissues, but primarily attacks the lungs. The ability of the pathogen to interact with the host surface structures is essential to further colonization, invasion, and growth. Epithelial cells may represent the first host barrier or the preferential site of entry of the fungus. For this reason, interactions between P. brasiliensis and Vero/A549 epithelial cells were evaluated, with an emphasis on the adherence, induction of cytoskeletal alterations, and differential signaling activity of the various surface molecules. The adhesion to and invasion of epithelial cells by P. brasiliensis may represent strategies employed to thwart the initial host immune response, and may help in the subsequent dissemination of the pathogen throughout the body.

Infección in vitro por diferentes cepas de Paracoccidioides brasiliensis / In vitro infection by different strains of Paracoccidioides brasiliensis

We analyzed the in vitro infection process by P. brasiliensis and the effect of extracellular factor(s) produced on monolayers of mammalian Vero cell lines. The yeast phase of four strains was studied: B339 (avirulent or slightly virulent), U, (intermediate virulence), 93745 and 63265 (both highly virulent). Strains of intermediate and high virulence had higher adherence at first contact (about 16). Strain B339 had a slower adherence at first contact (8) than the others during the same period. The production of extracellular proteases, soluble extracellular factor(s) and extracellular antigen gP43 showed no correlation with the in vitro physiopathogenicity of the analyzed strains. We demonstrate that the Vero model presented in this paper is a suitable system to study infection and virulence in vitro. We are currently assessing its usefulness as a tool for the analysis of the interaction between pathogen, host and antifungal agents.

The 213-amino-acid leucine-rich repeat region of the listeria monocytogenes InlB protein is sufficient for entry into mammalian cells, stimulation of PI 3-kinase and membrane ruffling.

The Listeria monocytogenes InlB protein is a 630-amino-acid surface protein that mediates entry of the bacterium into a wide variety of cell types, including hepatocytes, fibroblasts and epithelial cells such as Vero, HEp-2 and HeLa cells. Invasion stimulates host proteins tyrosine phosphorylation, PI 3-kinase activity and rearrangements in the actin cytoskeleton. We previously showed that InlB is sufficient for entry of InlB-coated latex beads into cells and recent results indicate that purified InlB can stimulate PI 3-kinase activity and is thus the first bacterial agonist of this lipid kinase. In this study, we identified the region of InlB responsible for entry and stimulation of signal transduction events. Eight monoclonal antibodies directed against InlB were raised and, of those, five inhibited bacterial entry. These five antibodies recognized epitopes within the leucine-rich repeat (LRR) region and/or the inter-repeat (IR) region. InlB-staphylococcal protein A (SPA) fusion proteins and recombinant InlB derivatives were generated and tested for their capacity to mediate entry into cultured mammalian cells. All the InlB derivatives that carried the amino-terminal 213-amino-acid LRR region conferred invasiveness to the normally non-invasive bacterium L. innocua or to inert latex beads and the corresponding purified polypeptides inhibited bacterial entry. In addition, the 213-amino-acid LRR region was able to stimulate PI 3-kinase activity and changes in the actin cytoskeleton (membrane ruffling). These properties were not detected with purified internalin, another invasion protein of L. monocytogenes that displays LRRs similar to those of InlB. Taken together, these results show that the first 213 amino acids of InlB are critical for its specific properties.

Isolamento de Rickettsia em cultura de células vero / Isolation of Rickettsia in vero cell culture

Embora o diagnóstico da febre maculosa baseie-se em sinais e sintomas característicos, o mesmo requer confirmaçäo laboratorial, pois existem alguns diagnósticos diferenciais possíveis como meningococcemia, leptospirose, infecçäo por enterovírus e febre tifóide. A confirmaçäo laboratorial pode ser feita através da pesquisa de anticorpos específicos, possível somente alguns dias após o aparecimento da doença, através do isolamento do agente em amostras de sangue e/ou biópsia de pele, e ainda, de amostras de carrapatos coletados do paciente ou de animais reservatório. O isolamento a partir de sangue ou biópsia de pele resulta em diagnóstico precoce da doença, pois na fase de rickettsemia ainda näo há anticorpos detectáveis no sangue. Assim, com o objetivo de facilitar o diagnóstico precoce da febre maculosa, estabelecemos um método de isolamento de rickettsia em cultura de células vero. Para a padronizaçäo foi inoculada amostra padräo de Rickettsia rickettsii, cepa Sheyla Smith, cedida pelo CDC. A identificaçäo foi feita através da reaçäo de imunofluorescência indireta. A presença de microrganismos verdes fluorescentes visualizados no interior do citoplasma das células caracterizou o crescimento do agente. Posteriormente, a metodologia foi confirmada pelo isolamento do agente da febre maculosa em amostras de biópsia de pele de paciente proveniente de área endêmica no Estado de Säo Paulo, bem como, de amostras de carrapato do gênero Amblyomma, considerado o reservatório e transmissor da doença no Brasil

Evaluation of an enzyme immunoassay for verotoxin detection in Escherichia coli.

Verotoxin-producing Escherichia coli strains (VTEC) cause hemorrhagic colitis and hemolytic-uremic syndrome in humans. Laboratory diagnosis by conventional methods is slow and cumbersome. The results of a new rapid enzyme immunoassay (EIA Premier EHEC) for verotoxin detection both in isolated strains and in clinical samples are presented, and they are compared with cell culture (CC) and polymerase chain reaction (PCR) techniques. Fifty-four strains have been analyzed by both EIA and PCR, and 33 by all three methods. The kit has also been evaluated for experimentally infected stool samples directly and after their enrichment on MacConkey broth. Nineteen, out of the 54 strains, were positive by EIA and 20 by PCR. The results of the 33 strains evaluated by the three techniques were coincident with one exception. The latter was uninterpretable by CC, negative by EIA and positive by PCR. The sensitivity of the kit for experimentally infected stool samples was approximately 5 x 10(7) bacteria/ml in the direct test, and 5 x 10(4) bacteria/ml after broth enrichment. EIA sensitivity and specificity were similar to those of CC and PCR. The diagnostic times were 18h for EIA, 3 days for PCR and 5 days for CC. Sensitivity, rapidity and ease of performance make this technique especially valuable for clinical diagnosis.

Effect of subinhibitory concentrations of Ciprofloxacin and gentamicin on the adherence of Pseudomonas aeruginosa to Vero cells and voided uroepithelial cells.

The effect of subinhibitory concentrations (1/8 x, 1/16 x, 1/32 x MIC) of ciprofloxacin or gentamicin on the adherence of Pseudomonas aeruginosa to Vero cells and voided uroepithelial cells was studied using two different assay methods. Strains studied included both reference strains and clinical strains, including mucoid and nonmucoid isolates. Sub-MICs of ciprofloxacin decreased adherence to Vero cells and voided uroepithelial cells in 3 of 4 ciprofloxacin-susceptible nonmucoid isolates but not in ciprofloxacin-resistant or mucoid isolates. Sub-MICs of gentamicin significantly reduced adherence in 5 of 7 nonmucoid strains but not in mucoid strains. Gentamicin effect on adherence did not correlate with susceptibility to ciprofloxacin. We conclude that ciprofloxacin susceptibility determines the effect of sub-MICs of ciprofloxacin on P. aeruginosa adherence to Vero cells and voided uroepithelial cells. In addition, both the Vero cell assay and voided uroepithelial cell assay demonstrated similar results.

Successful foscarnet therapy for acyclovir-resistant mucocutaneous infection with herpes simplex virus in a recipient of allogeneic BMT.

A 41-year-old recipient of matched unrelated BMT acquired a severe mucocutaneous herpes simplex virus (HSV) type I infection during acyclovir prophylaxis. He was subsequently treated with high-dose acyclovir, but the HSV infection continued. In vitro analysis of the HSV isolate, obtained before and after the administration of high-dose acyclovir, demonstrated marked resistance to acyclovir but sensitivity to the antiviral agent foscarnet. The mucocutaneous HSV infection healed completely to a 16 day course of foscarnet. However, relapse of the acyclovir-resistant HSV infection occurred 202 days after the first foscarnet treatment but he responded again to a second foscarnet course. These data indicate that, with the rising frequency of acyclovir-resistant HSV infections observed in immunocompromised hosts, viral isolates should be tested for susceptibility to different antiviral drugs in recipients of BMT with recurrent or persistent HSV infections.

Preliminary characterization of a monoclonal antibody specific for a viral 27 kD glycoprotein family synthesized in porcine epidemic diarrhoea virus infected cells.

We describe a new monoclonal antibody No. 204 (mcAb 204) which recognized a family of four polypeptides, consisting of a 27kD, a 24/23kD double band and a 19kD protein present within PEDV infected cell lysates. These proteins were identified by immunoprecipitation as well as by staining of immunoblots. In infected Vero cell cultures, the synthesis of the 27kD protein was initiated between 6 and 8 hours post inoculation. The 24/23kD double band and the 19kD protein were only detectable later. At least the 27 and the 24/23kD proteins were apparently glycosylated and present in purified virions. Pulse-chase as well as solubilization experiments indicated that the faster migrating bands represented processed products of the 27kD glycoprotein. The nature of the processing is not known at present. We suggest that the 27kD protein family may represent the integral membrane protein M of PEDV. Since this protein is highly abundant in virions as well as in infected cells, and since mcAb 204 is able to react with its antigen under various conditions, this monoclonal antibody may be useful to further studies of the M-protein of PEDV. In addition, it may provide a useful tool for routine diagnosis.

Palmitoylation of Semliki Forest virus glycoproteins in insect cells (C6/36) occurs in an early compartment and is coupled to the cleavage of the precursor p62.

The acylation of the envelope proteins of Semliki Forest virus by palmitic acid in infected mosquito (C6/36) cells was investigated. It is shown that in these cells palmitic acid was incorporated post-translationally via hydroxylamine-labile linkages onto cysteines in the inner domains of the viral envelope proteins. The kinetics of incorporation, however, differed considerably as compared to higher eukaryotic cells. (i) The precursor of the envelope proteins E2 and E3, p62, was weakly and incompletely palmitoylated irrespective of the duration of labeling. (ii) Under all conditions tested complete acylation of E2 was delayed as compared to E1. (iii) Heavy protein complexes were formed consisting of unacylated p62 and partially unacylated E1. From this data, we conclude that during the maturation of SFV glycoproteins in mosquito cells differently acylated intermediates of p62/E2 exist. Furthermore, acylation of p62/E2 and cleavage of p62 are coupled events, occurring in an early compartment and allowing the release of the envelope oligomers for transport.

The varicella-zoster virus immediate early protein, IE62, can positively regulate its cognate promoter.

Varicella-Zoster virus (VZV) is a neurotropic alphaherpes virus closely related to herpes simplex virus (HSV). However, unlike its close relative HSV, VZV lacks a functional alpha-TIF (alpha-gene transinducing factor) that activates the transcription of immediate early genes during the initial events of the virus life cycle. Hence, in the absence of a functional alpha-TIF, the mechanism triggering the expression of immediate early genes in VZV at present remains unclear. Accumulating evidence indicates that the gene product of the putative immediate early gene ORF62 (IE62) plays a pivotal role in activating VZV genes of all three putative kinetic classes, namely immediate early (alpha), early (beta), and late (gamma) classes of VZV genes. In the present study, we show that IE62 can positively autoregulate its cognate promoter using a transient transfection assay, both in lymphocytes and in neural cells. In the same system, we can also demonstrate activation of the VZV IE62 promoter by HSV ICP4. By deletion analysis and oligonucleotide-directed site-specific mutagenesis we have localized specific regions in the IE62 promoter/upstream sequences that mediate inducibility by IE62 and HSV ICP4, and provide evidence that this promoter activation by these two proteins may be through different mechanisms. These data, taken together with the recent demonstration of the presence of IE62 in the VZ virion tegument (Kinchington, P.R., Hoagland, J.K., Arvin, A.M., Ruyechan, W.T., and Hay, J. 1992. J. Virol. 66, 359-366) provides a possible mechanism by which the triggering of VZV gene expression occurs in the absence of a functional alpha-TIF protein.

Role of phospholipids in BK virus infection and haemagglutination.

The role of phospholipids in BK virus infection and haemagglutination was studied by competition binding experiments and by treatment of susceptible cells with phospholipases. Phospholipids extracted from Vero cells and some commercial phospholipids showed an inhibiting activity on both BK virus infectivity and haemagglutination. The treatment of Vero cells with phospholipases affected the binding of BK virus, but the addition of phospholipids to enzyme-treated cells restored their susceptibility to both viral infectivity and haemagglutination.

A 269-amino-acid segment with a pseudo-leucine zipper and a helix-turn-helix motif codes for the sequence-specific DNA-binding domain of herpes simplex virus type 1 origin-binding protein.

The UL9 gene of herpes simplex virus (HSV) codes for a DNA-binding protein (OBP) that interacts sequence specifically with the origin of replication. This protein is essential for HSV DNA replication in cultured cells. The UL9 gene was cloned into a plasmid vector downstream of the SP6 RNA polymerase promoter. By using in vitro transcription and translation systems, a full-length OBP was synthesized. This synthetic protein is recognized by an antiserum generated against the C-terminal decapeptide of OBP and is functionally active in binding to OriS sequence specifically. The in vitro-synthesized protein has sequence specificity for binding similar to that found for the in vivo-generated OBP. A total of 14 in-frame deletion and insertion mutants of the UL9 gene were generated and expressed in vitro. Using these deletion mutants, we determined that the 269-amino-acid stretch defined by amino acids 564 to 832 localizes the OriS-specific DNA-binding domain. The N-terminal boundary is between amino acids 565 and 596, while the C terminus lies between amino acids 833 and 805. This segment contains a helix-turn-helix moiety and a pseudo-leucine zipper, neither of which alone can support DNA binding. The other leucine zipper from amino acids 150 to 173 is not required for the in vitro sequence-specific DNA-binding activity of OBP.