Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells

BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

Interferon-γ inhibits group B Streptococcus survival within human endothelial cells

Endothelial dysfunction is a major component of the pathophysiology of septicaemic group B Streptococcus (GBS) infections. Although cytokines have been shown to activate human umbilical vein endothelial cells (HUVECs), the capacity of interferon (IFN)-γ to enhance the microbicidal activity of HUVECs against GBS has not been studied. We report that the viability of intracellular bacteria was reduced in HUVECs activated by IFN-γ. Enhanced fusion of lysosomes with bacteria-containing vacuoles was observed by acid phosphatase and the colocalisation of Rab-5, Rab-7 and lysosomal-associated membrane protein-1 with GBS in IFN-γ-activated HUVECs. IFN-γ resulted in an enhancement of the phagosome maturation process in HUVECs, improving the capacity to control the intracellular survival of GBS.

Prevalence of Group B Streptococcus serotypes III and V in pregnant women of Rio de Janeiro, Brazil

GBS serotypes III and V were the most prevalent in pregnant women and exhibited resistance to tetracycline, clindamycin and sulfamethoxazole/trimethoprim. Serotype III showed high sialic acid content and PFGE analysis discerned 33 heterogeneous profiles. Phenotypic and genotypic characterization could be relevant to control GBS infections unaffected by intra-partum chemoprophylaxis.

Aggregative adherent strains of Corynebacterium pseudodiphtheriticum enter and survive within HEp-2 epithelial cells

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

Difteria pelo Corynebacterium ulcerans: uma zoonose emergente no Brasil e no mundo / Corynebacterium ulcerans diphtheria: an emerging zoonosis in Brazil and worldwide / Difteria por Corynebacterium ulcerans: una zoonosis emergente en Brasil y en el mundo

O artigo revisa a literatura sobre a emergência de infecções humanas causadas por Corynebacterium ulcerans em diversos países, incluindo o Brasil. Foi realizada análise de artigos publicados entre 1926 e 2011 nas bases Medline/PubMed e SciELO, bem como artigos e informes do Ministério da Saúde. Apresenta-se um esquema de triagem, rápido, econômico e de fácil execução, capaz de permitir a realização do diagnóstico presuntivo de C. ulcerans e C. diphtheriae na maioria dos laboratórios brasileiros públicos e privados. A circulação de C. ulcerans em vários países, aliada aos recentes casos de isolamento do patógeno no Rio de Janeiro, é um alerta a clínicos, veterinários e microbiologistas sobre a ocorrência de difteria zoonótica e a circulação do C. ulcerans em regiões urbanas e rurais do território nacional e/ou da América Latina.

The article is a literature review on the emergence of human infections caused by Corynebacterium ulcerans in many countries including Brazil. Articles in Medline/PubMed and SciELO databases published between 1926 and 2011 were reviewed, as well as articles and reports of the Brazilian Ministry of Health. It is presented a fast, cost-effective and easy to perform screening test for the presumptive diagnosis of C. ulcerans and C. diphtheriae infections in most Brazilian public and private laboratories. C. ulcerans spread in many countries and recent isolation of this pathogen in Rio de Janeiro, southeastern Brazil, is a warning to clinicians, veterinarians, and microbiologists on the occurrence of zoonotic diphtheria and C. ulcerans dissemination in urban and rural areas of Brazil and/or Latin America.

El articulo revisa la literatura sobre la emergencia de infecciones humanas causadas por Corynebacterium ulcerans en diversos países, incluyendo Brasil. Se realizó análisis de artículos publicados entre 1926 y 2011 en las bases Medline/Pubmed y SciELO, así como artículos e informes del Ministerio Brasileño de la Salud. Se presenta un esquema de selección, rápido, económico y de fácil ejecución, capaz de permitir la realización del diagnóstico presuntivo de C. ulcerans y C. diphtheriae en la mayoría de los laboratorios brasileños públicos y privados. La circulación de C. ulcerans en varios países, aliada a los recientes casos de aislamiento del patógeno en Rio de Janeiro (Sureste de Brasil), es un alerta a clínicos, veterinarios y microbiólogos sobre la ocurrencia de difteria zoológica y la circulación de C. ulcerans en regiones urbanas y rurales del territorio nacional y/o de América Latina.

Fibrinogen binds to nontoxigenic and toxigenic Corynebacterium diphtheriae strains

The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.