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

Monica Cristina De Souza,Lídia Maria Buarque Oliveira Asad,Raphael Hirata Júnior,Ana Claúdia De Paula Rosa,Thereza Christina Ferreira Camello,Débora Leandro Rama Gomes,Louisy Sanches Dos Santos,Cinta Silva Dos Santos,Prescilla Emy Nagao,Ana Luiza De Mattos Guaraldi,Priscila Soares Sabbadini

doi:10.1590/s0074-02762012000400008

Abstract

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.

Highlights

More than half of all of the infectious diseases that affect mildly immunocompromised patients involve bacterial species that are commonly encountered in the environment or are found among the body’s normal flora
Bacterial strains and culture conditions - The C. pseudodiphtheriticum ATCC 10700 type strain, isolated from exudative pharyngitis (Izurieta et al 1997) and two clinical isolates previously obtained from patients (Camello et al 2009, Martins et al 2009) that were treated at a teaching hospital in the metropolitan area of Rio de Janeiro, Brazil were used in these experiments
Microorganisms were attached to the surfaces of the cultured epithelial cells at focal points and to exposed areas of the glass slide around the HEp-2 cells

Summary

Introduction

More than half of all of the infectious diseases that affect mildly immunocompromised patients involve bacterial species that are commonly encountered in the environment or are found among the body’s normal flora. Opportunistic pathogens, including Corynebacterium pseudodiphtheriticum, are frequently resistant to antimicrobial agents and there is no effective vaccine against this microbe. C. pseudodiphtheriticum has been reported to cause human disease in both immunocompromised and immunocompetent individuals, despite the fact that it is commonly found as a harmless commensal organism on the skin and in the upper respiratory tract (Ahmed et al 1995, Burke et al 1997, von Graevenitz et al 1998, Camello et al 2009, Martins et al 2009, Bittar et al 2010, Olender & Niemcewicz 2010). Additional studies are necessary to investigate whether C. pseudodiphtheriticum strains that have the (HA)-positive phenotype exist and, if so, to what extent they are involved in human infections. We investigated the adherence pattern, invasion, intracellular survival and persistence of C. pseudodiphtheriticum within human epithelial cells

Methods

Results

Conclusion