Renata Stavracakis Peixoto

Suche


Analysis of virulence factors of Corynebacterium diphtheriae

Principal investigator
Andreas Burkovski

Mentor

 

Phenotypic and genotypic characterization of Corynebacterium diphtheriae interactions with abiotic and biotic surfaces

Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, different clones have been also associated with invasive infections such as sepsis, endocarditis, septic arthritis and osteomyelitis. While the production of diphtheria toxin has been well-established as a major virulence factor, little is known about C. diphtheriae  factors crucial for colonization of the host, corresponding host receptors recognized by these factors, development of the infection and haematogenic dissemination, even though an essentials steps of pathogenicity. In this study, we evaluated the association and invasion mechanisms with human umbilical vein endothelial cells (HUVECs) and experimental arthritis in mice of endocarditis-associated strains and control non-invasive strains. C. diphtheriae  strains were able to adhere to and invade HUVECs at different levels. The endocarditis-associated strains displayed an aggregative adherence pattern and a higher number of internalized viable cells in HUVECs. Transmission electron microscopy (TEM) analysis revealed intracellular bacteria free in the cytoplasm and/or contained in a host-membrane-confined compartment as single micro-organisms. Data showed bacterial internalization dependent on microfilament and microtubule stability and involvement of protein phosphorylation in the HUVEC signalling pathway. A high number of affected joints and high arthritis index in addition to the histopathological features indicated a strain-dependent ability of C. diphtheriae  to cause severe polyarthritis. A correlation between the arthritis index and increased systemic levels of IL-6 and TNF-α was observed for endocarditis-associated strains. In conclusion, higher incidence of potential mechanisms by which C. diphtheriae  may access the bloodstream through the endothelial barrier and stimulate the production of pro-inflammatory cytokines such as IL-6 and TNF-a, in addition to the ability to affect the joints and induce arthritis through haematogenic spread are thought to be related to the pathogenesis of endocarditis-associated strains. While pathogen factors responsible for adhesion are well known, the molecular background of adhesion is more or less unclear. Since we were interested in this process, we started a functional genetics approach to identify a sortase anchored protein DIP2093, which shows weak similarity to members of the serine aspartate repeat (Sdr) family of adhesins from staphylococci (MSCRAMMs), described in Cerdeño-Tárraga et al, 2003 and D’Afonseca et al, 2011 as possible target to be used in development of vaccines.

Figure: Micrographs demonstrating the aggregative adherence pattern in HUVECs, characterized by clumps of bacteria with a ‘stacked-brick’ appearance of endocarditis-associated C. diphtheriae  subsp. mitis HC01 (a) and HC02 (b) blood isolates (3 h incubation). Magnification, _1000. TEM demonstrating intimate contact (c) and focal points of adherence (d) of C. diphtheriae  with the host cell membrane; (e, f) the HC01 strain internalization process in HUVECs after 3 h incubation; (g, h) electron-dense material surrounding adherent bacteria and formation of a cup-shaped process on the endothelial cell membrane underlying adherent bacteria. Internalized bacterial cells are located in a loose vacuole. (i, j) Micro-organisms appear to be free within partially degraded cytoplasm. Areas of interest are marked with black arrows. Scale bars represent 500 nm in (c), (e), (g) and (i), and 200nm in (f), (h) and (j).

 

Publications

 

Presentations

     
July 2015 7th Annual Retreat, Erlangen School of Molecular Communication, Schloss Hirschberg, Beilngries, Germany
”Phenotypic and genotypic characterization of Corynebacterium diphtheriae interations with abiotic and biotic surfaces”
Poster