Dulanthi Weerasekera

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Analysis of virulence factors of Corynebacterium diphtheriae

Principal investigator
Andreas Burkovski

Mentor
Anja Lührmann

 

Characterization of new virulence factors of Corynebacterium diphtheriae

Corynebacterium diphtheriae is the classical aetiological agent of diphtheria, a localized toxaemic infection of respiratory tract that can be fatal. It can also lead to other systemic infections such as endocarditis, bacteraemia, pneumonia, osteomyelitis etc. This indicates that C. diphtheriae is not only colonizes epithelia but also penetrate into deeper parts of the body and interact with various types of host cells. Besides the main virulence factor i.e, diphtheria toxin, there are few other virulence factors that have also been characterized in detail. These include mainly pili and a few other adhesion factors. However, little is known about the factors mediating the entry processes in the host cell and the receptors involved in recognition. An interesting protein in respect to its role in C. diphtheriae pathogenicity is DIP0733, which has an ability to interact with matrix proteins and cell surfaces participating in cellular internalization. Despite the different properties attributed to the protein, i.e, haemagglutination, binding to host cell receptors and induction of apoptosis, it has also been identified that the protein sequence is lacking annotation of functional domains within the protein. However, the molecular characterization of the protein DIP0733 has shown functionally astonishing and diverse characteristics with respect to its virulence properties and therefore it has been considered as a microbial surface components recognizing adhesive matrix molecule (MSCRAMM). The sequenced toxigenic strain CDC-E8392 of C. diphtheriae and its insertion mutant CAM-1 strain which showed a kanamycin-resistant phenotype was used in this study. As a more quantitative approach we are analyzing the differences between the wild-type and mutant strains by determining the pattern of adhesion and invasion in HeLa cells. Interestingly, the adhesion and invasion rates of mutant strain seems to be more attenuated than the wild type. Furthermore, for a more detailed molecular analysis of this multi-functional protein, extensive mutagenesis studies including the truncated DIP0733 forms need to be studied. Therefore, the aim of this project is to further investigate new adhesion and virulence factors of C. diphtheriae, elucidating the role of the gene encoding the protein DIP0733 and by tracking how the process of interaction with the host cell contributes as a virulence property.

Figure: Schematic illustration of DT like cellular intoxication pathways based on Simon et al. 2014. Diphtheria toxin (DT)-like toxins bind to host membrane-bound receptors and enter cells via receptor-mediated endocytosis. Protease cleavage of the peptide backbone results in a single disulphide-linked chain. Following endosomal acidification, the A domain of DT is translocated into the cytosol by insertion of the translocation domain into the endosomal membrane and reduction of the disulphide bond. Conversely, Pseudomonas aeruginosa exotoxin A (PE) undergoes retrograde trafficking to the ER, where reduction of the disulphide bond and interactions with ER-associated chaperone proteins, such as protein disulphide isomerase (PDI), promote the translocation of the A domain through a Sec61-like channel into the cytosol. Once in the cytosol, the A domains of DT-like toxins ADP-ribosylate elongation factor 2 (EF2), which inhibits protein synthesis and kills the cell. Cholix toxin (ChxA) can use low-density lipoprotein receptor-related protein 1 (LRP1) to enter cells and may follow the same intoxication pathway as PE. (Simon et al. 2014).

 

Publications

 

Presentations

October 2016 8th Annual Retreat, Erlangen School of Molecular Communication, Schloss Schney, Lichtenfels, Germany
“Characterization of new virulence factors of Corynebacterium diphtheriae and Corynebacterium ulcerans
Poster