Functional characterization of the collagen-binding protein DIP2093 and its influence on host–pathogen interaction and arthritogenic potential of Corynebacterium diphtheriae

Abstract

Corynebacterium diphtheriae is typically recognized as the a etiological agent of diphtheria, a toxaemic infection of the respiratory tract; however, both non-toxigenic and toxigenic strains are increasingly isolated from cases of invasive infections. The molecular mechanisms responsible for bacterial colonization and dissemination to host tissues remain only partially understood. In this report, we investigated the role of DIP2093, described as a putative adhesin of the serine-aspartate repeat (Sdr) protein family in host-pathogen interactions of C. diphtheriae wild-type strain NCTC13129. Compared to the parental strain, a DIP2093 mutant RN generated in this study was attenuated in its ability to bind to type I collagen, to adhere to and invade epithelial cells, as well as to survive within macrophages. Furthermore, DIP2093 mutant strain RN had a less detrimental impact on the viability of Caenorhabditis elegans as well as in the clinical severity of arthritis in mice. In conclusion, DIP2093 functions as a microbial surface component recognizing adhesive matrix molecules, and may be included among the factors that contribute to the pathogenicity of C. diphtheriae strains, independently of toxin production.

The C-terminal coiled-coil domain of Corynebacterium diphtheriae DIP0733 is crucial for interaction with epithelial cells and pathogenicity in invertebrate animal model systems

“…In previous studies, the interaction of C. diphtheriae with proteins of the extracellular matrix surrounding eukaryotic cells, e. g. collagen I and IV, was reported [12, 14, 19, 27]. Additionally, the interaction with fibrinogen, a major component of the human plasma, which is crucial for blood clot formation due to its conversion into insoluble fibrin, has also been investigated in corynebacteria [27–29].…”

“…ECM protein binding assays were performed based on the combination of two protocols [7, 18] as previously described [12]. In summary, 96-well polystyrene microtiter plates were coated with collagen type I, collagen type IV and fibrinogen (Sigma, Munich, Germany) dissolved in PBS (Phosphate Buffered Saline) and incubated at 4 °C for 24 h. The coated wells were washed twice with PBS and subsequently filled with 200 μl of resuspended bacterial cells in HI medium (OD 600 of 0.2).…”

“…Cellular interaction assays were performed using epithelial cells derived from human cervical carcinoma as previously described protocols [12, 14]. In short, HeLa cells were infected with C. diphtheriae strains, washed with PBS, detached with trypsin solution, lysed with Tween 20 and the number of colony forming units (CFU) for adhesion was determined.…”

“…The assays were performed as previously described [12, 19–23]. Briefly, C. elegans N2 were maintained and synchronized on plates containing nematode growth medium (NGM) agar for approximately four to seven days at 20 °C and used in infection assays with C. diphtheriae strains.…”

“…Besides classical diphtheria, an increasing number of systemic infections caused by non-toxigenic C. diphtheriae strains have been reported [3–7]. Moreover, it has been demonstrated that C. diphtheriae possesses various virulence factors that may act independently of diphtheria toxin [8–12]. Among these, only a few have been investigated in detail.…”