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

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.

Caenorhabditis elegans star formation and negative chemotaxis induced by infection with corynebacteria.

Caenorhabditis elegans is one of the major model systems in biology based on advantageous properties such as short life span, transparency, genetic tractability and ease of culture using an Escherichia coli diet. In its natural habitat, compost and rotting plant material, this nematode lives on bacteria. However, C. elegans is a predator of bacteria, but can also be infected by nematopathogenic coryneform bacteria such Microbacterium and Leucobacter species, which display intriguing and diverse modes of pathogenicity. Depending on the nematode pathogen, aggregates of worms, termed worm-stars, can be formed, or severe rectal swelling, so-called Dar formation, can be induced. Using the human and animal pathogens Corynebacterium diphtheriae and Corynebacterium ulcerans as well as the non-pathogenic species Corynebacterium glutamicum, we show that these coryneform bacteria can also induce star formation slowly in worms, as well as a severe tail-swelling phenotype. While C. glutamicum had a significant, but minor influence on survival of C. elegans, nematodes were killed after infection with C. diphtheriae and C. ulcerans. The two pathogenic species were avoided by the nematodes and induced aversive learning in C. elegans.

Characterization of DIP0733, a multi-functional virulence factor of Corynebacterium diphtheriae.

Corynebacterium diphtheriae is typically recognized as an extracellular pathogen. However, a number of studies revealed its ability to invade epithelial cells, indicating a more complex pathogen-host interaction. The molecular mechanisms controlling and facilitating internalization of Cor. diphtheriae are poorly understood. In this study, we investigated the role of DIP0733 as virulence factor to elucidate how it contributes to the process of pathogen-host cell interaction. Based on in vitro experiments, it was suggested recently that the DIP0733 protein might be involved in adhesion, invasion of epithelial cells and induction of apoptosis. A corresponding Cor. diphtheriae mutant strain generated in this study was attenuated in its ability to colonize and kill the host in a Caenorhabditis elegans infection model system. Furthermore, the mutant showed an altered adhesion pattern and a drastically reduced ability to adhere and invade epithelial cells. Subsequent experiments showed an influence of DIP0733 on binding of Cor. diphtheriae to extracellular matrix proteins such as collagen and fibronectin. Furthermore, based on its fibrinogen-binding activity, DIP0733 may play a role in avoiding recognition of Cor. diphtheriae by the immune system. In summary, our findings support the idea that DIP0733 is a multi-functional virulence factor of Cor. diphtheriae.

Tellurite resistance: a putative pitfall in Corynebacterium diphtheriae diagnosis?

Corynebacterium diphtheriae strains continue to circulate worldwide causing diphtheria and invasive diseases, such as endocarditis, osteomyelitis, pneumonia and catheter-related infections. Presumptive C. diphtheriae infections diagnosis in a clinical microbiology laboratory requires a primary isolation consisting of a bacterial culture on blood agar and agar containing tellurite (TeO3(2-)). In this study, nine genome sequenced and four unsequenced strains of C. diphtheriae from different sources, including three samples from a recent outbreak in Brazil, were characterized with respect to their growth properties on tellurite-containing agar. Levels of tellurite-resistance (Te(R)) were evaluated by determining the minimum inhibitory concentrations of potassium tellurite (K2TeO3) and by a viability reduction test in solid culture medium with K2TeO3. Significant differences in Te(R) levels of C. diphtheriae strains were observed independent of origin, biovar or presence of the tox gene. Data indicated that the standard initial screening with TeO3(2-)-selective medium for diphtheria bacilli identification may lead to false-negative results in C. diphtheriae diagnosis laboratories.

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans.

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO32-) is toxic to most microorganisms, TeO32--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO32--resistance (TeR) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative TeR determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO32- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the TeR and pathogenic potential of C. diphtheriae.

Corynebacterium ulcerans isolates from humans and dogs: fibrinogen, fibronectin and collagen-binding, antimicrobial and PFGE profiles.

Corynebacterium ulcerans has been increasingly isolated as an emerging zoonotic agent of diphtheria and other infections from companion animals. Since pets are able to act as symptomless carriers, it is also essential to identify virulence potential for humans of these isolates. In this work the ability of C. ulcerans to bind to fibrinogen (Fbg), fibronectin (Fn) and Type I collagen as well the genetic relationship among strains isolated from human and asymptomatic dogs in Rio de Janeiro (Brazil) were analyzed. Five pulsed-field gel electrophoresis (PFGE) profiles were demonstrated (I, II, III, IV and V). In addition, the IV and V profiles exhibiting ≥85 % similarity were expressed by the BR-AD41 and BR-AD61 strains from companion dogs living in the same neighborhood. Independent of the PFGE-types, human and dog isolates showed affinity to Fbg, Fn and collagen. Heterogeneity of PFGE profiles indicated endemicity of C. ulcerans in the Rio de Janeiro metropolitan area. Differences in the expression of adhesins to the human extracellular matrix may contribute to variations in the virulence and zoonotic potential of C. ulcerans strains.

Insights of OxyR role in mechanisms of host-pathogen interaction of Corynebacterium diphtheriae.

Corynebacterium diphtheriae, the leading causing agent of diphtheria, has been increasingly related to invasive diseases, including sepsis, endocarditis, pneumonia, and osteomyelitis. Oxidative stress defense is required not only for successful growth and survival under environmental conditions but also in the regulation of virulence mechanisms of human pathogenic species, by promoting mucosal colonization, survival, dissemination, and defense against the innate immune system. OxyR, functioning as a negative and/or positive transcriptional regulator, has been included among the major bacterial coordinators of antioxidant response. OxyR was first reported as a repressor of catalase expression in C. diphtheriae. However, the involvement of OxyR in C. diphtheriae pathogenesis remains unclear. Accordingly, this work aimed to investigate the role of OxyR in mechanisms of host-pathogen interaction of C. diphtheriae through the disruption of the OxyR of the diphtheria toxin (DT)-producing C. diphtheriae CDC-E8392 strain. The effects of OxyR gene disruption were analyzed through interaction assays with human epithelial cell lines (HEp-2 and pneumocytes A549) and by the induction of experimental infections in Caenorhabditis elegans nematodes and Swiss Webster mice. The OxyR disruption exerted influence on NO production and mechanism accountable for the expression of the aggregative-adherence pattern (AA) expressed by CDC-E8392 strain on human epithelial HEp-2 cells. Moreover, invasive potential and intracytoplasmic survival within HEp-2 cells, as well as the arthritogenic potential in mice, were found affected by the OxyR disruption. In conclusion, data suggest that OxyR is implicated in mechanisms of host-pathogen interaction of C. diphtheriae.

Pathogenic properties of a Corynebacterium diphtheriae strain isolated from a case of osteomyelitis.

Corynebacterium diphtheriae is typically recognized as a colonizer of the upper respiratory tract (respiratory diphtheria) and the skin (cutaneous diphtheria). However, different strains of Corynebacteriumdiphtheriae can also cause invasive infections. In this study, the characterization of a non-toxigenic Corynebacteriumdiphtheriae strain (designated BR-INCA5015) isolated from osteomyelitis in the frontal bone of a patient with adenoid cystic carcinoma was performed. Pathogenic properties of the strain BR-INCA5015 were tested in a Caenorhabditis elegans survival assay showing strong colonization and killing by this strain. Survival rates of 3.8±2.7 %, 33.6±7.3 % and 0 % were observed for strains ATCC 27010T, ATCC 27012 and BR-INCA5015, respectively, at day 7. BR-INCA5015 was able to colonize epithelial cells, showing elevated capacity to adhere to and survive within HeLa cells compared to other Corynebacteriumdiphtheriae isolates. Intracellular survival in macrophages (THP-1 and RAW 264.7) was significantly higher compared to control strains ATCC 27010T (non-toxigenic) and ATCC 27012 (toxigenic). Furthermore, the ability of BR-INCA5015 to induce osteomyelitis was confirmed by in vivo assay using Swiss Webster mice.

Corynebacterium diphtheriae putative tellurite-resistance protein (CDCE8392_0813) contributes to the intracellular survival in human epithelial cells and lethality of Caenorhabditis elegans

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO3^2-) is toxic to most microorganisms, TeO3^2--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO3^2--resistance (Te^R) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative Te^R determinant (CDCE8392_813gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO3^2- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegansand the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the Te^R and pathogenic potential of C. diphtheriae.