Caracterização funcional do gene dip1180 e influência na patogenicidade de cepas de Corynebacterium diphtheriae de origens diversas

Abstract

Diphtheria is an acute and immunopreventable disease caused by Corynebacterium diphtheriae. Besides classical diphtheria, C. diphtheriae has also been isolated from invasive infections, mainly caused by non-toxigenic strains, revealing the involvement of other virulence factors. The sequencing of the first pangenomic study of this species allowed the identification of 57 genomic islands, with highly differentiated genetic content, suggesting the horizontal transference of genetic material, and those differences could be involved in host-parasite interaction. The importance of hemolysins on the virulence of different bacterial species has been emphasized in the literature, alongside its possibility of transference between bacterial strains. Some C. diphtheriae strains may produce hemolysins, and some authors have classified C. diphtheriae strains according to its hemolytic activity. Nevertheless, little is still known about the virulence associated to hemolytic activity in C. diphtheriae, and this is the proposal of this investigation. At first, we have investigated the presence of putative protein sequences related to cytotoxin/hemolysin in the sequenced NCTC 13129, as a model to search for the sequences in other sequenced Brazilian strains deposited in NCBI; to predict the tridimensional structure and analyze the functional similarity to hemolysins of other species; to design primers and to evaluate the amplification after PCR reactions, and to construct a mutant strain through homologous recombination. The influence of this gene on the pathogenicity was evaluated based on hemolytic activity assays, interaction on both biotic an non-biotic surfaces, and evaluation of the strain to cause hematogenous infection and bone infection, in vivo, using mice model. The codifying sequence of hemolysin DIP 1180, was found in the wild type strain, and presented high similarity with other C. diphtheriae strains deposited in NCBI. The tridimensional structure was very similar to hemolysins of Streptococcus genera, reinforcing the probable hemolytic activity. The gene truncation by homologous recombination in the gene dip1180, demonstrated the hemolytic properties was related to the expression of this gene in C. diphtheriae. Non-toxigenic C. diphtheriae strains presented an intense hemolytic activity when compared to toxigenic strains. Capnophilic environment appeared to favor the hemolytic activity, except to non-hemolytic strains MA 136 and the mutant LDCIC-A1. A reduction in the capability to adhere to polystyrene and glass and to form biofilm was observed in the mutant LDCIC-A1 strain, demonstrating the influence of this gene in biofilm production. Iron deprivation in the culture medium was able to promote a significant reduction in biofilm production, demonstrating the relevance of this ion in the adhesion of C. diphtheriae, regardless of the presence of both tox and dip1180 genes. The in vivo analysis of the influence of the dip1180 gene on the arthritogenic activity of C. diphtheriae demonstrated a noticeable reduction in the severity of the lesions presented by the mice infected with the mutant strain. The data obtained indicate the important contribution of the dip1180 gene to the virulence of C. diphtheriae strains, especially in non-TD producing strains, which have long been neglected and associated with increasing frequency of invasive infections.