Avaliação da interação entre amostras de Corynebacterium spp. associadas à infecção do trato respiratório e células humanas simulando o microambiente alveolar in vitro

Abstract

Respiratory infections are among the leading causes of death recognized worldwide. Worryingly, bacteria of the genus Corynebacterium such as Corynebacterium diphtheriae, Corynebacterium ulcerans and Corynebacterium pseudodiphtheriticum have demonstrated the ability to infect the lower respiratory tract (LRT). Despite this, studies aimed at the factors involved in the pathogenesis of infections that affect the LRT of these three species are scarce, highlighting the importance of conducting investigations. Attempting to achieve this, the objective of this study was to evaluate the interaction of strains of C. diphtheriae, C. ulcerans and C. pseudodiphtheriticum with human cells in vitro. For this purpose, the representative samples of each species selected were: C. pseudodiphtheriticum 2802, C. pseudodiphtheriticum 2803 (both obtained from nasal cavity), C. pseudodiphtheriticum ATCC 10700T (exudative pharyngitis), C. ulcerans 809 (brochoalveolar lavage), C. ulcerans 2965 (oropharynx), C. diphtheriae ATCC 27010T (diphtheria) and its homologue C. diphtheriae ATCC 27012. The cytotoxicity of the metabolites produced by all strains used in this study on monoculture of alveolar epithelial cells (A549) was evaluated, as well as the adhesion pattern presented (aggregative, diffuse or localized), quantitative adhesion and ability to migrate to the intracellular compartment. Furthermore, an indirect culture model was developed using the A549 and THP-1 cell lines to simulate the alveolar microenvironment in vitro. The model was built in the Transwell® system and was intended solely for evaluating the immune response caused by Corynebacterium spp. The influence of the samples on each cell type separately was also evaluated. The results obtained showed a mixed-type performance pattern presented by C. diphtheriae 27012, while the other samples exhibited an aggregate-type pattern. Regarding the cytotoxicity of the metabolites, only the strains C. ulcerans 809 and C. pseudodiphtheriticum 2802 led to a significant reduction in cell vision. Therefore, three samples were selected for the other tests: C. diphtheriae ATCC 27010T, C. ulcerans 809 and C. pseudodiphtheriticum 2802. Through the quantitative adhesion test, all were able to adhere significantly to A549 cell line, varying only in intensity as a function of time. Through the exclusion test with amikacin, only C. diphtheriae ATCC 27010T and C. ulcerans 809 were able to migrate into the cells. A previous characterization of A549 epithelial cells cultured in the Transwell® system demonstrated that cultivation in contact with air provides greater resistance to external stimuli and better organization on the culture support. Corroborating the characterization findings, when stimulating epithelial cells cultured within the system with C. ulcerans 809 and C. diphtheriae ATCC 27010T, greater cytokine release was observed by cells cultured submerged than in contact with air. In the indirect coculture model developed in this study, A549 epithelial cells infected with C. ulcerans 809 appear to modulate IL-6 expression by THP-1-derived macrophages. These findings show that Corynebacterium species associated with TRI infections are capable of interacting with lung epithelial cells, with C. ulcerans 809 being highlighted here for its ability to induce severe immunological alterations and cell death.