| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1207-1 | ||||
Resumo:Despite being commonly found as part of the intestinal microbiota of humans and several warm-blooded animals, some Escherichia coli strains are potentially pathogenic. Among the various pathotypes of the species, extraintestinal pathogenic E. coli (ExPEC) stands out for its ability to commensally colonize the gut microbiota, but also for being pathogenic in other sites. Based on the current literature, it is known that ExPEC can be considered a relevant pathogen in some diseases that affect the respiratory system, such as aspiration pneumonia and cystic fibrosis. In the airways, mucus constitutes an essential barrier against infections, as it hinders the colonization of pathogens that might reach the site. Some pathogenic bacteria, however, can employ several mechanisms to overcome this barrier, thus favoring their growth and subsequent infection. It is currently known that some enteropathogenic strains of E. coli can degrade mucins, induce mucus secretion and use mucins as a source of carbon. However, how ExPEC strains interact with mucus is still an understudied issue. Therefore, this work aims to characterize and evaluate the interaction between five ExPEC strains isolated from bloodstream infections and pulmonary epithelial cells (A549) cultivated in vitro to evaluate the ability of these strains to degrade and use mucins as a carbon source, as well as assess the presence of mucinolytic enzymes in silico. For the assays, A549 cells were cultured in 24-well plates in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37°C to an 85-90% confluence. Adherence and invasion assays were performed with a 3 h incubation period. The bacterial invasion was assessed by CFU counting and by transmission electron microscopy (TEM). Mucin degradation tests were performed by adding Transwell inserts (PET membrane with 8.0 μm pores) filled with 50 μl of 10% mucin from porcine stomach and 150 μl of DMEM to 24-well plates. After a 3 h-incubation period, medium in the lower compartment was collected, serially diluted, and plated onto MacConkey agar for bacterial counting. The use of mucins as a carbon source was evaluated by comparing the growth curve following bacterial cultivation in M9 minimal medium (M9) and M9 supplemented with 0.3% mucin. The bacterial counts were performed as previously described. Lastly, a search for mucinolytic enzymes was performed on BLASTp in all strains’ genomes. All strains interacted with A549 cells, but two were cytotoxic to the cells. Cell invasion assays were then performed with the three non-cytotoxic strains, and two of them were able to invade the A549 cells. TEM images of the most invasive strain suggested that bacterial invasion may occur by intercellular spaces. Regarding the mucinolytic activity, all five strains could degrade mucins, with 105 to 107 CFU/mL successfully crossing the mucus barrier. Furthermore, all strains used mucin as a carbon source to varying degrees. Regarding mucolytic enzymes, SslE, Vat, and Tsh were identified in four, two, and one strains, respectively. One strain was devoid of all searched enzymes. Our results indicate that ExPEC strains present relevant pathogenicity against A549 cells, being able to degrade and use mucins as a carbon source. These results give some insights into how ExPEC strains might be able to colonize and cause infections in the respiratory tract. Palavras-chave: ExPEC, Lung infections, Mucin, Mucinase, Pathogenicity Agência de fomento:CAPES, FAPESP | |||||