| Congresso Brasileiro de Microbiologia 2023 | Resumo: 1209-1 | ||||
Resumo:Intestinal mucins are an important complex of glycoproteins responsible for preventing the establishment of pathogenic strains at the epithelial cell surface, protecting it from chemical and physical alterations. Among the groups of pathogenic Escherichia coli, only the enteroaggregative pathotype has shown the dual activity of degrading and inducing mucin secretion as part of its pathogenicity phenotype. In this study, we showed the ability of a subset of atypical enteropathogenic E. coli (aEPEC) strains to induce mucus hypersecretion in goblet-like cells and to degrade commercial mucin to penetrate mucus layers and use mucin as a carbon source. To this end, subcultures of 13 aEPEC strains were inoculated in 24-well plates containing LS174T cells at 70-80% confluence in Dulbecco’s Modified Eagle Medium (DMEM) plus 10% fetal bovine serum and a MOI of 50. After 5 h of incubation at 37ºC, the monolayers were washed, fixed, and stained with Alcian Blue (AB) and Periodic Acid-Schiff (PAS) staining kits. All images were obtained using a CMOS color camera and processed in ImageJ using a color deconvolution plugin. Moreover, gel-forming secreted mucins, MUC2 and MUC5AC, were detected on non-permeabilized cells by immunofluorescence with monoclonal antibodies and analyzed by confocal microscopy. Furthermore, to confirm whether the classical Attaching and Effacing (A/E) lesion was induced in goblet-like cells, z-sections from confocal images were evaluated, along with analysis of the monolayers by scanning electron microscopy (SEM). For the mucinolytic activity assessment, growth kinetics in M9 minimal medium (M9) without glycose and M9 supplemented with 3 g/L commercial mucin (porcine stomach type III) were evaluated after 2, 4, and 6 h of incubation at 37ºC, after which the bacterial cultures were serially diluted, plated and counted. Transwells with 8 µm pores coated with 100 g/L mucin and DMEM were mounted in 24-well plates containing Lysogeny Broth at the bottom chamber to determine the bacterial ability to penetrate through the mucin layer. An inoculum of 1x106 CFU of each bacterial subculture was inoculated over the mucin layer and after 3 h of incubation were counted as previously described. The staining techniques showed that four strains induced mucin hypersecretion, three belonging to a close phylogenetic cluster. Extracellular mucinous area estimates of infected cells showed significant secreted mucin differences ranging from 10.1 to 34.5% compared to 3.7% in the non-infected cells. The confocal Z-stack images confirmed the induction of hypersecretion of MUC2 and MUC5AC and revealed A/E lesion formation by these strains, also confirmed with SEM images. All strains showed considerable mucinolytic activity as estimated by mucin consumption as a carbon source in the M9 medium, with a growing rate 3 times higher, in some cases, than their growth kinetics in the absence of mucin. In addition, 10 strains penetrated the mucus layer extensively (above 104 UFC/mL). Summarizing, we hypothesize that mucus hypersecretion, degradation and penetration associated with A/E lesion formation on goblet cells should improve the aEPEC colonization in vivo. Together, these responses combined with an increased inflammatory response, it may result in a breakdown of immune tolerance, leading to a consequent tissue damage with loss of function. This approach brought new insights into the pathogenicity mechanisms of aEPEC strains for future studies. Palavras-chave: aEPEC, A/E lesion, goblet cells, mucin, mucinase Agência de fomento:Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), BCO – Pós-Doutorado (Nº 2022/12006-2), Projeto de Pesquisa Temático (2017/14821-7) | |||||