Macropinocytosis in Shiga toxin 1 uptake by human intestinal epithelial cells and transcellular transcytosis

Abstract

Shiga toxin 1 and 2 production is a cardinal virulence trait of enterohemorrhagic Escherichia coli infection that causes a spectrum of intestinal and systemic pathology. However, intestinal sites of enterohemorrhagic E. coli colonization during the human infection and how the Shiga toxins are taken up and cross the globotriaosylceramide (Gb3) receptor-negative intestinal epithelial cells remain largely uncharacterized. We used samples of human intestinal tissue from patients with E. coli O157:H7 infection to detect the intestinal sites of bacterial colonization and characterize the distribution of Shiga toxins. We further used a model of largely Gb3-negative T84 intestinal epithelial monolayers treated with B-subunit of Shiga toxin 1 to determine the mechanisms of non-receptor-mediated toxin uptake. We now report that E. coli O157:H7 were found at the apical surface of epithelial cells only in the ileocecal valve area and that both toxins were present in large amounts inside surface and crypt epithelial cells in all tested intestinal samples. Our in vitro data suggest that macropinocytosis mediated through Src activation significantly increases toxin endocytosis by intestinal epithelial cells and also stimulates toxin transcellular transcytosis. We conclude that Shiga toxin is taken up by human intestinal epithelial cells during E. coli O157:H7 infection regardless of the presence of bacterial colonies. Macropinocytosis might be responsible for toxin uptake by Gb3-free intestinal epithelial cells and transcytosis. These observations provide new insights into the understanding of Shiga toxin contribution to enterohemorrhagic E. coli-related intestinal and systemic diseases.

Large-Scale Screening and Identification of Novel Ebola Virus and Marburg Virus Entry Inhibitors

“…This low rate suggests that the compounds are acting specifically and may provide insight into their cellular targets. MLS000394177, the macropinocytosis inhibitor, was active in assays for inhibition of Janus kinase 2 (JNK2), Synuclein activity, and Shiga toxins A and B. Shiga toxins are known to be taken into cells by macropinocytosis (51,52). However, a role for JNK signaling or Synuclein in macropinocytosis has not been reported, but each is associated with cytoskeletal rearrangements that could promote macropinocytosis.…”

Mecanismos de virulencia de Escherichia coli enteropatógena

“…Inmediatamente después, el auto-transportador EatA (EatA: autotransporter A) incluida en el grupo de las serin-proteasas (SPATEs: serine protease autotransporter of Enterobacteriaceae) inhiben la actividad de EtpA, lo que da origen a la adhesión de ECET a los enterocitos por el loci toxigénico de invasión A (Tia: toxigenic invasion loci A), una proteína de membrana externa y el loci toxigénico de invasión B (TibA: toxigenic invasion loci B) (Figura 3) [69][70][71][72] . P) 50,51 , que contribuye a la formación de microcolonias y a la adhesión a células epiteliales de colon T84 51 (Figura 2b); b) Transcitosis: mediante vesículas la toxina pasa de un espacio extracelular a otro (Figura 2b) 52 . Este mecanismo aún está en estudio; no obstante, se conoce que aumenta debido a la MPC y facilita la propagación sistémica de la toxina hacia las células endoteliales que expresan Gb3, siendo el endotelio glomerular el que expresa los mayores niveles 10 .…”

“…Este mecanismo aún está en estudio; no obstante, se conoce que aumenta debido a la MPC y facilita la propagación sistémica de la toxina hacia las células endoteliales que expresan Gb3, siendo el endotelio glomerular el que expresa los mayores niveles 10 . Le siguen en su orden las células endoteliales del intestino y cerebro 50,52,53 ; c) Endocitosis: una vez se une la subunidad B de la toxina al receptor Gb3 y posteriormente, mediante invaginación de la membrana celular, la toxina es introducida al citoplasma 54 . Adicionalmente, el fragmento A 1 de la subunidad A, realiza actividad enzimática sobre el ARNr 28S, inhibe la síntesis proteica y conduce a la muerte celular 55 (Figura 2c).…”

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

“…The Stx1 family consists of Stx1, Stx1c, and Stx1d, and the Stx2 group consists of Stx2, Stx2c, Stx2c2, Stx2d, Stx2e, and Stx2f. EHEC adhering at the apical surface of fully differentiated T84 cells induces Src-dependent macropinocytosis that allows the delivered Stx1 to penetrate and traffic within the cells (635)(636)(637)(638) (Table 3). EHEC expressing Stx1 causes galectin 3 depletion in fully differentiated T84 cells by increasing the expression and secretion of apical galectin 3, which in turn impairs trafficking and apical protein mistargeting of several brush border structural proteins and transporters, including villin, DPP IV, and NHE2 (639,640).…”