Serine Protease EspP from Enterohemorrhagic Escherichia Coli Is Sufficient to Induce Shiga Toxin Macropinocytosis in Intestinal Epithelium

Abstract

Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells’ basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.

Zinc protects against shiga-toxigenic Escherichia coli by acting on host tissues as well as on bacteria

“…Tight junctions are known to be regulated by extracellular divalent metals, especially calcium and zinc [34,73-77]. Based on previous research, therefore, we believe the effect of zinc on Stx translocation seen in Figures  1 and 2, and in Phase 2 of Figure  7, is likely due do its protective effect on the paracellular pathway rather than the transcellular/macropinocytosis pathway for Stx translocation that has also been well described [29,30]. …”

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).…”