Pic Protein From Enteroaggregative E. coli Induces Different Mechanisms for Its Dual Activity as a Mucus Secretagogue and a Mucinase

Abstract

A hallmark of enteroaggregative Escherichia coli (EAEC) infection is the formation of an intestinal biofilm, which comprises a mucus layer with immersed bacteria. Pic is an autotransporter secreted by EAEC, and other E. coli pathotypes, and has been involved in two apparently contradictory phenotypes, as a mucus secretagogue and as a mucinase. Here, we investigated this Pic dual activity, mucus secretagogue capability and mucinolytic activity, in human goblet cells that secrete MUC2 and MUC5AC. Pic induced mucus hypersecretion directly in the goblet cells, without other intestinal cell types involved. At the same time, Pic exhibited strong proteolytic activity on the secreted mucins. These activities were independent since a mutation in the serine protease motif (PicS258I) abolished mucin degradation while maintaining the mucus secretagogue activity intact. Furthermore, deoxycholic acid (DCA)-induced mucins were proteolytically degraded when goblet cells were co-incubated with DCA/Pic, while co-incubation with DCA/PicS258I induced a synergistic effect on mucus hypersecretion. Pic was more efficient degrading MUC5AC than MUC2, but no degradation was detected with Pic inactivated at the active site by mutation or pharmacological inhibition. Remarkably, Pic cleaved MUC2 and MUC5AC in the C-terminal domain, leaving N-terminal subproducts, impacting the feature of gel-forming mucins and allowing mucus layer penetration by EAEC. Astonishingly, Pic stimulated rapid mucin secretion in goblet-like cells by activating the intracellular calcium pathway resulting from the PLC signal transduction pathway, leading to the production of DAG and releasing IP 3 , a second messenger of calcium signaling. Therefore, the dual activity of Pic, as a mucus secretagogue and a mucinase, is relevant in the context of carbon source generation and mucus layer penetration, allowing EAEC to live within the layer of mucus but also access epithelial cells.

Serine proteases autotransporter of Enterobacteriaceae: Structures, subdomains, motifs, functions, and targets

“…As mentioned above, a prototype class‐1 SPATE is Pet, where its cytotoxic activity has been extensively studied (Betancourt‐Sanchez & Navarro‐Garcia, 2009; Canizalez‐Roman & Navarro‐Garcia, 2003; Chavez‐Duenas et al, 2016; Navarro‐Garcia et al, 1999, 2001; Navarro‐Garcia, Canizalez‐Roman, Burlingame, et al, 2007; Navarro‐Garcia, Canizalez‐Roman, Vidal, et al, 2007). On the other hand, Pic is a prototype protein of SPATE class 2, where its phenotypes as a mucosal colonization and immunomodulation factor have also been extensively studied (Abreu et al, 2015, 2016; Bhullar et al, 2015; Durand et al, 2016; Dutta et al, 2002; Flores‐Sanchez et al, 2020; Gutierrez‐Jimenez et al, 2008; Harrington et al, 2009; Henderson et al, 1999; Navarro‐Garcia et al, 2010; Ruiz‐Perez et al, 2011). Therefore, the function of these two SPATEs will be revised in extension and compared with other members of these two SPATE classes.…”

“…Interestingly, these activities are independent since the mutation in the SPM (PicS258I) abolished mucin degradation while maintaining the mucus secretagogue activity. It was also elegantly demonstrated that deoxycholic acid (DCA)‐induced mucins were proteolytically degraded when goblet cells were co‐incubated with Pic (DCA/Pic), while co‐incubation with DCA/PicS258I induced a synergistic effect on mucus hypersecretion (Flores‐Sanchez et al, 2020).…”

“…Degradation of this large part of MUC5AC/MUC2 at the C‐terminal portion is critical for gel‐forming mucins, since these mucins form dimers in the far C‐terminal end, by the cysteine‐knot domain (Flores‐Sanchez et al, 2020), which would destroy the large net‐like sheets of the gel‐forming mucins. Indeed, Pic‐secreting EAEC are able to efficiently penetrate the mucus layer, but not variants with mutations in the SPM of Pic (Flores‐Sanchez et al, 2020).…”

“…PLC catalyzes PIP 2 conversion to IP 3 and DAG; IP 3 is the major contributor to intracellular calcium release (Tran et al, 2016) and DAG targets Munc13 (Zhu et al, 2007). DAG production was identified by PKCδ phosphorylation induced by Pic, cPKCs, and nPKCs are the best characterized targets for DAG; PKCδ activation enhances mucin secretion (Flores‐Sanchez et al, 2020).…”

“…On the other hand, Pic is a prototype protein of SPATE class 2, where its phenotypes as a mucosal colonization and immunomodulation factor have also been extensively studied (Abreu et al, 2015(Abreu et al, , 2016Bhullar et al, 2015;Durand et al, 2016;Dutta et al, 2002;Flores-Sanchez et al, 2020;Gutierrez-Jimenez et al, 2008;Harrington et al, 2009;Henderson et al, 1999;Navarro-Garcia et al, 2010;Ruiz-Perez et al, 2011). Therefore, the function of these two SPATEs will be revised in extension and compared with other members of these two SPATE classes.…”