Pathogenic Lifestyles of E. coli Pathotypes in a Standardized Epithelial Cell Model Influence Inflammatory Signaling Pathways and Cytokines Secretion

Abstract

Inflammatory response is key for the host defense against diarrheagenic Escherichia coli and contributes to the pathogenesis of the disease but there is not a comparative study among different diarrheagenic pathotypes. We analyzed the inflammatory response induced by five diarrheagenic pathotypes in a HT-29 cell infection model. The model was unified to reproduce the pathogenesis of each pathotype. To compare the inflammatory responses we evaluated: (i) nuclear NF-κB and ERK1/2 translocation by confocal microscopy; (ii) kinetics of activation by each pathway detecting p65 and ERK1/2 phosphorylation by Western blotting; (iii) pathways modulation through bacterial infections with or without co-stimulation with TNF-α or EGF; (iv) cytokine profile induced by each pathotype with and without inhibitors of each pathway. EHEC but mainly EPEC inhibited translocation and activation of p65 and ERK1/2 pathways, as well as cytokines secretion; inhibition of p65 and ERK1/2 phosphorylation prevailed in the presence of TNF-α and EGF, respectively. Intracellular strains, EIEC/Shigella flexneri, caused a strong translocation, activation, and cytokines secretion but they could not inhibit TNF-α and EGF stimulation. ETEC and mainly EAEC caused a moderate translocation, but a differential activation, and high cytokines secretion; interestingly TNF-α and EGF stimulation did no modify p65 and ERK1/2 activation. The use of inhibitors of NF-κB and/or ERK1/2 showed that NF-κB is crucial for cytokine induction by the different pathotypes; only partially depended on ERK1/2 activation. Thus, in spite of their differences, the pathotypes can also be divided in three groups according to their inflammatory response as those (i) that inject effectors to cause A/E lesion, which are able to inhibit NF-κB and ERK1/2 pathways, and cytokine secretion; (ii) with fimbrial adherence and toxin secretion with a moderate inhibition of both pathways but high cytokines secretion through autocrine cytokine regulation; and (iii) the intracellular bacteria that induce the highest pathways activation and cytokines secretion by using different activation mechanisms. This study provides a comprehensive analysis of how the different pathogenesis schemes of E. coli pathotypes manipulate inflammatory signaling pathways, which leads to a specific proinflammatory cytokine secretion in a cell model infection that reproduce the hallmarks of infection of each pathotype.

Escherichia coli O157:H7 suppresses host autophagy and promotes epithelial adhesion via Tir-mediated and cAMP-independent activation of protein kinase A

“… 36 Pharmacological inhibition of ERK1/2 phosphorylation with U0126 or PD98059 results in a decrease in TNF-induced autophagy in MCF-7 breast cancer cells. 37 Furthermore, ERK1/2 phosphorylation is blocked by either EPEC 38 or EHEC 37 , 39 infection. In our study, although the mRNA expressions of ERK1/2 were not altered by infection ( Supplementary Figure S2 ), the protein level of phosphorylated ERK1/2 was decreased.…”

EspH Suppresses Erk by Spatial Segregation from CD81 Tetraspanin Microdomains

“…Previous studies reported the ability of EPEC to manipulate the MAPK/Erk pathway. Some studies suggested that EPEC activates the pathway (27,31), while others showed an inhibitory effect (28)(29)(30). We believe that these discrepancies can be attributed to differences in the infection conditions, as EPEC has been shown to induce a transient activation of Erk (e.g., see reference 31).…”

“…Studies have shown that the tetraspanin CD81 activates extracellular signal-regulated kinase (Erk) signaling (26). It was also shown that EPEC infection of epithelial host cells can manipulate Erk activity in a T3SS-dependent manner (27)(28)(29)(30)(31). However, despite the potential importance of the process in the microbe's life cycle, its underlying mechanism has not been fully explored.…”

The Intriguing Evolutionary Journey of Enteroinvasive E. coli (EIEC) toward Pathogenicity

“…Following the discovery that EIEC strains carry a pINV plasmid identical to that of Shigella ( Harris et al, 1982 ; Sansonetti et al, 1982 ; Hale et al, 1983 ) and that they can display a Shigella -like invasive behavior ( Hale et al, 1985 ; Small and Falkow, 1988 ; Taylor et al, 1988 ), in vitro and in vivo studies have been extensively focused on Shigella , providing in-depth knowledge about its pathogenicity/virulence mechanisms. In recent years, the pathogenicity of EIEC has gained new interest and comparative analyses between EIEC and Shigella have been performed, aimed at understanding the different clinical outcome severity of the two infections ( Moreno et al, 2009 ; Bando et al, 2010 ; Sanchez-Villamil et al, 2016 ). Here we first present the invasive process as it has been inferred from studies on S. flexneri .…”

“…Conversely, although following a similar kinetics, the accumulation of phosphorylated ERK1/2 is much higher in cells infected with EIEC at 4 h post-infection. Despite these differences, HT-29 cells infected with EIEC or Shigella release comparable amounts of cytokines, as IL-8 and TNF-α with similar kinetics ( Sanchez-Villamil et al, 2016 ). The phosphorylation of ERK1/2 and p38 is controlled by the phosphothreonine lyase activity of OspF, to which both anti-inflammatory ( Arbibe et al, 2007 ) and pro-inflammatory roles ( Reiterer et al, 2011 ) have been attributed.…”