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Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen associated with persistent diarrhea in travelers, immunocompromised patients and children worldwide. However, the pathogenesis of this organism is yet to be established. In this study, the role of Toll-like receptors (TLRs) was evaluated in epidermal growth factor receptor (EGFR)-mediated IL-8 secretion by EAEC-infected human small intestinal and colonic epithelial cells (INT-407 and HCT-15, respectively). We observed that EAEC-induced upregulation of TLR2, TLR4 and TLR5 transcripts in both types of cells, and the maximum level of these transcripts was seen in cells infected with EAEC-T8 (an invasive clinical isolate). All these TLRs made a significant contribution to the EAEC-T8-mediated EGFR activation in these cells. Furthermore, these TLRs were found to be associated with activation of the downstream effectors (ERK-1/2, PI3 kinase and Akt) and transcription factors (NF-κB, c-Jun, c-Fos and STAT-3) of EGFR-mediated signal transduction pathways. Moreover, the involvement of these TLRs was also noted in IL-8 secretion by both EAEC-T8-infected cell types. Our findings suggest that EAEC-induced upregulation of TLR2, TLR4 and TLR5 is important for the IL-8 response via EGFR-mediated signal transduction pathways in these cells.

Emerging evidence have suggested that aberrant sialylation on cell-surface carbohydrate architecture may influence host–pathogen interactions. The α2,6-sialyltransferase (ST) enzymes were found to alter the glycosylation pattern of the pathogen-infected host cell-surface proteins, which could facilitate its invasion. In this study, we assessed the role of specific α2,6-ST enzymes in the regulation of enteroaggregative E. coli (EAEC)–induced cell signaling pathways in human intestinal epithelial cells. EAEC-induced expression of α2,6-ST family genes in HCT-15 and INT-407 cell lines was assessed at mRNA level by qRT-PCR. Specific esi-RNA was used to silence the target ST-gene in each of the EAEC-infected cell type. Subsequently, the role of these enzymes in regulation of EAEC-induced cell signaling pathways was unraveled by analyzing the expression of MAPkinases (ERK1/2, p38, JNK) and transcription factors (NFκB, cJun, cFos, STAT) at mRNA and protein levels by qRT-PCR and western immunoblotting, respectively, expression of selected sialoglycoproteins by western immunoblotting along with the secretory IL-8 response using sandwich ELISA. ST6GAL-1 and ST6GAL-2 were efficiently silenced in EAEC-infected HCT-15 and INT-407 cells, respectively. Significant reduction in EAEC-induced activation of MAPKs, transcription factors, sialoglycoproteins, and IL-8 secretion was noted in ST-silenced cells in comparison to the respective control cells. We propose that ST6GAL-1 and ST6GAL-2 are quintessential for EAEC-induced stimulation of MAPK-mediated pathways, resulting in activation of transcription factors, leading to an inflammatory response in the human intestinal epithelial cells. Our study may be helpful to design better therapeutic strategies to control EAEC- infection. Key points • EAEC induces α2,6-sialyltransferase (ST) upregulation in intestinal epithelial cells • Target STs (ST6GAL-1 & ST6GAL-2) were efficiently silenced using specific esiRNAs • Expression of MAPKs, transcription factors & IL-8 was reduced in ST silenced cells Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-022-12321-2.

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Enteric pathogens exploit the versatility of cytoskeletal system for internalization into non-phagocytic cells, as a crucial step in their pathogenic life cycle. Enteropathogenic Escherichia coli(EPEC) andEnterohemorrhagic Escherichia coli (EHEC) were shown to form actin pedestals in host cells using type-III secretion system. Earlier, we reported that EAEC induced increase in intracellular calcium ions might have crucial role in F-actin rearrangements in INT-407 cells leading to its invasion. It was suggested that EGFR might contribute in Rck-mediated adherence and invasion of Salmonella in host cells. In the present study, we assessed the role of EAEC induced activated EGFR in human intestinal epithelial cell lines (INT-407 & HCT-15). The presence of activated EGFR was detected in membrane fractions ofeach cell line, infected with two different strains of EAEC (EAEC-T8 & EAEC-O42) separatelyfor 3h in presence and absence of Tyrphostin AG1478 (EGFR-inhibitor), by western immunoblotting using p-EGFR (Y1068) antibody. Adherence and invasion of EAEC-T8 to each cell line were checked in presence of Tyrphostin AG1478. Further, the effect of Tyrphostin AG1478 on cytoskeletal F-actin rearrangement of EAEC-T8 infected cells was assessed under confocal microscope following staining with TRITC-phalloidin. EAEC-T8 induced maximum increase in EGFR autophosphorylation at Y1068. Adherence and invasion of EAEC-T8 as well as this organism induced cytoskeletal F-actin polymerization were found to be inhibited in presence of Tyrphostin AG1478. Our study revealed that EAEC induced activated EGFR might play a major role in host cell adherence and cytoskeletal rearrangements leading to invasion of the organism in these cells.