Interaction of pathogenic Escherichia coli carrying T6SS with an improved model of gut epithelial cells

Behnoush Asgari

doi:10.25907/00966

Escherichia coli is a versatile and genetically diverse bacterium that typically colonizes the human gastrointestinal (GI) tract shortly after birth as a commensal organism. While most E. coli strains are harmless, a subset can acquire mobile genetic elements that confer pathogenic traits, enabling them to cause diseases ranging from localized GI disorders to severe extraintestinal infections such as urinary tract infections (UTIs), neonatal meningitis, and sepsis. Based on disease-causing potential and genetic markers, E. coli strains are broadly classified into two major groups: intestinal pathogenic E. coli (InPEC) and extraintestinal pathogenic E. coli (ExPEC). In recent years, two emerging pathotypes have attracted attention for their pathogenic features and clinical relevance: (a) adherent-invasive E. coli (AIEC), implicated in pathogenesis of inflammatory bowel disease (IBD), and (b) translocating E. coli (TEC), capable of crossing the intestinal epithelium and entering systemic circulation via the bloodstream or mesenteric lymph nodes, potentially leading to sepsis. Despite their importance, the exact molecular mechanisms that underlie the translocation ability of TEC strains remain poorly understood. This PhD project focused on a prototypical TEC strain, HMLN-1, previously isolated from a patient with fatal pancreatitis and shown to translocate efficiently across epithelial barriers in both in vitro and in vivo models. Genomic comparison of HMLN-1 with 41 publicly available E. coli genomes revealed a distinct genomic island, GI-argU, which encodes a complete Type 6 Secretion System (T6SS), including structural components and two associated effectors: vgrG (valine-glycine repeat G protein) and hcp (hemolysin-coregulated protein). T6SS is increasingly recognized as a virulence mechanism that facilitates bacterial competition, intracellular survival, and host manipulation. To investigate the role of T6SS in translocation of HMLN-1 strain this project employed a co-culture of human intestinal epithelial cells (Caco-2 cells and mucus-producing HT29-MTX cells). This model mimics the physiological properties of the intestinal epithelium, including microvilli, tight junctions, and mucin secretion. HMLN-1 was allowed to interact with this co-culture at different intervals. Using qPCR, a significant upregulation of the vgrG gene and clpV, a T6SS-associated ATPase gene was observed during invasion and post-translocation stages. Increased expression of these genes suggests their possible involvement in the translocation process. Alongside gene expression profiling, this project assessed host responses using differential gene expression analysis of epithelial cells following HMLN-1 interaction. A marked upregulation of proinflammatory cytokines and chemokines, including CXCL1, CXCL8 (IL-8), and CCL20, was detected, confirming that HMLN-1 triggers robust immune signaling pathways associated with inflammation and barrier disruption. These findings align with the strain’s invasive behavior and reinforce the hypothesis that T6SS-mediated interactions contribute to epithelial damage and immune activation. To evaluate the potential of probiotic intervention in mitigating HMLN-1 effects, this project also challenged the HMLN-1 strain against the inhibitory effect of a recently identified probiotic strain, Limosilactobacillus reuteri M4-100 using the co-culture model of intestinal epithelium. Two approaches were employed for this challenge. In the first approach, equal numbers of both strains were added to the co-culture at the same time (co-inoculation approach). In the second approach L. reuteri M4-100 was inoculated 90 min before HMLN-1 challenge (pre-inoculation approach). Both approaches significantly reduced HMLN-1 translocation across the coculture cells and attenuated expression of proinflammatory genes involved in immune activation. However, pre-inoculation approach resulted in a greater reduction in bacterial translocation and inflammatory gene expression compared to co-inoculation approach. This enhanced efficacy likely reflects the probiotic’s ability to establish colonization of the coculture cells and modulate the epithelial environment in advance, thereby creating a competitive niche that impedes pathogen adhesion and invasion. The immunomodulatory properties of L. reuteri M4-100, characterized in previous studies by our group, also appear to dampen inflammatory signaling, ultimately preserving epithelial barrier integrity. These findings underscore the therapeutic potential of probiotic application as a preventative approach against translocation of TEC strains. The second part of the study tested the role of T6SS in translocation of a large collection of clinical uropathogenic E. coli (UPEC). The results indicated that UPEC strains carrying T6SS were able to translocate significantly higher than T6SS-negative strains in both renal cells A498, and in intestinal epithelial cells. The translocation ability of T6SS-positive strains was also shown to be cell type specific and was found certain clonal groups of UPEC. These results suggest that specific UPEC clonal types carrying T6SS may have adapted to translocate via different epithelial barriers, potentially contributing to systemic infections such as urosepsis and gut-associated septicaemia. In all, this thesis provides novel insights into the contribution of T6SS to the virulence and translocation ability of both TEC and UPEC strains. It also highlights the dual risk posed by certain E. coli strains capable of breaching either the urinary or GI barriers to cause bloodstream infections. Furthermore, the protective effect of L. reuteri M4-100 underscores the potential of this probiotic strain in mitigating inflammation and reducing epithelial disruption caused by translocating E. coli.

Interaction of pathogenic Escherichia coli carrying T6SS with an improved model of gut epithelial cells

Files and links (1)

Abstract

Details

Metrics