Escherichia coli Alpha-Hemolysin HlyA Induces Host Cell Polarity Changes, Epithelial Barrier Dysfunction and Cell Detachment in Human Colon Carcinoma Caco-2 Cell Model via PTEN-Dependent Dysregulation of Cell Junctions

Abstract

Escherichia coli (E. coli) of the B2 phylotype reside in human and animal intestines. The bacteria possess pathogenicity factors such as α-hemolysin (HlyA) that can induce intestinal epithelial leaks. We addressed the questions which host cell processes were dysregulated by E. coli HlyA that can potentiate intestinal diseases. The colon carcinoma cell line Caco-2 was infected by HlyA+ E. coli. Cell polarity regulation was analyzed by live cell imaging for the phosphatidylinositol-4,5-bisphosphate (PIP2) abundance. In Caco-2 monolayers, transepithelial electrical resistance was measured for characterization of barrier function. Cell proliferation and separation were assessed microscopically. Epithelial regulation and cell signaling were analyzed by RNA-Seq and Ingenuity Pathway Analysis (IPA). Our main findings from E. coli HlyA toxinogenicity in the colon carcinoma cell line are that (i) PIP2 at the membrane decrease, (ii) PTEN (phosphatase and tensin homolog) inhibition leads to cell polarity changes, (iii) epithelial leakiness follows these polarity changes by disruption of cell junctions and (iv) epithelial cell detachment increases. HlyA affected pathways, e.g., the PTEN and metastasis signaling, were identified by RNA-Seq bioinformatics calculations in IPA. In conclusion, HlyA affects cell polarity, thereby inducing epithelial barrier dysfunction due to defective tight junctions and focal leak induction as an exemplary mechanism for leaky gut.

UPEC Colonic-Virulence and Urovirulence Are Blunted by Proanthocyanidins-Rich Cranberry Extract Microbial Metabolites in a Gut Model and a 3D Tissue-Engineered Urothelium

“…Since the SHIME only mimics the microbial interactions and physicochemical parameters of the luminal part of the colon but does not include intestinal epithelial cells, we cannot truly claim UPEC as enterovirulent. Such concept of UPEC enterovirulence should be specifically tested in the presence of intestinal epithelial cells, as recently discussed in the work of Schultz et al, using an intestinal model of Caco-2 cells ( 22 ). The authors have shown that the strain UPEC 536, positive for the hemolysin toxin production (HlyA+), induced epithelial barrier dysfunction by compromising tight junctions as potentiator of the leaky gut syndrome ( 22 ).…”

“…Such concept of UPEC enterovirulence should be specifically tested in the presence of intestinal epithelial cells, as recently discussed in the work of Schultz et al, using an intestinal model of Caco-2 cells ( 22 ). The authors have shown that the strain UPEC 536, positive for the hemolysin toxin production (HlyA+), induced epithelial barrier dysfunction by compromising tight junctions as potentiator of the leaky gut syndrome ( 22 ). Our work concords with that of Schulz et al ( 22 ), highlighting the importance of studying UPEC upstream the famous urovirulent stage.…”

“…The authors have shown that the strain UPEC 536, positive for the hemolysin toxin production (HlyA+), induced epithelial barrier dysfunction by compromising tight junctions as potentiator of the leaky gut syndrome ( 22 ). Our work concords with that of Schulz et al ( 22 ), highlighting the importance of studying UPEC upstream the famous urovirulent stage.…”

Kingella kingae RtxA Cytotoxin in the Context of Other RTX Toxins

“…Later, the same group demonstrated that mice infected with HlyA-secreting E. coli showed an increase in focal leak areas in the colonized colons compared to the HlyA-deficient mutant, suggesting that HlyA impairs intestinal barrier function via induction of focal leaks in the epithelium in vivo [ 349 ]. Recently, the same group presented data that could explain the induction of focal leaks in the epithelial layer [ 366 ]. It was shown that the infection of the human colon carcinoma cell line Caco-2 with HlyA-secreting but not HlyA-deficient E. coli bacteria induced inhibition of PTEN (phosphatase and tensin homolog deleted on chromosome 10), a phosphatase that dephosphorylates membrane phosphatidylinositol-3,4,5-trisphosphate (PIP3) and plays a role in regulation of cell polarity.…”

“…This resulted in the disorganization of barrier-forming junctional complexes and increased epithelial permeability, followed by increased epithelial cell detachment. Thus, these host cell processes dysregulated by HlyA could induce focal leaks in the intestinal epithelium, potentiating intestinal diseases caused by pathogenic E. coli strains [ 366 ].…”