PI3K/Akt pathway restricts epithelial adhesion of Dr + Escherichia coli by down-regulating the expression of decay accelerating factor.

The urogenital microbial infection in pregnancy is an important cause of maternal and neonatal morbidity and mortality. Uropathogenic Escherichia coli strains which express Dr fimbriae (Dr+) are associated with unique gestational virulence and they utilize cell surface decay accelerating factor (DAF or CD55) as one of the cellular receptor before invading the epithelial cells. Previous studies in our laboratory established that nitric oxide reduces the rate of E. coli invasion by delocalizing the DAF protein from cell surface lipid rafts and down-regulating its expression. The phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) cell signal pathway plays an important role in host-microbe interaction because many bacteria including E. coli activate this pathway in order to establish infection. In the present study, we showed that the PI3K/Akt pathway negatively regulated the expression of DAF on the epithelial cell surface and thus inhibited the adhesion of Dr(+) E. coli to epithelial cells. Initially, using two human cell lines Ishikawa and HeLa which differ in constitutive activity of PI3K/Akt, we showed that DAF levels were associated with the PI3K/Akt pathway. We then showed that the DAF gene expression was up-regulated and the Dr(+) E. coli adhesion increased after the suppression of PI3K/Akt pathway in Ishikawa cells using inhibitor LY294002, and a plasmid which allowed the expression of PI3K/Akt regulatory protein PTEN. The down-regulation of PTEN protein using PTEN-specific siRNA activated the PI3K/Akt pathway, down-regulated the DAF, and decreased the adhesion of Dr(+) E. coli. We conclude that the PI3K/Akt pathway regulated the DAF expression in a nitric oxide independent manner.

Nitric oxide induces segregation of decay accelerating factor (DAF or CD55) from the membrane lipid-rafts and its internalization in human endometrial cells.

Recent studies suggest that DAF (decay accelerating factor), a complement regulatory protein, present in lipid rafts, is utilized by Dr fimbriated Escherichia coli for their binding and internalization. Previous studies in our laboratory have shown that NO (nitric oxide) can reduce the invasion of Dr(+) E. coli and the severity of uterine infection in pregnant rats. Also, the expression level of DAF both at the mRNA and protein levels has been shown to be reduced by NO. Therefore NO mediated down-regulation of DAF appears to be an important factor in reducing the susceptibility to E. coli infection. However, it is unclear if NO can actually modulate the membrane association of DAF and therefore initial bacterial binding to cells. We found that NO induces the delocalization of DAF from the G(M1)-rich lipid rafts. Using biochemical and cell biological approaches in a uterine epithelial cell model (Ishikawa cells), DAF accumulates in caveolae upon exposure to NO. Interaction of DAF with the caveolar protein, caveolin1, leads to their internalization by endosomes. NO-induced delocalization of DAF from the lipid raft and its accumulation in caveolae are mediated through a cGMP (cyclic guanosine monophosphate) pathway. The acute localized synthesis of NO and its influence on DAF localization may represent an important unrecognized phenomenon of host defence against Dr(+) E. coli bacteria, as well as many disease conditions that involve complement system.

Group B streptococcus exploits lipid rafts and phosphoinositide 3-kinase/Akt signaling pathway to invade human endometrial cells.

OBJECTIVE: We sought to determine the role lipid rafts and phosphoinositide 3-kinase (PI3K) in invasiveness of group B streptococci (GBS) to endometrial cells. STUDY DESIGN: Antibiotic protection assay and electron microscopy were used to evaluate the invasion of GBS to human endometrial Ishikawa cells cholesterol-depleted by using methyl-beta-cyclodextrin or treated with PI3K inhibitors: wortmannin or LY294002. Immunoblotting analysis of Akt phosphorylation and cellular imaging of GFP-Akt-PH probe were used to assess PI3Ks activation in infected cells. RESULTS: Infected Ishikawa cells streptococci are associated to membrane ruffles with morphological features of undergoing internalization. GBS remained attached but completely failed to invade to cholesterol-depleted human endometrial cells or displayed decreased invasiveness in the presence of PI3K inhibitors. Cholesterol depletion resulted in loss of membrane ruffling and dispersion of raft-associated molecules: monosialoganglioside GM1 and PI3K. CONCLUSION: This work provides the evidence that lipid rafts and raft-associated PI3K are implicated in GBS invasion to human endometrial cells.

Epithelial invasion by Escherichia coli bearing Dr fimbriae is controlled by nitric oxide-regulated expression of CD55.

We previously reported that inhibition of nitric oxide (NO) increases the rate of bacteremia and maternal mortality in pregnant rats with uterine infection by Escherichia coli expressing the Dr fimbria (Dr(+)). Epithelial binding and invasion by Dr(+) E. coli has also been shown to be dependent upon the expression level of the cellular receptor decay-accelerating factor (DAF; CD55). Here, we hypothesize that NO-related severity of infection could be mediated by changes in DAF expression and in the rate of epithelial invasion. The cellular basis of NO effects on epithelial invasion with Dr(+) E. coli was studied using Ishikawa endometrial carcinoma cells as an in vitro model of the human endometrial epithelium. Initially, we show that Ishikawa cells produce NO and express both NO synthase enzymes, NOS II and NOS III, and DAF protein. We next tested the abilities of both Dr(+) E. coli and a Dr(-) E. coli mutant to invade Ishikawa cells, and invasion was seen only with Dr(+) E. coli. Invasion by Dr(+) E. coli was decreased by elevated NO production and increased by NO inhibition. Elevated NO production significantly decreased DAF protein and mRNA expression in Ishikawa cells in a time- and dose-dependent manner. Here, we propose that in vitro invasion of an epithelial cell line is directly related to NO-regulated expression of DAF. The significance of NO-regulated receptor-ligand invasion is that it may represent a novel unrecognized phenomenon of epithelial defense against infection.