Abstract
Mucin is an important physical barrier against enteric pathogens. VvpE is an elastase encoded by Gram-negative bacterium Vibrio vulnificus; however, the functional role of VvpE in intestinal mucin (Muc) production is yet to be elucidated. The recombinant protein (r) VvpE significantly reduced the level of Muc2 in human mucus-secreting HT29-MTX cells. The repression of Muc2 induced by rVvpE was highly susceptible to the knockdown of intelectin-1b (ITLN) and sequestration of cholesterol by methyl-β-cyclodextrin. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into the membrane lipid rafts coupled with ITLN to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of ERK, which was downregulated by the silencing of the PKCδ. Moreover, rVvpE induced region-specific methylation in the Muc2 promoter to promote the transcriptional repression of Muc2. In two mouse models of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus exhibited an increased survival rate and maintained the level of Muc2 expression in intestine. These results demonstrate that VvpE inhibits Muc2 expression by hypermethylation via lipid raft-mediated ROS signaling in the intestinal epithelial cells.
Highlights
Methylation and histone modifications to regulate selective activation or silencing of specific host genes.[9]
To evaluate the role of VvpE in the regulation of Muc[2] expression, HT29-MTX cells were exposed to various concentrations (0 ~ 100 pg/ml) of recombinant protein of VvpE (rVvpE) for 120 min. rVvpE significantly reduced the expression of Muc[2] from 10 to 100 pg/ml compared with the cells with no treatment (Figure 1b)
Given that reactive oxygen species (ROS) has an important role as signal messengers in regulating cellular functions through activation of protein kinase C (PKC),[22] we examined whether rVvpE induces the phosphorylation and translocation of PKC as an important downstream intermediate of ROS in mucus-secreting HT29-MTX cells. rVvpE significantly induced PKC phosphorylation from 30 min (Figure 3a)
Summary
Methylation and histone modifications to regulate selective activation or silencing of specific host genes.[9]. Vibrio (V.) vulnificus is a Gram-negative food pathogen which causes septicemia, necrotizing wound infections, or gastroenteritis.[14] The majority of the virulence effect is known to derive from the secreted toxins encoded by cytolytic poreforming hemolysin (VvhA)[14] and the multifunctional autoprocessing repeats-in-toxin (MARTXVv).[14,15] VvpE is a homolog of the hemagglutinin/protease, which is suspected to be a major elastase produced by V. vulnificus.[16,17] Previous work reported that VvpE, a 45-kDa protein, consists of a 35-kDa N-terminal domain for catalytic activity and a 10-kDa domain for its attachment to the substrate.[18] the functions of VvhA and MARTXVv and their roles in pathogenesis have been well studied[14] owing to their significant fulminating and destructive actions in human tissues, the VvpE appeared to be dispensable for the virulence effect of V. vulnificus.[17] it has been reported that the expression of VvpE is regulated by the quorum-sensing regulator in promoting the pathogenesis of Abbreviations: Cont, Control; EDTA, Ethylenediaminetetraacetic acid; EGTA, ethylene glycol tetraacetic acid; ERK, extracellular signal-regulated kinases; FBS, fetal bovine serum; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; Ig, immunoglobulin; ITLN, intelectin-1b; JNK, c-Jun N-terminal kinases; MAPK, mitogenactivated protein kinases; MβCD, methyl-β-cyclodextrin; NAC, N-acetylcysteine; NCF1, neutrophil cytosolic factor 1; NOX2, NADPH oxidase 2; Pan-cad, pan-cadherin; PBS, phosphate-buffered saline; p38, p38 MAPK; PI, propidium iodide; PKC, protein kinase C; PVDF, polyvinylidene fluoride; RFI, relative fluorescence intensity. We investigate the pathogenic mechanism of VvpE and its related signaling pathway in the regulation of the Muc[2] expression
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