Abstract
Campylobacter jejuni is a major cause of bacterial diarrheal disease. Most enteropathogenic bacteria including C. jejuni can invade cultured eukaryotic cells via an actin- and/or microtubule-dependent and an energy-consuming uptake process. Recently, we identified a novel highly efficient C. jejuni invasion pathway that involves bacterial migration into the subcellular space of non-polarized epithelial cells (termed subvasion) followed by invasion from the cell basis. Here we report cellular requirements of this entry mechanism and the subsequent intracellular trafficking route of C. jejuni in polarized islands of Caco-2 intestinal epithelial cells. Advanced microscopy on infected cells revealed that C. jejuni invades the polarized intestinal cells via the subcellular invasion pathway. Remarkably, invasion was not blocked by the inhibitors of microtubule dynamics colchicine or paclitaxel, and was even enhanced after disruption of host cell actin filaments by cytochalasin D. Invasion also continued after dinitrophenol-induced cellular depletion of ATP, whereas this compound effectively inhibited the uptake of invasive Escherichia coli. Confocal microscopy demonstrated that intracellular C. jejuni resided in membrane-bound CD63-positive cellular compartments for up to 24 h. Establishment of a novel luciferase reporter-based bacterial viability assay, developed to overcome the limitations of the classical bacterial recovery assay, demonstrated that a subset of C. jejuni survived intracellularly for up to 48 h. Taken together, our results indicate that C. jejuni is able to actively invade polarized intestinal epithelial cells via a novel actin- and microtubule-independent mechanism and remains metabolically active in the intracellular niche for up to 48 hours.
Highlights
Campylobacter is the most common cause of bacterial diarrheal disease worldwide [1]
Control experiments with intact monolayers of Caco-2 cells rather than islands of cells yielded no invasive C. jejuni consistent with previous results that showed minimal bacterial penetration via the apical cell surface [19]. These results suggest that C. jejuni efficiently invades an intact layer of polarized epithelial cells from the basal cell side once an access point is available
The results suggest that C. jejuni subvasion requires intact microtubules, but that efficient C. jejuni invasion into polarized epithelial cells can occur via an actin- and microtubule-independent mechanism
Summary
Campylobacter is the most common cause of bacterial diarrheal disease worldwide [1]. It is estimated that each year up to 1% of the western population is infected with Campylobacter [2]. Campylobacter jejuni (C. jejuni) is the most prominent cause of human infections. Major infection sources are contaminated chicken and surface water. C. jejuni displays commensal behavior in chicken. The molecular basis of the difference in pathogenicity of C. jejuni in human and chicken still remains to be resolved. C. jejuni penetrates the mucus and colonizes the intestinal crypts in a very efficient manner [3]. The crypts seem to be an optimal growth environment for C. jejuni [4]. Several studies suggest that after colonization, C. jejuni can cross the mucosal barrier and invade intestinal cells [5,6,7,8]. The exact mechanism(s) of invasion and the intracellular processing of the bacteria are not well understood
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