Basolateral Invasion and Trafficking of Campylobacter jejuni in Polarized Epithelial Cells

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.

Inflammasome Activation by Campylobacter jejuni

“…After 6 h, the cells were washed twice, lysed with 13 reporter lysis buffer supplemented with 1% Triton-X100, and placed at 280˚C for at least 30 min. The cell lysate was analyzed for luciferase activity in a luminometer (TD20/20; Turner Designs) immediately after adding 50 ml Promega Luciferase Assay Agent to the sample as described previously (20). To measure intracellular survival, the medium was replaced after 6 h with medium containing gentamicin (250 mg/ml).…”

“…Therefore, we examined a possible correlation between the number of bacteria that infected the macrophages and IL-1b secretion for both taxis mutants and the parental strain 108 using a luciferase reporter assay (20). Macrophages were infected with different strains of C. jejuni-producing luciferase.…”

“…At some point the epithelial barrier is breached, resulting in acute inflammation accompanied by strong neutrophil recruitment and activation of T-and B-cell responses (15,16). Bacterial motility and chemotaxis are crucial for causing disease (17)(18)(19)(20). Other proposed virulence traits include the polysaccharide capsule, secreted proteins (Cia proteins, HtrA protease), type 6 secretion (T6SS) effector molecules, apoptosis-inducing proteins (cytolethal distending toxin, FspA2), and bacterial adhesion and invasion promoting factors (FlaC, PEB1, JlpA, CapA, and CadF) (for review, see Refs.…”

Pathogenesis of Human Enterovirulent Bacteria: Lessons from Cultured, Fully Differentiated Human Colon Cancer Cell Lines

“…During C. jejuni translocation across fully differentiated Caco-2 cell monolayers, the absence of any change in TER and inulin paracellular passage indicates that the bacteria translocated through the cytoplasm of the cells invade, rather than via intercellular spaces (355). C. jejuni invading, via an actin-and microtubule-independent mechanism, fully differentiated Caco-2 cells localized in at the periphery of large islets of cells have been found residing within CD63-positive vacuoles, in which they are metabolically active (356). The serine protease HtrA of C. jejuni plays a role in promoting the basolateral invasion of fully differentiated Caco-2 by cleaving E-cadherin (357).…”

A Glance at Prokaryotes and Eukaryotes Interplay and Campylobacter jejuni–Host Interaction

“…Curiously, C. jejuni displays commensal behavior in chicken while in the human intestine, C. jejuni penetrates the mucus and colonizes the intestinal crypts in a very efficient manner. The molecular basis of the difference in pathogenicity of C. jejuni in human and chicken still remains to be resolved (Bouwman et al 2013).…”