Elsevier

Microbial Pathogenesis

Volume 49, Issue 5, November 2010, Pages 260-272
Microbial Pathogenesis

Campylobacter fetus translocation across Caco-2 cell monolayers

https://doi.org/10.1016/j.micpath.2010.06.008Get rights and content

Abstract

Campylobacter fetus is a recognized pathogen of cattle and sheep, though human infection has also been reported. Ingestion of contaminated food or water is a proposed route of transmission for both humans and animals. The subsequent detection of the organism from extra-intestinal and systemic locations implies an ability to translocate across epithelial barriers. To determine how C. fetus disseminates from the intestine, Caco-2 cells cultured on porous membrane supports, were used as model intestinal epithelial cell monolayers. C. fetus was found to translocate equally well in both apical-to-basolateral and basolateral-to-apical directions for up to 24 h without altering Caco-2 cell monolayer permeability as assessed by transepithelial resistance and absence of paracellular diffusion of FITC-inulin. Using modified antibiotic protection assays, C. fetus was also observed to invade and subsequently egress from Caco-2 cells. Caco-2 cell invasion and translocation occurred independently of C. fetus S layer expression. Scanning and transmission electron microscopy revealed the presence of C. fetus associated with both apical and basal surfaces as well as in intracellular locations. C. fetus was, however, never observed in paracellular locations nor associated with Caco-2 cells junctions. Neither C. fetus invasion nor translocation across Caco-2 cell monolayers was impacted by latrunculin A, though translocation was enhanced in the presence of cytochalasin D which disrupted tight junctions. Tubulin cytoskeleton disrupting agents, colchicine and vinblastine, did inhibit C. fetus translocation though entry into Caco-2 cells remained unaffected. Together, translocation without disrupting monolayer integrity, invasion and egression from Caco-2 cells, electron microscopy observations and the requirement of a functional tubulin cytoskeleton for translocation, support a transcellular mechanism of C. fetus translocation across Caco-2 cell monolayers. The ability to invade and subsequently egress would contribute to establishment of an infecting C. fetus population in the host, while the demonstrated ability to translocate across model intestinal epithelial barriers accounts for the observed in vivo recovery of C. fetus from extra-intestinal locations.

Introduction

Campylobacter’s are resident intestinal flora of many birds and mammals though several species including Campylobacter jejuni, Campylobacter coli, Campylobacter lari, Campylobacter upsaliensis and Campylobacter fetus are human pathogens [1]. In Canada, C. jejuni is the most frequent cause of human enteritis with reports of C. fetus infection being substantially lower [2]. C. fetus infection however, is more often associated with bacteremia and invasive disease or prolonged, relapsing illness, especially in compromised individuals [1]. Both C. fetus subspecies are also recognized animal pathogens. C. fetus subsp. venerealis induces venereal infection in cattle which can result in infertility in cows [3] whereas C. fetus subsp. fetus causes septic abortion in cattle and sheep [3], [4]. Though C. fetus supsp. fetus typically initiates infection at mucosal surfaces, transmission remains poorly defined. It is proposed that most animal and human infections are initiated following ingestion of C. fetus subsp. fetus-contaminated food or water [3], [5], [6], [7]. That infection is frequently associated with transient bacteremia [4], [5], and that this organism may be recovered from extra-intestinal locations, implies an ability to translocate across the intestinal epithelial cell barrier. A mechanism for C. fetus dissemination from the intestine has, however, not been forth coming.

Bacterial translocation across intact epithelial barriers has been described in Ref. [8]. Three translocation mechanisms are relevant to the intestinal epithelium: exploitation of M cells, specialized intestinal epithelial cells which transport luminal antigens across the epithelial barrier to underlying lymphoid tissue; transcellular translocation or transcytosis, in which pathogens invade epithelial cells, migrate across the cytoplasm and egress from the opposite surface, and; paracellular translocation or paracytosis, in which bacteria travel extracellularly through tight junctions between adjacent epithelial cells. Translocation across intestinal epithelial barriers via all 3 mechanisms has been reported for C. jejuni [9], [10], [11], [12].

The invasive nature of C. fetus is well established in clinical and veterinary situations and uptake into cultured human intestinal epithelial cells (INT 407) has also been demonstrated [13]. Caco-2 cells, a human colonic cell line, differentiate to form distinct apical and basolateral surfaces separated by tight junctions and express markers characteristic of human small intestinal epithelium [14]. In the present study, Caco-2 cells cultured on permeable supports were used as a model system to investigate C. fetus translocation across intestinal epithelial cell barriers.

Section snippets

C. fetus translocates across Caco-2 cell monolayers

One hour following apical infection of differentiated Caco-2 monolayers, C. fetus 11686 was detected in the basolateral medium (Fig. 1A). Although numbers of translocating C. fetus increased each hour during the subsequent 5 h, transepithelial electrical resistance (TER) values remained at 700–900 Ω/cm2 indicating that monolayer integrity was maintained for the duration of the 6 h assay (Fig. 1B). Salmonella typhimurium was also detected in the basolateral medium 1 h p.i. (Fig. 1A), however

Discussion

Numerous mucosal pathogens translocate across epithelial barriers enroute to establishing disease states. The present study characterizes C. fetus translocation across model intestinal epithelial cell monolayers. Initial assays comparing C. fetus 11686 to S. typhimurium and E. coli, as positive and negative control bacteria, respectively, validated the use of Caco-2 cell monolayers as a model for C. fetus translocation. Consistent with previous reports [14], [15], translocation of S. typhimurium

Bacterial strains and culture conditions

Bacterial strains used in this study are presented in Table 1. Stock bacterial cultures were maintained in peptone–glycerol broth (10 g peptone/L, 5 g NaCl/L, 25% v/v glycerol) at −80 °C and for use in all studies, bacteria were passaged five or less times. C. fetus and C. jejuni were cultured on Columbia agar (Oxoid, Nepean, Ontario) supplemented with 5% v/v sheep blood (sBAP; Oxoid) at 37 °C for 48 h under microaerobic conditions (5% O2; 10% CO2; 85% N2). Isogenic S layer-deficient C. fetus

Acknowledgements

This work was funded by a Natural Sciences and Engineering Research Council Discovery Grant to LLG.

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    Present address: Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 1X5.

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