The intestinal spirochete Brachyspira pilosicoli attaches to cultured Caco-2 cells and induces pathological changes

Abstract

Background: Brachyspira pilosicoli is an anaerobic spirochete that has received relatively little study, partly due to its specialized culture requirements and slow growth. This bacterium colonizes the large intestine of various species, including humans; typically, a dense layer of spirochete cells may be found intimately attached by one cell end to the surface of colonic enterocytes. Colonized individuals may develop colitis, but the mechanisms involved are not understood. The current study aimed to develop an in vitro model to investigate this process.

Methodology/principal findings: Four strains of B. pilosicoli were incubated at a high multiplicity of infection with monolayers of a human colonic adenocarcinoma cell line (Caco-2 cells). One strain isolated from a pig (95/1000) and one from a human (WesB) attached to the monolayers. Colonization increased with time, with the Caco-2 cell junctions being the initial targets of attachment. By electron microscopy, individual spirochete cells could be seen to have one cell end invaginated into the Caco-2 cell membranes, with the rest of the spirochete draped over the Caco-2 cell surface. After 6 h incubation, the monolayer was covered with a layer of spirochetes. Colonized monolayers demonstrated a time-dependent series of changes: staining with labelled phalloidin identified accumulation of actin at the cell junctions; ZO-1 staining revealed a loss of Caco-2 tight junction integrity; and Hoechst staining showed condensation and fragmentation of nuclear material consistent with apoptosis. Using quantitative reverse transcription PCR, the colonized monolayers demonstrated a significant up-regulation of interleukin-1beta (IL-1beta) and IL-8 expression. B. pilosicoli sonicates caused significant up-regulation of IL-1beta, TNF-alpha, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not alter cytokine expression.

Conclusions/significance: The changes induced in the Caco-2 cells provide evidence that B. pilosicoli has pathogenic potential, and give insights into the likely in vivo pathogenesis.

Figure 1. Scanning electron micrographs of B. pilosicoli interacting with Caco-2 cells.

The cells were incubated for 6 h with DMEM (A), B. pilosicoli 95/1000 for 2 h (B), and 6 h (C), and WesB for 6 h (D). The non-infected cells show intact tight junctions with clear boundaries. After 2 h, B. pilosicoli 95/1000 mainly colonizes the cell boundaries (arrows), but by 6 h most of the cell surface is covered with spirochetes. The ends of the WesB cells can be seen penetrating the membrane of the Caco-2 cells (arrows), with the rest of the spirochete cell body lying on the Caco2 cell surface. The photographs were taken at magnifications of X 2,100 for panels A, B and C, and X 9,800 for panel D.

Figure 2. Transmission electron micrographs of B. pilosicoli interacting with Caco-2 cells.

Cross-sections and tangential-sections of B. pilosicoli can be seen at the cell junctions (A) and under the cell membranes (B) (arrows). Spirochete cells can be seen attached to the Caco-2 cell surface (C), and invaginating into pit-like structures (arrow) in the Caco-2 cell membrane (D). Compared to the nuclei of control cells (E), the nuclei of many cells in the infected monolayers show chromatin condensation and fragmentation (arrows), consistent with apoptosis (F). The photographs were taken at magnifications of X 5,800, 7,900, 33,800, 24,500, 5,800 and 5,800, respectively.

Figure 3. Epifluorescent micrographs illustrating ZO-1 integrity in Caco-2 cell monolayers.

Monolayers grown in DMEM (A), and exposed to B. pilosicoli 95/1000 for 6 h (B). In the control cells the ZO1 distribution is regular and limited to the junctions, which are intact. After 6 h incubation with B. pilosicoli the tight junctions are disrupted and the ZO-1 is punctuated and has migrated towards the cytoplasm (arrow). Photographs taken at a magnification of X 100.

Figure 4. Epifluorescent micrographs showing Hoechst staining of DNA in Caco-2 cells.

Monolayers either grown in DMEM (A), or exposed to a culture of B. pilosicoli 95/1000 (B) for 6 h. Exposure to B. pilosicoli has resulted in many nuclei appearing condensed, and some showing clear chromatin fragmentation, consistent with apoptosis (arrows). Photographs taken at a magnification of X 100.

Figure 5. Epifluorescent micrographs showing actin staining in Caco-2 monolayers.

Monolayers either grown in DMEM (A), or exposed to B. pilosicoli 95/1000 (B) for 6 h. In the control section there is regular distribution of FITC (phalloidin) over the monolayers. After 6 h incubation with B. pilosicoli the actin filaments are clearly mobilized and can be seen as round bodies on the junction of the Caco2 cells (arrows). Photographs taken at a magnification of X 100.