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 FindingsFour 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-1β (IL-1β) and IL-8 expression. B. pilosicoli sonicates caused significant up-regulation of IL-1β, TNF-α, and IL-6, but culture supernatants and non-pathogenic Brachyspira innocens did not alter cytokine expression.Conclusions/SignificanceThe changes induced in the Caco-2 cells provide evidence that B. pilosicoli has pathogenic potential, and give insights into the likely in vivo pathogenesis.
Highlights
The intestinal spirochete Brachyspira pilosicoli colonizes the large intestine of a variety of species of animals and birds, as well as human beings [1]
Humans may be colonized with the distinct species Brachyspira aalborgi, which attaches to colonic enterocytes by one cell end [13,14]
Attachment to Caco-2 Monolayers The Caco-2 monolayers exposed to Dulbecco’s modified Eagle medium (DMEM) for 6 h remained intact throughout the assays
Summary
The intestinal spirochete Brachyspira pilosicoli colonizes the large intestine of a variety of species of animals and birds, as well as human beings [1]. A characteristic feature of colonization with B. pilosicoli is the intimate end-on or ‘‘polar’’ attachment of spirochete cells to the luminal surface of colonic and rectal epithelial cells, in a condition called ‘‘intestinal spirochetosis’’ or ‘‘colonic spirochetosis’’ [1] This description was first made in colonic biopsy samples from humans where the associated dense layer of attached spirochetes was described as a ‘‘false brush border’’ [9]. 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. The current study aimed to develop an in vitro model to investigate this process
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