Interactions between Blastocystis subtype ST4 and gut microbiota in vitro

Abstract

Background: Blastocystis ST4 is a common protistan parasite of the gastrointestinal tract of humans and a wide range of animals. While it has been suggested that colonization with ST4 is associated with healthy gut microbiota, how ST4 influences the gut microbiota remains poorly studied. This study aimed to examine the interactions between ST4 and several intestinal bacteria using in vitro co-culture systems, and to further investigate the mechanism of interaction and its effect on the epithelial barrier integrity of HT-29 cells.

Methods: Seven intestinal bacteria Bacteroides fragilis, Bifidobacterium longum, Bacillus subtilis, Bacteroides vulgatus, Escherichia coli, Enterococcus faecalis, and Lactobacillus brevis were co-cultured with Blastocystis ST4 in vitro. Flow cytometry and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to determine the role of reactive oxygen species (ROS) and bacteria oxidoreductase genes, respectively, in response to Blastocystis co-incubation. Transepithelial electrical resistance (TEER) and flux assays were performed to assess the effect of microbiota representatives on the integrity of the intestinal epithelial barrier.

Results: Co-incubation with Blastocystis ST4 showed a beneficial influence on most intestinal bacteria, while ST4 significantly inhibited the growth of B. vulgatus, a common pathogen in the genus Bacteroides. The decrease in B. vulgatus when co-incubated with Blastocystis ST4 was associated with high levels of ROS and the upregulation of oxidative stress-related genes. Furthermore, co-incubation with Blastocystis ST4 was able to protect the intestinal epithelial barrier from damage by B. vulgatus.

Conclusions: This study demonstrated, for the first time, that Blastocystis ST4 has beneficial effects on intestinal commensal bacteria in vitro, and can inhibit the growth of pathogenic B. vulgatus. Combined with previous microbiome research on ST4, our data suggest that ST4 may be a beneficial commensal.

Keywords: Blastocystis; Co-incubation; Epithelial barrier; Gut microbiota; ROS.

Fig. 1

Intestinal bacteria have a positive effect on Blastocystis cell count in vitro. 10⁷ cells of Blastocystis ST4 were co-cultured with 10⁹ CFU of gut bacteria (B. fragilis, B. longum, B. subtilis, B. vulgatus, E. coli, E. faecalis, and L. brevis) in 1 ml of pre-reduced PBS for 24 h at 37 °C. a Blastocystis ST4-WR1 cell count. b Blastocystis ST4-WR2 cell count

Fig. 2

Blastocystis ST4 has a positive effect on some intestinal bacteria in vitro. a Most gut bacteria co-cultured with Blastocystis ST4 showed higher CFU count, while the CFU of B. vulgatus was significantly decreased in the presence of Blastocystis ST4-WR1. b Representative plates of colony-forming units (CFU)/ml

Fig. 3

Blastocystis induces oxidative stress to B. vulgatus in vitro. a ROS gating strategies. b The representative flow cytometry plots of DCFDA staining. Co-incubation of B. vulgatus with Blastocystis ST4 displayed significant higher DCFDA content indicating the presence of increased cellular ROS

Fig. 4

Oxidoreductase gene expression of B. vulgatus when incubated with Blastocystis ST4-WR1 and ST4-WR2

Fig. 5

Bacteroides vulgatus disrupts the integrity of the epithelial barrier, while co-incubation with Blastocystis ST4 prevented the disruption of epithelial integrity (a, b)