Abstract
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is an opportunistic pathogen of several species including humans. S. zooepidemicus is found on mucus membranes of healthy horses, but can cause acute and chronic endometritis. Recently S. zooepidemicus was found able to reside in the endometrium for prolonged periods of time. Thus, we hypothesized that an intracellular phase may be part of the S. zooepidemicus pathogenesis and investigated if S. zooepidemicus was able to invade and survive inside epithelial cells. HEp-2 and HeLa cell lines were co-cultured with two S. zooepidemicus strains (1-4a and S31A1) both originating from the uterus of mares suffering from endometritis. Cells were fixed at different time points during the 23 h infection assay and field emission scanning electron microscopy (FESEM) was used to characterize adhesion and invasion mechanisms. The FESEM images showed three morphologically different types of invasion for both bacterial strains. The main port of entry was through large invaginations in the epithelial cell membrane. Pili-like bacterial appendages were observed when the S. zooepidemicus cells were in close proximity to the epithelial cells indicating that attachment and invasion were active processes. Adherent and intracellular S. zooepidemicus, and bacteria in association with lysosomes was determined by immunofluorescence staining techniques and fluorescence microscopy. Quantification of intracellular bacteria was determined in penicillin protection assays. Both S. zooepidemicus strains investigated were able to invade epithelial cells although at different magnitudes. The immunofluorescence data showed significantly higher adhesion and invasion rates for strain 1-4a when compared to strain S31A1. S. zooepidemicus was able to survive intracellularly, but the survival rate decreased over time in the cell culture system. Phagosome-like compartments containing S. zooepidemicus at some stages fused with lysosomes to form a phagolysosome. The results indicate that an intracellular phase may be one way S. zooepidemicus survives in the host, and could in part explain how S. zooepidemicus can cause recurrent/persistent infections. Future studies should reveal the ability of S. zooepidemicus to internalize and survive in primary equine endometrial cells and during in vivo conditions.
Highlights
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a Gram-positive, β-hemolytic coccus belonging to the Lancefield group C
The endometrial lavage shows improved sensitivity for culturing bacteria compared to the swab, and is especially sensitive in diagnosing endometritis caused by E. coli, but there is a somewhat higher risk of contamination (LeBlanc et al, 2007; Leblanc and Causey, 2009), which has been lowed considerably after a double-guarded technique was introduced (Christoffersen et al, 2015)
S. zooepidemicus adhered to the cell (Figures 2A, 3A) and three morphologically different invasion mechanisms for both strains were visualized; (a) triggering cytoskeletal rearrangements, i.e., membrane ruffling (Figures 2B, 3B), (b) formation of large invaginations in the HEp-2 cell membrane which serve as invading portal for the bacteria (Figures 2C,D, 3C,D), and (c) invading with bacterial engulfment taking place in the middle part of an adherent streptococcal chain, with the host cell membrane forming protrusions overarching the bacterial chain and thereby internalizing S. zooepidemicus (Figures 2E, 3E)
Summary
Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is a Gram-positive, β-hemolytic coccus belonging to the Lancefield group C. The biopsy can be advantageous when evaluating chronically infected mares, as these mares can carry S. zooepidemicus deep within the endometrium indicating that at least some strains of S. zooepidemicus seem to have the ability to enter and hide within the tissue for prolonged periods of time (Petersen et al, 2009; Rasmussen et al, 2013) This is further supported by clinical studies in infertile mares that were tested bacteriologically negative, despite extensive diagnostic efforts, yet were shown to carry a silent endometritis, when instilled with a bacterial growth medium that apparently can activate dormant streptococci (Petersen et al, 2015). We hypothesize that S. zooepidemicus can be located intracellularly and the aim of the current study was to investigate if S. zooepidemicus can invade and survive intracellularly in epithelial cells and at least partly explain why this bacterium can be difficult to diagnose and cause chronic and recurrent infections
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