Streptococcus equi subsp. zooepidemicus Invades and Survives in Epithelial Cells

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.

Keywords: Streptococcus equi subsp. zooepidemicus; cell infection assay; equine endometritis; immunofluorescence microscopy; intracellular survival; quantitative analysis of immunofluorescence data; scanning electron microscopy.

Figure 1

Illustration of experimental setup. A model of an epithelial cell infected with S. zooepidemicus is shown. The green and red circles represent double immunofluorescence stained S. zooepidemicus, where adherent extracellular bacteria are green, whereas intracellular bacteria are red (A). Intracellular S. zooepidemicus are depicted in phagosome-like compartments. A majority of the compartments have no LAMP-1 signal (B). However, some of the compartments are LAMP-1 positive (turquoise circles), meaning that lysosomes have fused with the compartment (C). Few bacteria did not seem to be within a compartment, presented free in the cytoplasm (D). Gray circles represent bacteria that were observed at the surface by scanning electron microscopy, and bacterial pili-like protrusions were expressed, illustrated with light gray lines (E). Upper right corner a blood agar plate with colonies is shown to illustrate the growth and intracellular survival estimated by plating and retrieval of intracellular S. zooepidemicus after extracellular bacteria have been killed in the penicillin protection assay (F).

Figure 2

FESEM analysis of HEp-2 cells co-cultured with S. zooepidemicus strain 1-4a for 1.5 h and 3.5 h. Adherent bacteria (A) trigger three different types of invasion. Membrane ruffling and cytoskeletal rearrangements (B), large invaginations in the plasma membrane of the HEp-2 cells (C–D), and engulfment from the middle of the chain causing the cell membrane to form protrusions which overgrow the bacterial chain (^*) (E) were observed after 1.5 h of co-culturing. At the later time point (3.5 h) the bacterial cell surface showed expression of pili-like appendages (arrows) (F). Heat-inactivated strain 1-4a was adherent but showed no invasion (G) as the non-invasive control L. lactis (H) after 3.5 h of co-culturing. Bars represent 1 μm in (A–C) and (E–H) and 0.5 μm in (D).

Figure 3

FESEM analysis of HEp-2 cells co-cultured with S. zooepidemicus strain S31A1 for 1.5 and 3.5 h. Adherent bacteria (A) trigger three different types of invasion. Membrane ruffling (B), large invaginations in the plasma membrane of the HEp-2 cells (C,D), and engulfment from the middle of the chain causing the cell membrane to form protrusions which overgrow the bacterial chain(^*) (E) were observed after 1.5 h of co-culturing. Pili-like appendages can also be found (arrows) (F). The pili-like appendages were seen in connection to the large invaginations as well after 3.5 h (G). Heat-inactivated strain S31A1 adherent, but non-invasive after 3.5 h (H). Bars represent 1 μm in (A–C) and E-H and 0.5 μm in (D).

Figure 4

Adherent and intracellular S. zooepidemicus determined by double immunofluorescence staining and quantitative analysis of fluorescent intensities. The S. zooepidemicus strains were co-cultured with HeLa cells. The infection was stopped at various time points (1 h 45 m, 2 h 45 m, and 3 h 30 m) immunofluorescence stained and analyzed as described in Materials and Methods. (A) The graph shows bacterial fluorescent cell area per HeLa cell, were the extracellular/adherent signals are shown with green bars and the intracellular signals shown with red bars. Strain 1-4a bars are slashed, while strain S31A1 bars have no filling. Means ± SD from three independent experiments are shown. Green asterisks mark significant difference between extracellular/adherent bacterial signals, and red asterisk mark significant difference between intracellular bacterial signals. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001. (B) Overview image showing adherent (green and yellow) and intracellular (red) bacteria for strain S31A1. (C) Overview image showing adherent and intracellular bacteria for strain 1-4a. For (B) and (C) autofluorescence, demarking the HeLa cells, is shown in white, and for (B–E) DNA is shown in blue. (D) Maxprojection of a confocal stack showing a strain S31A1 infected cell (E) Maxprojection of a confocal stack showing a cell highly infected with strain 1-4a. (B,C) Axioscan images, scale bars are 20 μm. (D,E) confocal images, scale bars are 10 μm.

Figure 5

The S. zooepidemicus strains were co-cultured with HeLa cells. At 2h post-infection the cells were washed and Complete Media containing penicillin was added. The number of surviving bacteria was determined by plating 0, 2, 9, and 21 h post-penicillin addition. Results were expressed as Log(10) CFU/ml. Means ± SD of three independent experiments are shown. ^****p < 0.0001.

Figure 6

Intracellular trafficking and staining for human lysosomal-associated membrane protein, LAMP-1. The S. zooepidemicus strains were co-cultured with HeLa cells and samples from different time points were immunofluorescence stained for LAMP-1 (turquoise), and the bacteria were double immunofluorescence stained (intracellular red; extracellular/adherent green), DNA is shown in blue. (A) Strain 1-4a in phagosome-like compartments (marked with asterisks ^*), with varying degrees of LAMP-1 association (arrows), 11 h post-infection. (B) Strain S31A1 in phagosome-like compartments (marked with asterisks ^*), with some LAMP-1 association (arrows), 3 h 30 min. (C) Strain 1-4a in a phagolysosome-like compartment, 3 h 30 min. (D) Strain S31A1 in a phagolysosome-like compartment, 3 h 30 min. (E) Intracellular strain 1-4a with no clear LAMP-1 association, 23 h. (F) Intracellular strain S31A1 with no clear LAMP-1 association, 23 h. All images are recorded with a confocal microscope. (A,B) with a DIC overlay, scale bars are 10 μm. (C–F) 3D images created from Z-stacks in Zen 2 (blue edition), scale bars are in μm.