P. zopfii genotype-I and -II were previously isolated from milk samples of dairy cows suffering from bovine mastitis and were stored in our laboratory at College of Veterinary Medicine, China Agricultural University, Beijing (Gao et al., 2012). The strains were revived on sabouraud dextrose agar (SDA; Difco™, Becton Dickison, Sparks MD, USA) at 37°C for 72 h and were used to assess their ability to invade and to evaluate their apoptotic potential in bovine mammary epithelial cells (bMECs). Prior to each experiment, fresh P. zopfii type-I and -II suspensions were used as described formerly (Shahid et al., 2016). P. zopfii was very carefully handled and all the experimental steps were carried out in biological safety cabinets, and we ensured the biosafety level 2 (BSL 2) precautions according to guidelines of Centers of Disease Control and Prevention (CDC).

Colonies of the P. zopfii genotype-I and -II were resuspended in phosphate buffered saline with pH 7.4 (PBS) and evaluated using an optical microscope (CKX 41SF Olympus, Japan).

P. zopfii genotype-I and II were cultivated on SDA to observe the morphological characteristics of colonies, the cultured plates were incubated at 37°C under aerobic condition for 5–7 days.

The exponential growth curve features of P. zopfii were analyzed in triplicate. The strains of genotype-I and -II were inoculated into 10 mL sabouraud dextrose broth (SDB; Difco™, Becton Dickison, Sparks MD, USA) in 15 mL centrifuge tube incubated at 37°C on a rotary shaker (150 rpm). Algal growth was determined by counting CFU per 4 h for first 3 days, after that CFU was counted per 24 for 7–8 days on SDA.

Primary bMECs were isolated and characterized as described previously (Liu et al., 2014). Cells were cultivated in growth medium containing Dulbecco's modified Eagle's (DMEM)/F12 medium (HyClone, USA) supplemented with 10% Fetal Bovine Serum (FBS; Gibco, Grand island, NY, USA) and antimicrobial agents (100 U/mL penicillin, 100 mg/mL streptomycin and 1 mg/mL amphotericin B) in cell culture plates (Corning, Corning, NY, USA). Cells were incubated in 5% CO₂ at 37°C, and cells from 2–8 passages were used for experiments.

For infection experiments, P. zopfii genotype-I and -II were grown on SDA for 48 h at 37°C, single colony was sub-cultivated into SDB for 72 h, after that P. zopfii were collected and suspended in DMEM/F12. Finally, the concentrations genotype-I and -II was adjusted to 5 × 10⁵ CFU/mL for all experiments.

Adhesion capability and time course of the survival of P. zopfii genotypes-I and -II in bMECs was determined as described previously (Pereyra et al., 2016; Chen et al., 2017). The bMECs were infected with genotypes-I and –II at a 5:1 multiplicity of infection (MOI; ratio of P. zopfii to bMECs). To study the adhesion capability and survival of P. zopfii genotypes-I and -II in bMEC cells, samples were taken at 1, 4, 8, 12, and 24 h and cultured on SDA after infection.

MTT cell proliferation assay kit (Trevigen, Gaithersburg, MD, USA) was used to assess the cell viability according to Verma et al. (2010) with slight modification. The bMECs at a density of 1 × 10⁵ cells per well were cultured in 96-well plates. After being treated as mentioned above, the cells were washed three times with PBS (pH 7.2), and cells were incubated with 100 μL medium and 10 μL of the activated MTT solution at 37°C for 4 h, after that treated with 50 μL DMSO (Sigma-Aldrich), and the absorption was measured by microplate reader (SpectraMax 190, Molecular Devices Corporation, Sunnyvale, CA, USA) at 570 nm. Cell viability was determined as the percentages (%) of the control.

The infected bMECs with P. zopfii genotype-I and -II were stained with FITC annexin V/PI apoptosis detection kit (Becton Dickinson, Franklin Lakes, NJ, USA) as described previously (Liu et al., 2014). Apoptosis rate was expressed using a FACSAria flow cytometry (Becton Dickinson, USA).

To further validate the apoptosis in bMECs caused by P. zopfii genotypes, Bax protein expression was detected by western blot analysis. The bMECs were infected with P. zopfii genotype-I and -II for 0, 4, 12, and 24 h at 37°C with 5% CO₂. Total protein was extracted from protothecal infected cells with protein extraction kit (KeyGEN, Nanjing, China). The protein quantification was carried out using bicinchoninic acid (BCA) protein assay kit (Beyotime, Haimen, China). Equivalent proteins from each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (PVDF; Millipore, MA, USA). Subsequently, the membranes were blocked in 5% bovine serum albumin (BSA; Gibco) and incubated with specific primary antibody for Bax (1:200, Santa, USA) and β-actin (1:1,000, Cell Signaling Technology, USA) overnight at 4°C followed by incubation with HRP-conjugated secondary antibody (1:5,000) for 1 h at room temperature. Finally, the bands were visualized using an enhanced chemiluminescence system (ECL, Beyotime, Haimen, China). The results were normalized to β-actin using Image J (National Institutes of Mental Health, Bethesda, MD, USA).

To explore the host cells specificity of P. zopfii, we infected the mouse osteoblast cell line MC3T3-E1 with P. zopfii genotype-I and -II. The MC3T3-E1 was purchased from American Type Culture Collection (ATCC, Rockville, MD, USA). These cell lines were maintained in minimal essential medium (α-MEM medium; Gibco) in the presence of 10% (v/v) fetal bovine serum (FBS; Gibco) and antimicrobials (100 U/mL penicillin and 100 U/mL streptomycin), incubated at 37°C in the presence of 5% CO₂. The infection protocol and flow cytometry assay was same as described earlier.

The infected cells with P. zopfii genotype-I and -II were washed three times with phosphate buffer saline (PBS; pH 7.2) and the cells were fixed with 2.5% glutaraldehyde (Novon Scientific, China; pH 7.4) as described previously (Chen et al., 2017). Then cells were dehydrated by a graded ethanol from 30 to 100%, dried by critical-point drying method, then gold-coated with E-1010 Ion Sputter Coater (Hitachi, Japan). Finally, the changes in bMECs were observed by SEM (TESCAN 5136, Czech Republic). TEM was used for ultrastructure analysis of bMECs infected with P. zopfii genotype-I and -II. The bMECs were cultured and challenged as mentioned above and after washing with PBS cells were fixed with 2% glutaraldehyde-1% paraformaldehyde solution (Sinopharm Chemical Reagent Co., Ltd., China; pH 7.2) for 45 min at room temperature. After washing with PBS, the fixed cells were harvested with a rubber scraper (Fisher Scientific, Nepean, ON, Canada) in 1.5 mL microcentrifuge tubes. Further steps in the processing of the TEM samples were conducted at Electron Microscopy Unit of Beijing University of Agriculture, China. Briefly, the pellet was washed 5–7 times with phosphate buffer saline (pH 7.2), post-fixed in a 0.5% osmium tetroxide for 2 h, washed 5–7 times with PBS. After five times washing with PBS, the pellet was dehydrated with graded alcohol series (30, 50, 70, 80, 90, 95, and 100%). The pellet was embedded in 50/50 LR white embedding resin (Electron Microscopic Sciences, Hatfield, PA, USA) and pure ethanol solution for 1 h followed by a pure resin solution for overnight at 4°C and incubated for 1 h at 60°C for polymerization. Thin slices (100 nm) were cut by an Ultracutmicrotome (Leica EM, Germany) with a glass knife. Sections were positioned on copper grid, stained with 2% uranyl acetate and lead citrate and were viewed on a transmission electron microscopy (HITACHI H-7650, Japan) at 80 kV. The imaging was done using a 4 K Gatan CCD camera using the iTEM software.

Statistical evaluation for triplicate experiments was carried out using student's t-test. Data are presented as the mean values ± standard deviation. p < 0.05 was considered as significant, while p < 0.01 was highly significant.

Under optical microscope the cells of P. zopfii genotype-I were spherical in shape whereas that of genotype-II were oval, elliptical, and smaller in size than genotype-I. Figures 1A,B indicates the different phases of microbial growth, having numerous stem cells called sporangia, forming sporangiospores or endospores by internal division. Under the electron microscopy, there were different morphological feature of genotype-II and genotype-I sporangia. Genotype-II revealed typical features of sporangia with spherical to oval, containing sporangiospores as shown in Figure 2A; while type-I showed compact, round shaped and twice in size of type-II (Figure 2B).

The colonies of both P. zopfii showed typical morphological characteristics like in case of genotype-II grayish white in color, with a central protrusion with granular surface and serrated shape (Figures 3A,C), while genotype-I displayed the creamy white colonies with bulging smooth surface (Figures 3B,D).

The exponential growth of P. zopfii is shown in Figure 4. According to growth curve of CFU, genotype-I and -II entered in logarithmic growth phase after 30 and 12 h and achieved the peak level at 144 and 96 h, respectively. This indicated that the genotype-II has faster growth kinetics than genotype-I.

Morphological changes in the bMECs monolayers following infection with genotype-I and -II are shown in Figures 5A–G. In case of genotype-I infection, the bMECs appeared similar to the control (uninfected cells); whereas type-II showed slight detachment from bMECs monolayer after 12 h (Figure 5F) and affected the integrity of monolayer cells after 24 h, as obvious from bMECs detachment in Figure 5G. The results depicted that the numbers of P. zopfii genotypes-I and -II increased steadily with passage of time as elaborated in Figure 5H. The genotype-II exhibited stronger adhesion capacity as compared to type-I.

The results of the MTT assay revealed that viability of cells infected with P. zopfii genotype-II was significantly decreased after 12 and 24 h as compared with the uninfected cells (Figure 6), the cell viability values significantly declined from 85.3 ± 3.7% (p < 0.05) to 48.17 ± 10.38% (p < 0.01), respectively. In contrast, genotype-I didn't show significant effect on cell viability as compared with control.

The results of flow cytometry assays showed significant changes in cell profile after exposure to P. zopfii genotype-II for 12 and 24 h. These results revealed that genotype-II significantly induced apoptosis in bMECs. After exposure, genotype-II showed high rise in apoptosis in comparison with control group as apoptotic rate was increased from 14.91 ± 5.54% (p < 0.05) to 63.83 ± 23.28% (p < 0.01) at 12 to 24 h, respectively. While, in case of genotype-I, there was non-significant effect on cells apoptosis (Figures 7A,B).

The results of Bax protein expression, using western blotting, in bMECs infected with P. zopfii genotype-I and -II are shown in Figure 8. Genotype-I showed non-significant effects on the expression Bax in bMECs as compared to control (i.e., at 0 h of infection). However, bMECs infected with genotype-II for 4, 12, and 24 h resulted in significant (p < 0.01) increase in Bax level (Figures 8A,B). This also validates the apoptotic effects of genotype-II on bMECs.

Figure 9 shows the apoptosis rate in MC3T3-E1 cells infected with P. zopfii genotype-I and -II for 0, 4, 12, and 24 h. The results elaborated non-significant difference in apoptosis rate in MC3T3-E1 cells at different time intervals (Figures 9A,B). This might suggest that P. zopfii genotype-I and -II didn't possess specificity toward these cell lines.

Under electron microscopy there were no morphological changes in uninfected bMECs (Figure 10A). In genotype-I infected group, there was also no prominent pathological alterations in the structure of cells; however the genotype-I have the adhesive capability with bMECs without damaging the cell integrity, this adhesion of genotype-I increased with the passage of time (Figures 10B–I). Whereas, genotype-II has showed adhesion without any structural alteration at 4 h as shown in Figure 11B. There were marked morphological changes in bMECs after exposure at 12–24 h. At 12 h, strong adhesion to bMECs was observed, which increased with passage of time, and disruption of cytomembrane initiated, this indicates the start of apoptosis (Figures 11C–F). At 24 h of exposure, the adhesion of genotype-II further increased (Figure 11G) and the cytomembrane was totally damaged (Figures 11H,I). In addition, the extent of cell disruption, the disappearance of microvilli and rupture of cellular membrane were prominent in the genotype-II exposure at 24 h (Figures 11G–I).

The ultrastructural features of bMECs as observed under TEM demonstrated various stages of P. zopfii genotype-II infection and its cytopathic and apoptotic effects in comparison with genotype-I. The normal bMECs maintained in DMEM with 10% FBS exhibited normal structure like intact cytoplasm and organelles (including intact mitochondria and endoplasmic reticulum structures) as in Figures 12A–C. After the adhesion of genotype-II at 4 h, slight increase in size of mitochondria was noted (Figure 12D), whereas, some epithelial cells extended around the adherent algae with the appearance of pseudopod like structures (Figure 12E). At 12 h, there were vacuolization in cytoplasm that originated from the swelling of endoplasmic reticulum as shown in Figure 12F and some of the mitochondria were swollen (Figure 12I). The genotype-II sporangiospores enclosed within the endocytic vacuoles were also seen in cells (Figures 12G–H). At 24 h post-infection, as elaborated in Figures 13B–I, all ultrastructural characteristics of apoptosis were confronted in the infected bMECs, such as cytoplasmic cavitation (Figures 13C,D,G), pyknosis (Figure 13D), cytomembrane disruption (Figures 13D,E), and appearance of apoptotic bodies (Figures 13G,H) as compared to control group (Figure 13A). The monolayer of bMECs was damaged and the released algae were seen near the remnant of disrupted epithelial cells as shown in Figure 13E at 24 h. The swollen mitochondrial structures and mitochondrial disruption were also observed (Figures 13F,H,I), and the cytoplasm was replaced by phagocytic vacuoles (Figure 13I) at 24 h after P. zopfii genotype-II infection. The average increase in mitochondrial size was 1.11, 1.69, and 2.63 μm at 4, 12, and 24 h post infection, respectively, as compared to the mitochondrial size of 0.98 μm of the control bMECs. Whereas, some epithelial cells having large vacuoles without any algae were observed, these vacuoles were the swelling of the endoplasmic reticulum (Figures 13F–H).

In P. zopfii genotype-I exposure, there was no marked ultrastructural changes in the bMECs at 4 h (Figure 14C) and 12 h (Figure 14D), similar like control cells (Figures 14A,B). While at 24 h post-infection the cytoplasmic membrane remained intact (Figures 14E,F,I), with slight vacuolization with enclosed algae (Figures 14G,H).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.