Prototheca zopfii genotype II induces mitochondrial apoptosis in models of bovine mastitis

Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of P. zopfii (genotype I and II (GT-I and -II)), P. zopfii GT-II is the genotype associated with acute mastitis and decreased milk production, although its pathogenesis is not well known. The objective was to determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance (ΔΨm) was altered and modulation of mitochondrion-mediated apoptosis regulating genes changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more pronounced in macrophages compared to MECs. In a murine mammary infection model, P. zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-α, IL-1β and Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-causing pathogen that triggers severe inflammation and also mitochondrial apoptosis.


epithelial cell and macrophage culture. A bMEC line isolated from a cow (MAC-T) (Shanghai Jingma
Biological Technology Co., Ltd. China), murine macrophages derived from mouse BALB/c monocytes (J.774, provided by Dr. Eduardo R. Cobo, University of Calgary) and a murine mammary epithelial cells line (mMECs; HC11, provided by Dr. Eduardo R. Cobo, University of Calgary) were used. The bMECs and murine macrophages were cultured in HyClone TM DMEM/F12 medium (Thermo Fisher Scientific, South Logan, NH, USA) along with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), penicillin (100 U/mL; Thermo Fisher Scientific) and streptomycin (100 U/mL; Thermo Fisher Scientific) in cell culture plates (Corning Inc., Corning, NY, USA). The mMECs were cultured in RPMI (Thermo Fisher Scientific) medium along with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), penicillin (100 U/mL; HyClone ® , USA) and streptomycin (100 U/mL; Thermo Fisher Scientific). For experimental challenges, bMECs and macrophages (bovine and murine) were challenged with P. zopfii GT-I and GT-II suspended in DMEM/F12 to 5 × 10 5 and 1 × 10 5 CFU/mL, respectively, for up to 24 h at 37 °C with 5% CO 2 . P. zopfii cell internalization assay. Murine macrophages and bMECs were infected with P. zopfii for up to 8 h, washed with PBS (pH 7.4) and incubated for 2 h with gentamycin (200 μg/mL) to eliminate extracellular P. zopfii. Cells were washed with PBS to eliminate non-adherent bacteria and then lysed with 0.5% Triton X-100 (v/v) to determine CFU by 10-fold serial dilution 26 . Further confirmation of phagocytic activity of macrophages was done by actin inhibition (cytochalasin D; C8273, Sigma, USA; 1 h) before inoculation. transmission electron microscopy (teM). Bovine MECs infected with P. zopfii GT-I and -II were washed with PBS (pH 7.2), fixed with 2% glutaraldehyde and 1% paraformaldehyde (pH 7.2; Sinopharm Chemical Reagent Co., Shanghai, China) and processed for TEM 22 . Mitochondrial damage assay. After infection with P. zopfii, GT-I and -II, bovine MECs were collected to assess changes in mitochondrial membrane potential (ΔΨm), as determined by presence of JC-1 (Cat# M8650, Solarbio, Beijing, China) using flow cytometry and immunofluorescence microscopy. JC-1 is a dual-emission potential-sensitive probe that forms red-fluorescent aggregates in healthy mitochondria, but becomes a green-fluorescent monomer after membrane potential collapses.
Transcriptional gene expression of inflammatory and apoptotic genes. Total RNA was extracted from bMECs, mMECs and murine macrophages with TRIzol reagent (Invitrogen) and converted to cDNA (RevertAid First Strand cDNA synthesis kit, Thermo Scientific). Quality of resulting RNA and cDNA were evaluated by the absorbance ratio (A260/A280 ratio) (NanoVue Spectrophotometer, GE Healthcare Bio-Sciences, Little Chalfont, Buckinghamshire, UK) 27 , which was corrected to be ∼1.8-2.0 for an individual sample. Amplification of mRNA genes for TNF-α, IL-1β, IL-8/Cxcl-1, Bcl-2, Bax, Apaf-1, cytochrome-c, caspase-9 and caspase-3 was done using a CFX-96 real-time PCR system (BioRad, Hercules, CA, USA). The reaction mixture for each sample carried 2 µL of cDNA, 1X SsoAdvanced Universal SYBR Green Supermix (BioRad) and 0.5 μM of each specific primer, in a 10 μL final volume. Relative primers for bovine and murine genes are shown (Tables 1 and 2, respectively). Reaction mixtures were incubated at 95 °C for 5 min, followed by denaturation for 5 s at 95 °C and combined annealing/extension for 10 s at 60 °C (total of 40 cycles). All treatments were examined in duplicate in three independent experiments. Values of target mRNA were corrected relative to the normalizer GAPDH. Data were assessed using the 2−ΔΔCT method 27 and results presented as mean fold change of target mRNA levels in infected groups versus an uninfected control group 27 . tUneL apoptosis staining. Apoptosis of bMECs, mMECs, murine macrophages and mouse mammary gland after P. zopfii GT-II inoculation was assessed by in situ TUNEL staining (S7165 ApopTaq apoptosis detection kit, MilliporeSigma, Haverhill, MA, USA). Apoptotic indices were calculated as positive stained apoptotic cells per field, using five fields per sample at 400 × magnification.

Gene
Primer sequence (5′−3′) Table 2. Primer sequences of qPCR for murine genes. www.nature.com/scientificreports www.nature.com/scientificreports/ Statistical analyses. Data were analyzed in triplicate for reproducibility and were expressed as mean ± standard deviation (SD). Data from infected and uninfected groups were analyzed using a paired Student's t-test with a 95% confidence interval. Data were further analyzed by ANOVA and post hoc tests using SPSS 20.0 (International Business Machines Corporation, Armonk, NY, USA). For all analyses, P < 0.05 was considered significant.

P. zopfii Gt-ii induced mastitis and apoptosis in a mouse model. To investigate causative effects
of P. zopfii GT-II in protothecal mastitis, lactating mice were intramammarily challenged with P. zopfii GT-II isolated from a bovine clinical mastitis case. Round to oval sporangia with regular internal divisions compatible with P. zopfii were observed in the mammary gland of lactating mice at 4 dpi, as detected by PAS and GMS staining (Fig. 1A). Prototheca zopfii GT-II replicated in the murine mammary gland as it was recovered by culture in greater amounts at 4 dpi compared to the initial inoculum (mean 3.4 × 10 7 CFU/g tissue).
Prototheca zopfii GT-II induced acute mastitis with infiltration of leukocytes throughout the parenchyma and within lumina of alveoli. Prototheca zopfii GT-II were present both free within alveolar lumina and throughout the interstitium of the mammary tissue (Fig. 1A). Using immune detection, macrophages were demonstrated in the mammary interstitium and neutrophils diffusely distributed in P. zopfii GT-II-infected mice (Fig. 1A). The presence of P. zopfii GT-II upregulated gene activity and protein production of pro-inflammatory TNF-α, IL-1β and Cxcl-1 in mammary tissue at 4 dpi (Fig. 1B,C).

P. zopfii Gt-ii induced apoptosis in bovine mammary epithelial cells.
To verify apoptotic effects of P. zopfii in the target animal species (cattle), prototype bovine MECs with morphological and functional characteristics of normal mammary epithelial cells were challenged with P. zopfii GT-II and GT-I common commensals in farm environments (e.g., animal bedding, soil) 3 . Prototheca zopfii GT-I did not induce any apoptotic effects, but P. zopfii GT-II caused TUNEL-mediated apoptosis in a time-dependent manner (Fig. 4A). This occurred rapidly, as P. zopfii GT-II were internalized by bMECs in the first 4 hpi, as confirmed by culture (Fig. 4B) and TEM (Fig. 4C). Apoptotic effects induced by P. zopfii GT-II were likely of mitochondrial origin, as mitochondrial transmembrane depolarization was detected by immunofluorescence and flow cytometry (12-24 hpi; Fig. 4D-E).
Transcriptional expression of genes regulating mitochondrion-mediated apoptosis, including increased Bax and Apaf-1 ( Supplementary Fig. 2G,H) and decreased Bcl-2, were detected in bovine MECs inoculated with P. zopfii GT-II (Fig. 5A). Expression of caspase-9 mRNA at early points (4 hpi) followed by caspase-3 mRNA later (24 hpi), increased after P. zopfii GT-II infection (Fig. 5B,C). Likewise, cytochrome-c and cleaved caspase-9 and-3 were over time increasingly immune blotted (Fig. 5D) and immunolocalized (Fig. 5E) in bMECs infected with P. zopfii GT-II. Apart from decreased Bcl-2 expression after 24 hpi, no effect of P. zopfii GT-I on apoptotic genes in bMECs was observed (Fig. 5A).  Fig. 5F,H). However, GT-I did not modify any inflammatory cytokine response in bMECs and demonstrated the apthogenic nature of this Prototheca. Taken together, P. zopfii GT-II was demonstrated to cause udder disease by provoking apoptosis and inducing inflammatory cytokine expression in mammary epithelium.

Discussion
Previously, pathogenesis of protothecal mastitis and virulence of P. zopfii GT-II isolated from bovine milk were uncertain. In this study, we used a Prototheca spp. identified as P. zopfii GT-II following a taxomonic approach commonly accepted for Prototheca 16 and a cytb-based genotyping used for unambiguous Prototheca spp. identification 5 based on the protothecal phylogeny 20 and we described the pathogenic role of P. zopfii GT-II when initiating acute mastitis and mitochondrion-mediated apoptosis using a murine mastitis model and cultured mammary epithelial cells and macrophages. Our study demonstrated that P. zopfii GT-II invaded mammary parenchyma and caused acute mastitis, with severe infiltration of macrophages and neutrophils and marked epithelial damage. A destructive role of P. zopfii GT-II has been reported in the udder interstitium of cows and mammary acini of mice experimentally infected with P. zopfii GT-II 30,31 .
Mammary epithelial cells are essential in microbial infection for sensing pathogens and producing an array of inflammatory cytokines 32 . Pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6 and IL-8, have direct cytopathic effects leading to tissue damage 33 . Additionally, IL-1β and TNF-α can induce cell apoptosis 34    Depolarization of mitochondrial transmembrane (ΔΨm) causes the release of cytochrome-c, which may initiate caspase cascade. Cytochrome-c bonds with apoptotic protease-activating factor 1 (Apaf-1) and activates caspase-9, this cleaves and activates caspase-3, which triggers apoptosis. NF-κB subunit 65 transiting into the nucleus wherein it regulates transcription of pro-inflammatory genes, e.g. IL-1β and TNF-α.
Whereas P. zopfii has been reported to induce apoptosis in cultured bMECs 21,22 , we demonstrated the pro-apoptotic role of P. zopfii GT-II in a murine mastitis model. The pro-apoptotic character of P. zopfii GT-II was demonstrated by increased numbers of TUNEL-positive cells in P. zopfii GT-II-infected mice, along with reduced Bcl-2 levels and elevated transcriptomic levels of Bax, Apaf-1, caspase-3, and caspase-9. These all indicated apoptosis via the intrinsic pathway, with functional alterations in mitochondria in mammary epithelial cells infected with P. zopfii GT-II. Moreover, P. zopfii GT-II induced ROS generation 21 which triggers mitochondrial Bax, a proapoptotic element of the Bcl-2 family proteins 37 . Prototheca zopfii GT-II invaded bMECs and murine macrophages, and indeed, apoptotic effects were promoted by microbial internalization, but independent of phagocytosis. Prototheca zopfii GT-II had higher penetration capabilities in bMECs than P. zopfii GT-I. We propose that mitochondrial damage due to P. zopfii GT-II invasion released protein cytochrome-c from intermembrane spaces into cytosol, which bonded with Apaf-1 to initiate apoptosome formation and activation of caspase-9 and caspase-3 [38][39][40] . Such P. zopfii-driven apoptosis was not restricted to mammary epithelial cells but also applied to leukocytes, including murine macrophages. Whereas P. zopfii GT-II was a pathogenic type of Prototheca causing mastitis, studies with other Prototheca strains may elucidate the complexity of these algae and their interactions with host and enviroment. A hypothetical schematic illustration of mitochondrial caspase-induced apoptotic pathway and NF-κB subunit 65 transiting into the nucleus in protothecal mastitis (Fig. 6) was consistent with reports in bMECs, wherein P. zopfii GT-II regulated transcription of pro-inflammatory genes like IL-1β and TNF-α 35 . In conclusion, pathomorphological alteration caused by P. zopfii GT-II highlighted this gentoype as a mastitis pathogen capable of penetrating into mammary epithelial cells to induce inflammation and cell death, via mitochondrial-dependent apoptosis.