Prototheca zopfiiGenotype II induces mitochondrial apoptosis in models of bovine mastitis

Abstract

20Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of 21 P. zopfii (genotype I and II (GT-I and II)), P. zopfii GT-II is the genotype associated with acute 22 mastitis and decreased milk production by unknown mechanisms. The objective was to 23 determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial 24 cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. 25 Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells 26 (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase 27 mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven 28 apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance 29 (ΔΨm) was altered and modulation of mitochondrion-mediated apoptosis regulating genes 30 changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), 31 whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more 32 pronounced in macrophages compared to MECs. In a murine mammary infection model, P. 33zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration 34 of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-α, IL-1β and 35 Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-36 causing pathogen that triggers severe inflammation and also mitochondrial apoptosis. 37 38 39 40 3 41 Author summary 42 Bovine mastitis (inflammation of the udder) reduces milk production and quality, causing huge 43 economic losses in the dairy industry worldwide. Although the alga Prototheca zopfii is a major 44 cause of mastitis in dairy cows, mechanisms by which it damages mammary tissues are not well 45 known. Here, we used cell cultures and a mouse model of mastitis to determine how Prototheca 46 caused inflammation and cell death in mammary tissues. Prototheca invaded mammary gland 47 cells, from cattle and mice, as well as macrophages (white cells that take up and kill pathogens) 48 and caused cell death by interfering with mitochondria. Furthermore, Prototheca causes severe 49 inflammation and tissue damage when injected into the mammary glands of mice. Although 50 there are two genotypes of P. zopfii, only genotype II causes tissue damage, whereas gentotype I, 51 common in farm environments, does not damage mammary tissues. Since P. zopfii is an alga and 52 not a bacterium, antibiotic treatments, frequently used to treat mastitis in cattle, are not effective 53 against this organism. Understanding how P. zopfii damages mammary tissue and causes mastitis 54 is important new knowledge to promote future development of evidence-based approaches to 55 prevent and treat mammary gland infections with this organism. 56 57 4 63 protothecal mastitis can be clinical or subclinical. In clinical cases, symptoms include fever (up 64 to 40 ...

Prototheca bovis induces autophagy in bovine mammary epithelial cells via the HIF-1α and AMPKα/ULK1 pathway

“…Isolates of Prototheca bovis recovered from clinical mastitis milk samples on Chinese dairy farms were stored at 4°C in the College of Veterinary Medicine, China Agricultural University, Beijing, China (7). These isolates were multiplied by streaking on sabouraud dextrose agar (SDA) and incubating the plate at 37°C for 48 h. Then, a single colony was placed in sabouraud dextrose broth (SDB) and incubated for 72 h. Thereafter, organisms were diluted in DMEM to pre-defined concentrations.…”

“…Prototheca mastitis increases somatic cell counts and decreases milk production and milk quality, causing substantial economic losses (2,3). Infection of bovine mammary epithelial cells (bMECs) by P. bovis damages cellular and subcellular organelles (4). Autophagy is an integral part of resistance to various pathogenic infections, clearing pathogens and damaged organelles (5).…”

“…Autophagy is an integral part of resistance to various pathogenic infections, clearing pathogens and damaged organelles (5). Infection with P. bovis caused oxidative stress and apoptosis in bMECs (6,7); however, mechanisms underlying P. bovis-induced autophagy remains unclear.…”