Rapid baso-apical translocation of Mycobacterium avium ssp. paratuberculosis in mammary epithelial cells in the presence of Escherichia coli

Abstract

Infection of mammary gland cells with bacterial pathogens begins with adhesion, invasion, and persistence within the cells or systemic distribution. Some bacteria, such as Escherichia coli, are known to causes bovine mastitis, resulting in acute proinflammatory responses in the mammary tissue. Mycobacterium avium ssp. paratuberculosis (MAP), the etiological agent of paratuberculosis, is able to spread to distant organs after crossing intestinal cells, reaching the mammary gland and potentially being released in milk, infecting calves during suckling. Its exit from systemic sites may be influenced by preexisting inflammation such as that caused by E. coli mastitis. Interactions between E. coli and MAP in mammary epithelial cells have not yet been described. In this study, we posited that E. coli-infected bovine mammary epithelial cells would facilitate baso-apical translocation of MAP in an ex vivo model. We showed that the presence of E. coli in a bovine mammary epithelial cell line (MAC-T) increased baso-apical translocation of MAP to the apical side of the cells. Levels were significantly higher 30 min post-infection and decreased at 120 min post-infection. Cells previously infected with E. coli and MAP or with E. coli alone showed a significant increase in IL1B mRNA expression at 120 min. We detected no significant expression of p38 mitogen-activated protein kinase (mapkp38) or IL10, regardless of treatment. Thereby, the presence of E. coli in MAC-T cells alters the translocation of MAP through epithelial cells, enabling its rapid translocation to the cellular surface. Expression of IL1B was shown to influence the apical-basal translocation of MAP at 120 min. Findings from the current study suggest that MAP translocation into milk is likely enhanced by inflammatory states such as those induced during E. coli mastitis. This is the first report demonstrating the effect of E. coli under MAP coinfection in bovine mammary epithelial cells under experimental conditions.

Inhibition ofEscherichia coliinvasion into bovine mammary epithelial cells previously infected byMycobacterium aviumsubsp.paratuberculosis

“…Coinfection study of MAP and E. coli isolated from bovine mastitis was recently described by Schwarz et al (2018). In this study, the previous E. coli infection of bovine mammary epithelial cell line (MAC-T) increased a rapid baso-apical translocation of MAP to the supernatant of the cells.…”

“…paratuberculosis (MAP) even being released by milk, appear to poorly stimulate the local inflammatory response and is not considered as the etiologic agent of classic mastitis (Larsen and Miller 1978). Recently, a study showed that cows with E. coli mastitis potentially can attract MAP from distant sites and release it into milk more efficiently when compared with cows without mastitis (Schwarz et al 2018). However, the mechanisms of how a non-mastitic pathogen can infect the mammary gland and interfere in a subsequent infection by another mastitic pathogen are still unknown.…”

“…Li et al (2016) demonstrated that the yeast cell wall components reduced MAP binding to mammary epithelial cells, but this fact was partially attributed to decreased cell viability. Although some authors have worked with the MAP invasion process in MAC-T cells (Li et al 2016;Schwarz et al 2018), it is known that MAP is capable of inducing apoptosis in macrophages by a caspase-dependent or caspase-independent mechanism and mitochondrial damage (Periasamy et al 2013). In the present study, regardless of the time of exposure and the treatment (MAP and E.coli, MAP or E. coli) there was no reduction in viability of MAC-T cells.…”

“…MAP K-10 standard strain and an Escherichia coli strain (Schwarz et al 2018) isolated from milk from a case of bovine clinical mastitis (kindly provided by Dr. Srinand Sreevatsan from the Laboratory of Udder Health, Minnesota Veterinary Diagnostic Laboratory, University of Minnesota, Saint Paul) were used in this experiment. Culture of MAP-K10 was tested for purity using IS900 PCR and cultivation in Middlebrook 7H9 agar medium supplemented with 10% glycerol, 1% oleic acid-albumin-dextrose (OADC), and mycobactin J (2 mg/L) (Allied Monitor Inc., Fayette, MO, USA).…”

“…Approximately 3.0 x 10 5 MAC-T were seeded onto the apical side of 3.0-mm-pore-size TranswellV R -Clear inserts (Corning, Lowell, MA, USA) in DMEM containing 10% FBS and incubated at 37 C in a humidified chamber containing 5% CO 2 up to the confluence of the monolayer ($ 2 days). Cell monolayer integrity was verified as previously described (Patel et al 2006;Schwarz et al 2018). MAP culture (1.0 x 10 6 CFU/mL) was pelleted at 12,000 x g for 15 min, washed two times in PBS and resuspended in DMEM containing 10% FBS.…”