Interactions of Candidate Gene Expression and in vitro Infection with Streptococcus uberis in Bovine Mammary Epithelial Cells

Streptococcus uberis is an environmental pathogen that can internalize into mammary epithelial cells (MECs), evade degradation within cells, and cause mastitis. Our objective was to investigate interactions between S. uberis and MEC function relative to RAB4a (Ras-related protein RAB4a), HECTD4 (HECT Domain E3 Ubiquitin Protein Ligase 4), and PSAP (Prosaposin coding gene) which are involved with intracellular trafficking and degradation and were located next to SNPs associated with in vivo mastitis phenotypes. A common MEC-line (MACT) and a primary line (BOMEC) were inoculated with S. uberis strains isolated from a chronic (UT888 CFU Mean=4.5x10⁷) or acute (UT366 CFU Mean=2.4x10⁸) mastitis case. Candidate gene expression, cell viability, and intracellular survival of bacteria were evaluated at 0, 3, 9, and 27 hours for infected and non-infected cells using a mixed model ANOVA (SAS 9.4, Cary, NC). The fixed effects were cell type, strain, infection status, and time, with a random effect of time (cell type*strain). Cell viability was not associated with any fixed effects, with mean viability remaining above 88% at all time points. Percent of internalized UT366 (LSMean=0.006, SE=0.0016) was higher than UT888 (LSMean=0.0023, SE=0.0016) (p=0.0026). Percent internalized for both strains peaked at 3 hours (LSMean=0.012, SE=0.0018), decreased at 9 hours (LSMean=0.0007, SE=0.0018), and decreased again at 27 hours (LSMean=-0.0002, SE=0.0018) (pS. uberis and candidate genes, small interfering RNA was used to reduce expression in MACT prior to infection with UT888. The number of live cells collected was reduced with knockdown of HECTD4 (Control: Mean=1.1x10⁶ cells/mL SE=1.2x10⁵, KD: Mean=2.3x10⁵ cells/mL SE=4.6x10⁴, p6 cells/mL SE=8.4x10⁴, KD: Mean=9.2x10⁵ cells/mL SE=1.2x10⁵, p=0.0008), but not with PSAP (Control: Mean=1.2x10⁶ cells/mL SE=6.8x10⁴, KD: Mean=7.0x10⁵ cells/mL SE=1.1x10⁵, p=0.13). Reduction in cell viability also was observed after HECTD4 (Control: 90% SE=7.9, KD: 56% SE=6.8, pS. uberis indicate some potential relevance to intracellular survival of S. uberis.