Effect of Two Different Drug-Resistant Staphylococcus aureus Strains on the Physiological Properties of MAC-T Cells and Their Transcriptome Analysis

Abstract

Staphylococcus aureus (S. aureus) is one of the main pathogens causing mastitis in dairy cows. The current work mainly focuses on the pathway of apoptosis induction in MAC-T cells caused by S. aureus infection or other factors. However, the physiological characteristics of S. aureus infected MAC-T cells and the resulting mRNA expression profile remain unknown particularly in the case of diverse drug resistant strains. Methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains were used to infect MAC-T cells to investigate this issue. The adhesion, invasion and apoptosis ability of MRSA-infected group and MSSA-infected group was assessed over time (2, 4, 6, 8, and 12 h). After 8 h, the RNA sequencing was conducted on the MRSA-infected and the MSSA-infected with uninfected MAC-T cells as controls. The results showed that the adhesion and invasion ability of MRSA-infected and MSSA-infected to MAC-T cells increased and then decreased with infection time, peaking at 8 h. The adhesion and invasion rates of the MSSA-infected were substantially lower than those of the MRSA-infected, and the invasion rate of the MSSA-infected group was nearly non-existent. Then the apoptosis rate of MAC-T cells increased as the infection time increased. The transcriptome analysis revealed 549 differentially expressed mRNAs and 390 differentially expressed mRNAs in MRSA-infected and MSSA-infected MAC-T cells, respectively, compared to the uninfected MAC-T cells. According to GO analysis, these differentially expressed genes were involved in immune response, inflammation, apoptosis, and other processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated the following pathways were linked to adhesion, invasion inflammation and apoptosis, including AMPK, FOXO, HIF-1, IL-17, JAK-STAT, MAPK, mTOR, NF-κB, p53, PI3K-Akt, TNF, Toll-like receptor, Rap1, RAS, prion disease, the bacterial invasion of epithelial cells pathway. We found 86 DEGs from 41 KEGG-enriched pathways associated with adhesion, invasion, apoptosis, and inflammation, all of which were implicated in MAC-T cells resistance to MRSA and MSSA infection. This study offers helpful data toward understanding the effect of different drug-resistant S. aureus on dairy cow mammary epithelial cells and aid in the prevention of mastitis in the dairy industry.

Keywords: MAC-T cells; MRSA; MSSA; adhesion; apoptosis; invasion; transcriptome.

Figure 1

Adhesion and invasion rates in MRSA-infected group and MSSA-infected group. (A) The adhesion rate of MRSA-infected and MSSA-infected MAC-T cells. (B) The invasion rate of MRSA-infected and MSSA-infected MAC-T cells.

Figure 2

The apoptosis rates of MRSA-infected and MSSA-infected MAC-T cells at different time periods. (A) The apoptosis rate of cells measured by flow cytometry. Q1 indicates necrotic cells (Anexin V– PI+), Q2 indicates late apoptotic cells (Anexin V+ PI+), Q3 indicates surviving cells (Anexin V– PI–), and Q4 indicates early apoptotic cells (Anexin V+ PI–). (a) The application of gating strategy in the analysis of MAC-T cell apoptosis. (b) The apoptosis rate of uninfected MAC-T cells. (c)–(g) The apoptosis rates of MRSA-infected cells at different time periods (2, 4, 6, 8, and 12 h). (h)–(l) The apoptosis rates of MRSA-infected cells at different time periods (2, 4, 6, 8, and 12 h). (B) The percentage of MRSA and MSSA induced apoptosis in MAC-T cells with time. *p < 0.05, **p < 0.01.

Figure 3

The functional enrichment analysis of total DEGs. (A) and (B) The volcano plots of total differentially expressed genes of MRSA-infected and MSSA-infected MAC-T cells. Log₂ |Fold change| >1 and p < 0.05 are the thresholds. Red plots represent up-regulated DEGs and green plots represent down-regulated DEGs. (C) The Venn diagram of total differentially expressed genes. Purple represents MRSA-infected group. Yellow represents MSSA-infected group. (D) and (E) The GO functional enrichment pathway of MRSA-infected and MSSA-infected MAC-T cells.

Figure 4

The enrichment analysis of selected DEGs in MRSA and MSSA infection groups. (A) and (B) The KEGG enrichment analysis of selected DEGs in MRSA and MSSA infection groups. (C) The Venn diagram of screening DEGs. Purple indicates MRSA-infected, yellow indicates MSSA-infected. (D) The interaction network map of important DEGs. (E) The clustering of important differentially expressed genes. Red represents highly expressed genes and blue represents lowly expressed genes.

Figure 5

The comparison of the relative expression of DEGs screened by RT-qPCR and RNA-seq for the MRSA-infected and MSSA-infected groups. (A) RT-qPCR to detect the relative expression of DEGs screened from the MRSA-infected group. (B) RT-qPCR to detect the relative expression of DEGs screened from the MSSA-infected group.

Figure 6

The effects of MRSA-infected and MSSA-infected MAC-T cells on the expression levels of TNF-α, IL-6, Bcl-2, cleavage–Caspase3, Phospho-p65 and PrP. (A) and (B) The MRSA-infected group and MSSA-infected group on the expression levels of TNF-α, IL-6, Bcl-2, cleavage–Caspase3, Phospho-p65, and PrP by Western blotting. (C) and (D) Gray-scale analysis of MRSA-infected group and MSSA-infected group.