Proteomic changes in the milk of water buffaloes (Bubalus bubalis) with subclinical mastitis due to intramammary infection by Staphylococcus aureus and by non-aureus staphylococci

Abstract

Subclinical mastitis by Staphylococcus aureus (SAU) and by non-aureus staphylococci (NAS) is a major issue in the water buffalo. To understand its impact on milk, 6 quarter samples with >3,000,000 cells/mL (3 SAU-positive and 3 NAS-positive) and 6 culture-negative quarter samples with <50,000 cells/mL were investigated by shotgun proteomics and label-free quantitation. A total of 1530 proteins were identified, of which 152 were significantly changed. SAU was more impacting, with 162 vs 127 differential proteins and higher abundance changes (P < 0.0005). The 119 increased proteins had mostly structural (n = 43, 28.29%) or innate immune defence functions (n = 39, 25.66%) and included vimentin, cathelicidins, histones, S100 and neutrophil granule proteins, haptoglobin, and lysozyme. The 33 decreased proteins were mainly involved in lipid metabolism (n = 13, 59.10%) and included butyrophilin, xanthine dehydrogenase/oxidase, and lipid biosynthetic enzymes. The same biological processes were significantly affected also upon STRING analysis. Cathelicidins were the most increased family, as confirmed by western immunoblotting, with a stronger reactivity in SAU mastitis. S100A8 and haptoglobin were also validated by western immunoblotting. In conclusion, we generated a detailed buffalo milk protein dataset and defined the changes occurring in SAU and NAS mastitis, with potential for improving detection (ProteomeXchange identifier PXD012355).

Figure 1

SDS-PAGE profiles of skim milk samples before trypsinisation for shotgun analysis. Pos: culture-positive samples. Neg: culture-negative samples. SAU: milk samples positive for Staphylococcus aureus. NAS: milk samples positive for non-aureus staphylococci. M: molecular weight markers. Sample numbers correspond to those listed in Table 1. Molecular weight references are indicated on the left. Proteins with a molecular weight corresponding to the main electrophoretic bands are indicated on the right as a reference. One microliter of skim milk was loaded in each lane.

Figure 2

Correlation between the abundance of differential proteins in SAU-positive and NAS-positive samples. The scatter plot illustrates the correlation existing between common proteins increased in milk of buffaloes with subclinical mastitis due to SAU or NAS IMI and highlights the higher intensity of changes in SAU IMI (slope >1, p < 0.0005). X axis: R_SC values measured when considering only NAS IMI. Y axis: R_SC values measured when considering only SAU IMI. Only common proteins with R_SC ≥ 1.5 or R_SC ≤ −1.5 and p ≤ 0.05 are reported in the plot.

Figure 3

Function distribution of proteins increased and decreased in staphylococcus-positive milk when compared to healthy milk. The number of proteins belonging to each function is indicated. Green: all staphylococcus-positive samples; Red: SAU-positive samples; Blue: NAS-positive samples.

Figure 4

Reactome network according to STRING. Proteins associated with Neutrophil degranulation, Innate Immune System, Immune System, Regulation of actin dynamics for phagocytic cup formation, and Antimicrobial peptides, are indicated in red, pink, green, yellow, and cyan, respectively. Seven different coloured lines link nodes and represent seven types of evidence used in predicting associations. Green lines: neighbourhood evidence; red lines: presence of fusion evidence; blue lines: co-occurrence evidence; black lines: co-expression evidence; purple lines: experimental evidence; light blue lines: database evidence; yellow lines: text-mining evidence.

Figure 5

Western immunoblotting validation. Top. Distribution of normalised spectral abundance factor (NSAF) values measured in each sample by shotgun proteomics for (A) cathelicidin proteoforms, (B) S100A8, and (C) haptoglobin. Bottom. Western immunoblotting reactivity of the same samples with (D) anti-pan-cathelicidin, (E) anti-S100A8, and (F) anti-haptoglobin antibodies. SAU: milk samples positive for Staphylococcus aureus. NAS: milk samples positive for non-aureus staphylococci. Neg: culture-negative milk. Sample numbers correspond to those listed in Table 1. One microliter of milk was loaded in each lane. Images were cropped to report relevant information. The original experiment images are reported in Supplementary Fig. 1.