Comparative secretome analysis of Staphylococcus aureus strains with different within-herd intramammary infection prevalence

Abstract

Staphylococcus aureus is a major pathogen causing intramammary infection and mastitis in dairy cows. S. aureus genotypes (GT) can differ significantly in their ability to diffuse and persist in the herd; while the association of virulence gene carriage with epidemiological behavior remains unclear, a role for secreted proteins has been postulated. We characterized the secretome of six S. aureus strains belonging to two genotypes with opposite within-herd prevalence, GTB (high) and GTS (low), corresponding to sequence types (ST) 8 and 398, by high-resolution tandem mass spectrometry and differential analysis with Proteome Discoverer. Data are available via ProteomeXchange with identifier PXD029571. Out of 720 identified proteins, 98 were unique or more abundant in GTB/ST8 and 68 in GTS/ST398. GTB/ST8 released more immunoglobulin-binding proteins, complement and antimicrobial peptide inhibitors, enterotoxins, and metabolic enzymes, while GTS/ST398 released more leukocidins, hemolysins, lipases, and peptidases. Furthermore, GTB/ST8 released the von Willebrand factor protein, staphylokinase, and clumping factor B, while GTS released the staphylococcal coagulase and clumping factor A. Hence, GTB/ST8 secretomes indicated a higher propensity for immune evasion and chronicity and GTS/ST398 secretomes for cellular damage and inflammation, consistent with their epidemiological characteristics. Accordingly, GTS/ST398 secretions were significantly more cytotoxic against bovine PBMCs in vitro. Our findings confirm the crucial role of extracellular virulence factors in S. aureus pathogenesis and highlight the need to investigate their differential release adding to gene carriage for a better understanding of the relationship of S. aureus genotypes with epidemiological behavior and, possibly, disease severity.

Keywords: Proteomics; bacterial virulence; dairy cow; immune evasion; inflammation; intramammary infection; mammary gland; mastitis.

Figure 1.

Growth curves in brain-heart infusion broth (BHI) medium of the six Staphylococcus aureus GTB/ST8 (shades of blue) and GTS/ST398 (shades of Orange) strains used in this study. The curves report the bacterial colony forming units (Log₁₀ CFU)/mL as a function of time. Each point represents the mean (symbol) and standard deviation (bars) of three replicate CFU measurements. The X axis indicates the sampling times.

Figure 2.

SDS-PAGE profile of the proteins secreted in brain-heart infusion (BHI) broth by the six Staphylococcus aureus strains evaluated in this study. The GT/ST is indicated above the name of respective S. aureus strains. Protein load is 10 µg per lane.

Figure 3.

General results of the differential shotgun proteomics of the Staphylococcus aureus secretome obtained in brain-heart infusion (BHI) broth. (a) Principal Component Analysis based on the normalized protein abundances. (b) Venn diagram illustrating the distribution of the 720 proteins identified in the secretomes of the two GT (ST), showing shared proteins and differential proteins identified for each sample group.

Figure 4.

Distribution of the differential functions of the proteins secreted in brain-heart infusion (BHI) broth by the six Staphylococcus aureus strains evaluated in this study, classified according to the respective GT/ST. The smaller graphs illustrate the relative composition of the categories “Pathogenesis” (Orange) and “Metabolic enzyme” (blue). Abbreviations: Reg: regulation of gene expression; MDR: multidrug resistance; Amino: aminoacid metabolism; Carbo: carbohydrate metabolism; Lipid: lipid metabolism; Nucleo: nucleotide metabolism.

Figure 5.

Heatmap of the extracellular virulence factors showing significant differences between the two GT/ST, reported in order of abundance in the respective GT/ST group. The first and second columns report the protein accession number and the protein name and acronym. The last six columns illustrate in a heat map the average normalized protein abundance value/1000 calculated for each strain with Proteome Discoverer. Color intensity ranges from the highest observed value (dark red) to the lowest observed value (dark green). White: the protein was not detected. The proteins detected only in one genotype are marked with an asterisk.

Figure 6.

Heatmap of the extracellular metabolic enzymes showing significant differences between the two GT/ST, reported in order of abundance in the respective GT/ST group. The first and second columns report the protein accession number and the protein name and acronym. The third column indicates the metabolic pathway. The last six columns illustrate in a heat map the average normalized protein abundance value/1000 calculated for each strain with Proteome Discoverer. Color intensity ranges from the highest observed value (dark red) to the lowest observed value (dark green). White: the protein was not detected. The proteins detected only in one genotype are marked with an asterisk.

Figure 7.

Peripheral blood mononuclear cell (PBMC) viability after 18 h of incubation with the proteins secreted in brain-heart infusion (BHI) broth by the two Staphylococcus aureus GT (ST) evaluated in this work. The viability is expressed as fold-change compared to cells incubated without secreted bacterial proteins (control) in six technical replicates per condition. Significance was accepted at P < 0.05 (*) and P < 0.01 (**). The lines inside the boxes denote the median. The whiskers indicate the variability outside the upper and lower quartiles.