Metabolic Master Switch: Pyruvate Carboxylase Fuels Antimicrobial Resistance and Virulence in Foodborne Staphylococcus aureus

Abstract

Staphylococcus aureus, a major cause of foodborne illness globally, presents significant challenges due to its multidrug resistance and biofilm-forming capabilities. Pyruvate carboxylase (PycA), a metabolic master switch linking glycolysis and the tricarboxylic acid (TCA) cycle, is a potential target for controlling S. aureus. In this study, a pycA mutant was constructed and analyzed using phenotypic assays and proteomics to investigate its role in virulence and antimicrobial resistance. The results showed that deletion of pycA in the foodborne methicillin-resistant strain ATCC BAA1717 resulted in a 4- to 1024-fold reduction in resistance to β-lactams, aminoglycosides, and macrolides; a 23.24% impairment in biofilm formation; and a 22.32% decrease in staphyloxanthin production, a key antioxidant essential for survival in oxidative food environments. Proteomic analysis revealed downregulation of the TCA cycle, purine biosynthesis, surface adhesins (FnbA/B, SasG), and β-lactamase (BlaZ), linking PycA-mediated metabolism to phenotypes relevant to food safety. These findings underscore the importance of PycA as a metabolic regulator crucial for S. aureus resilience in food systems, suggesting novel strategies to combat foodborne staphylococcal infections through metabolic interference.

Keywords: Staphylococcus aureus; TCA cycle; antimicrobial resistance; biofilm; purine metabolism; pyruvate carboxylase.

Figure 1

The effect of PycA on the growth of S. aureus. (a) Growth curves of S. aureus strains in TSB medium; (b) L-ASP auxotrophy assay in MEM with or without 100 μg/mL L-ASP supplementation; (c) SEM images showing surface morphology at 10,000×, 20,000×, and 30,000× magnification; (d) TEM images of cellular ultrastructure at 11,000×, 22,000×, and 57,000× magnification. *** p < 0.001; n.s., not significant.

Figure 2

In vitro assessment of virulence-associated phenotypes in S. aureus strains. (a) Quantification of biofilm formation; (b) quantification of STX production; (c) total antioxidant capacity assessed using DPPH radical scavenging assay; (A) fluorescence microscopy of A549 cells after infection with S. aureus strains, in the absence (−) or presence (+) of 8 μg/mL ampicillin; and (B,C) LDH release assays measuring cytotoxicity of different S. aureus strains to A549 cells under (B) normal and (C) ampicillin-treated conditions. * p < 0.05; ** p < 0.01; *** p < 0.001 compared to the WT group.

Figure 3

Proteomic and functional analysis of the ΔpycA mutant in S. aureus. (a) PCA showing distinct clustering of WT and ΔpycA; (b) volcano plot showing differentially expressed proteins between WT and ΔpycA; (c) GO enrichment analysis of differentially expressed proteins; (d) KEGG pathway enrichment analysis of differentially expressed proteins; (e) measurement of intracellular ATP levels in S. aureus strains; and (f,g) qPCR (f) and RT-qPCR (g) analysis of repA and blaZ genes associated with resistance plasmids. * p < 0.05; *** p < 0.001 compared to the WT group.

Figure 4

Proposed regulatory role of PycA in coordinating metabolism, virulence, and antimicrobial resistance in S. aureus. Purple words represent phenotypic results; blue words represent proteomic results; black solid bars (⊥) indicate suppression; and black dashed lines indicate hypothesized relationships based on indirect evidence.