PurN Is Involved in Antibiotic Tolerance and Virulence in Staphylococcus aureus

Abstract

Staphylococcus aureus can cause chronic infections which are closely related to persister formation. Purine metabolism is involved in S. aureus persister formation, and purN, encoding phosphoribosylglycinamide formyltransferase, is an important gene in the purine metabolism process. In this study, we generated a ΔpurN mutant of the S. aureus Newman strain and assessed its roles in antibiotic tolerance and virulence. The ΔpurN in the late exponential phase had a significant defect in persistence to antibiotics. Complementation of the ΔpurN restored its tolerance to different antibiotics. PurN significantly affected virulence gene expression, hemolytic ability, and biofilm formation in S. aureus. Moreover, the LD₅₀ (3.28 × 10¹⁰ CFU/mL) of the ΔpurN for BALB/c mice was significantly higher than that of the parental strain (2.81 × 10⁹ CFU/mL). Transcriptome analysis revealed that 58 genes that were involved in purine metabolism, alanine, aspartate, glutamate metabolism, and 2-oxocarboxylic acid metabolism, etc., were downregulated, while 24 genes involved in ABC transporter and transferase activity were upregulated in ΔpurN vs. parental strain. Protein-protein interaction network showed that there was a close relationship between PurN and GltB, and SaeRS. The study demonstrated that PurN participates in the formation of the late exponential phase S. aureus persisters via GltB and regulates its virulence by activating the SaeRS two-component system.

Keywords: Staphylococcus aureus; persister; purN; purine metabolism; virulence.

Figure 1

Exposure assay results of S. aureus wild-type, ΔpurN to ampicillin (10 μg/mL, (A–C)), and levofloxacin (20 μg/mL, (D–F)) in cultures at different time points. 5 h point (A,D). 9 h point (B,E). 18 h point (C,F).

Figure 2

Drug exposure results of Newman::pRAB11, ∆purN::pRAB11, ∆purN::pRABpurN, and Newman::pRBpurN to ampicillin (A,B), vancomycin (C,D), gentamicin (E,F) and levofloxacin (G,H) at different culture times. 5-h culture (A,C,E,G); 9-h culture (B,D,F,H).

Figure 3

Comparison of the virulence of Newman::pRAB11, ∆purN::pRAB11, ∆purN::pRABpurN, and Newman::pRBpurN in S. aureus. (A) The virulence gene expression levels detected by RT-qPCR. (B) Variation of hemolysis in different strains. Hemolysis status of Newman::pRAB11 (a,e), ∆purN::pRAB11 (b,f), ∆purN::pRABpurN (c,g), and Newman::pRBpurN (d,h) cultured for 24 h (a–d) and 48 h (e–h) on blood TSA plates. The hemolysis assay of the four strains was measured in different time points cultures. (i)10 h, (j) 14 h, (k) 24 h, and (l) 48 h. (C) The biofilm formation abilities of the four S. aureus strains in 96-well plates. Comparison of OD₅₅₀ and biofilm images in 96-well plate of different strains. * p < 0.05, ** p < 0.01.

Figure 4

Comparative analyses of the transcriptomics of ΔpurN and wild-type, and the gltB, saeR, and saeS expression in Newman::pRAB11, ΔpurN::pRAB11, ΔpurN::pRBpurN, and Newman::pRBpurN strains. (A) DEGs and pathways involved in the comparison of ΔpurN and wild-type. The genes in the green box and red box are downregulated and upregulated genes, respectively. (B) Protein-protein interaction network of DEGs between ΔpurN and parental strain by STRING database. The line thickness of the network indicates the strength of association/binding. (C) Comparison of expression levels of the gltB, saeR, and saeS in the four S. aureus strains (* p < 0.05).

Figure 5

Antibiotics exposure results of Newman::pRAB11, ∆gltB::pRAB11, ΔgltB::pRBpurN and Newman::pRBpurN to ampicillin (A,B), levofloxacin (C,D), gentamicin (E,F) and vancomycin (G,H) at different culture times. 5-h culture (A,C,E,G); 9-h culture (B,D,F,H).

Figure 6

Pathways that indicate how purN is involved in persister formation and virulence in S. aureus.