Vancomycin-intermediate Staphylococcus aureus employs CcpA-GlmS metabolism regulatory cascade to resist vancomycin

Abstract

Vancomycin (VAN)-intermediate Staphylococcus aureus (VISA) is a critical cause of VAN treatment failure worldwide. Multiple genetic changes are reportedly associated with VISA formation, whereas VISA strains often present common phenotypes, such as reduced autolysis and thickened cell wall. However, how mutated genes lead to VISA common phenotypes remains unclear. Here, we show a metabolism regulatory cascade (CcpA-GlmS), whereby mutated two-component systems (TCSs) link to the common phenotypes of VISA. We found that ccpA deletion decreased VAN resistance in VISA strains with diverse genetic backgrounds. Metabolic alteration in VISA was associated with ccpA upregulation, which was directly controlled by TCSs WalKR and GraSR. RNA-sequencing revealed the crucial roles of CcpA in changing the carbon flow and nitrogen flux of VISA to promote VAN resistance. A gate enzyme (GlmS) that drives carbon flow to the cell wall precursor biosynthesis was upregulated in VISA. CcpA directly controlled glmS expression. Blocking CcpA sensitized VISA strains to VAN treatment in vitro and in vivo. Overall, this work uncovers a link between the formation of VISA phenotypes and commonly mutated genes. Inhibition of CcpA-GlmS cascade is a promising strategy to restore the therapeutic efficiency of VAN against VISA infections.

Keywords

catabolite control protein A / metabolic changes / Staphylococcus aureus / two-component system / vancomycin resistance

References

[1] IkutaKS, Swetschinski LR, Robles AguilarG, et al. Global mortality associated with 33 bacterial pathogens in 2019: a systematic analysis for the global burden of disease study 2019. Lancet. 2022;400(10369):2221-2248.

[2] TongSYC, DavisJS, EichenbergerE, HollandTL, Fowler Jr. VG. Staphylococcus aureus infections: epidemiology, pathophysiology, clinical manifestations, and management. Clin Microbiol Rev. 2015;28(3):603-661.

[3] LevineDP. Vancomycin: a history. Clin Infect Dis. 2006;42(Suppl 1):S5-S12.

[4] UnniS, Siddiqui TJ, BidaiseeS. Reduced susceptibility and resistance to vancomycin of Staphylococcus aureus: a review of global incidence patterns and related genetic mechanisms. Cureus. 2021;13(10):e18925.

[5] CongY, YangS, RaoX. Vancomycin resistant Staphylococcus aureus infections: a review of case updating and clinical features. J Adv Res. 2020;21:169-176.

[6] HuQ, PengH, RaoX. Molecular events for promotion of vancomycin resistance in vancomycin intermediate Staphylococcus aureus. Front Microbiol. 2016;7:1601.

[7] GardeteS, TomaszA. Mechanisms of vancomycin resistance in Staphylococcus aureus. J Clin Invest. 2014;124(7):2836-2840.

[8] SieradzkiK, TomaszA. Alterations of cell wall structure and metabolism accompany reduced susceptibility to vancomycin in an isogenic series of clinical isolates of Staphylococcus aureus. J Bacteriol. 2003;185(24):7103-7110.

[9] CuiL, Murakami H, Kuwahara-AraiK, HanakiH, Hiramatsu K. Contribution of a thickened cell wall and its glutamine nonamidated component to the vancomycin resistance expressed by Staphylococcus aureus Mu50. Antimicrob Agents Chemother. 2000;44(9):2276-2285.

[10] ZhangS, SunX, ChangW, Dai Y, MaX. Systematic review and meta-analysis of the epidemiology of vancomycin-intermediate and heterogeneous vancomycin-intermediate Staphylococcus aureus isolates. PLoS One. 2015;10(8):e0136082.

[11] ShenP, ZhouK, WangY, et al. High prevalence of a globally disseminated hypervirulent clone, Staphylococcus aureus CC121, with reduced vancomycin susceptibility in community settings in China. J Antimicrob Chemother. 2019;74(9):2537-2543.

[12] TacconelliE, Carrara E, SavoldiA, et al. Discovery, research, and development of new antibiotics: the WHO priority list of antibiotic-resistant bacteria and tuberculosis. Lancet Infect Dis. 2018;18(3):318-327.

[13] HowdenBP, DaviesJK, JohnsonPDR, Stinear TP, GraysonML. Reduced vancomycin susceptibility in Staphylococcus aureus, including vancomycin-intermediate and heterogeneous vancomycin-intermediate strains: resistance mechanisms, laboratory detection, and clinical implications. Clin Microbiol Rev. 2010;23(1):99-139.

[14] HowdenBP, McEvoyCRE, AllenDL, et al. Evolution of multidrug resistance during Staphylococcus aureus infection involves mutation of the essential two component regulator WalKR. PLoS Pathog. 2011;7(11):e1002359.

[15] FaitA, SeifY, MikkelsenK, et al. Adaptive laboratory evolution and independent component analysis disentangle complex vancomycin adaptation trajectories. Proc Natl Acad Sci. 2022;119(30):e2118262119.

[16] MatsuoM, CuiL, KimJ, Hiramatsu K. Comprehensive identification of mutations responsible for heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA)-to-VISA conversion in laboratory-generated VISA strains derived from hVISA clinical strain Mu3. Antimicrob Agents Chemother. 2013;57(12):5843-5853.

[17] MwangiMM, WuSW, ZhouY, et al. Tracking the in vivo evolution of multidrug resistance in Staphylococcus aureus by whole-genome sequencing. Proc Natl Acad Sci. 2007;104(22):9451-9456.

[18] PengH, HuQ, ShangW, et al. WalK(S221P), a naturally occurring mutation, confers vancomycin resistance in VISA strain XN108. J Antimicrob Chemother. 2017;72(4):1006-1013.

[19] HuJ, ZhangX, LiuX, ChenC, SunB. Mechanism of reduced vancomycin susceptibility conferred by walK mutation in community-acquired methicillin-resistant Staphylococcus aureus strain MW2. Antimicrob Agents Chemother. 2015;59(2):1352-1355.

[20] CuiL, NeohH, ShojiM, Hiramatsu K. Contribution of vraSR and graSR point mutations to vancomycin resistance in vancomycin-intermediate Staphylococcus aureus. Antimicrob Agents Chemother. 2009;53(3):1231-1234.

[21] HowdenBP, Stinear TP, AllenDL, JohnsonPDR, WardPB, DaviesJK. Genomic analysis reveals a point mutation in the two-component sensor gene graS that leads to intermediate vancomycin resistance in clinical Staphylococcus aureus. Antimicrob Agents Chemother. 2008;52(10):3755-3762.

[22] GardeteS, KimC, HartmannBM, et al. Genetic pathway in acquisition and loss of vancomycin resistance in a methicillin resistant Staphylococcus aureus (MRSA) strain of clonal type USA300. PLoS Pathog. 2012;8(2):e1002505.

[23] StokesJM, Lopatkin AJ, LobritzMA, CollinsJJ. Bacterial metabolism and antibiotic efficacy. Cell Metab. 2019;30(2):251-259.

[24] ZampieriM, EnkeT, ChubukovV, Ricci V, PiddockL, SauerU. Metabolic constraints on the evolution of antibiotic resistance. Mol Syst Biol. 2017;13(3):917.

[25] LopatkinAJ, BeningSC, MansonAL, et al. Clinically relevant mutations in core metabolic genes confer antibiotic resistance. Science. 2021;371(6531):eaba0862.

[26] GardnerSG, Marshall DD, DaumRS, PowersR, Somerville GA. Metabolic mitigation of Staphylococcus aureus vancomycin intermediate-level susceptibility. Antimicrob Agents Chemother. 2018;62(1):e01608-17.

[27] AlexanderEL, Gardete S, BarHY, WellsMT, TomaszA, RheeKY. Intermediate-type vancomycin resistance (VISA) in genetically-distinct Staphylococcus aureus isolates is linked to specific, reversible metabolic alterations. PLoS One. 2014;9(5):e97137.

[28] NelsonJL, RiceKC, SlaterSR, et al. Vancomycin-intermediate Staphylococcus aureus strains have impaired acetate catabolism: implications for polysaccharide intercellular adhesin synthesis and autolysis. Antimicrob Agents Chemother. 2007;51(2):616-622.

[29] IbarraJA, Pérez-Rueda E, CarrollRK, ShawLN. Global analysis of transcriptional regulators in Staphylococcus aureus. BMC Genomics. 2013;14:126.

[30] PoudelS, HefnerY, SzubinR, et al. Coordination of CcpA and CodY regulators in Staphylococcus aureus USA300 strains. mSystems. 2022;7(6):e0048022.

[31] SeidlK, StuckiM, RueggM, et al. Staphylococcus aureus CcpA affects virulence determinant production and antibiotic resistance. Antimicrob Agents Chemother. 2006;50(4):1183-1194.

[32] PengH, RaoY, YuanW, et al. Reconstruction of the vancomycin-susceptible Staphylococcus aureus phenotype from a vancomycin-intermediate S. aureus XN108. Front Microbiol. 2018;9:2955.

[33] DubracS, MsadekT. Identification of genes controlled by the essential YycG/YycF two-component system of Staphylococcus aureus. J Bacteriol. 2004;186(4):1175-1181.

[34] FalordM, Mäder U, HironA, DébarbouilléM, MsadekT. Investigation of the Staphylococcus aureus GraSR regulon reveals novel links to virulence, stress response and cell wall signal transduction pathways. PLoS One. 2011;6(7):e21323.

[35] KatayamaY, SekineM, HishinumaT, Aiba Y, HiramatsuK. Complete reconstitution of the vancomycin-intermediate Staphylococcus aureus phenotype of strain Mu50 in vancomycin-susceptible S. aureus. Antimicrob Agents Chemother. 2016;60(6):3730-3742.

[36] KomatsuzawaH, Fujiwara T, NishiH, et al. The gate controlling cell wall synthesis in Staphylococcus aureus. Mol Microbiol. 2004;53(4):1221-1231.

[37] YangY, ZhangL, HuangH, et al. A flexible binding site architecture provides new insights into CcpA global regulation in Gram-positive bacteria. mBio. 2017;8(1):e02004-16.

[38] HuangQ, ZhangZ, LiH, et al. Identification of a novel inhibitor of catabolite control protein A from Staphylococcus aureus. ACS Infect Dis. 2020;6(3):347-354.

[39] ZhuX, LiuC, GaoS, LuY, ChenZ, Sun Z. Vancomycin intermediate-resistant Staphylococcus aureus (VISA) isolated from a patient who never received vancomycin treatment. Int J Infect Dis. 2015;33:185-190.

[40] BisicchiaP, BuiNK, AldridgeC, Vollmer W, DevineKM. Acquisition of vanb-type vancomycin resistance by Bacillus subtilis: the impact on gene expression, cell wall composition and morphology. Mol Microbiol. 2011;81(1):157-178.B

[41] GonchevaMI, Flannagan RS, SterlingBE, et al. Stress-induced inactivation of the Staphylococcus aureus purine biosynthesis repressor leads to hypervirulence. Nat Commun. 2019;10(1):775.

[42] CuiL, MaX, SatoK, et al. Cell wall thickening is a common feature of vancomycin resistance in Staphylococcus aureus. J Clin Microbiol. 2003;41(1):5-14.

[43] KriegeskorteA, BlockD, DrescherM, et al. Inactivation of thyA in Staphylococcus aureus attenuates virulence and has a strong impact on metabolism and virulence gene expression. mBio. 2014;5(4):e01447-14.

[44] Mengin-LecreulxD, Van Heijenoort J. Copurification of glucosamine-1-phosphate acetyltransferase and N-acetylglucosamine-1-phosphate uridyltransferase activities of Escherichia coli: characterization of the glmU gene product as a bifunctional enzyme catalyzing two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis. J Bacteriol. 1994;176(18):5788-5795.

[45] DaiJK, DanWJ, CaoYD, Gao JX, WangJR, WanJB. Discovery of new quaternized norharmane dimers as potential anti-MRSA agents. J Adv Res. 2023;S2090-1232(23):00328-4.

[46] QuD, HouZ, LiJ, et al. A new coumarin compound DCH combats methicillin-resistant Staphylococcus aureus biofilm by targeting arginine repressor. Sci Adv. 2020;6(30):eaay9597.

[47] RoseW, FantlM, GeriakM, Nizet V, SakoulasG. Current paradigms of combination therapy in methicillin-resistant Staphylococcus aureus (MRSA) bacteremia: does it work, which combination, and for which patients? Clin Infect Dis. 2021;73(12):2353-2360.

[48] ShatalinK, Nuthanakanti A, KaushikA, et al. Inhibitors of bacterial H₂S biogenesis targeting antibiotic resistance and tolerance. Science. 2021;372(6547):1169-1175.

[49] TranN, RybakMJ. β-Lactam combinations with vancomycin show synergistic activity against vancomycin-susceptible Staphylococcus aureus, vancomycin-Intermediate S. aureus (VISA), and heterogeneous VISA. Antimicrob Agents Chemother. 2018;62(6):e00157-18.

[50] ZhangX, HuQ, YuanW, et al. First report of a sequence type 239 vancomycin-intermediate Staphylococcus aureus isolate in Mainland China. Diagn Microbiol Infect Dis. 2013;77(1):64-68.

[51] LiuM, FengM, YangK, et al. Transcriptomic and metabolomic analyses reveal antibacterial mechanism of astringent persimmon tannin against methicillin-resistant Staphylococcus aureus isolated from pork. Food Chem. 2020;309:125692.

[52] LangmeadB, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nature Methods. 2012;9(4):357-359.

[53] ValihrachL, Demnerova K. Impact of normalization method on experimental outcome using RT-qPCR in Staphylococcus aureus. J Microbiol Meth. 2012;90(3):214-216.

[54] RaoY, PengH, ShangW, et al. A vancomycin resistance-associated WalK(S221P) mutation attenuates the virulence of vancomycin-intermediate Staphylococcus aureus. J Adv Res. 2022;40:167-178.

[55] LiuH, ShangW, HuZ, et al. A novel SigB(Q225P) mutation in Staphylococcus aureus retains virulence but promotes biofilm formation. Emerg Microbes Infect. 2018;7(1):1-12.

[56] BrücknerR. Gene replacement in Staphylococcus carnosus and Staphylococcus xylosus. FEMS Microbiol Lett. 1997;151(1):1-8.

[57] XueT, ZhaoL, SunB. LuxS/AI-2 system is involved in antibiotic susceptibility and autolysis in Staphylococcus aureus NCTC 8325. Int J Antimicro Ag. 2013;41(1):85-89.

[58] ZhaoX, ShenM, JiangX, et al. Transcriptomic and metabolomics profiling of phage-host interactions between phage PaP1 and Pseudomonas aeruginosa. Front Microbiol. 2017;8:548.

[59] YuanW, HuQ, ChengH, et al. Cell wall thickening is associated with adaptive resistance to amikacin in methicillin-resistant Staphylococcus aureus clinical isolates. J Antimicrob Chemother. 2013;68(5):1089-1096.

[60] Martinez-IrujoJJ, Villahermosa ML, AlberdiE, SantiagoE. A checkerboard method to evaluate interactions between drugs. Biochem Pharmacol. 1996;51(5):635-644.

[61] den HollanderJG, MoutonJW, VerbrughHA. Use of pharmacodynamic parameters to predict efficacy of combination therapy by using fractional inhibitory concentration kinetics. Antimicrob Agents Chemother. 1998;42(4):744-748.

[62] LiM, DuX, VillaruzAE, et al. MRSA epidemic linked to a quickly spreading colonization and virulence determinant. Nat Med. 2012;18(5):816-819.

RIGHTS & PERMISSIONS

2024 The Author(s). MedComm - Future Medicine published by John Wiley & Sons Australia, Ltd on behalf of Sichuan International Medical Exchange & Promotion Association (SCIMEA).