Metabolic Analysis of the Mode of Action and Mode of Resistance for Novobiocin in Staphylococcus aureus

Abstract

Objective: Methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant Staphylococcus aureus (VRSA) are critical pathogens identified by the WHO for their significant drug resistance. Targeting of bacterial gyrase, specifically the gyrB subunit, is a promising approach because of this enzyme’s essential role in bacterial DNA replication and its absence in higher eukaryotes. However, understanding of the mode of action of gyrB inhibitors remains largely incomplete. This study explored the resistance mechanisms of Staphylococcus aureus (S. aureus) to novobiocin, a gyrB inhibitor.

Methods: Through adaptive laboratory evolution, key resistance mutations (in gyrB, potB, and fpgS) in S. aureus were identified after repeated exposure to novobiocin. Further metabolomic analysis revealed the function of the major mutation (in gyrB) in relation to the potential mechanism through which S. aureus responds to novobiocin.

Results: Through whole genome sequencing, three mutations of S. aureus in gyrB, potB, and fpgS were identified. The gyrB mutation was the primary driver of resistance, and was associated with changes in growth, survival under surface and oxidative stress, cell wall permeability, and coagulation functions. Metabolomic analysis demonstrated compensatory metabolic adjustments affecting protein synthesis and DNA replication in the resistant strain.

Conclusions: These findings provide insights into the complex resistance mechanisms of S. aureus to novobiocin and highlight the metabolic costs associated with gyrB mutations, thereby potentially informing future antibacterial strategy development.

Keywords

Staphylococcus aureus / novobiocin / drug resistance / metabolomics

References

[1] Ferrero L, Cameron B, Crouzet J. Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus . Antimicrob Agents Chemother. 1995; 39(7): 1554-1558.

[2] Heisig P. Genetic evidence for a role of parC mutations in development of high-level fluoroquinolone resistance in Escherichia coli . Antimicrob Agents Chemother. 1996; 40(4): 879-885.

[3] Yoshida H, Bogaki M, Nakamura M, Nakamura S. Quinolone resistance-determining region in the DNA gyrase gyrA gene of Escherichia coli . Antimicrob Agents Chemother. 1990; 34(6): 1271-1272.

[4] Yoshida H, Bogaki M, Nakamura M, Yamanaka LM, Nakamura S. Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli . Antimicrob Agents Chemother. 1991; 35(8): 1647-1650.

[5] Takei M, Fukuda H, Kishii R, Hosaka M. Target preference of 15 quinolones against Staphylococcus aureus, based on antibacterial activities and target inhibition . Antimicrob Agents Chemother. 2001; 45(12): 3544-3547.

[6] Alovero FL, Pan XS, Morris JE, Manzo RH, Fisher LM. Engineering the specificity of antibacterial fluoroquinolones: benzenesulfonamide modifications at C-7 of ciprofloxacin change its primary target in Streptococcus pneumoniae from topoisomerase IV to gyrase. Antimicrob Agents Chemother. 2000; 44(2): 320-325.

[7] Gellert M, O’Dea MH, Itoh T, Tomizawa J. Novobiocin and coumermycin inhibit DNA supercoiling catalyzed by DNA gyrase. Proc Natl Acad Sci U S A. 1976; 73(12): 4474-4478.

[8] Fournier B, Hooper DC. Mutations in topoisomerase IV and DNA gyrase of Staphylococcus aureus: novel pleiotropic effects on quinolone and coumarin activity . Antimicrob Agents Chemother. 1998; 42(1): 121-128.

[9] Muñoz R, Bustamante M, de la Campa AG. Ser-127-to-Leu substitution in the DNA gyrase B subunit of Streptococcus pneumoniae is implicated in novobiocin resistance. J Bacteriol. 1995; 177(14): 4166-4170.

[10] Stieger M, Angehrn P, Wohlgensinger B, Gmünder H. GyrB mutations in Staphylococcus aureus strains resistant to cyclothialidine, coumermycin, and novobiocin . Antimicrob Agents Chemother 1996; 40(4): 1060-1062.

[11] Gu H, Carroll PA, Du J, Zhu J, Neto FC, Eisenman RN, et al. Quantitative method to investigate the balance between metabolism and proteome biomass: starting from glycine. Angew Chem Int Ed Engl. 2016; 55(50): 15646-15650.

[12] Sierra JM, Marco F, Ruiz J, Jiménez de Anta MT, Vila J. Correlation between the activity of different fluoroquinolones and the presence of mechanisms of quinolone resistance in epidemiologically related and unrelated strains of methicillin-susceptible and -resistant Staphylococcus aureus . Clin Microbiol Infect. 2002; 8(12): 781-790.

[13] Sulaiman JE, Lam H. Novel daptomycin tolerance and resistance mutations in methicillin-resistant Staphylococcus aureus from adaptive laboratory evolution . mSphere. 2021; 6(5): e00692-21.

[14] Gostev V, Kalinogorskaya O, Sopova J, Sulian O, Chulkova P, Velizhanina M, et al. Adaptive laboratory evolution of Staphylococcus aureus resistance to vancomycin and daptomycin: mutation patterns and cross-resistance . Antibiotics (Basel). 2023; 12(5): 928.

[15] Sweeney MT, Gunnett L, Kumar DM, Lunt BL, Moulin V, Barrett M, et al. Antimicrobial susceptibility of mastitis pathogens isolated from North American dairy cattle, 2011-2022. Vet Microbiol. 2024; 291: 110015.

[16] Huang X, Duan N, Xu H, Xie TN, Xue YR, Liu CH. CTAB-PEG DNA extraction from fungi with high contents of polysaccharides. Mol Biol. 2018; 52(4): 621-628.

[17] Wu N, Wu Y, Chu Y, Ren Z, Li H, Rong C, et al. The first rare case of Candida palmioleophila infection reported in China and its genomic evolution in a human host environment . Front Microbiol. 2023; 14: 1165721.

[18] Lei T, Mao Q, Chen C, Ji Y. Metabolomic profiling of Staphylococcus aureus . In Methicillin-Resistant Staphylococcus Aureus (MRSA) Protocols: Cutting-Edge Technologies and Advancements. Edited by Ji Y. New York, NY: Springer US; 2020: 177-186.

[19] Lobritz MA, Belenky P, Porter CB, Gutierrez A, Yang JH, Schwarz EG, et al. Antibiotic efficacy is linked to bacterial cellular respiration. Proc Natl Acad Sci U S A. 2015; 112(27): 8173-8180.

[20] Thai VC, Lim TK, Le KPU, Lin Q, Nguyen TTH. iTRAQ-based proteome analysis of fluoroquinolone-resistant Staphylococcus aureus . J Glob Antimicrob Resist. 2017; 8: 82-89.

[21] Kashiwagi K. Polyamine transport in Escherichia coli and eukaryotic cells . Yakugaku Zasshi. 1996; 116(3): 175-191.

[22] Mathieu M, Debousker G, Vincent S, Viviani F, Bamas-Jacques N, Mikol V . Escherichia coli FolC structure reveals an unexpected dihydrofolate binding site providing an attractive target for anti-microbial therapy . J Biol Chem. 2005; 280(19): 18916-18922.

[23] Fisher JF, Mobashery S. Constructing and deconstructing the bacterial cell wall. Protein Sci. 2020; 29(3): 629-646.

[24] Peacock SJ, Paterson GK. Mechanisms of methicillin resistance in Staphylococcus aureus . Annu Rev Biochem. 2015; 84: 577-601.

[25] McGuinness WA, Malachowa N, DeLeo FR. Vancomycin resistance in Staphylococcus aureus . Yale J Biol Med. 2017; 90(2): 269-281.

[26] Das B, Bhadra RK. (p)ppGpp metabolism and antimicrobial resistance in bacterial pathogens. Front Microbiol. 2020; 11: 563944.

[27] Abranches J, Martinez AR, Kajfasz JK, Chávez V, Garsin DA, Lemos JA. The molecular alarmone (p)ppGpp mediates stress responses, vancomycin tolerance, and virulence in Enterococcus faecalis. J Bacteriol. 2009; 191(7): 2248-2256.

[28] Corrigan RM, Bellows LE, Wood A, Gründling A. ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in gram-positive bacteria. Proc Natl Acad Sci U S A. 2016; 113(12): E1710-E1719.

[29] Frank MW, Whaley SG, Rock CO . Branched-chain amino acid metabolism controls membrane phospholipid structure in Staphylococcus aureus . J Biol Chem. 2021; 297(5): 101255.

[30] Zivkovic I, Gruic-Sovulj I. Exploring mechanisms of mupirocin resistance and hyper-resistance. Biochem Soc Trans. 2024; 52(3): 1109-1120.

[31] Moscoso M, García P, Cabral MP, Rumbo C, Bou G. A D-alanine auxotrophic live vaccine is effective against lethal infection caused by Staphylococcus aureus . Virulence. 2018; 9(1): 604-620.

[32] Zeng D, Debabov D, Hartsell TL, Cano RJ, Adams S, Schuyler JA, et al. Approved glycopeptide antibacterial drugs: mechanism of action and resistance. Cold Spring Harb Perspect Med. 2016; 6(12): a026989.

[33] Jeong B, Shah MA, Roh E, Kim K, Park I, Bae T. Staphylococcus aureus does not synthesize arginine from proline under physiological conditions . J Bacteriol. 2022; 204(6): e00018-22.

[34] Freiberg JA, Reyes Ruiz VM, Gimza BD, Murdoch CC, Green ER, Curry JM, et al. Restriction of arginine induces antibiotic tolerance in Staphylococcus aureus . Nat Commun. 2024; 15(1): 6734.

[35] Pozdeev G, Mogre A, Dorman CJ. Consequences of producing DNA gyrase from a synthetic gyrBA operon in Salmonella enterica serovar Typhimurium. Mol Microbiol. 2021; 115(6): 1410-1429.

[36] Gardete S, Tomasz A. Mechanisms of vancomycin resistance in Staphylococcus aureus . J Clin Invest. 2014; 124(7): 2836-2840.

[37] Peng B, Su YB, Li H, Han Y, Guo C, Tian YM, et al. Exogenous alanine and/or glucose plus kanamycin kills antibiotic-resistant bacteria. Cell Metab. 2015; 21(2): 249-262.

[38] Schelli K, Zhong F, Zhu J. Comparative metabolomics revealing Staphylococcus aureus metabolic response to different antibiotics . Microb Biotechnol. 2017; 10(6): 1764-1774.

[39] Heide L. New aminocoumarin antibiotics as gyrase inhibitors. Int J Med Microbiol. 2014; 304(1): 31-36.

[40] Dwyer DJ, Kohanski MA, Hayete B, Collins JJ. Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli . Mol Syst Biol. 2007; 3: 91.

[41] Luo Z, Chen M, Chen T, She P, Wu Y. Lactic acid produced by glycolysis contributed to Staphylococcus aureus aggregation induced by glucose . Curr Microbiol. 2019; 76(5): 607-612.

[42] Sevag MG, Green MN. The mechanism of resistance to sulfonamides: III. Pantothenic acid and tryptophane metabolism: the role of pantothenic acid in the synthesis of tryptophane by Staphylococcus aureus and the effect of vitamins on tryptophane in exercising antagonism to sulfonamides . J Bacteriol. 1944; 48(6): 631-638.

[43] Santhaseelan H, Dinakaran VT, Sakthivel B, Somasundaram M, Thanamegam K, Devendiran V, et al. Bioactive efficacy of novel carboxylic acid from halophilic Pseudomonas aeruginosa against methicillin-resistant Staphylococcus aureus . Metabolites. 2022; 12(11): 1094.

[44] Jensen PA, Zhu Z, van Opijnen T. Antibiotics disrupt coordination between transcriptional and phenotypic stress responses in pathogenic bacteria. Cell Rep. 2017; 20(7): 1705-1716.

[45] Bhargava P, Collins JJ. Boosting bacterial metabolism to combat antibiotic resistance. Cell Metab. 2015; 21(2): 154-155.

[46] Kohanski MA, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ. A common mechanism of cellular death induced by bactericidal antibiotics. Cell. 2007; 130(5): 797-810.

[47] Munita JM, Arias CA. Mechanisms of antibiotic resistance. Microbiol Spectr. 2016; 4(2): 1-24.

[48] Maisnier-Patin S, Berg OG, Liljas L, Andersson DI . Compensatory adaptation to the deleterious effect of antibiotic resistance in Salmonella typhimurium. Mol Microbiol. 2002; 46(2): 355-366.

[49] Maisnier-Patin S, Andersson DI. Adaptation to the deleterious effects of antimicrobial drug resistance mutations by compensatory evolution. Res Microbiol. 2004; 155(5): 360-369.

[50] Touati A, Bellil Z, Barache D, Mairi A. Fitness cost of antibiotic resistance in Staphylococcus aureus: a systematic review . Microb Drug Resist. 2021; 27(9): 1218-1231.

[51] Gupta SK, Pfeltz RF, Wilkinson BJ, Gustafson JE. Transcriptomic and metabolomic analysis of a fusidic acid-selected fusA mutant of Staphylococcus aureus . Antibiotics (Basel). 2022; 11(8): 1051.