Novel antibiotics against Staphylococcus aureus without detectable resistance by targeting proton motive force and FtsH

Abstract

The increased prevalence of methicillin-resistant Staphylococcus aureus (MRSA) and its biofilms poses a great threat to human health. Especially, S. aureus-related osteomyelitis was hardly cured even by conventional antibiotics combined with surgical treatment. The development of novel structural antibiotics is urgently needed. By high-throughput screening and rational design, we identified a small molecule C218-0546 and its optimized analog STK848198 with great antimicrobial potential against MRSA avoiding resistance occurrence. And significant synergistical antimicrobial effects were found between the molecules and conventional antibiotics. Mechanisms studies by transcriptomics, fluorescent probes, molecule dynamics, and plasma surface resonance indicated that the proton motive force as well as FtsH are the main potential targets of these molecules. The compounds exhibited excellent in vivo pharmacokinetics, toxicity profiles, and antimicrobial activities in the abscess model as well as the peritonitis-sepsis model. In addition, STK848198 was found to be effective against MRSA biofilms by interacting with the quorum sensing system. STK848198 also showed in vivo efficacy in the periprosthetic joint infection model. In all, our study identified a class of antimicrobials with novel scaffolds that could be potential alternatives for the treatment of MRSA and its biofilm-related infections.

Keywords

antibiotic development / methicillin-resistant Staphylococcus aureus / biofilm / proton motive force / FtsH / in vivo

References

[1] Lowy FD. Staphylococcus aureus infections. N Engl J Med. 1998; 339(8): 520-532.

[2] Kwiecinski JM, Horswill AR. Staphylococcus aureus bloodstream infections: pathogenesis and regulatory mechanisms. Curr Opin Microbiol. 2020; 53: 51-60.

[3] Lee AS, de Lencastre H, Garau J, et al. Methicillin-resistant Staphylococcus aureus. Nat Rev Dis Primers. 2018; 4: 18033.

[4] Cosgrove SE, Sakoulas G, Perencevich EN, Schwaber MJ, Karchmer AW, Carmeli Y. Comparison of mortality associated with methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clin Infect Dis. 2003; 36(1): 53-59.

[5] McCallum N, Berger-Bächi B, Senn MM. Regulation of antibiotic resistance in Staphylococcus aureus. Int J Med Microbiol. 2010; 300(2-3): 118-129.

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

[7] Bhattacharya M, Wozniak DJ, Stoodley P, Hall-Stoodley L. Prevention and treatment of Staphylococcus aureus biofilms. Expert Rev Anti Infect Ther. 2015; 13(12): 1499-1516.

[8] Ellington JK, Harris M, Webb L, et al. Intracellular Staphylococcus aureus. A mechanism for the indolence of osteomyelitis. J Bone Joint Surg Br. 2003; 85(6): 918-921.

[9] Calhoun JH, Manring MM, Shirtliff M. Osteomyelitis of the long bones. Semin Plast Surg. 2009; 23(2): 59-72.

[10] Funk SS, Copley LA. Acute hematogenous osteomyelitis in children: pathogenesis, diagnosis, and treatment. Orthop Clin North Am. 2017; 48(2): 199-208.

[11] Arciola CR. Why focus on implant infections?. Int J Artif Organs. 2005; 28(11): 1060-1061.

[12] Law MD Jr, Stein RE. Late infection in healed fractures after open reduction and internal fixation. Orthop Rev. 1993; 22(5): 545-552.

[13] Kavanagh N, Ryan EJ, Widaa A, et al. Staphylococcal osteomyelitis: disease progression, treatment challenges, and future directions. Clin Microbiol Rev. 2018; 31(2): e00084-17.

[14] van de Belt H, Neut D, Schenk W, van Horn JR, van der Mei HC, Busscher HJ. Infection of orthopedic implants and the use of antibiotic-loaded bone cements. A review. Acta Orthop Scand. 2001; 72(6): 557-571.

[15] Krauss JL, Roper PM, Ballard A, et al. Staphylococcus aureus infects osteoclasts and replicates intracellularly. mBio. 2019; 10(5): e02447-19.

[16] Tice AD, Hoaglund PA, Shoultz DA. Outcomes of osteomyelitis among patients treated with outpatient parenteral antimicrobial therapy. Am J Med. 2003; 114(9): 723-728.

[17] Noskin GA, Rubin RJ, Schentag JJ, et al. The burden of Staphylococcus aureus infections on hospitals in the United States: an analysis of the 2000 and 2001 Nationwide Inpatient Sample Database. Arch Intern Med. 2005; 165(15): 1756-1761.

[18] Wculek SK, Dunphy G, Heras-Murillo I, Mastrangelo A, Sancho D. Metabolism of tissue macrophages in homeostasis and pathology. Cell Mol Immunol. 2022; 19(3): 384-408.

[19] Jeannin P, Paolini L, Adam C, Delneste Y. The roles of CSFs on the functional polarization of tumor-associated macrophages. FEBS J. 2018; 285(4): 680-699.

[20] Schenker ML, Yannascoli S, Baldwin KD, Ahn J, Mehta S. Does timing to operative debridement affect infectious complications in open long-bone fractures? A systematic review. J Bone Joint Surg Am. 2012; 94(12): 1057-1064.

[21] Mitchell P. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature. 1961; 191: 144-148.

[22] Yang B, Tong Z, Shi J, Wang Z, Liu Y. Bacterial proton motive force as an unprecedented target to control antimicrobial resistance. Med Res Rev. 2023; 43(4): 1068-1090.

[23] Berry BJ, Trewin AJ, Amitrano AM, Kim M, Wojtovich AP. Use the protonmotive force: mitochondrial uncoupling and reactive oxygen species. J Mol Biol. 2018; 430(21): 3873-3891.

[24] Ma W, Zhang D, Li G, et al. Antibacterial mechanism of daptomycin antibiotic against Staphylococcus aureus based on a quantitative bacterial proteome analysis. J Proteomics. 2017; 150: 242-251.

[25] Hubbard AT, Barker R, Rehal R, Vandera KA, Harvey RD, Coates AR. Mechanism of action of a membrane-active quinoline-based antimicrobial on natural and model bacterial membranes. Biochemistry. 2017; 56(8): 1163-1174.

[26] Liu Y, Ding S, Dietrich R, Märtlbauer E, Zhu K. A biosurfactant-inspired heptapeptide with improved specificity to kill MRSA. Angew Chem Int Ed Engl. 2017; 56(6): 1486-1490.

[27] Makowski M, Felício MR, Fensterseifer ICM, Franco OL, Santos NC, Gonçalves S. EcDBS1R4, an antimicrobial peptide effective against Escherichia coli with in vitro fusogenic ability. Int J Mol Sci. 2020; 21(23): 9104.

[28] Sannasiddappa TH, Lund PA, Clarke SR. In vitro antibacterial activity of unconjugated and conjugated bile salts on Staphylococcus aureus. Front Microbiol. 2017; 8: 1581.

[29] Wu SC, Han F, Song MR, et al. Natural flavones from Morus alba against methicillin-resistant Staphylococcus aureus via targeting the proton motive force and membrane permeability. J Agric Food Chem. 2019; 67(36): 10222-10234.

[30] Ito K, Akiyama Y. Cellular functions, mechanism of action, and regulation of FtsH protease. Annu Rev Microbiol. 2005; 59: 211-231.

[31] Barkad MA, Bayraktar A, Doruk T, Tunca S. Effect of lon protease overexpression on endotoxin production and stress resistance in Bacillus thuringiensis. Curr Microbiol. 2021; 78(9): 3483-3493.

[32] Bittner LM, Arends J, Narberhaus F. Mini review: ATP-dependent proteases in bacteria. Biopolymers. 2016; 105(8): 505-517.

[33] Langklotz S, Baumann U, Narberhaus F. Structure and function of the bacterial AAA protease FtsH. Biochim Biophys Acta. 2012; 1823(1): 40-48.

[34] Dalbey RE, Wang P, van Dijl JM. Membrane proteases in the bacterial protein secretion and quality control pathway. Microbiol Mol Biol Rev. 2012; 76(2): 311-330.

[35] Yeo WS, Anokwute C, Marcadis P, et al. A membrane-bound transcription factor is proteolytically regulated by the AAA+ protease FtsH in Staphylococcus aureus. J Bacteriol. 2020; 202(9): e00019-20.

[36] Liu Q, Hu M, Yeo WS, et al. Rewiring of the FtsH regulatory network by a single nucleotide change in saeS of Staphylococcus aureus. Sci Rep. 2017; 7(1): 8456.

[37] Wang W, Jiang J, Chen H, Zhang Y, Liu Q. FtsH is required for protein secretion homeostasis and full bacterial virulence in Edwardsiella piscicida. Microb Pathog. 2021; 161(Pt A):105194.

[38] Lithgow JK, Ingham E, Foster SJ. Role of the hprT-ftsH locus in Staphylococcus aureus. Microbiology (Reading). 2004; 150(Pt 2): 373-381.

[39] Pengfei S, Yaqian L, Lanlan X, et al. L007-0069 kills Staphylococcus aureus in high resistant phenotypes. Cell Mol Life Sci. 2022; 79(11): 552.

[40] Xie Z, Ou Z, Zhang M, et al. Indole-3-acetic acid regulating the initial adhesion of microalgae in biofilm formation. Environ Res. 2024; 252(Pt 4):119093.

[41] Ohyama T, Mugikura S, Nishikawa M, Igarashi K, Kobayashi H. Osmotic adaptation of Escherichia coli with a negligible proton motive force in the presence of carbonyl cyanide m-chlorophenylhydrazone. J Bacteriol. 1992; 174(9): 2922-2928.

[42] Lapashina AS, Feniouk BA. ADP-inhibition of H+-F_OF₁-ATP synthase. Biochemistry (Mosc). 2018; 83(10): 1141-1160.

[43] Nakamura H, Hisano T, Rahman MM, Tosha T, Shirouzu M, Shiro Y. Structural basis for heme detoxification by an ATP-binding cassette-type efflux pump in gram-positive pathogenic bacteria. Proc Natl Acad Sci USA. 2022; 119(27): e2123385119.

[44] Kamble E, Pardesi K. Antibiotic tolerance in biofilm and stationary-phase planktonic cells of Staphylococcus aureus. Microb Drug Resist. 2021; 27(1): 3-12.

[45] Guo H, Tong Y, Cheng J, et al. Biofilm and small colony variants-an update on Staphylococcus aureus strategies toward drug resistance. Int J Mol Sci. 2022; 23(3): 1241.

[46] Mansson M, Nielsen A, Kjærulff L, et al. Inhibition of virulence gene expression in Staphylococcus aureus by novel depsipeptides from a marine photobacterium. Mar Drugs. 2011; 9(12): 2537-2552.

[47] Tan L, Li SR, Jiang B, Hu XM, Li S. Therapeutic targeting of the Staphylococcus aureus accessory gene regulator (agr) system. Front Microbiol. 2018; 9: 55.

[48] Sully EK, Malachowa N, Elmore BO, et al. Selective chemical inhibition of agr quorum sensing in Staphylococcus aureus promotes host defense with minimal impact on resistance. PLoS Pathog. 2014; 10(6): e1004174.

[49] Schindler BD, Kaatz GW. Multidrug efflux pumps of Gram-positive bacteria. Drug Resist Updat. 2016; 27: 1-13.

[50] Alav I, Sutton JM, Rahman KM. Role of bacterial efflux pumps in biofilm formation. J Antimicrob Chemother. 2018; 73(8): 2003-2020.

[51] AlMatar M, Albarri O, Makky EA, Köksal F. Efflux pump inhibitors: new updates. Pharmacol Rep. 2021; 73(1): 1-16.

[52] Stokes JM, Yang K, Swanson K, et al. A deep learning approach to antibiotic discovery. Cell. 2020; 180(4): 688-702.e13.

[53] Dombach JL, Quintana JLJ, Detweiler CS. Staphylococcal bacterial persister cells, biofilms, and intracellular infection are disrupted by JD1, a membrane-damaging small molecule. mBio. 2021; 12(5): e0180121.

[54] Mohiuddin SG, Ghosh S, Kavousi P, Orman MA. Proton motive force inhibitors are detrimental to methicillin-resistant Staphylococcus aureus strains. Microbiol Spectr. 2022; 10(4): e0202422.

[55] She P, Li S, Zhou L, et al. Repurposing Eltrombopag as an antimicrobial agent against methicillin-resistant Staphylococcus aureus. Front Microbiol. 2022; 12: 790686.

[56] Ernst R, Kueppers P, Stindt J, Kuchler K, Schmitt L. Multidrug efflux pumps: substrate selection in ATP-binding cassette multidrug efflux pumps–first come, first served?. FEBS J. 2010; 277(3): 540-549.

[57] Nelson N, Perzov N, Cohen A, Hagai K, Padler V, Nelson H. The cellular biology of proton-motive force generation by V-ATPases. J Exp Biol. 2000; 203(Pt 1): 89-95.

[58] Illigmann A, Thoma Y, Pan S, Reinhardt L, Brötz-Oesterhelt H. Contribution of the Clp protease to bacterial survival and mitochondrial homoeostasis. Microb Physiol. 2021; 31(3): 260-279.

[59] Wenk M, Ba Q, Erichsen V, et al. A universally conserved ATPase regulates the oxidative stress response in Escherichia coli. J Biol Chem. 2012; 287(52): 43585-43598.

[60] Jayasekera MM, Foltin SK, Olson ER, Holler TP. Escherichia coli requires the protease activity of FtsH for growth. Arch Biochem Biophys. 2000; 380(1): 103-107.

[61] Suno R, Niwa H, Tsuchiya D, Zhang X, Yoshida M, Morikawa K. Structure of the whole cytosolic region of ATP-dependent protease FtsH. Mol Cell. 2006; 22(5): 575-585.

[62] Chen Y, Guo D, Wang X, et al. Structural insights into the mechanotransducing mechanism of FtsEX in cell division. MedComm. 2024; 5(11): e688.

[63] Masters EA, Hao SP, Kenney HM, et al. Distinct vasculotropic versus osteotropic features of S. agalactiae versus S. aureus implant-associated bone infection in mice. J Orthop Res. 2021; 39(2): 389-401.

[64] Urish KL, Cassat JE. Staphylococcus aureus osteomyelitis: bone, bugs, and surgery. Infect Immun. 2020; 88(7): e00932-19.

[65] Nishitani K, Sutipornpalangkul W, de Mesy Bentley KL, et al. Quantifying the natural history of biofilm formation in vivo during the establishment of chronic implant-associated Staphylococcus aureus osteomyelitis in mice to identify critical pathogen and host factors. J Orthop Res. 2015; 33(9): 1311-1319.

RIGHTS & PERMISSIONS

2025 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.