[en] Microbial lipopeptides are synthesized by nonribosomal peptide synthetases and are composed of a hydrophobic fatty acid chain and a hydrophilic peptide moiety. These structurally diverse amphiphilic molecules can interact with biological membranes and possess various biological activities, including antiviral properties. This study aimed to evaluate the cytotoxicity and antiviral activity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of 15 diverse lipopeptides to understand their structure-activity relationships. Non-ionic lipopeptides were generally more cytotoxic than charged ones, with cationic lipopeptides being less cytotoxic than anionic and non-ionic variants. At 100 µg/mL, six lipopeptides reduced SARS-CoV-2 RNA to undetectable levels in infected Vero E6 cells, while six others achieved a 2.5- to 4.1-log reduction, and three had no significant effect. Surfactin, white line-inducing principle (WLIP), fengycin, and caspofungin emerged as the most promising anti-SARS-CoV-2 agents. Detailed analysis revealed that these four lipopeptides affected various stages of the viral life cycle involving the viral envelope. Surfactin and WLIP significantly reduced viral RNA levels in replication assays, comparable to neutralizing serum. Surfactin uniquely inhibited viral budding, while fengycin impacted viral binding after pre-infection treatment of the cells. Caspofungin demonstrated a lower antiviral effect compared to the others. Key structural traits of lipopeptides influencing their cytotoxic and antiviral activities were identified. Lipopeptides with a high number of amino acids, especially charged (preferentially anionic) amino acids, showed potent anti-SARS-CoV-2 activity. This research paves the way for designing new lipopeptides with low cytotoxicity and high antiviral efficacy, potentially leading to effective treatments.
IMPORTANCE: This study advances our understanding of how lipopeptides, which are molecules mostly produced by bacteria, with both fat and protein components, can be used to fight viruses like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). By analyzing 15 different lipopeptides, researchers identified key structural features that make some of these molecules particularly effective at reducing viral levels while being less harmful to cells. Specifically, lipopeptides with certain charged amino acids were found to have the strongest antiviral effects. This research lays the groundwork for developing new antiviral treatments that are both potent against viruses and safe for human cells, offering hope for better therapeutic options in the future.
Disciplines :
Biotechnology Veterinary medicine & animal health Biochemistry, biophysics & molecular biology
Author, co-author :
Hoste, Alexis ; Université de Liège - ULiège > Département GxABT > Microbial technologies
Smeralda, Willy ; Université de Liège - ULiège > Département GxABT > Microbial technologies
Cugnet, Aurélien ; Université de Liège - ULiège > Département GxABT > Microbial technologies
Brostaux, Yves ; Université de Liège - ULiège > TERRA Research Centre > Modélisation et développement
Deleu, Magali ; Université de Liège - ULiège > TERRA Research Centre > Chemistry for Sustainable Food and Environmental Systems (CSFES)
Garigliany, Mutien-Marie ; Université de Liège - ULiège > Département de morphologie et pathologie (DMP) > Pathologie générale et autopsies
Jacques, Philippe ; Université de Liège - ULiège > TERRA Research Centre
Language :
English
Title :
The structure of lipopeptides impacts their antiviral activity and mode of action against SARS-CoV-2 in vitro.
We thank Romain Thomas and Germain Bonhomme for technical support (University of Li\u00E8ge). This work was supported by the FNRS-funded SURFACOVID project.We thank Romain Thomas and Germain Bonhomme for technical support (University of Li\u00E8ge). This work was supported by the FNRS-funded SURFACOVID project. A.C.R.H. and A.C. performed the experiments. A.C.R.H. wrote the original draft of the manuscript. M.D., M.G., and P.J. conceived the original idea and supervised the project. W.S., M.D., Y.B., M.G., and P.J. substantially revised and edited the manuscript. All authors read and approved the final manuscript.
Drosten C, Günther S, Preiser W, van der Werf S, Brodt H-R, Becker S, Rabenau H, Panning M, Kolesnikova L, Fouchier RAM, et al. 2003. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348:1967–1976. https://doi.org/10.1056/NEJMoa030747
Zaki AM, van Boheemen S, Bestebroer TM, Osterhaus A, Fouchier RAM. 2012. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N Engl J Med 367:1814–1820. https://doi.org/10.1056/ NEJMoa1211721
Zhu N, Zhang D, Wang W, Li X, Yang B, Song J, Zhao X, Huang B, Shi W, Lu R, Niu P, Zhan F, Ma X, Wang D, Xu W, Wu G, Gao GF, Tan W, China Novel Coronavirus Investigating and Research Team. 2020. A novel coronavirus from patients with pneumonia in China, 2019. N Engl J Med 382:727–733. https://doi.org/10.1056/NEJMoa2001017
Polack FP, Thomas SJ, Kitchin N, Absalon J, Gurtman A, Lockhart S, Perez JL, Pérez Marc G, Moreira ED, Zerbini C, et al. 2020. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med 383:2603–2615. https://doi.org/10.1056/NEJMoa2034577
Voysey M, Clemens SAC, Madhi SA, Weckx LY, Folegatti PM, Aley PK, Angus B, Baillie VL, Barnabas SL, Bhorat QE, et al. 2021. Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK. Lancet 397:99–111. https://doi.org/10.1016/ S0140-6736(20)32661-1
Li G, Hilgenfeld R, Whitley R, De Clercq E. 2023. Therapeutic strategies for COVID-19: progress and lessons learned. Nat Rev Drug Discov 22:449–475. https://doi.org/10.1038/s41573-023-00672-y
Hoffmann M, Kleine-Weber H, Schroeder S, Krüger N, Herrler T, Erichsen S, Schiergens TS, Herrler G, Wu N-H, Nitsche A, Müller MA, Drosten C, Pöhlmann S. 2020. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181:271–280. https://doi.org/10.1016/j.cell.2020.02.052
Snijder EJ, Limpens R, de Wilde AH, de Jong AWM, Zevenhoven-Dobbe JC, Maier HJ, Faas F, Koster AJ, Bárcena M. 2020. A unifying structural and functional model of the coronavirus replication organelle: tracking down RNA synthesis. PLoS Biol 18:e3000715. https://doi.org/10.1371/journal.pbio.3000715
Cortese M, Lee J-Y, Cerikan B, Neufeldt CJ, Oorschot VMJ, Köhrer S, Hennies J, Schieber NL, Ronchi P, Mizzon G, et al. 2020. Integrative imaging reveals SARS-CoV-2-induced reshaping of subcellular morphologies. Cell Host Microbe 28:853–866. https://doi.org/10.1016/j.chom.2020.11.003
V’kovski P, Kratzel A, Steiner S, Stalder H, Thiel V. 2021. Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol 19:155–170. https://doi.org/10.1038/s41579-020-00468-6
Ghosh S, Dellibovi-Ragheb TA, Kerviel A, Pak E, Qiu Q, Fisher M, Takvorian PM, Bleck C, Hsu VW, Fehr AR, Perlman S, Achar SR, Straus MR, Whittaker GR, de Haan CAM, Kehrl J, Altan-Bonnet G, Altan-Bonnet N. 2020. βcoronaviruses use lysosomes for egress instead of the biosynthetic secretory pathway. Cell 183:1520–1535. https://doi.org/10.1016/j.cell.2020.10.039
Hammond J, Leister-Tebbe H, Gardner A, Abreu P, Bao W, Wisemandle W, Baniecki M, Hendrick VM, Damle B, Simón-Campos A, Pypstra R, Rusnak JM, EPIC-HR Investigators. 2022. Oral nirmatrelvir for high-risk, nonhospitalized adults with Covid-19. N Engl J Med 386:1397–1408. https://doi.org/10.1056/NEJMoa2118542
WHO Solidarity Trial Consortium. 2022. Remdesivir and three other drugs for hospitalised patients with COVID-19: final results of the WHO Solidarity randomised trial and updated meta-analyses. Lancet 399:1941–1953. https://doi.org/10.1016/S0140-6736(22)00519-0
Ader F, Bouscambert-Duchamp M, Hites M, PeifferSmadja N, Poissy J, Belhadi D, Diallo A, Lê M-P, Peytavin G, Staub T, Greil R, Guedj J, Paiva JA, Costagliola D, Yazdanpanah Y, Burdet C, Mentré F, DisCoVeRy Study Group. 2022. Remdesivir plus standard of care versus standard of care alone for the treatment of patients admitted to hospital with COVID-19 (DisCoVeRy): a phase 3, randomised, controlled, open-label trial. Lancet Infect Dis 22:209–221. https://doi.org/10.1016/S1473-3099(21)00485-0
Zhou S, Hill CS, Sarkar S, Tse LV, Woodburn BMD, Schinazi RF, Sheahan TP, Baric RS, Heise MT, Swanstrom R. 2021. β-d-N4-hydroxycytidine inhibits SARS-CoV-2 through lethal mutagenesis but is also mutagenic to mammalian cells. J Infect Dis 224:415–419. https://doi.org/10.1093/infdis/jiab247
Cox M, Peacock TP, Harvey WT, Hughes J, Wright DW, Willett BJ, Thomson E, Gupta RK, Peacock SJ, Robertson DL, Carabelli AM, COVID-19 Genomics UK (COG-UK) Consortium. 2023. SARS-CoV-2 variant evasion of monoclonal antibodies based on in vitro studies. Nat Rev Microbiol 21:112–124. https://doi.org/10.1038/s41579-022-00809-7
Hoste ACR, Görgen S, Jacques P. 2023. Increasing the natural biodiversity of microbial lipopeptides using a synthetic biology approach, p 203–247. In Soberon-Chavez G (ed), Biosurfactants: research and development foundations and frontiers in enzymology, 1st ed. Academic Press.
Wu X-C, Shen X-B, Ding R, Qian C-D, Fang H-H, Li O. 2010. Isolation and partial characterization of antibiotics produced by Paenibacillus elgii B69. FEMS Microbiol Lett 310:32–38. https://doi.org/10.1111/j.1574-6968.2010.02040.x
Ding R, Wu X-C, Qian C-D, Teng Y, Li O, Zhan Z-J, Zhao Y-H. 2011. Isolation and identification of lipopeptide antibiotics from Paenibacillus elgii B69 with inhibitory activity against methicillin-resistant Staphylococcus aureus. J Microbiol 49:942–949. https://doi.org/10.1007/s12275-011-1153-7
Kim J, Il Kim P, Bong KM, Il Kim J, Shin SY, Song J, Min HJ, Lee CW. 2018. Isolation and structural elucidation of pelgipeptin E, a novel pore-forming pelgipeptin analog from Paenibacillus elgii with low hemolytic activity. J Antibiot 71:1008–1017. https://doi.org/10.1038/s41429-018-0095-2
Biria D, Maghsoudi E, Roostaazad R, Dadafarin H, Sahebghadam Lotfi A, Amoozegar MA. 2010. Purification and characterization of a novel biosurfactant produced by Bacillus licheniformis MS3. World J Microbiol Biotechnol 26:871–878. https://doi.org/10.1007/s11274-009-0246-5
Matsuyama T, Murakami T, Fujita M, Fujita S, Yano I. 1986. Extracellular vesicle formation and biosurfactant production by Serratia marcescens. Microbiol (Reading) 132:865–875. https://doi.org/10.1099/00221287-132-4-865
Li H, Tanikawa T, Sato Y, Nakagawa Y, Matsuyama T. 2005. Serratia marcescens gene required for surfactant serrawettin W1 production encodes putative aminolipid synthetase belonging to nonribosomal peptide synthetase family. Microbiol Immunol 49:303–310. https://doi.org/10.1111/j.1348-0421.2005.tb03734.x
Ballio A, Barra D, Bossa F, Collina A, Grgurina I, Marino G, Moneti G, Paci M, Pucci P, Segre A. 1991. Syringopeptins, new phytotoxic lipodepsipeptides of Pseudomonas syringae pv. syringae. FEBS Lett 291:109–112. https://doi.org/10.1016/0014-5793(91)81115-o
Theatre A, Hoste ACR, Rigolet A, Benneceur I, Bechet M, Ongena M, Deleu M., Jacques P. 2021. Bacillus sp.: a remarkable source of bioactive lipopeptides, p 123–180. In Biosurfactants for the biobased economy advances in biochemical engineering/biotechnology
Kracht M, Rokos H, Ozel M, Kowall M, Pauli G, Vater J. 1999. Antiviral and hemolytic activities of surfactin isoforms and their methyl ester derivatives. J Antibiot (Tokyo) 52:613–619. https://doi.org/10.7164/antibiotics.52.613
Vollenbroich D, Ozel M, Vater J, Kamp RM, Pauli G. 1997. Mechanism of inactivation of enveloped viruses by the biosurfactant surfactin from Bacillus subtilis. Biologicals 25:289–297. https://doi.org/10.1006/biol.1997.0099
Huang X, Lu Z, Zhao H, Bie X, Lü F, Yang S. 2006. Antiviral activity of antimicrobial lipopeptide from Bacillus subtilis fmbj against pseudorabies virus, porcine parvovirus, Newcastle disease virus and infectious bursal disease virus in vitro. Int J Pept Res Ther 12:373–377. https://doi.org/10.1007/s10989-006-9041-4
Wang X, Hu W, Zhu L, Yang Q. 2017. Bacillus subtilis and surfactin inhibit the transmissible gastroenteritis virus from entering the intestinal epithelial cells. Biosci Rep 37:1–10. https://doi.org/10.1042/BSR20170082
Johnson BA, Hage A, Kalveram B, Mears M, Plante JA, Rodriguez SE, Ding Z, Luo X, Bente D, Bradrick SS, Freiberg AN, Popov V, Rajsbaum R, Rossi S, Russell WK, Menachery VD. 2019. Peptidoglycan-associated cyclic lipopeptide disrupts viral infectivity. J Virol 93:e01282-19. https://doi.org/10.1128/JVI.01282-19
Yuan L, Zhang S, Wang Y, Li Y, Wang X, Yang Q. 2018. Surfactin inhibits membrane fusion during invasion of epithelial cells by enveloped viruses. J Virol 92:1–19. https://doi.org/10.1128/JVI.00809-18
Yuan L, Zhang S, Peng J, Li Y, Yang Q. 2019. Synthetic surfactin analogues have improved anti-PEDV properties. PLoS One 14:e0215227. https://doi.org/10.1371/journal.pone.0215227
Baindara P, Chowdhury T, Roy D, Mandal M, Mandal SM. 2023. Surfactin-like lipopeptides from Bacillus clausii efficiently bind to spike glycoprotein of SARS-CoV-2. J Biomol Struct Dyn 41:14152–14163. https://doi.org/10.1080/07391102.2023.2196694
Xia B, Luo M, Pang L, Liu X, Yi Y. 2021. Lipopeptides against COVID-19 RNA-dependent RNA polymerase using molecular docking. Biomed J 44:S15–S24. https://doi.org/10.1016/j.bj.2021.11.010
Vergoten G, Bailly C. 2021. In silico analysis of echinocandins binding to the main proteases of coronaviruses PEDV (3CLpro) and SARS-CoV-2 (Mpro). In Silico Pharmacol 9:1–10. https://doi.org/10.1007/s40203-021-00101-1
Crovella S, de Freitas LC, Zupin L, Fontana F, Ruscio M, Pena EPN, Pinheiro IO, Calsa Junior T. 2022. Surfactin bacterial antiviral lipopeptide blocks in vitro replication of SARS-CoV-2. Appl Microbiol 2:680–687. https://doi.org/10.3390/applmicrobiol2030052
Nakajima S, Ohashi H, Akazawa D, Torii S, Suzuki R, Fukuhara T, Watashi K. 2023. Antiviral activity of micafungin and its derivatives against SARS-CoV-2 RNA replication. Viruses 15:1–8. https://doi.org/10.3390/ v15020452
Shekunov EV, Zlodeeva PD, Efimova SS, Muryleva AA, Zarubaev VV, Slita AV, Ostroumova OS. 2023. Cyclic lipopeptides as membrane fusion inhibitors against SARS-CoV-2: new tricks for old dogs. Antiviral Res 212:105575. https://doi.org/10.1016/j.antiviral.2023.105575
Devos T, Van Thillo Q, Compernolle V, Najdovski T, Romano M, Dauby N, Jadot L, Leys M, Maillart E, Loof S, et al. 2022. Early high antibody titre convalescent plasma for hospitalised COVID-19 patients: DAWn-plasma. Eur Respir J 59:2101724. https://doi.org/10.1183/13993003.01724-2021
Touré Y, Ongena M, Jacques P, Guiro A, Thonart P. 2004. Role of lipopeptides produced by Bacillus subtilis GA1 in the reduction of grey mould disease caused by Botrytis cinerea on apple. J Appl Microbiol 96:1151–1160. https://doi.org/10.1111/j.1365-2672.2004.02252.x
Debois D, Ongena M, Cawoy H, De Pauw E. 2013. MALDI-FTICR MS imaging as a powerful tool to identify Paenibacillus antibiotics involved in the inhibition of plant pathogens. J Am Soc Mass Spectrom 24:1202–1213. https://doi.org/10.1007/s13361-013-0620-2
Landy M, Warren GH, RosenmanM SB, Colio LG. 1948. Bacillomycin: an antibiotic from Bacillus subtilis active against pathogenic fungi. Exp Biol Med (Maywood) 67:539–541. https://doi.org/10.3181/00379727-67-16367
Paulus H, Gray E. 1964. The biosynthesis of polymyxin B by growing cultures of Bacillus polymyxa. J Biol Chem 239:865–871. https://doi.org/10.1016/S0021-9258(18)51670-7
Lu X, Wang L, Sakthivel SK, Whitaker B, Murray J, Kamili S, Lynch B, Malapati L, Burke SA, Harcourt J, Tamin A, Thornburg NJ, Villanueva JM, Lindstrom S. 2020. US CDC real-time reverse transcription PCR panel for detection of severe acute respiratory syndrome coronavirus 2. Emerg Infect Dis 26:1654–1665. https://doi.org/10.3201/eid2608.201246
Piorkowski G, Baronti C, de Lamballerie X, de Fabritus L, Bichaud L, Pastorino BA, Bessaud M. 2014. Development of generic Taqman PCR and RT-PCR assays for the detection of DNA and mRNA of β-actinencoding sequences in a wide range of animal species. J Virol Methods 202:101–105. https://doi.org/10.1016/j.jviromet.2014.02.026
Reed LJ, Muench H. 1938. A simple method of estimating fifty per cent endpoints. Am J Epidemiol 27:493–497. https://doi.org/10.1093/oxfordjournals.aje.a118408
Jangra M, Randhawa HK, Kaur M, Srivastava A, Maurya N, Patil PP, Jaswal P, Arora A, Patil PB, Raje M, Nandanwar H. 2018. Purification, characterization and in vitro evaluation of polymyxin A from Paenibacillus dendritiformis: an underexplored member of the polymyxin family. Front Microbiol 9:2864. https://doi.org/10.3389/fmicb.2018.02864
Azad MAK, Finnin BA, Poudyal A, Davis K, Li J, Hill PA, Nation RL, Velkov T, Li J. 2013. Polymyxin B induces apoptosis in kidney proximal tubular cells. Antimicrob Agents Chemother 57:4329–4335. https://doi.org/10.1128/AAC.02587-12
Naghmouchi K, Baah J, Hober D, Jouy E, Rubrecht C, Sané F, Drider D. 2013. Synergistic effect between colistin and bacteriocins in controlling gram-negative pathogens and their potential to reduce antibiotic toxicity in mammalian epithelial cells. Antimicrob Agents Chemother 57:2719–2725. https://doi.org/10.1128/AAC.02328-12
Mishra NM, Stolarzewicz I, Cannaerts D, Schuermans J, Lavigne R, Looz Y, Landuyt B, Schoofs L, Schols D, Paeshuyse J, Hickenbotham P, Clokie M, Luyten W, Van der Eycken EV, Briers Y. 2018. Iterative chemical engineering of vancomycin leads to novel vancomycin analogs with a high in vitro therapeutic index. Front Microbiol 9:1175. https://doi.org/10.3389/fmicb.2018.01175
Wegner B, Baer P, Gauer S, Oremek G, Hauser IA, Geiger H. 2005. Caspofungin is less nephrotoxic than amphotericin B in vitro and predominantly damages distal renal tubular cells. Nephrol Dial Transplant 20:2071–2079. https://doi.org/10.1093/ndt/gfh948
Gales AC, Jones RN, Sader HS. 2011. Contemporary activity of colistin and polymyxin B against a worldwide collection of Gram-negative pathogens: results from the SENTRY antimicrobial surveillance program (2006-09). J Antimicrob Chemother 66:2070–2074. https://doi.org/10. 1093/jac/dkr239
Roberts KD, Azad MAK, Wang J, Horne AS, Thompson PE, Nation RL, Velkov T, Li J. 2015. Antimicrobial activity and toxicity of the major lipopeptide components of polymyxin B and colistin: last-line antibiotics against multidrug-resistant Gram-negative bacteria. ACS Infect Dis 1:568–575. https://doi.org/10.1021/acsinfecdis.5b00085
Kourmentza K, Gromada X, Michael N, Degraeve C, Vanier G, Ravallec R, Coutte F, Karatzas KA, Jauregi P. 2020. Antimicrobial activity of lipopeptide biosurfactants against foodborne pathogen and food spoilage microorganisms and their cytotoxicity. Front Microbiol 11:561060. https://doi.org/10.3389/fmicb.2020.561060
Khodavirdipour A, Chamanrokh P, Alikhani MY, Alikhani MS. 2022. Potential of Bacillus subtilis against SARS-CoV-2 – a sustainable drug development perspective. Front Microbiol 13:718786. https://doi.org/10.3389/fmicb.2022.718786
Hartenian E, Nandakumar D, Lari A, Ly M, Tucker JM, Glaunsinger BA. 2020. The molecular virology of coronaviruses. J Biol Chem 295:12910–12934. https://doi.org/10.1074/jbc.REV120.013930
Groupe V, PughLH, WeissD, KochiM. 1951. Observations on antiviral activity of viscosin. Proc Soc Exp Biol Med 78:354–358. https://doi.org/10.3181/00379727-78-19071
Silva DS e, Castro CC de, Silva F da S e, Sant’anna V, Vargas GD, Lima M de, Fischer G, Brandelli A, Motta A de S da, Hübner S de O. 2014. Antiviral activity of a Bacillus sp. P34 peptide against pathogenic viruses of domestic animals. Braz J Microbiol 45:1089–1094. https://doi.org/10.1590/S1517-83822014000300043
Maget-Dana R, Thimon L, Peypoux F, Ptak M. 1992. Surfactin/iturin A interactions may explain the synergistic effect of surfactin on the biological properties of iturin A. Biochimie 74:1047–1051. https://doi.org/10.1016/0300-9084(92)90002-v
Desmyttere H, Deweer C, Muchembled J, Sahmer K, Jacquin J, Coutte F, Jacques P. 2019. Antifungal activities of Bacillus subtilis lipopeptides to two Venturia inaequalis strains possessing different tebuconazole sensitivity. Front Microbiol 10:2327. https://doi.org/10.3389/fmicb.2019. 02327
Leconte A, Tournant L, Muchembled J, Paucellier J, Héquet A, Deracinois B, Deweer C, Krier F, Deleu M, Oste S, Jacques P, Coutte F. 2022. Assessment of lipopeptide mixtures produced by Bacillus subtilis as biocontrol products against apple scab (Venturia inaequalis). Microorganisms 10:1810. https://doi.org/10.3390/microorganisms10091810
