{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T03:53:41Z","timestamp":1775879621323,"version":"3.50.1"},"posted":{"date-parts":[[2025,1,6]]},"group-title":"Microbiology","reference-count":50,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,1,6]],"date-time":"2025-01-06T00:00:00Z","timestamp":1736121600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,1,6]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Bacterial genomic mutations in\n                  <jats:italic>Staphylococcus aureus (S. aureus)<\/jats:italic>\n                  have been detected in isolated resistant clinical strains, yet their mechanistic effect on the development of antimicrobial resistance remains unclear. The resistance-associated regulatory systems acquire adaptive mutations under stress conditions that may lead to a gain of function effect and contribute to the resistance phenotype. Here, we investigate the effect of a single-point mutation (T331I) in VraS histidine kinase, part of the VraSR two-component system in\n                  <jats:italic>S. aureus.<\/jats:italic>\n                  VraSR senses and responds to environmental stress signals by upregulating gene expression for cell wall synthesis. A combination of enzyme kinetics, microbiological, and transcriptomic analysis revealed the mechanistic effect of the mutation on VraS and\n                  <jats:italic>S. aureus<\/jats:italic>\n                  . Michaelis Menten\u2019s kinetics show that the VraS mutation caused an increase in the autophosphorylation rate of VraS and enhanced its catalytic efficiency. The introduction of the mutation through recombineering coupled with CRISPR-Cas9 counterselection to the Newman strain wild-type (WT) genome doubled the minimum inhibitory concentration of three cell wall-targeting antibiotics. The mutation caused an enhanced\n                  <jats:italic>S. aureus<\/jats:italic>\n                  growth rate at sub-lethal doses of the antibiotics, confirming the causative effect of mutation on bacterial persistence. Transcriptomic analysis showed a genome-wide alteration in gene expression levels and protein-protein interaction network of the mutant compared to the WT strain after exposure to vancomycin. The results suggest that\n                  <jats:italic>vraS<\/jats:italic>\n                  mutation causes several mechanistic changes at the protein and cellular levels that favor bacterial survival under antibiotic stress and cause the mutation-harboring strains to become the dominant population during infection.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>Importance<\/jats:title>\n                  <jats:p>\n                    Rising antimicrobial resistance (AMR) is a global health problem. Mutations in the two- component system have been linked to drug- resistance in\n                    <jats:italic>Staphylococcus aureus<\/jats:italic>\n                    , yet the exact mechanism through which these mutations work is understudied. We investigated the T331I mutation in the\n                    <jats:italic>vraS<\/jats:italic>\n                    gene linked to sensing and responding to cell wall stress. The mutation caused changes at the protein level by increasing the catalytic efficiency of VraS kinase activity. Introducing the mutation to the genome of an\n                    <jats:italic>S. aureus<\/jats:italic>\n                    strain resulted in changes in the phenotypic antibiotic susceptibility, growth kinetics, and genome-wide transcriptomic alterations. By a combination of enzyme kinetics, microbiological, and transcriptomic approaches, we highlight how small genetic changes can significantly impact bacterial physiology and survival under antibiotic stress. Understanding the mechanistic basis of antibiotic resistance is crucial to guide the development of novel therapeutic agents to combat AMR.\n                  <\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.01.06.631495","type":"posted-content","created":{"date-parts":[[2025,1,7]],"date-time":"2025-01-07T02:45:33Z","timestamp":1736217933000},"source":"Crossref","is-referenced-by-count":1,"title":["A mechanistic understanding of the effect of\n                  <i>Staphylococcus aureus<\/i>\n                  VraS histidine kinase single point mutation on antibiotic resistance"],"prefix":"10.64898","author":[{"given":"Liaqat","family":"Ali","sequence":"first","affiliation":[{"name":"Fisch College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Salima","family":"Karki","sequence":"additional","affiliation":[{"name":"Fisch College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gunavanthi D","family":"Boorgula","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, School of Medicine, University of Texas at Tyler, Tyler, Texas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amir","family":"Mekakda","sequence":"additional","affiliation":[{"name":"Fisch College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Brittnee","family":"Cagle-White","sequence":"additional","affiliation":[{"name":"Fisch College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shrijan","family":"Bhattarai","sequence":"additional","affiliation":[{"name":"Fisch College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Robert","family":"Beaudoin","sequence":"additional","affiliation":[{"name":"Fisch College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aryanna","family":"Blakeney","sequence":"additional","affiliation":[{"name":"Fisch College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sanjay","family":"Singh","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, School of Medicine, University of Texas at Tyler, Tyler, Texas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Shashikant","family":"Srivastava","sequence":"additional","affiliation":[{"name":"Division of Infectious Diseases, Department of Medicine, School of Medicine, University of Texas at Tyler, Tyler, Texas, USA"},{"name":"Department of Cellular and Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4852-9806","authenticated-orcid":false,"given":"May H.","family":"Abdelaziz","sequence":"additional","affiliation":[{"name":"Fisch College of Pharmacy, University of Texas at Tyler, Tyler, TX, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026041020384504000_2025.01.06.631495v1.1","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-018-0147-4"},{"key":"2026041020384504000_2025.01.06.631495v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-021-99269-x"},{"key":"2026041020384504000_2025.01.06.631495v1.3","unstructured":"2022 SPECIAL REPORT: COVID-19 U.S. Impact on Antimicrobial Resistance | Enhanced Reader. Retrieved 28 October 2024."},{"key":"2026041020384504000_2025.01.06.631495v1.4","doi-asserted-by":"publisher","DOI":"10.1093\/cid\/ciq146"},{"key":"2026041020384504000_2025.01.06.631495v1.5","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.40.11.3970-3975.2002"},{"key":"2026041020384504000_2025.01.06.631495v1.6","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0609839104"},{"key":"2026041020384504000_2025.01.06.631495v1.7","doi-asserted-by":"publisher","DOI":"10.1128\/JB.01410-13"},{"key":"2026041020384504000_2025.01.06.631495v1.8","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01173-08"},{"key":"2026041020384504000_2025.01.06.631495v1.9","doi-asserted-by":"crossref","unstructured":"Sharkey LKR , Guerillot R , Walsh CJ , Turner AM , Lee JYH , Neville SL , Klatt S , Baines SL , Pidot SJ , Rossello FJ , Seemann T , McWilliam HEG , Cho E , Carter GP , Howden BP , McDevitt CA , Hachani A , Stinear TP , Monk IR . 2023. The two-component system WalKR provides an essential link between cell wall homeostasis and DNA replication in Staphylococcus aureus. mBio 14.","DOI":"10.1128\/mbio.02262-23"},{"key":"2026041020384504000_2025.01.06.631495v1.10","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01139-12"},{"key":"2026041020384504000_2025.01.06.631495v1.11","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00042-09"},{"key":"2026041020384504000_2025.01.06.631495v1.12","doi-asserted-by":"publisher","DOI":"10.1186\/s12866-021-02146-w"},{"key":"2026041020384504000_2025.01.06.631495v1.13","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dks171"},{"key":"2026041020384504000_2025.01.06.631495v1.14","first-page":"e00370","article-title":"Staphylococcus aureus susceptibility to complestatin and corbomycin depends on the VraSR two-component system","volume":"11","year":"2023","journal-title":"Microbiol Spectr"},{"key":"2026041020384504000_2025.01.06.631495v1.15","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2958.2003.03599.x"},{"key":"2026041020384504000_2025.01.06.631495v1.16","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00356-06"},{"key":"2026041020384504000_2025.01.06.631495v1.17","first-page":"452547","article-title":"Heterogeneous vancomycin- intermediate staphylococcus aureus uses the vrasr regulatory system to modulate autophagy for increased intracellular survival in macrophage-like cell line RAW264.7","volume":"10","year":"2019","journal-title":"Front Microbiol"},{"key":"2026041020384504000_2025.01.06.631495v1.18","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijantimicag.2016.02.003"},{"key":"2026041020384504000_2025.01.06.631495v1.19","doi-asserted-by":"publisher","DOI":"10.1038\/ja.2015.42"},{"key":"2026041020384504000_2025.01.06.631495v1.20","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01173-08"},{"key":"2026041020384504000_2025.01.06.631495v1.21","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00834-08"},{"key":"2026041020384504000_2025.01.06.631495v1.22","first-page":"411098","article-title":"Daptomycin resistant staphylococcus aureus clinical strain with novel non-synonymous mutations in the mprF and vraS genes: A new insight into daptomycin resistance","volume":"9","year":"2018","journal-title":"Front Microbiol"},{"key":"2026041020384504000_2025.01.06.631495v1.23","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M710010200"},{"key":"2026041020384504000_2025.01.06.631495v1.24","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.00720-10"},{"key":"2026041020384504000_2025.01.06.631495v1.25","doi-asserted-by":"crossref","unstructured":"Bhattarai S , Marsh L , Knight K , Ali L , Gomez A , Sunderhaus A , Abdel Aziz MH . 2023. NH125 Sensitizes Staphylococcus aureus to Cell Wall-Targeting Antibiotics through the Inhibition of the VraS Sensor Histidine Kinase. Microbiol Spectr 11.","DOI":"10.1128\/spectrum.04861-22"},{"key":"2026041020384504000_2025.01.06.631495v1.26","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0267226"},{"key":"2026041020384504000_2025.01.06.631495v1.27","doi-asserted-by":"crossref","unstructured":"Penewit K , Holmes EA , McLean K , Ren M , Waalkes A , Salipante SJ . 2018. Efficient and scalable precision genome editing in Staphylococcus aureus through conditional recombineering and CRISPR\/Cas9-mediated counterselection. mBio 9.","DOI":"10.1128\/mBio.01839-18"},{"key":"2026041020384504000_2025.01.06.631495v1.28","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkg269"},{"key":"2026041020384504000_2025.01.06.631495v1.29","doi-asserted-by":"publisher","DOI":"10.1021\/acsinfecdis.9b00096"},{"key":"2026041020384504000_2025.01.06.631495v1.30","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/28.1.27"},{"key":"2026041020384504000_2025.01.06.631495v1.31","doi-asserted-by":"crossref","first-page":"1774","DOI":"10.12688\/f1000research.20887.2","article-title":"RCy3: Network biology using Cytoscape from within R","volume":"8","year":"2019","journal-title":"F1000Res"},{"key":"2026041020384504000_2025.01.06.631495v1.32","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2018.01086"},{"key":"2026041020384504000_2025.01.06.631495v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/S0140-6736(97)07324-8"},{"key":"2026041020384504000_2025.01.06.631495v1.34","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dkp394"},{"key":"2026041020384504000_2025.01.06.631495v1.35","doi-asserted-by":"publisher","DOI":"10.1016\/j.ijantimicag.2023.106971"},{"key":"2026041020384504000_2025.01.06.631495v1.36","doi-asserted-by":"crossref","unstructured":"Ali L , Aziz MHA . 2024. Crosstalk involving two-component systems in Staphylococcus aureus signaling networks. J Bacteriol 206.","DOI":"10.1128\/jb.00418-23"},{"key":"2026041020384504000_2025.01.06.631495v1.37","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M110.147843"},{"key":"2026041020384504000_2025.01.06.631495v1.38","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-017-02088-w"},{"key":"2026041020384504000_2025.01.06.631495v1.39","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0236198"},{"key":"2026041020384504000_2025.01.06.631495v1.40","first-page":"1362","article-title":"Molecular Determinants of \u03b2-Lactam Resistance in Methicillin- Resistant Staphylococcus aureus (MRSA): An Updated Review","volume":"2023","year":"2023","journal-title":"Antibiotics"},{"key":"2026041020384504000_2025.01.06.631495v1.41","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01121-07"},{"key":"2026041020384504000_2025.01.06.631495v1.42","doi-asserted-by":"crossref","unstructured":"Hort M , Bertsche U , Nozinovic S , Dietrich A , Schr\u00f6tter AS , Mildenberger L , Axtmann K , Berscheid A , Bierbaum G . 2021. The Role of \u03b2-Glycosylated Wall Teichoic Acids in the Reduction of Vancomycin Susceptibility in Vancomycin-Intermediate Staphylococcus aureus. Microbiol Spectr 9.","DOI":"10.1128\/Spectrum.00528-21"},{"key":"2026041020384504000_2025.01.06.631495v1.43","doi-asserted-by":"publisher","DOI":"10.1093\/jac\/dku297"},{"key":"2026041020384504000_2025.01.06.631495v1.44","doi-asserted-by":"crossref","unstructured":"Lee JJ , Lee SK , Song N , Nathan TO , Swarts BM , Eum SY , Ehrt S , Cho SN , Eoh H . 2019. Transient drug-tolerance and permanent drug-resistance rely on the trehalose-catalytic shift in Mycobacterium tuberculosis. Nature Communications 2019 10:1 10:1\u201312.","DOI":"10.1038\/s41467-019-10975-7"},{"key":"2026041020384504000_2025.01.06.631495v1.45","doi-asserted-by":"publisher","DOI":"10.1128\/JB.00645-07"},{"key":"2026041020384504000_2025.01.06.631495v1.46","doi-asserted-by":"crossref","unstructured":"Lopes AA , Yoshii Y , Yamada S , Nagakura M , Kinjo Y , Mizunoe Y , Okuda Kichi . 2019. Roles of Lytic Transglycosylases in Biofilm Formation and \u03b2-Lactam Resistance in Methicillin- Resistant Staphylococcus aureus. Antimicrob Agents Chemother 63.","DOI":"10.1128\/AAC.01277-19"},{"key":"2026041020384504000_2025.01.06.631495v1.47","doi-asserted-by":"publisher","DOI":"10.1038\/S41598-021-00490-5"},{"key":"2026041020384504000_2025.01.06.631495v1.48","doi-asserted-by":"publisher","DOI":"10.1128\/AAC.01651-12"},{"key":"2026041020384504000_2025.01.06.631495v1.49","doi-asserted-by":"publisher","DOI":"10.1128\/JB.187.5.1815-1824.2005"},{"key":"2026041020384504000_2025.01.06.631495v1.50","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-4939-9740-4_14"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.01.06.631495","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,11]],"date-time":"2026-04-11T03:38:57Z","timestamp":1775878737000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.01.06.631495"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,1,6]]},"references-count":50,"URL":"https:\/\/doi.org\/10.1101\/2025.01.06.631495","relation":{},"subject":[],"published":{"date-parts":[[2025,1,6]]},"subtype":"preprint"}}