{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,29]],"date-time":"2026-05-29T10:38:59Z","timestamp":1780051139639,"version":"3.53.1"},"reference-count":46,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2025,2,25]],"date-time":"2025-02-25T00:00:00Z","timestamp":1740441600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82272341"],"award-info":[{"award-number":["82272341"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Staphylococcus aureus is a vital pathogen causing clinical infections. Capsules are important virulence factors for S. aureus. This study investigates the regulatory mechanisms underlying capsule production in S. aureus. Bacterial strains XN108 and Newman were used, and combined approaches like RNA sequencing (RNA-seq), RT-qPCR, transmission electron microscopy (TEM), gene reporter, and electrophoretic mobility shift assay (EMSA) were performed to test the role and mechanism of WalK(S221P) mutation in S. aureus capsule production. RNA-seq showed an increased expression of cap genes in the WalK(S221P)-carried S. aureus XN108 relative to the mutation-cured XN108-R. TEM and capsular polysaccharide determination demonstrated that XN108 produced more capsules than XN108-R did. Similar results were presented in the WalK(S221P)-contained K-Newman versus the wild-type Newman. RT-qPCR screening showed an increasing expression of the mgrA gene in XN108 versus XN108-R. Gene reporter and EMSA analysis revealed that WalK(S221P) mutation promoted S. aureus capsule production through MgrA. Deletion of mgrA decreased the WalK(S221P)-mediated capsule yield. Moreover, WalK(S221P) mutation remarkably increased the tolerance of S. aureus to whole blood killing and microphage phagocytosis. Overall, these data provide mechanistic insights into the effect of WalK(S221P) on the capsule production of S. aureus, which may set down foundations for future S. aureus virulence investigations.<\/jats:p>","DOI":"10.3390\/microorganisms13030502","type":"journal-article","created":{"date-parts":[[2025,2,25]],"date-time":"2025-02-25T03:40:12Z","timestamp":1740454812000},"page":"502","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["WalK(S221P) Mutation Promotes the Production of Staphylococcus aureus Capsules Through an MgrA-Dependent Pathway"],"prefix":"10.3390","volume":"13","author":[{"given":"Zuwen","family":"Guo","sequence":"first","affiliation":[{"name":"Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing, Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-5828-8764","authenticated-orcid":false,"given":"Huagang","family":"Peng","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing, Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Weilong","family":"Shang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing, Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yi","family":"Yang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing, Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhen","family":"Hu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing, Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yifan","family":"Rao","sequence":"additional","affiliation":[{"name":"Department of Emergency Medicine, Xinqiao Hospital, Army Medical University (Third Military Medical University), Chongqing 400037, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaonan","family":"Huang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing, Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianxiong","family":"Dou","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing, Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zihui","family":"Xu","sequence":"additional","affiliation":[{"name":"Department of Integrative Medicine, Xinqiao Hospital, Army Medical University (Third Military Medical University), Chongqing 400037, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9905-760X","authenticated-orcid":false,"given":"Xiancai","family":"Rao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Microbial Engineering under the Educational Committee in Chongqing, Department of Microbiology, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing 400038, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,2,25]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2449085","DOI":"10.1080\/22221751.2024.2449085","article-title":"Long-term impact of molecular epidemiology shifts of methicillin-resistant Staphylococcus aureus on severity and mortality of bloodstream infection","volume":"14","author":"Kaku","year":"2025","journal-title":"Emerg. Microbes Infect."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"120","DOI":"10.1038\/s41564-021-01013-8","article-title":"Identification of structurally diverse menaquinone-binding antibiotics with in vivo activity against multidrug-resistant pathogens","volume":"7","author":"Li","year":"2022","journal-title":"Nat. Microbiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1080\/23744235.2021.1985732","article-title":"High rate of bacterial respiratory tract co-infections upon admission amongst moderate to severe COVID-19 patients","volume":"54","author":"Cohen","year":"2022","journal-title":"Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1199","DOI":"10.1016\/S0140-6736(24)01867-1","article-title":"Global burden of bacterial antimicrobial resistance 1990\u20132021: A systematic analysis with forecasts to 2050","volume":"404","author":"Naghavi","year":"2024","journal-title":"Lancet"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"106683","DOI":"10.1016\/j.micpath.2024.106683","article-title":"Protease SfpB plays an important role in cell membrane stability and immune system evasion in Streptococcus agalactiae","volume":"192","author":"Li","year":"2024","journal-title":"Microb Pathog."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e00062-20","DOI":"10.1128\/MMBR.00062-20","article-title":"Emerging roles of functional bacterial amyloids in gene regulation, toxicity, and immunomodulation","volume":"85","author":"Salinas","year":"2020","journal-title":"Microbiol. Mol. Biol. Rev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"380","DOI":"10.1038\/s41579-023-00852-y","article-title":"Staphylococcus aureus host interactions and adaptation","volume":"21","author":"Howden","year":"2023","journal-title":"Nat. Rev. Microbiol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Jiang, J., Cameron, D.R., Nethercott, C., Aires-de-Sousa, M., and Peleg, A.Y. (2023). Virulence attributes of successful methicillin-resistant Staphylococcus aureus lineages. Clin. Microbiol. Rev., 36.","DOI":"10.1128\/cmr.00148-22"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1253","DOI":"10.1093\/infdis\/jiae188","article-title":"The capsular polysaccharide obstructs wall teichoic acid functions in Staphylococcus aureus","volume":"230","author":"Lehmann","year":"2024","journal-title":"J. Infect. Dis."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"480","DOI":"10.4161\/hv.23223","article-title":"Capsular polysaccharides are an important immune evasion mechanism for Staphylococcus aureus","volume":"9","author":"Nanra","year":"2013","journal-title":"Hum. Vaccines Immunother."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"783","DOI":"10.1039\/C9OB02546D","article-title":"Staphylococcus aureus capsular polysaccharides: A structural and synthetic perspective","volume":"18","author":"Visansirikul","year":"2020","journal-title":"Org. Biomol. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1128\/CMR.17.1.218-234.2004","article-title":"Staphylococcus aureus capsular polysaccharides","volume":"17","author":"Lee","year":"2004","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"9365","DOI":"10.1073\/pnas.161175598","article-title":"Structural rationale for the modulation of abscess formation by Staphylococcus aureus capsular polysaccharides","volume":"98","author":"Tzianabos","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1404","DOI":"10.1038\/s41467-019-09356-x","article-title":"Coordination of capsule assembly and cell wall biosynthesis in Staphylococcus aureus","volume":"10","author":"Rausch","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Keinhorster, D., George, S.E., Weidenmaier, C., and Wolz, C. (2019). Function and regulation of Staphylococcus aureus wall teichoic acids and capsular polysaccharides. Int. J. Med. Microbiol., 309.","DOI":"10.1016\/j.ijmm.2019.151333"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1083","DOI":"10.1111\/mmi.14347","article-title":"Revisiting the regulation of the capsular polysaccharide biosynthesis gene cluster in Staphylococcus aureus","volume":"112","author":"Salzer","year":"2019","journal-title":"Mol. Microbiol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"3666","DOI":"10.1128\/JB.00640-15","article-title":"RbsR activates capsule but represses the rbsUDK operon in Staphylococcus aureus","volume":"197","author":"Lei","year":"2015","journal-title":"J. Bacteriol."},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Jutras, B.L., Chenail, A.M., Rowland, C.L., Carroll, D., Miller, M.C., Bykowski, T., and Stevenson, B. (2013). Eubacterial SpoVG homologs constitute a new family of site-specific DNA-binding proteins. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0066683"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"e00495-20","DOI":"10.1128\/JB.00495-20","article-title":"MgrA activates staphylococcal capsule via SigA-dependent promoter","volume":"203","author":"Lei","year":"2020","journal-title":"J. Bacteriol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"3123","DOI":"10.1099\/mic.0.29177-0","article-title":"The arl locus positively regulates Staphylococcus aureus type 5 capsule via an mgrA-dependent pathway","volume":"152","author":"Luong","year":"2006","journal-title":"Microbiology"},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Crosby, H.A., Schlievert, P.M., Merriman, J.A., King, J.M., Salgado-Pab\u00f3n, W., and Horswill, A.R. (2016). The Staphylococcus aureus global regulator MgrA modulates clumping and virulence by controlling surface protein expression. PLoS Pathog., 12.","DOI":"10.1371\/journal.ppat.1005604"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"e00294-18","DOI":"10.1128\/JB.00294-18","article-title":"MsaB and CodY interact to regulate Staphylococcus aureus capsule in a nutrient-dependent manner","volume":"200","author":"Batte","year":"2018","journal-title":"J. Bacteriol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1128\/JB.00987-10","article-title":"Staphylococcus aureus ClpC divergently regulates capsule via sae and codY in strain Newman but activates capsule via codY in strain UAMS-1 and in strain Newman with repaired saeS","volume":"193","author":"Luong","year":"2011","journal-title":"J. Bacteriol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1128\/IAI.70.2.444-450.2002","article-title":"Regulation of Staphylococcus aureus capsular polysaccharide expression by agr and sarA","volume":"70","author":"Luong","year":"2002","journal-title":"Infect. Immun."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"8257","DOI":"10.1128\/JB.00645-07","article-title":"New insights into the WalK\/WalR (YycG\/YycF) essential signal transduction pathway reveal a major role in controlling cell wall metabolism and biofilm formation in Staphylococcus aureus","volume":"189","author":"Dubrac","year":"2007","journal-title":"J. Bacteriol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1016\/j.jare.2021.11.015","article-title":"A vancomycin resistance-associated WalK(S221P) mutation attenuates the virulence of vancomycin-intermediate Staphylococcus aureus","volume":"40","author":"Rao","year":"2022","journal-title":"J. Adv. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"64","DOI":"10.1016\/j.diagmicrobio.2013.06.008","article-title":"First report of a sequence type 239 vancomycin-intermediate Staphylococcus aureus isolate in Mainland China","volume":"7","author":"Zhang","year":"2013","journal-title":"Diagn. Microbiol. Infect. Dis."},{"key":"ref_28","first-page":"1006","article-title":"WalK(S221P), a naturally occurring mutation, confers vancomycin resistance in VISA strain XN108","volume":"72","author":"Peng","year":"2017","journal-title":"J. Antimicrob. Chemother."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Peng, H., Rao, Y., Yuan, W., Zheng, Y., Shang, W., Hu, Z., Yang, Y., Tan, L., Xiong, K., and Li, S. (2018). Reconstruction of the vancomycin-susceptible Staphylococcus aureus phenotype from a vancomycin-intermediate S. aureus XN108. Front. Microbiol., 9.","DOI":"10.3389\/fmicb.2018.02955"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e70007","DOI":"10.1002\/mef2.70007","article-title":"Vancomycin-intermediate Staphylococcus aureus employs CcpA-GlmS metabolism regulatory cascade to resist vancomycin","volume":"3","author":"Peng","year":"2024","journal-title":"MedComm Future Med."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Zheng, M., Zhu, K., Peng, H., Shang, W., Zhao, Y., Lu, S., Rao, X., Li, M., Zhou, R., and Li, G. (2022). CcpA regulates Staphylococcus aureus biofilm formation through direct repression of staphylokinase expression. Antibiotics, 11.","DOI":"10.3390\/antibiotics11101426"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"867","DOI":"10.1111\/j.1462-5822.2004.00409.x","article-title":"Non-encapsulated strains reveal novel insights in invasion and survival of Streptococcus suis in epithelial cells","volume":"6","author":"Benga","year":"2004","journal-title":"Cell. Microbiol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1089\/mdr.2015.0226","article-title":"Comparative fitness and determinants for the characteristic drug resistance of ST239-MRSA-III-t030 and ST239-MRSA-III-t037 strains isolated in China","volume":"22","author":"Shang","year":"2016","journal-title":"Microb. Drug. Resist."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/S0167-7012(02)00151-3","article-title":"Quantitative determination of pneumococcal capsular polysaccharide serotype 14 using a modification of phenol-sulfuric acid method","volume":"52","author":"Cuesta","year":"2003","journal-title":"J. Microbiol. Methods."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/j.ab.2004.12.001","article-title":"Carbohydrate analysis by a phenol\u2013sulfuric acid method in microplate format","volume":"339","author":"Masuko","year":"2005","journal-title":"Anal. Biochem."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Echaniz-Aviles, G., Velazquez-Meza, M.E., Rodriguez-Arvizu, B., Carnalla-Barajas, M.N., and Nogueron, A.S. (2022). Detection of capsular genotypes of methicillin-resistant Staphylococcus aureus and clonal distribution of the cap5 and cap8 genes in clinical isolates. Arch Microbiol., 204.","DOI":"10.1007\/s00203-022-02793-1"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"2367","DOI":"10.1128\/iai.58.7.2367-2374.1990","article-title":"Synthesis and immunologic properties in mice of vaccines composed of Staphylococcus aureus type 5 and type 8 capsular polysaccharides conjugated to Pseudomonas aeruginosa exotoxin A","volume":"58","author":"Fattom","year":"1990","journal-title":"Infect. Immun."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"5183","DOI":"10.1128\/IAI.66.11.5183-5189.1998","article-title":"Staphylococcus aureus serotype 5 capsular polysaccharide is antiphagocytic and enhances bacterial virulence in a murine bacteremia model","volume":"11","author":"Thakker","year":"1998","journal-title":"Infect. Immun."},{"key":"ref_39","first-page":"917","article-title":"Staphylococcus aureus Cap5O has UDP-mannac dehydrogenase activity and is essential for capsule expression","volume":"2","author":"Kiser","year":"2001","journal-title":"Infect. Immun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1367","DOI":"10.1086\/606012","article-title":"Role of rsbU and staphyloxanthin in phagocytosis and intracellular growth of Staphylococcus aureus in human macrophages and endothelial cells","volume":"200","author":"Olivier","year":"2009","journal-title":"J. Infect. Dis."},{"key":"ref_41","doi-asserted-by":"crossref","unstructured":"Fortuna, A., Collalto, D., Schiaffi, V., Pastore, V., Visca, P., Ascenzioni, F., Rampioni, G., and Leoni, L. (2022). The Pseudomonas aeruginosa DksA1 protein is involved in H2O2 tolerance and within-macrophages survival and can be replaced by DksA2. Sci. Rep., 12.","DOI":"10.1038\/s41598-022-14635-7"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"e00203-18","DOI":"10.1128\/JB.00203-18","article-title":"Repression of capsule production by XdrA and CodY in Staphylococcus aureus","volume":"200","author":"Lei","year":"2018","journal-title":"J. Bacteriol."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Hu, Q., Peng, H., and Rao, X. (2016). Molecular events for promotion of vancomycin resistance in vancomycin intermediate Staphylococcus aureus. Front. Microbiol., 7.","DOI":"10.3389\/fmicb.2016.01601"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1007\/978-0-387-78885-2_15","article-title":"Tearing down the wall: Peptidoglycan metabolism and the WalK\/WalR (YycG\/YycF) essential two-component system","volume":"631","author":"Dubrac","year":"2008","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1111\/mmi.14404","article-title":"The Staphylococcus aureus ArlRS two-component system regulates virulence factor expression through MgrA","volume":"113","author":"Crosby","year":"2020","journal-title":"Mol. Microbiol."},{"key":"ref_46","doi-asserted-by":"crossref","unstructured":"Brissac, T., Mart\u00ednez, E., Kruckow, K.L., Riegler, A.N., Ganaie, F., Im, H., Bakshi, S., Arroyo-Diaz, N.M., Spencer, B.L., and Saad, J.S. (2021). Capsule promotes intracellular survival and vascular endothelial cell translocation during invasive pneumococcal disease. mBio, 12.","DOI":"10.1128\/mBio.02516-21"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/3\/502\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T16:41:48Z","timestamp":1760028108000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/13\/3\/502"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,2,25]]},"references-count":46,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2025,3]]}},"alternative-id":["microorganisms13030502"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms13030502","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,2,25]]}}}