{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,13]],"date-time":"2026-02-13T15:20:42Z","timestamp":1770996042905,"version":"3.50.1"},"reference-count":51,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2021,3,15]],"date-time":"2021-03-15T00:00:00Z","timestamp":1615766400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["R\/N02687X\/1"],"award-info":[{"award-number":["R\/N02687X\/1"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100000265","name":"Medical Research Council","doi-asserted-by":"publisher","award":["MR\/R001871\/1"],"award-info":[{"award-number":["MR\/R001871\/1"]}],"id":[{"id":"10.13039\/501100000265","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Vaccines"],"abstract":"<jats:p>Rationale: Nasopharyngeal administration of live virulence-attenuated Streptococcus pneumoniae strains is a potential novel preventative strategy. One target for creating reduced virulence S. pneumoniae strains is the capsule, but loss of the capsule reduces the duration of S. pneumoniae colonisation in mice which could impair protective efficacy against subsequent infection. Objectives: To assess protective efficacy of nasopharyngeal administration of unencapsulated S. pneumoniae strains in murine infection models. Methods: Strains containing cps locus deletions combined with the S. pneumoniae virulence factors psaA (reduces colonisation) or proABC (no effect on colonisation) were constructed and their virulence phenotypes and ability to prevent recolonisation or invasive infection assessed using mouse infection models. Serological responses to colonisation were compared between strains using ELISAs, immunoblots and 254 S. pneumoniae protein antigen array. Measurements and Main Results: The \u2206cps\/piaA and \u2206cps\/proABC strains were strongly attenuated in virulence in both invasive infection models and had a reduced ability to colonise the nasopharynx. ELISAs, immunoblots and protein arrays showed colonisation with either strain stimulated weaker serological responses than the wild type strain. Mice previously colonised with these strains were protected against septicaemic pneumonia but, unlike mice colonised with the wild type strain, not against S. pneumoniae recolonisation. Conclusions: Colonisation with the \u2206cps\/piaA and \u2206cps\/proABC strains prevented subsequent septicaemia, but in contrast, to published data for encapsulated double mutant strains they did not prevent recolonisation with S. pneumoniae. These data suggest targeting the cps locus is a less effective option for creating live attenuated strains that prevent S. pneumoniae infections.<\/jats:p>","DOI":"10.3390\/vaccines9030261","type":"journal-article","created":{"date-parts":[[2021,3,15]],"date-time":"2021-03-15T22:16:54Z","timestamp":1615846614000},"page":"261","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Protective Effect of Nasal Colonisation with \u2206cps\/piaA and \u2206cps\/proABCStreptococcus pneumoniae Strains against Recolonisation and Invasive Infection"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0803-2755","authenticated-orcid":false,"given":"Elisa","family":"Ramos-Sevillano","sequence":"first","affiliation":[{"name":"Centre for Inflammation and Tissue Repair, UCL Respiratory, Division of Medicine, University College London, Rayne Institute, London WC1E 6JF, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7326-1680","authenticated-orcid":false,"given":"Giuseppe","family":"Ercoli","sequence":"additional","affiliation":[{"name":"Centre for Inflammation and Tissue Repair, UCL Respiratory, Division of Medicine, University College London, Rayne Institute, London WC1E 6JF, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6593-403X","authenticated-orcid":false,"given":"Jos\u00e9 Afonso","family":"Guerra-Assun\u00e7\u00e3o","sequence":"additional","affiliation":[{"name":"Great Ormond Street Institute of Child Health, University College London (UCL), London WC1N 1EH, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Philip","family":"Felgner","sequence":"additional","affiliation":[{"name":"Vaccine Research and Development Center, Department of Physiology and Biophysics, University of California Irvine, Irvine, CA 92697-4560, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rafael","family":"Ramiro de Assis","sequence":"additional","affiliation":[{"name":"Vaccine Research and Development Center, Department of Physiology and Biophysics, University of California Irvine, Irvine, CA 92697-4560, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rie","family":"Nakajima","sequence":"additional","affiliation":[{"name":"Vaccine Research and Development Center, Department of Physiology and Biophysics, University of California Irvine, Irvine, CA 92697-4560, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Goldblatt","sequence":"additional","affiliation":[{"name":"Immunobiology Section, UCL Great Ormond Street Institute of Child Health, NIHR Biomedical Research Centre, London WC1N 1EH, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Kevin Kweku Adjei","family":"Tetteh","sequence":"additional","affiliation":[{"name":"Faculty of Infectious and Tropical Diseases, London School of Tropical Medicine and Hygiene, London WC1E 7HT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4573-449X","authenticated-orcid":false,"given":"Robert Simon","family":"Heyderman","sequence":"additional","affiliation":[{"name":"Research Department of Infection, Division of Infection and Immunity, University College London, Rayne Institute, London WC1E 6JF, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6576-1116","authenticated-orcid":false,"given":"Stephen Brian","family":"Gordon","sequence":"additional","affiliation":[{"name":"Malawi-Liverpool-Wellcome Trust Clinical Research Programme, Blantyre 30096, Malawi"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniela Mulari","family":"Ferreria","sequence":"additional","affiliation":[{"name":"Department of Clinical Sciences, Liverpool School of Tropical Medicine, Liverpool L3 5QA, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5650-5361","authenticated-orcid":false,"given":"Jeremy Stuart","family":"Brown","sequence":"additional","affiliation":[{"name":"Centre for Inflammation and Tissue Repair, UCL Respiratory, Division of Medicine, University College London, Rayne Institute, London WC1E 6JF, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2021,3,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1136\/thorax.56.4.296","article-title":"Study of community acquired pneumonia aetiology (SCAPA) in adults admitted to hospital: Implications for management guidelines","volume":"56","author":"Lim","year":"2001","journal-title":"Thorax"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1016\/j.jinf.2005.02.008","article-title":"The current burden of pneumococcal disease in England and Wales","volume":"52","author":"Melegaro","year":"2006","journal-title":"J. Infect."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1075","DOI":"10.1136\/thoraxjnl-2012-201924","article-title":"Changes in prevalence and load of airway bacteria using quantitative PCR in stable and exacerbated COPD","volume":"67","author":"Garcha","year":"2012","journal-title":"Thorax"},{"key":"ref_4","first-page":"CD000422","article-title":"Vaccines for preventing pneumococcal infection in adults","volume":"2013","author":"Moberley","year":"2013","journal-title":"Cochrane Database Syst. Rev."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1114","DOI":"10.1056\/NEJMoa1408544","article-title":"Polysaccharide conjugate vaccine against pneumococcal pneumonia in adults","volume":"372","author":"Bonten","year":"2015","journal-title":"N. Engl. J. Med."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Abdullahi, O., Karani, A., Tigoi, C.C., Mugo, D., Kungu, S., Wanjiru, E., Jomo, J., Musyimi, R., Lipsitch, M., and Scott, J.A.G. (2012). The prevalence and risk factors for pneumococcal colonization of the nasopharynx among children in Kilifi District, Kenya. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0030787"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"760","DOI":"10.1016\/S1473-3099(11)70090-1","article-title":"Herd immunity and serotype replacement 4 years after seven-valent pneumococcal conjugate vaccination in England and Wales: An observational cohort study","volume":"11","author":"Miller","year":"2011","journal-title":"Lancet Infect. Dis."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Balsells, E., Guillot, L., Nair, H., and Kyaw, M.H. (2017). Serotype distribution of Streptococcus pneumoniae causing invasive disease in children in the post-PCV era: A systematic review and meta-analysis. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0177113"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Bradshaw, J.L., Pipkins, H.R., Keller, L.E., Pendarvis, J.K., and McDaniel, L.S. (2018). Mucosal infections and invasive potential of nonencapsulated Streptococcus pneumoniae are enhanced by oligopeptide binding proteins AliC and AliD. mBio, 9.","DOI":"10.1128\/mBio.02097-17"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"18977","DOI":"10.1038\/s41598-020-75691-5","article-title":"Divergent serotype replacement trends and increasing diversity in pneumococcal disease in high income settings reduce the benefit of expanding vaccine valency","volume":"10","author":"Lochen","year":"2020","journal-title":"Sci. Rep."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Wilson, R., Cohen, J.M., Reglinski, M., Jos\u00e9, R.J., Chan, W.Y., Marshall, H., De Vogel, C., Gordon, S., Goldblatt, D., and Petersen, F.C. (2017). Naturally acquired human immunity to pneumococcus is dependent on antibody to protein antigens. PLoS Pathog., 13.","DOI":"10.1371\/journal.ppat.1006259"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Lipsitch, M., Whitney, C.G., Zell, E., Kaijalainen, T., Dagan, R., and Malley, R. (2005). Are anticapsular antibodies the primary mechanism of protection against invasive pneumococcal disease?. PLoS Med., 2.","DOI":"10.1371\/journal.pmed.0020015"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"2328","DOI":"10.1016\/j.vaccine.2013.03.013","article-title":"Lack of cross-protection against invasive pneumonia caused by heterologous strains following murine Streptococcus pneumoniae nasopharyngeal colonisation despite whole cell ELISAs showing significant cross-reactive IgG","volume":"31","author":"Cohen","year":"2013","journal-title":"Vaccine"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"4453","DOI":"10.1016\/j.vaccine.2012.04.080","article-title":"Contributions of capsule, lipoproteins and duration of colonisation towards the protective immunity of prior Streptococcus pneumoniae nasopharyngeal colonisation","volume":"30","author":"Cohen","year":"2012","journal-title":"Vaccine"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"2469","DOI":"10.1128\/IAI.01972-06","article-title":"Live attenuated Streptococcus pneumoniae strains induce serotype-independent mucosal and systemic protection in mice","volume":"75","author":"Roche","year":"2007","journal-title":"Infect. Immun."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Wright, A.K.A., Ferreira, D.M., Gritzfeld, J.F., Wright, A.D., Armitage, K., Jambo, K.C., Bate, E., El Batrawy, S., Collins, A., and Gordon, S.B. (2012). Human nasal challenge with Streptococcus pneumoniae is immunising in the absence of carriage. PLoS Pathog., 8.","DOI":"10.1371\/journal.ppat.1002622"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Wright, A.K.A., Bangert, M., Gritzfeld, J.F., Ferreira, D.M., Jambo, K.C., Wright, A.D., Collins, A.M., and Gordon, S.B. (2013). Experimental human pneumococcal carriage augments IL-17A-dependent T-cell defence of the lung. PLoS Pathog., 9.","DOI":"10.1371\/journal.ppat.1003274"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"726","DOI":"10.1111\/imcb.12257","article-title":"Enhanced safety and immunogenicity of a pneumococcal surface antigen A mutant whole-cell inactivated pneumococcal vaccine","volume":"97","author":"David","year":"2019","journal-title":"Immunol. Cell Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1084\/jem.20011576","article-title":"The immune response to pneumococcal proteins during experimental human carriage","volume":"195","author":"McCool","year":"2002","journal-title":"J. Exp. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"627","DOI":"10.1038\/mi.2014.95","article-title":"Protection against Streptococcus pneumoniae lung infection after nasopharyngeal colonization requires both humoral and cellular immune responses","volume":"8","author":"Wilson","year":"2015","journal-title":"Mucosal Immunol."},{"key":"ref_21","first-page":"1899","article-title":"Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice","volume":"119","author":"Zhang","year":"2009","journal-title":"J. Clin. Investig."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Cohen, J.M., Khandavilli, S., Camberlein, E., Hyams, C., Baxendale, H.E., and Brown, J.S. (2011). Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-cell responses to nasopharyngeal colonisation. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0025558"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Ramos-Sevillano, E., Ercoli, G., Felgner, P., De Assis, R.R., Nakajima, R., Goldblatt, D., Heyderman, R.S., Gordon, S.B., Ferreira, D.M., and Brown, J.S. (2020). Preclinical development of virulence attenuated Streptococcus pneumoniae strains able to enhance protective immunity against pneumococcal infection. Am. J. Respir. Crit. Care Med.","DOI":"10.1164\/rccm.202011-4161LE"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1164\/rccm.201212-2277OC","article-title":"Controlled human infection and rechallenge with Streptococcus pneumoniae reveals the protective efficacy of carriage in healthy adults","volume":"187","author":"Ferreira","year":"2013","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4523","DOI":"10.1172\/JCI128865","article-title":"Innate and adaptive nasal mucosal immune responses following experimental human pneumococcal colonization","volume":"129","author":"Jochems","year":"2019","journal-title":"J. Clin. Investig."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"4965","DOI":"10.1128\/IAI.05923-11","article-title":"Infection with conditionally virulent Streptococcus pneumoniae \u2206pab strains induces antibody to conserved protein antigens but does not protect against systemic infection with heterologous strains","volume":"79","author":"Chimalapati","year":"2011","journal-title":"Infect. Immun."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1016\/j.vaccine.2018.11.027","article-title":"Immunization with attenuated non-transformable pneumococcal pep27 and comD mutant provides serotype-independent protection against pneumococcal infection","volume":"37","author":"Kim","year":"2019","journal-title":"Vaccine"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1002\/emmm.201202150","article-title":"A live-attenuated pneumococcal vaccine elicits CD4+ T-cell dependent class switching and provides serotype independent protection against acute otitis media","volume":"6","author":"Rosch","year":"2014","journal-title":"EMBO Mol. Med."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Chimalapati, S., Cohen, J.M., Camberlein, E., Macdonald, N., Durmort, C., Vernet, T., Hermans, P.W.M., Mitchell, T., and Brown, J.S. (2012). Effects of deletion of the Streptococcus pneumoniae lipoprotein diacylglyceryl transferase gene lgt on ABC transporter function and on growth in vivo. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0041393"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"5255","DOI":"10.1128\/iai.64.12.5255-5262.1996","article-title":"Sequence heterogeneity of PsaA, a 37-kilodalton putative adhesin essential for virulence of Streptococcus pneumoniae","volume":"64","author":"Berry","year":"1996","journal-title":"Infect. Immun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1635","DOI":"10.1128\/IAI.70.3.1635-1639.2002","article-title":"Virulence of Streptococcus pneumoniae: PsaA mutants are hypersensitive to oxidative stress","volume":"70","author":"Tseng","year":"2002","journal-title":"Infect. Immun."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2541","DOI":"10.1101\/gr.137430.112","article-title":"A fine scale phenotype-genotype virulence map of a bacterial pathogen","volume":"22","author":"Camilli","year":"2012","journal-title":"Genome Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"11140","DOI":"10.1073\/pnas.92.24.11140","article-title":"An unmodified heptadecapeptide pheromone induces competence for genetic transformation in Streptococcus pneumoniae","volume":"92","author":"Havarstein","year":"1995","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1128\/IAI.00881-09","article-title":"The Streptococcus pneumoniae capsule inhibits complement activity and neutrophil phagocytosis by multiple mechanisms","volume":"78","author":"Hyams","year":"2010","journal-title":"Infect. Immun."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1038\/s41541-018-0090-4","article-title":"A recombinant conjugated pneumococcal vaccine that protects against murine infections with a similar efficacy to Prevnar-13","volume":"3","author":"Reglinski","year":"2018","journal-title":"NPJ Vaccines"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Chan, W.-Y., Entwisle, C., Ercoli, G., Ramos-Sevillano, E., McIlgorm, A., Cecchini, P., Bailey, C., Lam, O., Whiting, G., and Green, N. (2019). A Novel, multiple-antigen pneumococcal vaccine protects against lethal Streptococcus pneumoniae challenge. Infect. Immun., 87.","DOI":"10.1128\/IAI.00846-18"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"E357","DOI":"10.1073\/pnas.1613937114","article-title":"Diverse evolutionary patterns of pneumococcal antigens identified by pangenome-wide immunological screening","volume":"114","author":"Croucher","year":"2017","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Ercoli, G., Ramos-Sevillano, E., Nakajima, R., de Assis, R.R., Jasinskas, A., Goldblatt, D., Felgner, P., Weckbecker, G., and Brown, J. (2021). The influence of B cell depletion therapy on naturally acquired immunity to Streptococcus pneumoniae. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.611661"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1366","DOI":"10.3389\/fimmu.2018.01366","article-title":"Streptococcus pneumoniae\u2019s virulence and host immunity: Aging, diagnostics, and prevention","volume":"9","author":"Brooks","year":"2018","journal-title":"Front. Immunol."},{"key":"ref_40","doi-asserted-by":"crossref","unstructured":"Bidossi, A., Mulas, L., Decorosi, F., Colomba, L., Ricci, S., Pozzi, G., Deutscher, J., Viti, C., and Oggioni, M.R. (2012). A functional genomics approach to establish the complement of carbohydrate transporters in Streptococcus pneumoniae. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0033320"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"7","DOI":"10.7554\/eLife.37015","article-title":"Panproteome-wide analysis of antibody responses to whole cell pneumococcal vaccination","volume":"7","author":"Campo","year":"2018","journal-title":"Elife"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"387","DOI":"10.1086\/431524","article-title":"Antibody responses to nasopharyngeal carriage of Streptococcus pneumoniae in adults: A longitudinal household study","volume":"192","author":"Goldblatt","year":"2005","journal-title":"J. Infect. Dis."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1086\/599364","article-title":"Epidemiological evidence for serotype-independent acquired immunity to pneumococcal carriage","volume":"200","author":"Granat","year":"2009","journal-title":"J. Infect. Dis."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1511","DOI":"10.1086\/587941","article-title":"Epidemiologic evidence for serotype-specific acquired immunity to pneumococcal carriage","volume":"197","author":"Weinberger","year":"2008","journal-title":"J. Infect. Dis."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1376","DOI":"10.1126\/science.1215947","article-title":"Niche and neutral effects of acquired immunity permit coexistence of pneumococcal serotypes","volume":"335","author":"Cobey","year":"2012","journal-title":"Science"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1164\/rccm.201910-2047ED","article-title":"Improving Pulmonary Immunity to Bacterial Pathogens through Streptococcus pneumoniae Colonization of the Nasopharynx","volume":"201","author":"Brown","year":"2020","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"335","DOI":"10.1164\/rccm.201903-0607OC","article-title":"Nasal pneumococcal density is associated with microaspiration and heightened human alveolar macrophage responsiveness to bacterial pathogens","volume":"201","author":"Mitsi","year":"2020","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"572","DOI":"10.1046\/j.1365-2958.2001.02414.x","article-title":"A Streptococcus pneumoniae pathogenicity island encoding an ABC transporter involved in iron uptake and virulence","volume":"40","author":"Brown","year":"2001","journal-title":"Mol. Microbiol."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"4389","DOI":"10.1128\/JB.183.14.4389-4392.2001","article-title":"Multiple genes for the last step of proline biosynthesis in Bacillus subtilis","volume":"183","author":"Belitsky","year":"2001","journal-title":"J. Bacteriol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1684","DOI":"10.1128\/JCM.00265-07","article-title":"Association of the pneumococcal pilus with certain capsular serotypes but not with increased virulence","volume":"45","author":"Basset","year":"2007","journal-title":"J. Clin. Microbiol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"2453","DOI":"10.1128\/IAI.74.4.2453-2456.2006","article-title":"RrgA and RrgB are components of a multisubunit pilus encoded by the Streptococcus pneumoniae rlrA pathogenicity islet","volume":"74","author":"LeMieux","year":"2006","journal-title":"Infect. Immun."}],"container-title":["Vaccines"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-393X\/9\/3\/261\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T05:35:53Z","timestamp":1760160953000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-393X\/9\/3\/261"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,3,15]]},"references-count":51,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2021,3]]}},"alternative-id":["vaccines9030261"],"URL":"https:\/\/doi.org\/10.3390\/vaccines9030261","relation":{},"ISSN":["2076-393X"],"issn-type":[{"value":"2076-393X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,3,15]]}}}