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A proteomics-driven reverse vaccinology approach has delivered multiple gonorrhea vaccine candidates; however, the biological functions of many of them remain to be elucidated. Herein, the functions of six gonorrhea vaccine candidates\u2014NGO2121, NGO1985, NGO2054, NGO2111, NGO1205, and NGO1344\u2014in cell envelope homeostasis were probed using phenotype microarrays under 1,056 conditions and a \u0394\n            <jats:italic>bamE<\/jats:italic>\n            mutant (\u0394\n            <jats:italic>ngo1780<\/jats:italic>\n            ) as a reference of perturbed outer membrane integrity. Optimal growth conditions for an\n            <jats:named-content content-type=\"genus-species\">N. gonorrhoeae<\/jats:named-content>\n            phenotype microarray assay in defined liquid medium were developed, which can be useful in other applications, including rapid and thorough antimicrobial susceptibility assessment. Our studies revealed 91 conditions having uniquely positive or negative effects on one of the examined mutants. A cluster analysis of 37 and 57 commonly beneficial and detrimental compounds, respectively, revealed three separate phenotype groups: NGO2121 and NGO1985; NGO1344 and BamE; and the trio of NGO1205, NGO2111, and NGO2054, with the last protein forming an independent branch of this cluster. Similar phenotypes were associated with loss of these vaccine candidates in the highly antibiotic-resistant WHO X strain. Based on their extensive sensitivity phenomes, NGO1985 and NGO2121 appear to be the most promising vaccine candidates. This study establishes the principle that phenotype microarrays can be successfully applied to a fastidious bacterial organism, such as\n            <jats:named-content content-type=\"genus-species\">N. gonorrhoeae<\/jats:named-content>\n            .\n          <\/jats:p>\n          <jats:p>\n            <jats:bold>IMPORTANCE<\/jats:bold>\n            Innovative approaches are required to develop vaccines against prevalent and neglected sexually transmitted infections, such as gonorrhea. Herein, we have utilized phenotype microarrays in the first such investigation into\n            <jats:named-content content-type=\"genus-species\">Neisseria gonorrhoeae<\/jats:named-content>\n            to probe the function of proteome-derived vaccine candidates in cell envelope homeostasis. Information gained from this screening can feed the vaccine candidate decision tree by providing insights into the roles these proteins play in membrane permeability, integrity, and overall\n            <jats:named-content content-type=\"genus-species\">N. gonorrhoeae<\/jats:named-content>\n            physiology. The optimized screening protocol can be applied in investigations into the function of other hypothetical proteins of\n            <jats:named-content content-type=\"genus-species\">N. gonorrhoeae<\/jats:named-content>\n            discovered in the expanding number of whole-genome sequences, in addition to revealing phenotypic differences between clinical and laboratory strains.\n          <\/jats:p>","DOI":"10.1128\/jb.00037-17","type":"journal-article","created":{"date-parts":[[2017,6,20]],"date-time":"2017-06-20T00:30:35Z","timestamp":1497918635000},"update-policy":"http:\/\/dx.doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":14,"title":["Deciphering the Function of New Gonococcal Vaccine Antigens Using Phenotypic Microarrays"],"prefix":"10.1128","volume":"199","author":[{"given":"Benjamin I.","family":"Baarda","sequence":"first","affiliation":[{"name":"Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sarah","family":"Emerson","sequence":"additional","affiliation":[{"name":"Department of Statistics, Oregon State University, Corvallis, Oregon, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Philip J.","family":"Proteau","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksandra E.","family":"Sikora","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, College of Pharmacy, Oregon State University, Corvallis, Oregon, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"235","reference":[{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1101\/cshperspect.a000414"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6976.2007.00094.x"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1093\/femsre\/fuu006"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1111\/febs.12041"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.M116.058800"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1074\/mcp.M113.029538"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1128\/microbiolspec.EI10-0009-2015"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1128\/CMR.00010-14"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1136\/sextrans-2013-051225"},{"key":"e_1_3_3_11_2","first-page":"124","article-title":"Vaccines for gonorrhea: can we rise to the challenge?","volume":"2","author":"Zhu W","year":"2011","unstructured":"Zhu W Chen CJ Thomas CE Anderson JE Jerse AE Sparling PF . 2011. 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