71a0b2fdf9373cd678d2cdfc1c50a8db pmc_9431570.pdf ab19d032ce793b21c5a652eba89065f32066b67d pmc_9431570.pdf 1d4aa0487fdcb16463393ec1e9fd75d9500afe084a22805e17a3e4e40131f705 pmc_9431570.pdf Title: An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates Subject: Research Article Keywords: Pseudomonas aeruginosa; quorum sensing; virulence; diagnostic; antibodies; immunoassay; HQNO-2-heptyl-4-quinoline N-oxide; MvfR; PqsR; virulence factors Author: Enrique J. Montagut;Juan Raya;M.-Teresa Martin-Gomez;Lluïsa Vilaplana;Barbara Rodriguez-Urretavizcaya;M.-Pilar Marco Creator: PTC Arbortext Advanced Print Publisher 11.1.4012/W Producer: Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version) CreationDate: Fri Aug 19 04:22:08 2022 CEST ModDate: Fri Aug 19 20:35:18 2022 CEST Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 12 Encrypted: no Page size: 584.957 x 782.986 pts Page rot: 0 File size: 1071277 bytes Optimized: no PDF version: 1.4 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- EALIIJ+AdvOTce3d9a73 Type 1C WinAnsi yes yes no 133 0 EALIIK+AdvOT56309c18.I Type 1C WinAnsi yes yes no 134 0 EALIKK+AdvOT07517017 Type 1C WinAnsi yes yes no 400 0 EALIKL+AdvOT07517017+fb Type 1C Custom yes yes yes 401 0 EALIKM+AdvOT07517017+20 Type 1C WinAnsi yes yes no 402 0 EALILN+AdvOT3b30f6db.B Type 1C WinAnsi yes yes no 39 0 EALILO+AdvOT46dcae81 Type 1C WinAnsi yes yes no 40 0 EALILP+AdvOT46dcae81+fb Type 1C Custom yes yes yes 41 0 EALIMA+AdvP80675 Type 1C WinAnsi yes yes no 62 0 EALIMB+AdvOT65f8a23b.I Type 1C WinAnsi yes yes no 42 0 EALIOB+AdvOTd0125be5.BI Type 1C WinAnsi yes yes no 33 0 EALIOC+AdvOT46dcae81+20 Type 1C WinAnsi yes yes no 43 0 EALIOD+AdvEURO Type 1C WinAnsi yes yes no 36 0 EALJDC+AdvP7DA6 Type 1C WinAnsi yes yes no 100 0 EALKCN+AdvP4C4E74 Type 1C Custom yes yes no 152 0 Jhove (Rel. 1.22.1, 2019-04-17) Date: 2023-06-30 02:36:30 CEST RepresentationInformation: pmc_9431570.pdf ReportingModule: PDF-hul, Rel. 1.12.1 (2019-04-17) LastModified: 2022-09-24 02:13:49 CEST Size: 1071277 Format: PDF Version: 1.4 Status: Well-Formed, but not valid SignatureMatches: PDF-hul ID: PDF-HUL-122 ErrorMessage: edu.harvard.hul.ois.jhove.module.pdf.PdfInvalidException: Invalid indirect destination - 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Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@4eb7f003 Item: Title: Synthesis of the immunizing hapten 6-(2-carboxyethyl)-2-heptyl-4-hydroxyquinoline N-oxide (16). Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@eafc191 Item: Title: Synthesis of the bioconjugates HQNO-KLH and HQNO-BSA. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612fc6eb Item: Title: ELISA. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1060b431 Item: Title: Implementation of the ELISA to the analysis of clinical isolates. Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@612679d6 Item: Title: SUPPLEMENTAL MATERIAL Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@11758f2a Item: Title: ACKNOWLEDGMENTS Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@e720b71 Item: Title: REFERENCES Destination: edu.harvard.hul.ois.jhove.module.pdf.PdfSimpleObject@1b26f7b2 Info: Title: An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates Author: Enrique J. Montagut;Juan Raya;M.-Teresa Martin-Gomez;Lluïsa Vilaplana;Barbara Rodriguez-Urretavizcaya;M.-Pilar Marco Subject: Research Article Keywords: Pseudomonas aeruginosa; quorum sensing; virulence; diagnostic; antibodies; immunoassay; HQNO-2-heptyl-4-quinoline N-oxide; MvfR; PqsR; virulence factors Creator: PTC Arbortext Advanced Print Publisher 11.1.4012/W Producer: Adobe LiveCycle PDF Generator; modified using iText® 5.5.13.2 ©2000-2020 iText Group NV (AGPL-version) CreationDate: Fri Aug 19 04:52:08 CEST 2022 ModDate: Fri Aug 19 21:05:18 CEST 2022 ID: 0xf611cb02fd8bde88c68069d14cca858c, 0xc1504e34554ea37dea5212e50af6e98f Filters: FilterPipeline: FlateDecode Images: Image: NisoImageMetadata: FormatName: image/png ImageWidth: 67 ImageHeight: 67 BitsPerSample: 8 BitsPerSampleUnit: integer Filter: FlateDecode Fonts: Type1: Font: BaseFont: EALIOB+AdvOTd0125be5.BI FontSubset: true FirstChar: 80 LastChar: 80 FontDescriptor: FontName: EALIOB+AdvOTd0125be5.BI Flags: Nonsymbolic FontBBox: -95, -250, 930, 889 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALIOD+AdvEURO FontSubset: true FirstChar: 101 LastChar: 101 FontDescriptor: FontName: EALIOD+AdvEURO Flags: Nonsymbolic FontBBox: 19, 9, 666, 729 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALJDC+AdvP7DA6 FontSubset: true FirstChar: 109 LastChar: 115 FontDescriptor: FontName: EALJDC+AdvP7DA6 Flags: Nonsymbolic FontBBox: -20, -208, 1000, 770 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALIIJ+AdvOTce3d9a73 FontSubset: true FirstChar: 37 LastChar: 239 FontDescriptor: FontName: EALIIJ+AdvOTce3d9a73 Flags: Nonsymbolic FontBBox: -42, -250, 858, 889 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALIIK+AdvOT56309c18.I FontSubset: true FirstChar: 80 LastChar: 117 FontDescriptor: FontName: EALIIK+AdvOT56309c18.I Flags: Nonsymbolic FontBBox: -103, -250, 914, 882 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALILN+AdvOT3b30f6db.B FontSubset: true FirstChar: 40 LastChar: 122 FontDescriptor: FontName: EALILN+AdvOT3b30f6db.B Flags: Nonsymbolic FontBBox: -39, -250, 875, 897 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALILO+AdvOT46dcae81 FontSubset: true FirstChar: 37 LastChar: 252 FontDescriptor: FontName: EALILO+AdvOT46dcae81 Flags: Nonsymbolic FontBBox: -46, -250, 838, 879 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALILP+AdvOT46dcae81+fb FontSubset: true FirstChar: 1 LastChar: 3 FontDescriptor: FontName: EALILP+AdvOT46dcae81+fb Flags: Symbolic FontBBox: 0, 0, 742, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: EALIMB+AdvOT65f8a23b.I FontSubset: true FirstChar: 45 LastChar: 121 FontDescriptor: FontName: EALIMB+AdvOT65f8a23b.I Flags: Nonsymbolic FontBBox: -112, -250, 894, 873 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALIOC+AdvOT46dcae81+20 FontSubset: true FirstChar: 146 LastChar: 150 FontDescriptor: FontName: EALIOC+AdvOT46dcae81+20 Flags: Nonsymbolic FontBBox: -9, -205, 1126, 868 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALIKK+AdvOT07517017 FontSubset: true FirstChar: 40 LastChar: 242 FontDescriptor: FontName: EALIKK+AdvOT07517017 Flags: Nonsymbolic FontBBox: -49, -250, 816, 867 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALIKL+AdvOT07517017+fb FontSubset: true FirstChar: 1 LastChar: 3 FontDescriptor: FontName: EALIKL+AdvOT07517017+fb Flags: Symbolic FontBBox: 0, 0, 683, 721 FontFile3: true EncodingDictionary: Differences: true ToUnicode: true Font: BaseFont: EALIKM+AdvOT07517017+20 FontSubset: true FirstChar: 146 LastChar: 146 FontDescriptor: FontName: EALIKM+AdvOT07517017+20 Flags: Nonsymbolic FontBBox: -6, -209, 1046, 868 FontFile3: true Encoding: WinAnsiEncoding Font: BaseFont: EALKCN+AdvP4C4E74 FontSubset: true FirstChar: 1 LastChar: 1 FontDescriptor: FontName: EALKCN+AdvP4C4E74 Flags: Symbolic FontBBox: -20, -958, 1114, 770 FontFile3: true EncodingDictionary: Differences: true Font: BaseFont: EALIMA+AdvP80675 FontSubset: true FirstChar: 44 LastChar: 54 FontDescriptor: FontName: EALIMA+AdvP80675 Flags: Nonsymbolic FontBBox: -20, -229, 1010, 812 FontFile3: true Encoding: WinAnsiEncoding XMP: An Immunochemical Approach to Detect the Quorum Sensing-Regulated Virulence Factor 2-Heptyl-4-Quinoline N-Oxide (HQNO) Produced by Pseudomonas aeruginosa Clinical Isolates 10.1128/spectrum.01073-21 Enrique J. Montagut Juan Raya M.-Teresa Martin-Gomez Lluïsa Vilaplana Barbara Rodriguez-Urretavizcaya M.-Pilar Marco Research Article Research Article Pseudomonas aeruginosa quorum sensing virulence diagnostic antibodies immunoassay HQNO-2-heptyl-4-quinoline N-oxide MvfR PqsR virulence factors American Society for Microbiology Montagut et al. Copyright © 2022 Montagut et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. 20220725 2022 ABSTRACT Understanding quorum sensing (QS) and its role in the development of pathogenesis may provide new avenues for diagnosing, surveillance, and treatment of infectious diseases. For this purpose, the availability of reliable and efficient analytical diagnostic tools suitable to specifically detect and quantify these essential QS small molecules and QS regulated virulence factors is crucial. Here, we reported the development and evaluation of antibodies and an enzyme-linked immunosorbent assay (ELISA) for HQNO (2-heptyl-4-quinoline N-oxide), a QS product of the PqsR system, which has been found to act as a major virulence factor that interferes with the growth of other microorganisms. Despite the nonimmunogenic character of HQNO, the antibodies produced showed high avidity and the microplate-based ELISA developed could detect HQNO in the low nM range. Hence, a limit of detection (LOD) of 0.60 ± 0.13 nM had been reached in Müeller Hinton (MH) broth, which was below previously reported levels using sophisticated equipment based on liquid chromatography coupled to mass spectrometry. The HQNO profile of release of different Pseudomonas aeruginosa clinical isolates analyzed using this ELISA showed significant differences depending on whether the clinical isolates belonged to patients with acute or chronic infections. These data point to the possibility of using HQNO as a specific biomarker to diagnose P. aeruginosa infections and for patient surveillance. Considering the role of HQNO in inhibiting the growth of coinfecting bacteria, the present ELISA will allow the investigation of these complex bacterial interactions underlying infections. IMPORTANCE Bacteria use quorum sensing (QS) as a communication mechanism that releases small signaling molecules which allow synchronizing a series of activities involved in the pathogenesis, such as the biosynthesis of virulence factors or the regulation of growth of other bacterial species. HQNO is a metabolite of the Pseudomonas aeruginosa-specific QS signaling molecule PQS (Pseudomonas quinolone signal). In this work, the development of highly specific antibodies and an immunochemical diagnostic technology (ELISA) for the detection and quantification of HQNO was reported. The ELISA allowed profiling of the release of HQNO by clinical bacterial isolates, showing its potential value for diagnosing and surveillance of P. aeruginosa infections. Moreover, the antibodies and the ELISA reported here may contribute to the knowledge of other underlying conditions related to the pathology, such as the role of the interactions with other bacteria of a particular microbiota environment. 20210729 20220621 2165-0497 Pages: Page: Label: 1 Annotations: Annotation: Subtype: Link Rect: 302, 565, 363, 574 Annotation: Subtype: Link Rect: 50, 554, 106, 563 Annotation: Subtype: Link Rect: 417, 124, 513, 131 Annotation: Subtype: Link Rect: 400, 115, 456, 122 Annotation: Subtype: Link Rect: 444, 24, 526, 31 Annotation: Subtype: Link Rect: 508, 683, 544, 719 Page: Label: 2 Annotations: Annotation: Subtype: Link Rect: 198, 653, 203, 662 Destination: 195 Annotation: Subtype: Link Rect: 202, 616, 207, 625 Destination: 195 Annotation: Subtype: Link Rect: 352, 592, 357, 601 Destination: 195 Annotation: Subtype: Link Rect: 357, 592, 361, 601 Destination: 195 Annotation: Subtype: Link Rect: 361, 592, 366, 601 Destination: 195 Annotation: Subtype: Link 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