Cristina Elías-López, Montserrat Muñoz-Rosa, Julia Guzmán-Puche, Elena Pérez-Nadales, Eduardo Chicano-Galvez, Luis Martínez-Martínez
{"title":"肺炎克雷伯氏菌临床分离物中茯苓蛋白的表达:SDS-PAGE 和 MALDI-TOF/MS 的比较以及全基因组测序分析的局限性。","authors":"Cristina Elías-López, Montserrat Muñoz-Rosa, Julia Guzmán-Puche, Elena Pérez-Nadales, Eduardo Chicano-Galvez, Luis Martínez-Martínez","doi":"10.1186/s12941-024-00761-9","DOIUrl":null,"url":null,"abstract":"<p><strong>Background: </strong>The permeability of the outer membrane barrier modulates the susceptibility of microorganisms to antimicrobial agents. Loss or structural alterations of porins contribute to decreased antibiotic concentration of multiple antimicrobial agents. Precise definition of porin profiles is of critical importance to understand the role of porins in antimicrobial resistance. The objectives of this study are to compare the expression patterns of major outer membrane proteins (OMP) of clinical isolates of Klebsiella pneumoniae obtained with Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF/MS), with those obtained with sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and to correlate porin expression patterns with the sequences of porins genes defined with whole genome sequencing (WGS).</p><p><strong>Methods: </strong>The OMP profiles of 26 clinical isolates of K. pneumoniae and of strain ATCC 13883 (wild-type) and ATCC 700603 (producing SHV-18) have been determined using both SDS-PAGE and MALDI-TOF/MS. SDS-PAGE was performed using both homemade and commercial gels, and protein bands were identified by liquid chromatography coupled to mass spectrometry. A rapid extraction method was used to analyse OMPs by MALDI-TOF/MS. The sequences of porin genes were obtained by WGS and mutations were defined by BLAST.</p><p><strong>Results: </strong>Same results were obtained for all strains either using SDS-PAGE or MALDI-TOF/MS. SDS-PAGE showed protein bands of ~ 35, ~36, and ~ 37 kDa, identified as OmpA, OmpK36 and OmpK35, respectively. By MALDI-TOF/MS, peaks at ~ 35,700 (OmpA), ~ 37,000 (OmpK35), and ~ 38,000 (OmpK36) m/z were detected. ompK35 was intact in nine wild-type isolates and was truncated in 13 isolates, but OmpK35 was not observed in 3 isolates without mutations in ompK35. One point mutation was detected in another isolate and multiple mutations were detected in the remaining isolate. ompK36 was truncated in two isolates lacking this protein and presented one point mutation (n = 1) or multiple mutations in the remaining isolates.</p><p><strong>Conclusion: </strong>MALDI-TOF/MS was reliable for porin detection, but because of the complex regulation of porin genes, WGS cannot always anticipate protein expression, as observed with SDS-PAGE and MALDI-TOF/MS.</p>","PeriodicalId":8052,"journal":{"name":"Annals of Clinical Microbiology and Antimicrobials","volume":"23 1","pages":"103"},"PeriodicalIF":4.6000,"publicationDate":"2024-11-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11587576/pdf/","citationCount":"0","resultStr":"{\"title\":\"Porin expression in clinical isolates of Klebsiella pneumoniae: a comparison of SDS-PAGE and MALDI-TOF/MS and limitations of whole genome sequencing analysis.\",\"authors\":\"Cristina Elías-López, Montserrat Muñoz-Rosa, Julia Guzmán-Puche, Elena Pérez-Nadales, Eduardo Chicano-Galvez, Luis Martínez-Martínez\",\"doi\":\"10.1186/s12941-024-00761-9\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><strong>Background: </strong>The permeability of the outer membrane barrier modulates the susceptibility of microorganisms to antimicrobial agents. Loss or structural alterations of porins contribute to decreased antibiotic concentration of multiple antimicrobial agents. Precise definition of porin profiles is of critical importance to understand the role of porins in antimicrobial resistance. The objectives of this study are to compare the expression patterns of major outer membrane proteins (OMP) of clinical isolates of Klebsiella pneumoniae obtained with Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF/MS), with those obtained with sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and to correlate porin expression patterns with the sequences of porins genes defined with whole genome sequencing (WGS).</p><p><strong>Methods: </strong>The OMP profiles of 26 clinical isolates of K. pneumoniae and of strain ATCC 13883 (wild-type) and ATCC 700603 (producing SHV-18) have been determined using both SDS-PAGE and MALDI-TOF/MS. SDS-PAGE was performed using both homemade and commercial gels, and protein bands were identified by liquid chromatography coupled to mass spectrometry. A rapid extraction method was used to analyse OMPs by MALDI-TOF/MS. The sequences of porin genes were obtained by WGS and mutations were defined by BLAST.</p><p><strong>Results: </strong>Same results were obtained for all strains either using SDS-PAGE or MALDI-TOF/MS. SDS-PAGE showed protein bands of ~ 35, ~36, and ~ 37 kDa, identified as OmpA, OmpK36 and OmpK35, respectively. By MALDI-TOF/MS, peaks at ~ 35,700 (OmpA), ~ 37,000 (OmpK35), and ~ 38,000 (OmpK36) m/z were detected. ompK35 was intact in nine wild-type isolates and was truncated in 13 isolates, but OmpK35 was not observed in 3 isolates without mutations in ompK35. One point mutation was detected in another isolate and multiple mutations were detected in the remaining isolate. ompK36 was truncated in two isolates lacking this protein and presented one point mutation (n = 1) or multiple mutations in the remaining isolates.</p><p><strong>Conclusion: </strong>MALDI-TOF/MS was reliable for porin detection, but because of the complex regulation of porin genes, WGS cannot always anticipate protein expression, as observed with SDS-PAGE and MALDI-TOF/MS.</p>\",\"PeriodicalId\":8052,\"journal\":{\"name\":\"Annals of Clinical Microbiology and Antimicrobials\",\"volume\":\"23 1\",\"pages\":\"103\"},\"PeriodicalIF\":4.6000,\"publicationDate\":\"2024-11-24\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11587576/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Annals of Clinical Microbiology and Antimicrobials\",\"FirstCategoryId\":\"3\",\"ListUrlMain\":\"https://doi.org/10.1186/s12941-024-00761-9\",\"RegionNum\":2,\"RegionCategory\":\"医学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q1\",\"JCRName\":\"MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Annals of Clinical Microbiology and Antimicrobials","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.1186/s12941-024-00761-9","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"MICROBIOLOGY","Score":null,"Total":0}
Porin expression in clinical isolates of Klebsiella pneumoniae: a comparison of SDS-PAGE and MALDI-TOF/MS and limitations of whole genome sequencing analysis.
Background: The permeability of the outer membrane barrier modulates the susceptibility of microorganisms to antimicrobial agents. Loss or structural alterations of porins contribute to decreased antibiotic concentration of multiple antimicrobial agents. Precise definition of porin profiles is of critical importance to understand the role of porins in antimicrobial resistance. The objectives of this study are to compare the expression patterns of major outer membrane proteins (OMP) of clinical isolates of Klebsiella pneumoniae obtained with Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight mass spectrometry (MALDI-TOF/MS), with those obtained with sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and to correlate porin expression patterns with the sequences of porins genes defined with whole genome sequencing (WGS).
Methods: The OMP profiles of 26 clinical isolates of K. pneumoniae and of strain ATCC 13883 (wild-type) and ATCC 700603 (producing SHV-18) have been determined using both SDS-PAGE and MALDI-TOF/MS. SDS-PAGE was performed using both homemade and commercial gels, and protein bands were identified by liquid chromatography coupled to mass spectrometry. A rapid extraction method was used to analyse OMPs by MALDI-TOF/MS. The sequences of porin genes were obtained by WGS and mutations were defined by BLAST.
Results: Same results were obtained for all strains either using SDS-PAGE or MALDI-TOF/MS. SDS-PAGE showed protein bands of ~ 35, ~36, and ~ 37 kDa, identified as OmpA, OmpK36 and OmpK35, respectively. By MALDI-TOF/MS, peaks at ~ 35,700 (OmpA), ~ 37,000 (OmpK35), and ~ 38,000 (OmpK36) m/z were detected. ompK35 was intact in nine wild-type isolates and was truncated in 13 isolates, but OmpK35 was not observed in 3 isolates without mutations in ompK35. One point mutation was detected in another isolate and multiple mutations were detected in the remaining isolate. ompK36 was truncated in two isolates lacking this protein and presented one point mutation (n = 1) or multiple mutations in the remaining isolates.
Conclusion: MALDI-TOF/MS was reliable for porin detection, but because of the complex regulation of porin genes, WGS cannot always anticipate protein expression, as observed with SDS-PAGE and MALDI-TOF/MS.
期刊介绍:
Annals of Clinical Microbiology and Antimicrobials considers good quality, novel and international research of more than regional relevance. Research must include epidemiological and/or clinical information about isolates, and the journal covers the clinical microbiology of bacteria, viruses and fungi, as well as antimicrobial treatment of infectious diseases.
Annals of Clinical Microbiology and Antimicrobials is an open access, peer-reviewed journal focusing on information concerning clinical microbiology, infectious diseases and antimicrobials. The management of infectious disease is dependent on correct diagnosis and appropriate antimicrobial treatment, and with this in mind, the journal aims to improve the communication between laboratory and clinical science in the field of clinical microbiology and antimicrobial treatment. Furthermore, the journal has no restrictions on space or access; this ensures that the journal can reach the widest possible audience.