Nielsen, Jeppe Lund

Nielsen, Kåre Lehmann

Nielsen, Per Halkjær

Thomsen, Trine Rolighed

Xu, Yijuan

English

    Aalborg UniversitetRNA-seq profiling of pathogens in prosthetic joint infectionXu, Yijuan; Nielsen, Kåre Lehmann; Nielsen, Per Halkjær; Nielsen, Jeppe Lund; Thomsen,Trine RolighedPublication date:2013Document VersionEarly version, also known as pre-printLink to publication from Aalborg UniversityCitation for published version (APA):Xu, Y., Nielsen, K. L., Nielsen, P. H., Nielsen, J. L., & Thomsen, T. R. (2013). RNA-seq profiling of pathogens inprosthetic joint infection. Poster presented at Danmarks Mikrobiologiske Selskab symposium, Denmark.General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: August 23, 2021                                                                                                                                            RNA-seq profiling of pathogens in prosthetic joint infection                                                          I n t r o d u c t i o n                             C o n c l u s i o n s This study indicates that RNA-seq  is a challenging but powerful tool to profile the gene expression of pathogens in vivo.  R e s u l t s                             Aim                                                         M e t h o d s To study the gene expression of pathogens directly in PJI in a human host using RNA-seq.  xuyijuan@bio.aau.dk Xu, Yijuan1; Nielsen, Kåre L.1; Nielsen, Per H.1; Nielsen, Jeppe L.1; Thomsen, Trine R. 1,2 1: Center for Microbial Communities, Aalborg University, Denmark  2: Danish Technological Institute, Denmark Prosthetic joint infections (PJIs) are serious complications of joint alloplasties. These device-associated infections are typically caused by bacteria growing in biofilms, which protect them from antimicrobial agents and host immune responses in poorly vascularized joints. Our current knowledge of how pathogens cope with the complex conditions within the host is based on in vitro studies and animal models, which can differ substantially from their behavior in the human host. Studying gene expression of pathogens during infection is a way to understand pathogenesis. RNA sequencing (RNA-seq) is capable of characterizing the entire transcriptome, both quantitatively and qualitatively, of an organism.  Swab Sonication 16S rRNA gene cloning 16S rRNA gene amplicon sequencing Identification Synovial fluid RNA-seq culture Total RNA extraction RiboPureTM-Bacteria Kit Bioinformatics analysis Illumina paired-end sequencing Sequencing library preparation TruSeq® RNA Sample Preparation Bacterial rRNA removal MCIROBExpressTM Kit Human RNA removal MICROBEnrichTM Kit  Identification Monomicrobial infection: Staphylococcus aureus was identified by 3 independent methods (culture, 16S rRNA gene cloning, and 16S rRNA gene amplicon sequencing).   Mapping statistics Number of reads Number of paired reads Total reads 23.000.000 11.500.000 S. aureus reads1 270.000 (1.17%) 113.000  (0.98%) S. aureus mRNA reads 17.000 (0.074%) 7.000 (0.061%) 1Reference genome: S. aureus N315, 2.84 Mb, 2694 genes, 2613 proteins Top 100 genes (ordered by RPKM)  • 52 Essential genes  • 22 Hypothetical proteins  • 8 Infection related genes   Virulence factors:       SaeS Agr quorum sensing signal  +P SaeR Virulence genes Virulence regulation genes:  saeS and saeR   S. aureus cell membrane • hlgA, hlgB and hlgC : form pores in immune cells • Sbi: interferes with the host complement system    Vancomycin resistance sensor/regulator:  vraS and vraR  VraS Cell wall stress stimulon  +P VraR Cell wall biosynthesis S. aureus cell membrane Peptidoglycan biosynthesis Acknowledgement  The study is part of the Danish “Prosthesis-related Infection and Pain” innovation Project, supported by a grant from the Danish Agency of Science and Technology (no. 09-052174). www.joint-prosthesis-infection-pain.dk 

RNA-seq profiling of pathogens in prosthetic joint infection

    Aalborg UniversitetRNA-seqprofiling of pathogens in prosthetic joint infectionXu, Yijuan; Nielsen, Kåre Lehmann; Nielsen, Per Halkjær; Nielsen, Jeppe Lund; Thomsen,Trine RolighedPublication date:2013Document VersionEarly version, also known as pre-printLink to publication from Aalborg UniversityCitation for published version (APA):Xu, Y., Nielsen, K. L., Nielsen, P. H., Nielsen, J. L., & Thomsen, T. R. (2013). RNA-seqprofiling of pathogens inprosthetic joint infection. Abstract from The Danish Microbiological Society, Copenhagen, Denmark.http://www.infection-research.de/events/view/detail/event/danish_microbiological_society_2013/General rightsCopyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright ownersand it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights.            ? Users may download and print one copy of any publication from the public portal for the purpose of private study or research.            ? You may not further distribute the material or use it for any profit-making activity or commercial gain            ? You may freely distribute the URL identifying the publication in the public portal ?Take down policyIf you believe that this document breaches copyright please contact us at vbn@aub.aau.dk providing details, and we will remove access tothe work immediately and investigate your claim.Downloaded from vbn.aau.dk on: August 23, 2021RNA-seq profiling of pathogens in prosthetic joint infectionI n t r o d u c t i o n C o n c l u s i o n sThis study indicates that RNA-seqis a challenging but powerful tool to profile the gene expression of pathogens in vivo. R e s u l t sAimM e t h o d sTo study the gene expression of pathogens directly in PJI in a human host using RNA-seq.xuyijuan@bio.aau.dkXu, Yijuan1; Nielsen, Kåre L.1; Nielsen, Per H.1; Nielsen, Jeppe L.1; Thomsen, Trine R. 1,21: Center for Microbial Communities, Aalborg University, Denmark 2: Danish Technological Institute, DenmarkProsthetic joint infections (PJIs) are serious complications of joint alloplasties. These device-associated infections are typically caused by bacteria growing in biofilms, which protect them from antimicrobial agents and host immune responses in poorly vascularized joints. Our current knowledge of how pathogens cope with the complex conditions within the host is based on in vitro studies and animal models, which can differ substantially from their behavior in the human host. Studying gene expression of pathogens during infection is a way to understand pathogenesis. RNA sequencing (RNA-seq) is capable of characterizing the entire transcriptome, both quantitatively and qualitatively, of an organism.SwabSonication16S rRNAgene cloning16S rRNA gene amplicon sequencingIdentificationSynovial fluidRNA-seqcultureTotal RNA extractionRiboPureTM-Bacteria KitBioinformatics analysisIllumina paired-end sequencingSequencing library preparationTruSeq® RNA Sample PreparationBacterial rRNA removalMCIROBExpressTM KitHuman RNA removalMICROBEnrichTM KitIdentificationMonomicrobial infection: Staphylococcus aureus was identified by 3 independent methods (culture, 16S rRNAgene cloning, and 16S rRNA gene amplicon sequencing).Mapping statisticsNumber of reads Number of paired readsTotal reads 23.000.000 11.500.000S. aureus reads1 270.000 (1.17%) 113.000  (0.98%)S. aureus mRNA reads17.000 (0.074%) 7.000 (0.061%)1Reference genome: S. aureus N315, 2.84 Mb, 2694 genes, 2613 proteinsTop 100 genes (ordered by RPKM)• 52 Essential genes • 22 Hypothetical proteins • 8 Infection related genesVirulence factors: SaeSAgr quorum sensing signal+PSaeR Virulence genesVirulence regulation genes: saeS and saeRS. aureus cell membrane• hlgA, hlgB and hlgC : form pores in immune cells• Sbi: interferes with the host complement system   Vancomycin resistance sensor/regulator:vraS and vraRVraSCell wall stress stimulon+PVraR Cell wall biosynthesisS. aureus cell membranePeptidoglycan biosynthesisAcknowledgementThe study is part of the Danish “Prosthesis-related Infection and Pain”innovation Project, supported by a grant from the Danish Agency of Scienceand Technology (no. 09-052174).www.joint-prosthesis-infection-pain.dk

RNA-seqprofiling of pathogens in prosthetic joint infection

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RNA-seqprofiling of pathogens in prosthetic joint infection

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