Synthesis of the accessory gene regulator autoinducing peptide in Staphylococcus aureus PDF
Preview Synthesis of the accessory gene regulator autoinducing peptide in Staphylococcus aureus
University of Iowa Iowa Research Online Theses and Dissertations Spring 2012 Synthesis of the accessory gene regulator autoinducing peptide in Staphylococcus aureus Matthew James Thoendel University of Iowa Copyright 2012 Matthew James Thoendel This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/2999 Recommended Citation Thoendel, Matthew James. "Synthesis of the accessory gene regulator autoinducing peptide in Staphylococcus aureus." PhD (Doctor of Philosophy) thesis, University of Iowa, 2012. https://ir.uiowa.edu/etd/2999. Follow this and additional works at:https://ir.uiowa.edu/etd Part of theMicrobiology Commons SYNTHESIS OF THE ACCESSORY GENE REGULATOR AUTOINDUCING PEPTIDE IN STAPHYLOCOCCUS AUREUS by Matthew James Thoendel An Abstract Of a thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Microbiology in the Graduate College of The University of Iowa May 2012 Thesis Supervisor: Associate Professor Alexander R. Horswill 1 ABSTRACT The accessory gene regulator (agr) quorum-sensing system is one of the major regulators of virulence factor production in the pathogen Staphylococcus aureus. Activation of the system depends on the production and sensing of a cyclic peptide signal called the autoinducing peptide (AIP). The biosynthesis of AIP depends on the coordinated action of the AgrB integral membrane endopeptidase and SpsB signal peptidase to process the peptide precursor AgrD into the final signal structure. The primary goal of this dissertation was to gain further insight on the role of AgrD and AgrB in the AIP biosynthesis mechanism. Studies in Chapter II were undertaken to better understand the role of AgrD domains in AgrB-mediated processing. A series of truncation and site-directed mutagenesis studies identified key residues in the AgrD C-terminus that were essential for AgrB processing and AIP production. In parallel, genetic manipulation of the N- terminal leader and AIP-encoding sequence revealed a role for these segments in AIP processing. For the first time, a complex of AgrD covalently linked to AgrB was identified, supporting proposals that this intermediate is an important precursor to AIP production. In Chapter III structure-function studies were performed on AgrB to gain further insight into the AIP biosynthetic mechanism. Initially, the agrBD genes were subjected to random mutagenesis and screened for deficiencies in AIP production. Single-site mutations at 20 different residues within AgrB and another 14 in AgrD were isolated. Interestingly, new mutations in the AgrD N-terminal leader were identified that affect AIP biosynthesis at different steps. In AgrB, most of the mutations blocked peptidase activity, but charge alterations to the K129-K131 region were defective in a later pathway step, separating the peptidase function from AIP ring formation and transport. To localize the AgrB mutations, we reevaluated the membrane topology using the substituted 2 cysteine accessibility method. Our new model predicts four transmembrane helices and a reentrant loop, with both termini located outside of the cell. Finally, co- immunoprecipitation studies indicate that AgrB forms oligomeric structures within the membrane. Taken together, these findings provide a better understanding of the functional role of specific AgrD and AgrB regions in AIP biosynthesis. Abstract Approved: ____________________________________ Thesis Supervisor ____________________________________ Title and Department ____________________________________ Date SYNTHESIS OF THE ACCESSORY GENE REGULATOR AUTOINDUCING PEPTIDE IN STAPHYLOCOCCUS AUREUS by Matthew James Thoendel A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Microbiology in the Graduate College of The University of Iowa May 2012 Thesis Supervisor: Associate Professor Alexander R. Horswill Graduate College The University of Iowa Iowa City, Iowa CERTIFICATE OF APPROVAL _______________________ PH.D. THESIS _______________ This is to certify that the Ph.D. thesis of Matthew James Thoendel has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Microbiology at the May 2012 graduation. Thesis Committee: ___________________________________ Alexander R. Horswill, Thesis Supervisor ___________________________________ Timothy L. Yahr ___________________________________ Jerrold P. Weiss ___________________________________ John R. Kirby ___________________________________ Michael J. Welsh To all my family, friends and labmates who have stuck by my side and made this such an enjoyable process. ii Nothing is impossible! Not if you believe in it. That's what being a scientist is all about! Professor Farnsworth A Clone of My Own iii ACKNOWLEDGMENTS I would like to start by thanking Alex for his excellent mentorship during my time in the lab. His wealth of knowledge and willingness to let me pursue my interests has led to this being possible. I must also thank him for assembling the best possible group of lab members anybody could ever hope to work with. They have provided a fun and absolutely wonderful environment that I looked forward to every day. Whether it be lessons about science, rants about hemoglobin and hockey, stories of heaven, laughing at good jokes, shaking heads at bad ones or putting up with the mean ones, talks about cooking and brewing, the countless ice cream pies and chili, Mootzing things up, sharing homebrews in the evening, or lending an ear during a trip to Java House, I cannot imagine making it through these last few years without all of these. iv ABSTRACT The accessory gene regulator (agr) quorum-sensing system is one of the major regulators of virulence factor production in the pathogen Staphylococcus aureus. Activation of the system depends on the production and sensing of a cyclic peptide signal called the autoinducing peptide (AIP). The biosynthesis of AIP depends on the coordinated action of the AgrB integral membrane endopeptidase and SpsB signal peptidase to process the peptide precursor AgrD into the final signal structure. The primary goal of this dissertation was to gain further insight on the role of AgrD and AgrB in the AIP biosynthesis mechanism. Studies in Chapter II were undertaken to better understand the role of AgrD domains in AgrB-mediated processing. A series of truncation and site-directed mutagenesis studies identified key residues in the AgrD C-terminus that were essential for AgrB processing and AIP production. In parallel, genetic manipulation of the N- terminal leader and AIP-encoding sequence revealed a role for these segments in AIP processing. For the first time, a complex of AgrD covalently linked to AgrB was identified, supporting proposals that this intermediate is an important precursor to AIP production. In Chapter III structure-function studies were performed on AgrB to gain further insight into the AIP biosynthetic mechanism. Initially, the agrBD genes were subjected to random mutagenesis and screened for deficiencies in AIP production. Single-site mutations at 20 different residues within AgrB and another 14 in AgrD were isolated. Interestingly, new mutations in the AgrD N-terminal leader were identified that affect AIP biosynthesis at different steps. In AgrB, most of the mutations blocked peptidase activity, but charge alterations to the K129-K131 region were defective in a later pathway step, separating the peptidase function from AIP ring formation and transport. To localize the AgrB mutations, we reevaluated the membrane topology using the substituted v
Description:The list of books you might like
