Staphylococcus aureus is a remarkably pathogenic bacterium that is widely prevalent among the human population. It is the leading agent of skin and soft tissue infections, and is also responsible for causing an array of severe and life threatening diseases. The invasiveness of the pathogen, coupled with increasing antibiotic resistance seen for S. aureus infections, makes this bacterium a prominent public health concern. The extended pathogenicity of S. aureus is largely due to its repertoire of virulence factors, which are typically characterized by being bound to the cell wall, or secreted into the extracellular environment. Previously, our lab identified a leucine aminopeptidase mutant (pepZ) as being strongly attenuated in virulence for both systemic and localized models of infection. Importantly, PepZ marked the first intracellular bacterial aminopeptidase found to be involved in pathogenesis in Gram-positive bacteria. In this study, we set out to explore the role of the remaining eleven, non-essential, uncharacterized aminopeptidase enzymes in S. aureus disease causation, and present two additional aminopeptidase genes, pepT1 and pepT2, which are important for virulence in both human ex vivo models, and murine in vivo models of disease. Interestingly, these enzymes do not appear to be necessary for the utilization of free peptides for cellular nutrition and metabolism, which is a typical characteristic of aminopeptidases. Transcriptional analysis reveals maximal expression of pepT1 and pepT2 during early exponential growth phase, while localization mapping demonstrates that the PepT1 and PepT2 enzymes are found in the bacterial cytoplasm during all stages of growth. To explore these findings on a global level, an in-depth proteomic investigation of cleavage properties and cellular substrates identified several proteins as having significant changes in N-terminal peptide abundance in pepT1 and pepT2 mutant proteomes. We identified a number of putative independent and shared targets for the PepT1 and PepT2 enzymes that are known to impact cellular fitness and pathogenesis, including: DnaK, a heat shock protein conserved throughout all organisms, which functions to help deal with mis-folded and aggregated proteins that have accumulated as a result of cellular stress; ArlR, the response regulator of the ArlRS two-component system, which is an important regulator of agglutination and virulence in S. aureus; and of special interest, ClpC, an ATP-dependent protease chaperone which is a component of the primary machinery by which protein degradation occurs in S. aureus. Collectively, our data proves the importance of the PepT aminopeptidase enzymes in S. aureus pathogenesis, and indicates these enzymes are likely involved in bacterial stress response and virulence by functioning through the bioactivation/inactivation of key cellular proteins.

京都大学 / 新制・課程博士 / 博士(薬科学) / 甲第23135号 / 薬科博第134号 / 新制||薬科||15(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 石濱 泰, 教授 松﨑 勝巳, 教授 加藤 博章 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

La thématique des maladies infectieuses représente un réel enjeu politique, économique et de santé publique. Les objectifs de mes travaux de thèse étaient de développer des approches protéomiques pour identifier, détecter, caractériser et/ou quantifier des protéines et de les appliquer à l’étude de maladies infectieuses pour lesquelles des données de protéomique pourraient ouvrir à de nouvelles approches thérapeutiques et/ou diagnostiques. Les stratégies d’identification et de détection des protéines développées pour l’étude de la maladie de Lyme ont permis de prouver la faisabilité du diagnostic par spectrométrie de masse et de proposer des protéines candidates-vaccin. Les stratégies de quantification mises en place pour l’étude de la toxoplasmose ont permis d’identifier et de quantifier un complexe protéique clef. Les stratégies de caractérisation du N-terminome pour l’étude du paludisme ont permis d’apporter des preuves expérimentales des processus de maturation et d’adressage des protéines. / Infectious diseases represent a real challenge in terms of politic, economic and public health. The purposes of my thesis works were to develop proteomic approaches able to identify, to detect, to characterize and/or to quantify proteins and to apply these approaches to the study of infectious diseases for which proteomic data cou Id open to new therapeutic and/or diagnostic strategies. Identification and specific detection strategies developed in the study of Lyme's disease allowed to prove the feasibility of diagnosis by mass spectrometry and to propose new vaccine-candidate proteins. Quantification strategies developed in the study of toxoplasmosis allowed us to identify and to quantify a key protein complex of the parasite. N-terminome characterization strategy developed in the study of malaria allowed us to bring experimental proofs of proteins processing and trafficking.