Development of comparative secretomic analyses for discovering Streptococcus pyogenes novel virulence factors

  • 温 耀增

Student thesis: Doctoral Thesis


Streptococcus pyogenes is an important human pathogen Numerous virulence factors secreted by S pyogenes interact directly a with host tissue to cause various diseases However how S pyogenes manipulate its secretome in response to different infected environments remains unclear Herein we proposed gel-based and gel-free quantitative proteomic analyses for identification and quantification of S pyogenes novel secreted virulence factors In the gel-based quantitative proteomic study we focus on an important peroxide regulator (PerR) which is associated with the peroxide resistance response and pathogenesis But it is little known about its regulation of the secretome involved in virulence To investigate how PerR regulates the expression of the S pyogenes secretome involved in virulence a perR deficient mutant was used for comparative secretomics analysis with a wild-type strain The conditioned medium (CM) containing secreted proteins of a wild-type strain and a perR deficient mutant at the stationary phase were collected for 2DGE analysis Differentially-expressed protein spots were identified by LC-ESI-MS/MS After further functional analyses we found a novel PerR-regulated extracellular virulence factor mitogen factor3 (MF3) In gel-free proteomic study we investigated the influence of environmental acidification on S pyogenes secretome To investigate temporal secretome changes under acidic environment a comparative secretomics approach using label-free LC-MS/MS was undertaken to analyze the secretome in acidic and neutral conditions The dynamic label-free LC-MS/MS profiling identified 45 predicted secreted proteins which are influenced by environmental acidification Mga and RofA/Nra were found that can respond to environmental acidification and growth-phase cues In addition we provided the first evidence that acidic stimuli and growth-phase cues are crucial for classical protein secretion in S pyogenes The protein-protein interaction data indicate that acid stimuli influence nutrient uptake protein synthesis and pilus formation There are 21 pathogenesis-related secreted proteins influencing immune evasion hemolysis adhesion tissue damage and nutrient acquisition In this study we demonstrated that an acid-induced secreted protein is a novel virulence factor and is involved in invasion and immune evasion We believe that our investigation provides vital information for documenting the broad influences and mechanisms underlying the invasive behavior of group A streptococci in an acidified environment In this dissertation I developed two different secretomic analytical approaches to discover novel virulence factors in S pyogenes It is efficient to find novel and important virulence factors for realizing the other new mechanisms of pathogenesis Besides these proteomic data might lead to the development of chemotherapeutic strategies to prevent or treat severe streptococcal diseases
Date of Award2014 Jun 26
Original languageEnglish
SupervisorPao-Chi Liao (Supervisor)

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