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Type: Theses
Title: Staphylococcus aureus: stress response and its roles in pathogenesis
Author: Bui, Long Minh Giao
Issue Date: 2015
School/Discipline: School of Biological Sciences
Abstract: Staphylococcus aureus has an incredible ability to survive, either by adapting to environmental conditions or defending against exogenous stresses. In part this ability is provided by the breadth of lifestyles or modes of growth S. aureus can adopt. Key to an understanding of chronic, persistent and relapsing S. aureus infections is determining the basis for their switch to quasi-dormant lifestyles. Across different bacterial species these alternative lifestyles form a population known as persister cells. It has been proposed that while within their host, a sub-population of S. aureus survives in host-generated and therapeutic antimicrobial stress by inducing biofilm growth on host tissue or by growing as Small Colony Variants (SCVs). These stresses include limited nutrition, reactive oxygen species, reactive nitrogen species, other toxic metabolites, cationic peptides, fluctuating pH and osmolarity, several antibiotics, and others that are generated from various host tissues and niches during an infection. In a multicellular biofilm, the metabolically quiescent bacterial community produces a highly protective extracellular polymeric substance (EPS). The EPS is variously composed of polysaccharides, extracellular DNA (eDNA), and protein, and its protection results in persistent bacterial infections. S. aureus forms biofilms in different human tissues, and to some degree the associated EPS has been studied. Alternatively, there exists a diversity of phenotypes and cell-types that translate to a particular bacterial lifestyle. In clinical settings, SCV of S. aureus have been observed for many years and when cultured, these cells are non-pigmented colonies ca. 10 times smaller than their counterparts on agar plates. Mutations in hemin and menadione biosynthesis (hemB and menA) have produced laboratory-generated SCVs and these and other mutations have been studied extensively. The presence of gentamicin has also been shown to impede S. aureus metabolism and results in SCVs. Both methods result in stable forms of SCV but are artificially generated. Various genotypic factors (single nucleotide polymorphisms, mutations, gene deletions) have been identified to attempt to characterize S. aureus SCVs as well as environmental stresses are also considered to be important inducers. Our project was aimed to investigate the lifestyle switching of S. aureus clinical isolates as a stress response to environmental stresses that help these organisms to survive. The results showed that S. aureus clinical isolates had relatively similar growth rates but were different in their response to chemical stresses. There were specific strains that responded to stresses by changing their lifestyles to form a biofilm and/or SCVs in harsh conditions (but still sub-lethal levels of stress) but not in lower levels of stress. These results implied that phenotype switching depends on bacterial and host factors and suggests some specific strains may possess a unique pathway involved in surviving when stressed. In addition, studying native characteristics of SCV has been problematic due to their reversion to the parental, rapid growing lifestyle. Using specific host-representative, steady-state growth conditions with low nutrients and growth rates over a prolonged time with methylglyoxal - a naturally resident chemical that is found in the host-pathogen environment, we uniquely induced a S. aureus clinical isolate (WCH-SK2) into a stable SCV cell-type. The stable SCV phenotype did not revert after numerous cycles of subculturing and analysis revealed it possessed a metabolic and surface profile different from either previously described SCV or biofilm cells. The existence of the stable SCVs was verified and its features were analyzed by genomic, transcriptomic and surface protein profile studies. Stable SCVs produced an extracellular matrix of protein and extracellular DNA; but not polysaccharide. Compared to its parental cell-type, the stable SCV cells increased expression of certain surface proteins (such as Ebh; host extracellular matrix binding protein homologue) and lantibiotic synthesis while down-regulating factors that stimulate the host immune response (leukocidins, capsule, carotenoids). This cell-type is consistent with a lifestyle protected by a matrix and hidden from immune responses. Genome sequencing revealed a set of genetic changes from the parental to stable SCV cell-type, including the transcription factors RsbB and MrgA, as well as a change in the methylome. Collectively, our data shows that there is heterogeneity within a S. aureus population as shown by a diverse scope of cell-types; by growing the cells in conditions that resemble long-term survival in the host, colonization or persistence, we have identified a previously unnoticed S. aureus lifestyle. These stable SCV are molecularly distinct in nature to SCV or biofilm cells and this cell-type provides a new understanding of S. aureus persistence in the host.
Advisor: Kidd, Stephen Peter
Zilm, Peter Stephen
Morona, Renato
Dissertation Note: Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Biological Sciences, 2015.
Keywords: biofilms
small colony variants
stress response
Provenance: This electronic version is made publicly available by the University of Adelaide in accordance with its open access policy for student theses. Copyright in this thesis remains with the author. This thesis may incorporate third party material which has been used by the author pursuant to Fair Dealing exceptions. If you are the owner of any included third party copyright material you wish to be removed from this electronic version, please complete the take down form located at:
Copyright material removed from digital thesis. See print copy in University of Adelaide Library for full text.
Appears in Collections:Research Theses

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