Effect of Altered Sulfur Metabolism on the Growth and Gene Expression of Staphylococcus aureus strain RN 4220.

摘要

The requirement of sulfur as an essential bio-element for various cellular processes has been established for all cell-based forms of life. The sulfur-containing amino acid cysteine is an essential pre-requisite for protein synthesis and the active source of several essential metabolites (e.g., coenzyme A). Sulfur metabolism is an important process to analyze in a common human pathogen, such as Staphylococcus aureus because direct links between sulfur uptake and expression of virulence factors have been reported in recent literature. Defining how the microbe's metabolism responds to manipulation of sulfur sources (and analogs) should provide important predictive information for developing and evaluating hypothetical models of the host /parasite infection system.;The aims of this project were to study changes in growth pattern and gene expression of Staphylococcus aureus strain RN 4220 in response to: (i) the absence of a sulfur source, (ii) changes in metabolic sulfur sources, and (iii) the addition of non-metabolic sulfur sources i.e. sulfur-analogs. In order to evaluate biochemical compounds as potential sulfur sources (or otherwise), a minimal defined medium previously developed was utilized. This defined medium contained Cysteine, Cystine or Glutathione, as supported sulfur sources. A null medium (devoid of any metabolizable sulfur source) was used to test stress due to sulfur limitation/manipulation. By supplementing this null medium with each of the supported sulfur sources along with one each of the following sulfur source analogs known to occur in nature: (i) Cysteine sulfinic acid, (ii) Cysteic acid, (iii) Cysteamine, (iv) 2-mercaptoethanol, and (v) Taurine, the lack of nutritive effect of these analogs on the microbe's growth was demonstrated, and their ability to inhibit utilization of known sulfur sources was tested. Quantitative polymerase chain reaction (qPCR) was then used to analyze the transcriptomic effects of the defined stress conditions to identify genes for which transcription was induced, repressed or not changed by sulfur limitation/manipulation. Observed results on gene expression may help define how this microbe's metabolism responds to the manipulation of sulfur nutrition sources and to the presence of naturally occurring sulfur source analogs. Genes whose protein products are involved in glycolysis (Glucose-6-phosphate isomerase), virulence, and biofilm production (Ica R) were selected for initial analysis by qPCR.;Co-supplementations of the null sulfur medium with Cysteine and Glutathione as sulfur source, along with the sulfur source analogs Cysteine sulfinic acid and Cysteic acid respectively, resulted in a statistically significant reduction of microbial growth over 4 days post supplementation. A preliminary study of the effects of these source-analog test systems at the transcriptome level revealed a strong down-regulation in the Ica R gene involved in the regulation of biofilm synthesis. This result has potential clinical significance because biofilm formation is an important adaptive tool for S. aureus to evade the host's defenses and has also been associated with enhanced virulence. Taken together, these studies showed that the manipulation of sulfur assimilation has a direct effect on the growth, metabolism and gene expression of S. aureus. These conclusions can help in the development of strategic therapies against this common pathogen through the elucidation of metabolic responses to sulfur stress, the ability of sulfur source analogs to influence expression of specific genes involved in the microbe's virulence and host colonization potential.