Function Of Two-Component System And LuxS/Al-2Quorum Sensing In Staphylococcus Aureus

Bacteria have a variety of signal transduction pathways which allow bacteria to respond to different environmental changes and stimuli. Two-component system （TCS） mainly functions as a basic stimulus-response mechanism that allows bacteria to sense and respond to changes in many different environmental conditions. A typical TCS is composed of two proteins:a sensor histidine kinase, which acts as a sensor of stimuli, and a cognate response regulator, which modulates gene expression. TCS widely occures in prokaryotes, sensing variety of environmental signals and playing a very important role in regulating bacterial physiology.Quorum sensing is a system of stimulus and response correlated to population density and modulates expression of certain genes when bacteria reach high density in order to adjust bacterial life pattern. The discovery of the phenomenon of quorum sensing made people realize that bacteria do not act alone. Instead, there are mechanisms for bacteria to communicate and cooperates at the population level. Quorum sensing, as a common signal pathway in bacteria, regulates various physiological activity to coordinate bacteria to change their life pattern in between low and high population density.Staphylococcus aureus is a common opportunistic pathogen that widely exists in the environment as well as in human body. When confronted with hypo immunity, injure, surgery, and foods contaminated by S. aureus, S. aureus can cause various diseases including abscesses, bacteremia, myelitis, and endocarditis, some of which are lethal. The abuse of use of antibiotics, has led to the emergence and spread of antibiotic resistant S. aureus. It’s more and more difficult to deal S. aureus infection with traditional antibiotics. The S. aureus virulence factors are under strict control of many regulators responding to environmental changes. Our study has focused on the signal transduction pathway of S. aureus, hoping to reveal new regulatory mechanisms and to find clues in the treatment of S. aureus infection.This paper has two parts of study.1. Functional study of TCSs in S. aureusThere are16TCSs in S. aureus and some of them remain to be characterized. We first deleted these TCSs by homologous recombination, and then compared the expression profile between the mutant and the wild-type, then analyze series of phenotype to find downstream targets, and finally tested if the regulators directly binds to targets genes’promoters by electrophoretic mobility shift assay （EMSA）.Following this plan, we have found that the TCS in S. aureus NCTC8325（SAOUHSC₀0184-185, which we defined as UhpAB） is able to sense extracellular glucose-6-phosphate and activate a glucose-6-phosphate transporter UhpT to import glucose-6-phosphate as a carbon source. In a medium which glucose-6-phosphate is the main carbon source, the UhpAB mutant showed growth defect. The extracellular glucose-6-phosphate can induce expression of UhpT in wild-type but not in UhpAB mutant. EMS A showed binding of UhpA to uhpT promoter, suggesting that UhpAB direct regulates uhpT. All these experiments suggest UhpAB is a important regulator of glucose-6-phosphate metabolism in S. aureus.And we found that another TCS, AirSR is related to cell wall metabolism. More than20genes that are related to cell wall metabolism were down-regulated in the airSR mutant. These genes include pbp1, ddl, lytM, and cap. EMS A showed that AirR can bind to the promoter of these four genes. Due to the attenuation of cell wall metabolism, airSR mutant are more sensitive to vancomycin and has a slow autolysis rate. These finding suggest that AirSR is a regulator which enhances cell wall synthesis and degradation.2. Investigation of the AI-2receptor in S. aureusAI-2is synthesized by the LuxS which exists in many bacteria, and many bacteria can produce AI-2. AI-2quorum sensing system is considered to be a interspecies communication mechanism for bacteria. Our previous study found that in S. aureus, the LuxS/AI-2system can regulate biofilm formation, capsular polysaccharide synthesis, and antibiotic resistance, indicating that in S. aureus the LuxS/AI-2system is functional. We are aiming to find the AI-2receptor, in order to reveal more details of this signaling pathway. We carried out a series of in vitro experiments of the suspected receptor SaaR, which could possibly bind to AI-2, but have not got a positive result. However, we did find that SaaR is in the downstream of the signal pathway in which LuxS/AI-2system regulates PIA depended biofilm. We are still searching for the missing parts between AI-2and SaaR.Through these studies, we have obtained a better understanding of S. aureus signal regulatory networks, In particular, we filled the blank of functions of some TCSs. As for the LuxS/AI-2system, we known more of its pathway, but the receptor of AI-2needs further study.