Two-Component Signal Transduction System Saers Positively Regulates Staphylococcus Epidermidis Glucose Metabolism
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Hindawi Publishing Corporation e Scientific World Journal Volume 2014, Article ID 908121, 12 pages http://dx.doi.org/10.1155/2014/908121 Research Article Two-Component Signal Transduction System SaeRS Positively Regulates Staphylococcus epidermidis Glucose Metabolism Qiang Lou,1 Yijun Qi,1 Yuanfang Ma,1 and Di Qu2 1 Laboratory of Cellular and Molecular Immunology, Henan University, Kaifeng 475004, China 2 Key laboratory of Medical Molecular Virology of Ministry of Education and Ministry of Public Health, Institute of Medical Microbiology and Institutes of Biomedical Sciences, Shanghai Medical College of Fudan University, 138 Yixueyuan Road, Shanghai, 200032, China Correspondence should be addressed to Yuanfang Ma; [email protected] and Di Qu; [email protected] Received 30 August 2013; Accepted 21 November 2013; Published 23 January 2014 Academic Editors: D. Maiorano, H. Okamura, and M. Shiraishi Copyright © 2014 Qiang Lou et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Staphylococcus epidermidis, which is a causative pathogen of nosocomial infection, expresses its virulent traits such as biofilm and autolysis regulated by two-component signal transduction system SaeRS. In this study, we performed a proteomic analysis of differences in expression between the S. epidermidis 1457 wild-type and saeRS mutant to identify candidates regulated by saeRS using two-dimensional gel electrophoresis (2-DE) combined with matrix-assisted laser desorption/lonization mass spectrometry (MALDI-TOF-MS). Of 55 identified proteins that significantly differed in expression between the two strains, 15 were upregulated and 40 were downregulated. The downregulated proteins included enzymes related to glycolysis and TCA cycle, suggesting that glucose is not properly utilized in S. epidermidis when saeRS was deleted. The study will be helpful for treatment of S. epidermidis infection from the viewpoint of metabolic modulation dependent on two-component signal transduction system SaeRS. 1. Background Glucose can induce the S. aureus biofilm formation by inducing ica gene. The addition of glucose to growth media With all kinds of artificial medical materials commonly used, triggered biofilm formation in S. aureus strain SA113 [6], the opportunistic pathogen Staphylococcus epidermidis has increased the expression of genes and enzymes of the gly- become a major pathogen of nosocomial infection [1, 2]. S. colytic pathway, while genes and proteins of the tricarboxylic epidermidis pathogenesis is associated with its ability to form acid (TCA) cycle, required for the complete oxidation of biofilms on the surface of medical indwelling materials [3]. glucose, were repressed via catabolite control protein A The biofilms have the ability of resistance to antibiotics and (CcpA) [7]. Deletion of CcpA, which regulates gene expres- the host immune system. sion in response to the carbon source, abolished the capacity Biofilm formation proceeds in two distinct developmen- of SA113 to form a biofilm. Biofilm formation is closely tal phases: primary attachment of staphylococcal cells to a related to bacteria energy metabolism and cell wall synthesis polystyrene surface followed by bacterial accumulation in [5]. Disintegration of S. epidermidis biofilms under glucose- multiple layers. During the initial stage of biofilm formation, limiting conditions depends on the activity of the alternative extracellular DNA (eDNA) is critical for bacterial attachment sigmafactorsigmaB[8]. [4]. Extracellular DNA release from S. epidermidis is related Glycolysis that is the principal pathway of glucose met- to AtlE-mediated bacterial autolysis. During the bacterial abolism occurs in the bacterial cytoplasm where glucose is accumulation phase in S. epidermidis, biofilm formation oxidized to pyruvate (in aerobic condition) or lactate (in ana- is mediated by polysaccharide intercellular adhesin (PIA) erobic condition) and generates energy in the form of ATP. synthesized by icaADBC operon-encoded enzymes [5]. The accumulation of organic acids in the culture medium 2 The Scientific World Journal occurred coincident with a decreased pH. The pH change aliquots and the protein concentrations were determined by ∘ coincided with changes in cell viability, cell ATP, and the the Bradford assay. Then, aliquots were stored at −80 Cfor ability to produce acidity by glucose fermentation [9]. Glycer- further analysis. aldehyde 3-phosphate dehydrogenase is an essential enzyme that catalyzes the sixth step of glycolysis and thus serves to break down glucose for energy and carbon molecules 2.3. Two-Dimensional Gel Electrophoresis and Image Analysis. [10]. Glucose metabolism of Escherichia coli strain NP315 The 2-DE gels were performed using 24 cm IPG strips (pH fda (encoding fructose-1, 6-diphosphate aldolase) and gnd 4–7, GE Healthcare) in Ettan IPGphor isoelectric focusing (encoding 6-phosphogluconate dehydrogenase) mutant was system (Amersham Biosciences) plus Ettan-Dalt Six system decreased [11]. (Amersham Biosciences) according to the manufacturer’s Staphylococcal biofilm formation is a complicated pro- instructions. To compensate the variability of gel elec- cess that is regulated by two-component signal transduction trophoresis, at least three replicate gels were performed for each group. In the first dimensional isoelectric focusing (IEF), systems (TCSs) [12, 13]. Prokaryotic TCSs modulate gene expression in response to different environmental signals 120 g proteins of each sample were diluted to 450 Lwith [14]. SaeRS TCS was originally identified in Staphylococcus rehydration buffer containing 8 M urea, 2% (w/v) CHAPS, aureus as a transposon mutant system deficient in the synthe- 50 mM DTT, and 0.5% (v/v) ampholyte (pH 4–7, Amersham sis of several exoproteins, including -and-hemolysin and Biosciences), and IPG strips were allowed to rehydrate in coagulase [15]. In S. aureus COL, proteomic approach showed the above solution under mineral oil. IEF was performed that extracellular proteins were influenced by a mutation in as follows: 30 V for 6 h (active rehydration), 60 V for 6 h saeS [16]. In a previous study of the S. epidermidis SaeRS (active rehydration), 500 V for 2 h, rapid, 1,000 V for 2 h, TCS, a saeR deletion mutant exhibited a lower anaerobic rapid, 4,000 V for 2 h, linear, linear ramping to 8,000 V for 2h,andfinally8,000Vforabout7hwithatotalof64KVh growth rate, a significantly reduced rate of nitrate utilization, ∘ and a slightly higher biofilm-forming ability compared to the at 20 C. Then the IPG strips were incubated in equilibration parental strain [17]. In S. epidermidis, the TCS SaeRS is known buffer (75 mM Tris-HCl (pH 8.8), 6 M urea, 29.3% (v/v) to be involved in biofilm formation. Furthermore, SaeRS was glycerol, 2% (w/v) SDS, and 0.002% (w/v) bromophenol blue) foundtobeinvolvedinS. epidermidis autolysis, resulting containing 2% (w/v) DTT for 15min with gentle agitation, from altered bacterial eDNA release [12]. followed by incubation in the same equilibration buffer In the present study, the influence of the TCS SaeRS on supplemented with 2.5% (w/v) iodoacetamide for 15 min at global gene expression in S. epidermidis was investigated by room temperature. The second dimension SDS-PAGE was proteomic approach. SaeRS positively regulates the proteins performed on 1 mm thick 12.5% polyacrylamide vertical gels in Ettan-Dalt Six system using 5 W/gel for 30 min and expression involved in glucose metabolism. Furthermore, we ∘ show that glucose metabolism by decreasing the pH value followed by 12 W/gel at 10 Cuntilthebromophenolblue affects biofilm-forming ability and bacterial cell viability. dye front reached the end of the gels. The gels were stained by a modified silver staining method compatible with MS analysis and scanned at 300 dpi (dots/inch) using ImageScan- 2. Materials and Methods ner (UMAX, Amersham Biosciences). Images were captured andanalyzedbyImageMaster2Dplatinum6.0software 2.1. Bacterial Strains, Plasmids, and Media. The bacterial str- (Amersham Biosciences). The percentage of the volume of ains used in this study are S. epidermidis wild-type (WT), S. the spots representing a certain protein was determined in epidermidis saeRS mutant (SAE), and S. epidermidis saeRS co- comparison with the total proteins present in the 2-DE gel. mplemental strain (SAEC). S. epidermidis cells were grown at ∘ To select differentially expressed protein spots, quantitative 37 C in BM medium (Tryptone 10 g, Yeast extract 5 g, NaCl analysis was performed using the Student’s t-test to compare 5g,K2HPO4 1 g, and Glucose 1 g/L medium) or tryptic soy br- the percentage volumes of spots between SAE and WT oth (TSB) (Oxiod, Basingstoke, Hampshire, England) supple- groups. The differentially expressed protein spots with mented with antibiotics when necessary. Spectinomycin (spc) values less than 0.05 were considered significant differences, was used at 300 g/mL for S. epidermidis. and at least 1.5-fold difference in percentage of the volume for each spot was set as a threshold. These protein spots 2.2. Sample Preparation for Two-Dimensional Gel Electropho- were selected and subjected to in-gel tryptic digestion and resis (2-DE). SAE and WT were grown until the exponential identification by MS. phase (OD600 = 1.6) in TSB medium and the bacteria (7.56 × 9 10 cells of each) were harvested by centrifugation at 7,000 g 2.4. In-Gel Tryptic