The Study of Stress Conditions on Growth and Proteome of Raoultella Planticola: A New Emerging Pathogen

All bacteria can survive and adapt to different stresses such as fluctuations in temperature, pH oxidative, and osmotic pressure occurring in their surrounding environments. This study aims to evaluate the effects of a variety of stress conditions on the growth, and proteome of Raoultella planticola PTCC 1598. R. planticola cells were exposed to different values of temperatures, sodium chloride, pH, and hydrogen peroxide stresses. Amongst the stress conditions, oxidative stress upon exposure to hydrogen peroxide (H2O2) at 4000 ppm concentration was selected for proteomics analysis in detail. Approximately 1400 spots were identified in two-dimensional gel electrophoresis (2-DE). Among the identified spots, 85 spots were repeatable using 2D-Platinum software and eye confirmation and, nine protein spots were differentially expressed. Among nine proteins, six proteins identified successfully with a MASCOT score greater than 40 (p<0.05) were 2, 3-dihydroxybenzoate-2, 3-dehydrogenase (oxidoreductase family), D-galactose-binding periplasmic protein, uridine phosphorylase (glycosyltransferases), uridine phosphorylase, a single peptide match to cysteine-binding periplasmic protein, and NADP(H) nitroreductase. All identified proteins showed decreased level expression. Based on the obtained results, we concluded that hydrogen peroxide as an antiseptic compound could affect cell growth and proteomics of R.planticola. So, we recommend using an antiseptic solution containing H2O2 to prevent the spread of R.planticola as a new emerging pathogen.

Score Model Based on Forward-Reverse Databases for Phosphopeptides Identification

Identification of phosphorylated peptides takes into account factors relevant in matching, building models, and score algorithms. In the paper we make a detailed comparative analysis among various phosphorylation identification methods, and study current mainstream algorithms in database searching and identification, and compares various aspects and methods of algorithms in site assessment. Based on the theory of forward-reverse databases searching, It is proposed a new score model to ensure the quality of identification. Our result shows that PTM and Mascot score models were strongly correlated and complementated in their differentiation abilities. Therefore, PTM and Mascot score models can be combined to filter peptide.

Indole-3-Glycerol-Phosphate Synthase Is Recognized by a Cold-Inducible Group II Chaperonin in Thermococcus kodakarensis

ABSTRACTThermococcus kodakarensisoptimally grows at 85°C and possesses two chaperonins, cold-inducible CpkA and heat-inducible CpkB. Gene disruptants DA1 (ΔcpkA) and DB1 (ΔcpkB) showed decreased cell growth at 60°C and 93°C, respectively. The DB2 mutant (ΔcpkA∷cpkBΔcpkB), whosecpkBgene was expressed under the control of thecpkApromoter, did not grow at 60°C, and the DB3 mutant [ΔcpkA(1–524)∷cpkB(1–524) ΔcpkB], whose CpkA amino acid residues 1 to 524 were replaced with corresponding CpkB residues that maintained the C-terminal region intact, grew at 60°C, implying that the CpkA C-terminal region plays a key role in cell growth at 60°C. To screen for specific CpkA target proteins, comparative pulldown studies with anti-Cpk were performed using cytoplasmic fractions from DA1 cells cultivated at 93°C and DB1 cells cultivated at 60°C. Among the proteins coprecipitated with anti-Cpk, TK0252, encoding indole-3-glycerol-phosphate synthase (TrpC), showed the highest Mascot score. Counter-pulldown experiments were also performed on DA1 and DB1 extracts using anti-TrpC. CpkA coimmunoprecipitated with anti-TrpC while CpkB did not. The results obtained indicate that TrpC is a specific target for CpkA. The effects of Cpks on denatured TrpC were then examined. The refolding of partially denatured TrpC was accelerated by the addition of CpkA but not by adding CpkB. DA1 cells grew optimally in minimal medium only in the presence of tryptophan but hardly grew in the absence of tryptophan at 60°C. It has been suggested that a lesion of functional TrpC is caused bycpkAdisruption, resulting in tryptophan auxotrophy.

Proteomics Used To Identify the Target of a Platelet Aggregating Antibody in a Patient with HUS.

Hemolytic Uremic Syndrome (HUS) has considerable overlap with Thrombotic Thrombocytopenia Purpura (TTP). Recently, evidence is emerging to suggest that TTP, like HIT, is an immune mediated disorder. We have investigated the plasmas of patients with Hemolytic Uremic Syndrome to determine the presence of antibodies and the specificity of the related antigens. Methods: A 20 year old female presented with repeated episodes of HUS. She is dialysis dependent. Plasma was taken for VWF, multimers, platelet aggregation, metalloprotease and inhibitor, western blot and 2D PAGE analysis. Results: At the time of the acute episodes, the VWF was increased three fold; the VWF multimer patterns on two occasions showed the presence of unusually large VWF forms. The metalloprotease was normal at all times and no inhibitor was present. Her platelets showed a normal response to aggregation, however, the plasma caused spontaneous aggregation of normal washed human platelets. This reactivity was removed after absorption with Staph A. On western blot analysis of platelet and microvascular endothelial cell lysate vs. patient plasma, multiple reactions were detected with a strong and persistent reactivity against antigens of 200,00 kDa and 55 kDa. Two dimensional PAGE of the whole platelet proteome identified strong immunoreactivity with two target spots in the 55 kDa area. Mass spectroscopy, using the nano-LC-MS/MS analysis, with the MASCOT search program confirmed the target antigen as beta fibrin with a molecular weight of 50.731 and an isoelectric point of 7.95 with a MASCOT score of 859 and 750 respectively. Conclusions: Anti-platelet antibodies which cross-react with platelet and microvascular endothelial cells are present in the plasma of patients with recurrent episodes of HUS. These antibodies cause aggregation of normal human platelets and are removed following specific absorption of IgG from the plasma. 2D proteomic analysis indicates that the antibody in our patient was directed against a specific antigen, B fibrin. The data suggests that HUS, like TTP and HIT has an immunological basis.