Proteome analysis of membrane and cell wall associated proteins from Staphylococcus aureus

Pathogenesis of Staphylococcus aureus, an opportunistic human pathogen, is complex and involves many virulence factors including an array of surface proteins (adhesins) that promote bacterial interactions with extracellular matrix components. A better understanding of these interactions can be achieved by studying the expression of membrane and cell wall associated proteins using a proteome analysis approach. To accomplish this, our goal here was to construct a reference map of membrane and cell wall associated proteins for S. aureus. Various lytic and solubilization methods have been tested to identify a suitable methodology for detection of these proteins in two-dimensional electrophoresis (2DE). Results demonstrate that cell lysis with lysostaphin, which lyses staphylococcal peptidoglycan, followed by solubilization with urea, thiourea, amidosulfobetaine 14 (ASB 14) and dithiothreitol (DTT) is an effective method, yielding a sample comprising proteins of wide molecular ranges and isoelectric points with minimum contamination from cytosolic proteins. Mass spectrometric analysis was employed to identify the membrane and cell surface proteins present in the sample and consequently an initial proteomic map of membrane and cell wall associated proteins for S. aureus is presented.     

Purification and characterization of an extracellular alpha-glucosidase protein from Trichoderma viride which degrades a phytotoxin associated with sheath blight disease in rice

AIMS: To purify and characterize an extracellular alpha-glucosidase from Trichoderma viride capable of inactivating a host-specific phytotoxin, designated RS toxin, produced by the rice sheath blight pathogen, Rhizoctonia solani Kühn. METHODS AND RESULTS: The host-specific RS toxin was purified from both culture filtrates (culture filtrate toxin, CFTox) and R. solani-inoculated rice sheaths (sheath blight toxin, SBTox). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis analyses of extracellular proteins, purified from a biocontrol fungus T. viride (TvMNT7) grown on SBTox and CFTox separately, were carried out. The antifungal activity of the purified high molecular weight protein (110 kDa) was studied against RS toxin as well as on the sclerotial germination and mycelial growth of R. solani. Enzyme assay and Western blot analysis with the antirabbit TvMNT7 110-kDa protein indicated that the protein was an alpha-glucosidase. The 110-kDa protein was highly specific to RS toxin and its Michaelis-Menten constant value was 0.40 mmol l-1 when p-nitrophenyl alpha-D-glucopyranoside was used as the substrate. The isoelectric point of the protein was 5.2. N-terminal sequencing of the alpha-glucosidase protein showed that its amino acid sequence showed no homology with other known alpha-glucosidases. CONCLUSION: This appears to be the first report of the purification and characterization of an alpha-glucosidase capable of inactivating a host-specific toxin of fungal origin. The alpha-glucosidase is specific to RS toxin and is different from the known alpha-glucosidases. SIGNIFICANCE AND IMPACT OF THE STUDY: As RS toxin could be inactivated by the microbial alpha-glucosidase enzyme, isolation of the gene that codes for the enzyme from T. viride and transfer of the gene to rice plants would lead to enhanced resistance against sheath blight pathogen by inactivation of RS toxin.     

Inhibition of Telomerase Recruitment and Cancer Cell Death

Continued proliferation of human cells requires maintenance of telomere length, usually accomplished by telomerase. Telomerase is recruited to chromosome ends by interaction with a patch of amino acids (the TEL patch, for TPP1 glutamate (E) and leucine (L)-rich patch) on the surface of telomere protein TPP1. In previous studies, interruption of this interaction by mutation prevented telomere extension in HeLa cells, but the cell culture continued to grow. We now show that the telomerase inhibitor BIBR1532 acts together with TEL patch mutations to inhibit the growth of HeLa cell lines and that apoptosis is a prominent mechanism of death of these cells. Survivor cells take over the population beginning around 40 days in culture. These cells no longer express the TEL patch mutant TPP1, apparently because of silencing of the expression cassette, a survival mechanism that would not be available to cancer cells. These results provide hope that inhibiting the binding of telomerase to the TEL patch of TPP1, perhaps together with a modest inhibition of the telomerase enzyme, could comprise an effective anticancer therapy for the ∼90% of human tumors that are telomerase-positive.     

PD-1 modulates regulatory T cells and suppresses T-cell responses in HCV-associated lymphoma

T regulatory (T(R)) cells suppress T-cell responses that are critical in the development of chronic viral infection and associated malignancies. Programmed death-1 (PD-1) also has a pivotal role in regulation of T-cell functions during chronic viral infection. To examine the role of PD-1 pathway in regulating T(R)-cell functions that inhibit T-cell responses during virus-associated malignancy, T(R) cells were investigated in the setting of hepatitis C virus-associated lymphoma (HCV-L), non-HCV-associated lymphoma (non-HCV-L), HCV infection alone and healthy subjects (HS). Relatively high numbers of CD4(+)CD25(+) and CD8(+)CD25(+) T(R) cells, as well as high levels of PD-1 expressions on these T(R) cells were found in the peripheral blood of subjects with HCV-L compared with those from non-HCV-L or HCV alone or HS. T(R) cells from the HCV-L subjects were capable of suppressing the autogeneic lymphocyte response, and depletion of T(R) cells in peripheral blood mononuclear cells from HCV-L improved T-cell proliferation. Additionally, the suppressed T-cell activation and proliferation in HCV-L was partially restored by blocking the PD-1 pathway ex vivo, resulting in both a reduction in T(R)-cell number and the ability of T(R) to suppress the activity of effector T cells. This study suggests that the PD-1 pathway is involved in regulating T(R) cells that suppress T-cell functions in the setting of HCV-associated B-cell lymphoma.     

Dramatic accumulation of triglycerides and precipitation of cardiac hemodynamic dysfunction during brief caloric restriction in transgenic myocardium expressing human calcium-independent phospholipase A2gamma

Previously, we identified calcium-independent phospholipase A2gamma (iPLA2gamma) with multiple translation initiation sites and dual mitochondrial and peroxisomal localization motifs. To determine the role of iPLA2gamma in integrating lipid and energy metabolism, we generated transgenic mice containing the alpha-myosin heavy chain promoter (alphaMHC) placed proximally to the human iPLA2gamma coding sequence that resulted in cardiac myocyte-restricted expression of iPLA2gamma (TGiPLA2gamma). TGiPLA2gamma mice possessed multiple phenotypes including: 1) a dramatic approximately 35% reduction in myocardial phospholipid mass in both the fed and mildly fasted states; 2) a marked accumulation of triglycerides during brief caloric restriction that represented 50% of total myocardial lipid mass; and 3) acute fasting-induced hemodynamic dysfunction. Biochemical characterization of the TGiPLA2gamma protein expressed in cardiac myocytes demonstrated over 25 distinct isoforms by two-dimensional SDS-PAGE Western analysis. Immunohistochemistry identified iPLA2gamma in the peroxisomal and mitochondrial compartments in both wild type and transgenic myocardium. Electron microscopy revealed the presence of loosely packed and disorganized mitochondrial cristae in TGiPLA2gamma mice that were accompanied by defects in mitochondrial function. Moreover, markedly elevated levels of 1-hydroxyl-2-arachidonoyl-sn-glycero-3-phosphocholine and 1-hydroxyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine were prominent in the TGiPLA2gamma myocardium identifying the production of signaling metabolites by this enzyme in vivo. Collectively, these results identified the participation of iPLA2gamma in the remarkable lipid plasticity of myocardium, its role in generating signaling metabolites, and its prominent effects in modulating energy storage and utilization in myocardium in different metabolic contexts.     

Proteomics of filamentous fungi

Proteomic analysis, defined here as the global assessment of cellular proteins expressed in a particular biological state, is a powerful tool that can provide a systematic understanding of events at the molecular level. Proteomic studies of filamentous fungi have only recently begun to appear in the literature, despite the prevalence of these organisms in the biotechnology industry, and their importance as both human and plant pathogens. Here, we review recent publications that have used a proteomic approach to develop a better understanding of filamentous fungi, highlighting sample preparation methods and whole-cell cytoplasmic proteomics, as well as subproteomics of cell envelope, mitochondrial and secreted proteins.     

Marginal bone loss around axial and straight implants supported with prefabricated SFI-Bar with mandibular overdentures

To assess the role of prefabricated SFI-Bar in peri-implant bone loss around immediately axially loaded and straight implants. This study comprised of 40 complete denture wearer patients who received two axially parallel implants connected by SFI-Bars in group I and two 15° mesially tilted implants connected by SFI-Bars in group II. Peri- implant bone loss (PiBL) was measured at 1 year, 2 years and 3 years. The mean PiBL at 1 year in group I was 0.21 mm and I group II was 0.22, at 2 years in group I was 0.26 mm and in group II was 0.23 mm and at 3 years, in group I was 0.29 mm and in group II was 0.34 mm. The difference was significant at 3 years (P< 0.05). The mean mesial PIBL at 1 year in group I was 0.18 mm, in group II was 0.20 mm, at 2 years in group I was 0.19 mm and in group II was 0.07 mm and at 3 years, in group I was 0.25 mm and in group II was 0.29 mm. The difference found to be significant in each time duration in both groups (P< 0.05).The mean distal PIBL at 1 year in group I was 0.23 mm, in group II was 0.22 mm, at 2 years in group I was 0.33 mm and in group II was 0.39 mm and at 3 years, in group I was 0.34 mm and in group II was 0.39 mm. The difference found to be significant at 2 and 3 years in both groups (P< 0.05). Authors found that mandibular overdentures retained with Prefabricated SFI-Bar with axial and straight inserted implants may be useful in patients with reduced bone height.     

Overlapping and distinct roles of GRK5 in TLR2-, and TLR3-induced inflammatory response in vivo

G-protein coupled receptor kinase-5 (GRK5) is a recently described NFκB regulator in TLR4 signaling pathway. To determine whether the role of GRK5 is MyD88- or TRIF-dependent, we injected wild type and GRK5 knockout mice with Pam3CSK4 (MyD88-dependent TLR1/2 ligand) and Poly(I:C) (TRIF-dependent TLR3 ligand) and examined the in vivo systemic inflammatory response. Our results demonstrate that GRK5 regulates IL-12p40 and G-CSF via a mechanism that is common to both MyD88 and TRIF. However, GRK5 regulates IL-5 and MCP-1 in a MyD88-dependent but TNFα in a TRIF-dependent manner. Together, our results demonstrate multiple roles of GRK5 in TLR signaling.