A Coordinated Interdependent Protein Circuitry Stabilizes the Kinetochore Ensemble to Protect CENP-A in the Human Pathogenic Yeast Candida albicans

Unlike most eukaryotes, a kinetochore is fully assembled early in the cell cycle in budding yeasts Saccharomyces cerevisiae and Candida albicans. These kinetochores are clustered together throughout the cell cycle. Kinetochore assembly on point centromeres of S. cerevisiae is considered to be a step-wise process that initiates with binding of inner kinetochore proteins on specific centromere DNA sequence motifs. In contrast, kinetochore formation in C. albicans, that carries regional centromeres of 3-5 kb long, has been shown to be a sequence independent but an epigenetically regulated event. In this study, we investigated the process of kinetochore assembly/disassembly in C. albicans. Localization dependence of various kinetochore proteins studied by confocal microscopy and chromatin immunoprecipitation (ChIP) assays revealed that assembly of a kinetochore is a highly coordinated and interdependent event. Partial depletion of an essential kinetochore protein affects integrity of the kinetochore cluster. Further protein depletion results in complete collapse of the kinetochore architecture. In addition, GFP-tagged kinetochore proteins confirmed similar time-dependent disintegration upon gradual depletion of an outer kinetochore protein (Dam1). The loss of integrity of a kinetochore formed on centromeric chromatin was demonstrated by reduced binding of CENP-A and CENP-C at the centromeres. Most strikingly, Western blot analysis revealed that gradual depletion of any of these essential kinetochore proteins results in concomitant reduction in cellular protein levels of CENP-A. We further demonstrated that centromere bound CENP-A is protected from the proteosomal mediated degradation. Based on these results, we propose that a coordinated interdependent circuitry of several evolutionarily conserved essential kinetochore proteins ensures integrity of a kinetochore formed on the foundation of CENP-A containing centromeric chromatin.

Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria

Most bacterial genomes harbor restriction-modification systems, encoding a REase and its cognate MTase. On attack by a foreign DNA, the REase recognizes it as nonself and subjects it to restriction. Should REases be highly specific for targeting the invading foreign DNA? It is often considered to be the case. However, when bacteria harboring a promiscuous or high-fidelity variant of the REase were challenged with bacteriophages, fitness was maximal under conditions of catalytic promiscuity. We also delineate possible mechanisms by which the REase recognizes the chromosome as self at the noncanonical sites, thereby preventing lethal dsDNA breaks. This study provides a fundamental understanding of how bacteria exploit an existing defense system to gain fitness advantage during a host-parasite coevolutionary ``arms race.''

Regulation of lipid biosynthesis, sliding motility, and biofilm formation by a membrane-anchored nucleoid-associated protein of mycobacterium tuberculosis

Bacteria use a number of small basic proteins for organization and compaction of their genomes. By their interaction with DNA, these nucleoid-associated proteins (NAPs) also influence gene expression. Rv3852, a NAP of Mycobacterium tuberculosis, is conserved among the pathogenic and slow-growing species of mycobacteria. Here, we show that the protein predominantly localizes in the cell membrane and that the carboxy-terminal region with the propensity to form a transmembrane helix is necessary for its membrane localization. The protein is involved in genome organization, and its ectopic expression in Mycobacterium smegmatis resulted in altered nucleoid morphology, defects in biofilm formation, sliding motility, and change in apolar lipid profile. We demonstrate its crucial role in regulating the expression of KasA, KasB, and GroEL1 proteins, which are in turn involved in controlling the surface phenotypes in mycobacteria.

Biological sulfate reduction of a sulfate-rich industrial waste liquor using sulfate reducing bacteria

An industrial waste liquor having high sulfate concentrations was subjected to biological treatment using the sulfate-reducing bacteria (SRB) Desulfovibrio desulfuricans. Toxicity levels of different sulfate, cobalt and nickel concentrations toward growth of the SRB with respect to biological sulfate reduction kinetics was initially established. Optimum sulfate concentration to promote SRB growth amounted to 0.8 - 1 g/L. The strain of D. desulfuricans used in this study initially tolerated up to 4 -5 g/L of sulfate or 50 mg/L of cobalt and nickel, while its tolerance could be further enhanced through adaptation by serial subculturing in the presence of increasing concentrations of sulfate, cobalt and nickel. From the waste liquor, more than 70% of sulfate and 95% of cobalt and nickel could be precipitated as sulfides, using a preadapted strain of D. desulfuricans. Probable mechanisms involving biological sulfide precipitation and metal adsorption onto precipitates and bacterial cells are discussed.

Synergistic effect of static magnetic field and HA-Fe3O4 magnetic composites on viability of S. aureus and E. coli bacteria

In addressing the issue of prosthetic infection, this work demonstrated the synergistic effect of the application of static magnetic field (SMF) and ferrimagnetic substrate properties on the bactericidal property in vitro. This aspect was studied using hydroxyapatite (HA)-xFe(3)O(4) (x=10, 20, and 40 wt.%) substrates, which have different saturation magnetization properties. During bacteria culture experiments, 100 mT SMF was applied to growth medium (with HA-xFe(3)O(4) substrate) in vitro for 30, 120, and 240 min. A combination of MTT assay, membrane rupture assays, live/dead assay, and fluorescence microscopic analysis showed that the bactericidal effect of SMF increases with the exposure duration as well as increasing Fe3O4 content in biomaterial substrates. Importantly, the synergistic bactericidal effect was found to be independent of bacterial cell type, as similar qualitative trend is measured with both gram negative Escherichia coli (E. coli) and gram positive Staphylococcus aureus (S. aureus) strains. The reduction in E. coli viability was 83% higher on HA-40 Wt % Fe3O4 composite after 4 h exposure to SMF as compared to nonexposed control. Interestingly, any statistically significant difference in ROS was not observed in bacterial growth medium after magnetic field exposure, indicating the absence of ROS enhancement due to magnetic field. Overall, this study illustrates significant role being played by magnetic substrate compositions towards bactericidal property than by magnetic field exposure alone. (c) 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 524-532, 2014.

Hot pressed silver doped hydroxyapatite biomaterials with bactericidal properties against magnetotactic bacteria

Hydroxyapatite (HA) is widely being researched for hard tissue replacement for its good osseointegration and biocompatibility property. However, the inferior antibacterial property of HA often results in infection at host site, and this leads to rejection of the implant. The antibacterial property of silver (Ag) is well known and in the past decade or so, the application of Ag is reinvented in medicinal applications like catheters, vascular grafts and orthopaedic implants. In this respect, the present work reports the synthesis of Ag doped HA using hot pressing in argon atmosphere. This work also reports the effect of HA-Ag composition on bacterial colonisation during in vitro study. The bactericidal property of Ag doped HA has been investigated against magnetotactic bacteria, a `magnetite' containing bacteria. Magnetotactic bacteria were seeded onto pellets, and the adhesion of bacteria was evaluated using scanning electron microscopy. It was confirmed that incorporation of Ag in HA leads to improved bactericidal property.

Biogenic nanosilver incorporated reverse osmosis membrane for antibacterial and antifungal activities against selected pathogenic strains: An enhanced eco-friendly water disinfection approach

Reverse osmosis (RO) membranes have been used extensively in water desalination plants, waste water treatment in industries, agricultural farms and drinking water production applications. The objective of this work is to impart antibacterial and antifungal activities to commercially available RO membrane used in water purification systems by incorporating biogenic silver nanoparticles (AgNPs) synthesized using Rosa indica wichuriana hybrid leaf extract. The morphology and surface topography of uncoated and AgNPs-coated RO membrane were studied using Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM). Elemental composition of the AgNPs-coated RO membrane was analyzed by energy-dispersive X-ray spectroscopy (EDAX). The functional groups were identified by Fourier Transform Infrared spectroscopy (FT-IR). Hydrophilicity of the uncoated and AgNPs-coated RO membrane was analyzed using water contact angle measurements. The thermal properties were studied by thermogravimetric analysis (TGA). The AgNPs incorporated RO membrane exhibited good antibacterial and antifungal activities against pathogenic bacterial strains such as E. coli, S. aureus, M. luteus, K. pneumoniae, and P. aeruginosa and fungal strains such as Candida tropicalis, C. krusei, C. glabrata, and C. albicans.

A Mycobacterial Phosphoribosyltransferase Promotes Bacillary Survival by Inhibiting Oxidative Stress and Autophagy Pathways in Macrophages and Zebrafish

Mycobacterium tuberculosis employs various strategies to modulate host immune responses to facilitate its persistence in macrophages. The M. tuberculosis cell wall contains numerous glycoproteins with unknown roles in pathogenesis. Here, by using Concanavalin A and LC-MS analysis, we identified a novel mannosylated glycoprotein phosphoribosyltransferase, encoded by Rv3242c from M. tuberculosis cell walls. Homology modeling, bioinformatic analyses, and an assay of phosphoribosyltransferase activity in Mycobacterium smegmatis expressing recombinant Rv3242c (MsmRv3242c) confirmed the mass spectrometry data. Using Mycobacterium marinum-zebrafish and the surrogate MsmRv3242c infection models, we proved that phosphoribosyltransferase is involved in mycobacterial virulence. Histological and infection assays showed that the M. marinum mimG mutant, an Rv3242c orthologue in a pathogenic M. marinum strain, was strongly attenuated in adult zebrafish and also survived less in macrophages. In contrast, infection with wild type and the complemented Delta mimG: Rv3242c M. marinum strains showed prominent pathological features, such as severe emaciation, skin lesions, hemorrhaging, and more zebrafish death. Similarly, recombinant Msm Rv3242c bacteria showed increased invasion in non-phagocytic epithelial cells and longer intracellular survival in macrophages as compared with wild type and vector control M. smegmatis strains. Further mechanistic studies revealed that the Rv3242c- and mimG-mediated enhancement of intramacrophagic survival was due to inhibition of autophagy, reactive oxygen species, and reduced activities of superoxide dismutase and catalase enzymes. Infection with MsmRv3242c also activated the MAPK pathway, NF-kappa B, and inflammatory cytokines. In summary, we show that a novel mycobacterial mannosylated phosphoribosyltransferase acts as a virulence and immunomodulatory factor, suggesting that it may constitute a novel target for antimycobacterial drugs.

Novel AgBr/Ag3PO4 Decorated Ceria Nanoflake Composites for Enhanced Photocatalytic Activity toward Dyes and Bacteria under Visible Light

The aim of this study was to develop heterogeneous visible light active photocatalysts using AgBr and Ag3PO4 using CeO2 nanoflakes as an efficient substrate. Ascorbic acid was employed as a fuel to synthesize fine ceria nanoflakes by a facile solution combustion process. AgBr and Ag3PO4 were decorated on ceria to prepare AgBr/Ag3PO4/ceria nanocomposites. The structure of the composite was determined by X-ray diffraction analysis. Novel flakelike morphology was revealed using electron microscopy techniques. The nanocomposites exhibit excellent photocatalytic activity under visible light compared to pristine ceria nanoparticles. The nanocomposite catalyst particles degraded both anionic and cationic dyes. It also exhibited efficient antimicrobial activity under visible light. The AgBr/Ag3PO4/ceria nanocomposite was characterized using X-ray diffraction analysis, diffuse reflectance spectroscopy, electron microscopy, BET surface area analysis, and X-ray photoelectron spectroscopy, and the reasons for enhanced photocatalytic activity were elucidated. The presence of silver based semiconductors on ceria has shown to decrease charge recombination through photoluminescence analysis that attributed for enhanced photocatalytic activity. The AgBr/Ag3PO4/ceria nanocomposite has shown a stable performance after many repeated cycles.

Haloarchaeal gas vesicle nanoparticles displaying Salmonella antigens as a novel approach to vaccine development

A safe, effective, and inexpensive vaccine against typhoid and other Salmonella diseases is urgently needed. In order to address this need, we are developing a novel vaccine platform employing buoyant, self-adjuvanting gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1, bioengineered to display highly conserved Salmonella enterica antigens. As the initial antigen for testing, we selected SopB, a secreted inosine phosphate effector protein injected by pathogenic S. enterica bacteria during infection into the host cells. Two highly conserved sopB gene segments near the 3'- region, named sopB4 and sopB5, were each fused to the grIpC gene, and resulting SopB-GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and SopB5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of SopB-GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ApmrG-H111-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-y, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Thl response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were also found to be stable at elevated temperatures for extended periods without refrigeration. The results show that bioengineered GVNPs are likely to represent a valuable platform for antigen delivery and development of improved vaccines against Salmonella and other diseases.

Epigenetics and miRNA during bacteria-induced host immune responses

Various cellular processes including the pathogen-specific immune responses, host-pathogen interactions and the related evasion mechanisms rely on the ability of the immune cells to be reprogrammed accurately and in many cases instantaneously. In this context, the exact functions of epigenetic and miRNA-mediated regulation of genes, coupled with recent advent in techniques that aid such studies, make it an attractive field for research. Here, we review examples that involve the epigenetic and miRNA control of the host immune system during infection with bacteria. Interestingly, many pathogens utilize the epigenetic and miRNA machinery to modify and evade the host immune responses. Thus, we believe that global epigenetic and miRNA mapping of such host-pathogen interactions would provide key insights into their cellular functions and help to identify various determinants for therapeutic value.

Green Nanosilver as Reinforcing Eco-Friendly Additive to Epoxy Coating for Augmented Anticorrosive and Antimicrobial Behavior

Epoxy resin GY250 representing diglycidyl ethers of bisphenol-A (DGEBA) was reinforced with 1, 3 and 5 wt % of surface functionalized silver nanoparticles (F-AgNPs) which were synthesized using Couroupita guianensis leaves extract with a view of augmenting the corrosion control property of the epoxy resin and also imparting antimicrobial activity to epoxy coatings on mild steel. Corrosion resistance of the coatings was evaluated by EIS, potentiodynamic polarization studies and cross scratch tests. AFM, SEM, HRTEM and EDX were utilized to investigate the surface topography, morphology and elemental composition of the coatings on MS specimens. Results showed that the corrosion resistance, hardness and T-g of the DGEBA/F-AgNPs coatings increased at 1 wt % of F-AgNPs. The DGEBA/F-AgNPs coatings also offered manifold antimicrobial protection to the MS surfaces by inhibiting the growth of biofilm forming bacteria like P. aeruginosa, B. subtilis, the most common human pathogen E. coli and the most virulent human pathogenic yeast C. albicans.

Novel synergistic photocatalytic degradation of antibiotics and bacteria using V-N doped TiO2 under visible light: the state of nitrogen in V-doped TiO2

We report the synthesis of vanadium and nitrogen co-doped TiO2 for photocatalysis mainly emphasizing the state of nitrogen doping into TiO2 in the presence of vanadium ions. Considering the increase in antibiotic resistance developed by microbes due to the excess of pharmaceutical waste in the ecosystem, the photocatalytic activity was measured by degrading an antibiotic, chloramphenicol. A novel experiment was conducted by degrading the antibiotic and bacteria in each other's vicinity to focus on their synergistic photo-degradation by V-N co-doped TiO2. The catalysts were characterized using XRD, DRS, PL, TEM, BET and XPS analysis. Both interstitial and substitutional nitrogen doping were achieved with V-TiO2, showing high efficiency under visible light for antibiotic and bacterial degradation. In addition, the effect of doping concentration of nitrogen and vanadium in TiO2 and catalyst loading was studied thoroughly. Reusability experiments show that the prepared V-N co-doped TiO2 was stable for many cycles.