Interaction of Silver Nanoparticles with Serum Proteins Affects Their Antimicrobial Activity In Vivo

The emergence of multidrug-resistant bacteria is a global threat for human society. There exist recorded data that silver was used as an antimicrobial agent by the ancient Greeks and Romans during the 8th century. Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities, with minimal cytotoxic effects on the cells. However, very few reports have shown the usage of AgNPs for antibacterial therapy in vivo. In this study, we deciphered the importance of the chosen methods for synthesis and capping of AgNPs for their improved activity in vivo. The interaction of AgNPs with serum albumin has a significant effect on their antibacterial activity. It was observed that uncapped AgNPs exhibited no antibacterial activity in the presence of serum proteins, due to the interaction with bovine serum albumin (BSA), which was confirmed by UV-Vis spectroscopy. However, capped AgNPs with citrate or poly(vinylpyrrolidone)] exhibited antibacterial properties due to minimized interactions with serum proteins. The damage in the bacterial membrane was assessed by flow cytometry, which also showed that only capped AgNPs exhibited antibacterial properties, even in the presence of BSA. In order to understand the in vivo relevance of the antibacterial activities of different AgNPs, a murine salmonellosis model was used. It was conclusively proved that AgNPs capped with citrate or PVP exhibited significant antibacterial activities in vivo against Salmonella infection compared to uncapped AgNPs. These results clearly demonstrate the importance of capping agents and the synthesis method for AgNPs in their use as antimicrobial agents for therapeutic purposes.

Quorum sensing and pathogenesis: role of small signalling molecules in bacterial persistence

The pathogenesis of Mycobacterium tuberculosis is associated with its ability to survive inside the human host and the bacteria use a variety of mechanism to evade the host's defence. A clearer understanding of the host pathogen interaction is needed to follow the pathogenicity and virulence. Recent advances in the study of inter and intra-cellular communication in bacteria had prompted us to study the role of quorum sensing in bacterial survival and pathogenicity. The cell cell communication in bacteria (quorum sensing) is mediated through the exchange of small molecules called as autoinducers that allow bacteria to modulate their gene expression in response to change in cell-population density. It is a coordinated response that confers multicellularity to a bacterial population in response to stress from external environment. Quorum sensing molecules are the global regulators and regulate a wide range of physiological processes including biofilm formation, motility, cell differentiation, long-term survival and many others. Many bacterial pathogens require quorum sensing to produce the virulence factors in response to host pathogen interaction. Here, we summarize our current understanding on small molecule signalling and their role in the bacterial persistence. New discoveries in these areas have enriched our knowledge on intracellular signalling and their role in the long-term survival of mycobacteria under nutrient starvation.

Impact of heavy metals on bacterial communities from mangrove soils of the Mahanadi Delta (India)

This study aimed to assess soil nutrient status and heavy metal content and their impact on the predominant soil bacterial communities of mangroves of the Mahanadi Delta. Mangrove soil of the Mahanadi Delta is slightly acidic and the levels of soil nutrients such as carbon, nitrogen, phosphorous and potash vary with season and site. The seasonal average concentrations (g/g) of various heavy metals were in the range: 14810-63370 (Fe), 2.8-32.6 (Cu), 13.4-55.7 (Ni), 1.8-7.9 (Cd), 16.6-54.7 (Pb), 24.4-132.5 (Zn) and 13.3-48.2 (Co). Among the different heavy metals analysed, Co, Cu and Cd were above their permissible limits, as prescribed by Indian Standards (Co=17g/g, Cu=30 g/g, Cd=3-6 g/g), indicating pollution in the mangrove soil. A viable plate count revealed the presence of different groups of bacteria in the mangrove soil, i.e. heterotrophs, free-living N-2 fixers, nitrifyers, denitrifyers, phosphate solubilisers, cellulose degraders and sulfur oxidisers. Principal component analysis performed using multivariate statistical methods showed a positive relationship between soil nutrients and microbial load. Whereas metal content such as Cu, Co and Ni showed a negative impact on some of the studied soil bacteria.

Infection of Human Endothelial Cells by Japanese Encephalitis Virus: Increased Expression and Release of Soluble HLA-E

Japanese encephalitis virus (JEV) is a single stranded RNA virus that infects the central nervous system leading to acute encephalitis in children. Alterations in brain endothelial cells have been shown to precede the entry of this flavivirus into the brain, but infection of endothelial cells by JEV and their consequences are still unclear. Productive JEV infection was established in human endothelial cells leading to IFN-beta and TNF-alpha production. The MHC genes for HLA-A, -B, -C and HLA-E antigens were upregulated in human brain microvascular endothelial cells, the endothelial-like cell line, ECV 304 and human foreskin fibroblasts upon JEV infection. We also report the release/shedding of soluble HLA-E (sHLA-E) from JEV infected human endothelial cells for the first time. This shedding of sHLA-E was blocked by an inhibitor of matrix metalloproteinases (MMP). In addition, MMP-9, a known mediator of HLA solubilisation was upregulated by JEV. In contrast, human fibroblasts showed only upregulation of cell-surface HLA-E. Addition of UV inactivated JEV-infected cell culture supernatants stimulated shedding of sHLA-E from uninfected ECV cells indicating a role for soluble factors/cytokines in the shedding process. Antibody mediated neutralization of TNF-alpha as well as IFNAR receptor together not only resulted in inhibition of sHLA-E shedding from uninfected cells, it also inhibited HLA-E and MMP-9 gene expression in JEV-infected cells. Shedding of sHLA-E was also observed with purified TNF-alpha and IFN-beta as well as the dsRNA analog, poly (I:C). Both IFN-beta and TNF-alpha further potentiated the shedding when added together. The role of soluble MHC antigens in JEV infection is hitherto unknown and therefore needs further investigation.

Physical understanding of pore formation on supported lipid bilayer by bacterial toxins

Pore forming toxins are being classified in the protein community based on their ability of forming pores in living cell membranes. Some initial study has apparently pointed out the crystallographic pathway rather can be viewed as a structural as well as morphological changes of proteins in terms of self assembly before and during the pore formation process in surfactant medium. Being a water soluble compound, it changes its conformation and originates some pre-pore complex, which later partially goes inside the cell membrane causing a pore. The physical mechanism for this whole process is still unknown. In this study we have tried to understand these types of biological processes from physical point of view by using supported lipid bilayer as a model system.

Estimating the fraction of progeny virions that must incorporate APOBEC3G for suppression of productive HIV-1 infection

The contest between the host factor APOBEC3G (A3G) and the HIV-1 protein Vif presents an attractive target of intervention. The extent to which the A3G-Vif interaction must be suppressed to tilt the balance in favor of A3G remains unknown. We employed stochastic simulations and mathematical modeling of the within-host dynamics and evolution of HIV-1 to estimate the fraction of progeny virions that must incorporate A3G to render productive infection unsustainable. Using three different approaches, we found consistently that a transition from sustained infection to suppression of productive infection occurred when the latter fraction exceeded similar to 0.8. The transition was triggered by A3G-induced hypermutations that led to premature stop codons compromising viral production and was consistent with driving the basic reproductive number, R-o, below unity. The fraction identified may serve as a quantitative guideline for strategies targeting the A3G-Vif axis. (C) 2013 Elsevier Inc. All rights reserved.

Inactivation of the Bacterial RNA Polymerase Due to Acquisition of Secondary Structure by the omega Subunit

The widely conserved omega subunit encoded by rpoZ is the smallest subunit of Escherichia coli RNA polymerase (RNAP) but is dispensable for bacterial growth. Function of omega is known to be substituted by GroEL in omega-null strain, which thus does not exhibit a discernable phenotype. In this work, we report isolation of omega variants whose expression in vivo leads to a dominant lethal phenotype. Studies show that in contrast to omega, which is largely unstructured, omega mutants display substantial acquisition of secondary structure. By detailed study with one of the mutants, omega(6) bearing N60D substitution, the mechanism of lethality has been deciphered. Biochemical analysis reveals that omega(6) binds to beta ` subunit in vitro with greater affinity than that of omega. The reconstituted RNAP holoenzyme in the presence of omega(6) in vitro is defective in transcription initiation. Formation of a faulty RNAP in the presence of mutant omega results in death of the cell. Furthermore, lethality of omega(6) is relieved in cells expressing the rpoC2112 allele encoding beta ` (2112), a variant beta ` bearing Y457S substitution, immediately adjacent to the beta ` catalytic center. Our results suggest that the enhanced omega(6)-beta ` interaction may perturb the plasticity of the RNAP active center, implicating a role for omega and its flexible state.

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.

Flocculation behaviour of hematite-kaolinite suspensions in presence of extracellular bacterial proteins and polysaccharides

Cells of Bacillus subtilis exhibited higher affinity towards hematite than to kaolinite. Bacterial cells were grown and adapted in the presence of hematite and kaolinite. Higher amounts of mineral-specific proteinaceous compounds were secreted in the presence of kaolinite while hematite-grown cells produced higher amounts of exopolysaccharides. Extracellular proteins (EP) exhibited higher adsorption density on kaolinite which was rendered more hydrophobic. Hematite surfaces were rendered more hydrophilic due to increased adsorption of extracellular polysaccharides (ECP). Significant surface chemical changes were produced due to interaction between minerals and extracellular proteins and polysaccharides. Iron oxides such as hematite could be effectively removed from kaolinite clays using selective bioflocculation of hematite after interaction with EP and ECP extracted from mineral-grown cells. (C) 2013 Elsevier B.V. All rights reserved.

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.

CONFORMATIONAL FEATURES OF SIGMA FACTOR/ANTI-SIGMA COMPLEXES

The association of a factors with the RNA polymerase dictates the expression profile of a bacterial cell. Major changes to the transcription profile are achieved by the use of multiple sigma factors that confer distinct promoter selectivity to the holoenzyme. The cellular concentration of a sigma factor is regulated by diverse mechanisms involving transcription, translation and post-translational events. The number of sigma factors varies substantially across bacteria. The diversity in the interactions between sigma factors also vary-ranging from collaboration, competition or partial redundancy in some cellular or environmental contexts. These interactions can be rationalized by a mechanistic model referred to as the partitioning of a space model of bacterial transcription. The structural similarity between different sigma/anti-sigma complexes despite poor sequence conservation and cellular localization reveals an elegant route to incorporate diverse regulatory mechanisms within a structurally conserved scaffold. These features are described here with a focus on sigma/anti-sigma complexes from Mycobacterium tuberculosis. In particular, we discuss recent data on the conditional regulation of sigma/anti-sigma factor interactions. Specific stages of M. tuberculosis infection, such as the latent phase, as well as the remarkable adaptability of this pathogen to diverse environmental conditions can be rationalized by the synchronized action of different a factors.

Influenza hemagglutinin stem-fragment immunogen elicits broadly neutralizing antibodies and confers heterologous protection

Influenza hemagglutinin (HA) is the primary target of the humoral response during infection/vaccination. Current influenza vaccines typically fail to elicit/boost broadly neutralizing antibodies (bnAbs), thereby limiting their efficacy. Although several bnAbs bind to the conserved stem domain of HA, focusing the immune response to this conserved stem in the presence of the immunodominant, variable head domain of HA is challenging. We report the design of a thermotolerant, disulfide-free, and trimeric HA stem-fragment immunogen which mimics the native, prefusion conformation of HA and binds conformation specific bnAbs with high affinity. The immunogen elicited bnAbs that neutralized highly divergent group 1 (H1 and H5 subtypes) and 2 (H3 subtype) influenza virus strains in vitro. Stem immunogens designed from unmatched, highly drifted influenza strains conferred robust protection against a lethal heterologous A/Puerto Rico/8/34 virus challenge in vivo. Soluble, bacterial expression of such designed immunogens allows for rapid scale-up during pandemic outbreaks.

Suramin is a potent and selective inhibitor of Mycobacterium tuberculosis RecA protein and the SOS response: RecA as a potential target for antibacterial drug discovery

In eubacteria, RecA is essential for recombinational DNA repair and for stalled replication forks to resume DNA synthesis. Recent work has implicated a role for RecA in the development of antibiotic resistance in pathogenic bacteria. Consequently, our goal is to identify and characterize small-molecule inhibitors that target RecA both in vitro and in vivo. We employed ATPase, DNA strand exchange and LexA cleavage assays to elucidate the inhibitory effects of suramin on Mycobacterium tuberculosis RecA. To gain insights into the mechanism of suramin action, we directly visualized the structure of RecA nucleoprotein filaments by atomic force microscopy. To determine the specificity of suramin action in vivo, we investigated its effect on the SOS response by pull-down and western blot assays as well as for its antibacterial activity. We show that suramin is a potent inhibitor of DNA strand exchange and ATPase activities of bacterial RecA proteins with IC50 values in the low micromolar range. Additional evidence shows that suramin inhibits RecA-catalysed proteolytic cleavage of the LexA repressor. The mechanism underlying such inhibitory actions of suramin involves its ability to disassemble RecA-single-stranded DNA filaments. Notably, suramin abolished ciprofloxacin-induced recA gene expression and the SOS response and augmented the bactericidal action of ciprofloxacin. Our findings suggest a strategy to chemically disrupt the vital processes controlled by RecA and hence the promise of small molecules for use against drug-susceptible as well as drug-resistant strains of M. tuberculosis for better infection control and the development of new therapies.

Avoiding acidic region streaking in two-dimensional gel electrophoresis: Case study with two bacterial whole cell protein extracts

Acidic region streaking (ARS) is one of the lacunae in two-dimensional gel electrophoresis (2DE) of bacterial proteome. This streaking is primarily caused by nucleic acid (NuA) contamination and poses major problem in the downstream processes like image analysis and protein identification. Although cleanup and nuclease digestion are practiced as remedial options, these strategies may incur loss in protein recovery and perform incomplete removal of NuA. As a result, ARS has remained a common observation across publications, including the recent ones. In this work, we demonstrate how ultrasound wave can be used to shear NuA in plain ice-cooled water, facilitating the elimination of ARS in the 2DE gels without the need for any additional sample cleanup tasks. In combination with a suitable buffer recipe, IEF program and frequent paper-wick changing approach, we are able to reproducibly demonstrate the production of clean 2DE gels with improved protein recovery and negligible or no ARS. We illustrate our procedure using whole cell protein extracts from two diverse organisms, Escherichia coli and Mycobacterium smegmatis. Our designed protocols are straightforward and expected to provide good 2DE gels without ARS, with comparable times and significantly lower cost.