Ketha V. K. Mohan

Detection technologies for Bacillus anthracis: prospects and challenges.

Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological agent of acute infectious disease anthrax, a lethal but rare disease of animals and humans in nature. With recent use of anthrax as a bioweapon, a number of techniques have been recently developed and evaluated to facilitate its rapid detection of B. anthracis in the environment as well as in point-of-care settings for humans suspected of exposure to the pathogen. Complex laboratory methods for B. anthracis identification are required since B. anthracis has similarities with other Bacillus species and its existence in both spore and vegetative forms. This review discusses current challenges and various improvements associated with anthrax agent detection.

Membrane array–based differential profiling of platelets during storage for 52 miRNAs associated with apoptosis

BACKGROUND: Enucleated platelets (PLTs) utilize posttranscriptional gene (mRNA) regulation (PTGR) for their normal morphologic and physiologic functions, which are altered in their ex vivo storage, also collectively referred to as storage lesions. While cellular micro‐RNAs (miRNAs) play a significant role in posttranscriptional gene (mRNA) regulation by binding to their target mRNAs, comprehensive analysis of apoptosis‐associated miRNAs and global changes in their profiles during PLT storage have not been evaluated to date.

An Atypical Protein Disulfide Isomerase from the Protozoan Parasite Leishmania Containing a Single Thioredoxin-like Domain

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.

Antiviral activity of selected antimicrobial peptides against vaccinia virus

Abstract Antimicrobial peptides (AMPs) are gaining importance as effective therapeutic alternatives to conventional antibiotics. Recently we have shown that a set of nine synthetic antimicrobial peptides, four originating from thrombin-induced human platelet-derived antimicrobial proteins named PD1–PD4 and five synthetic repeats of arginine-tryptophan (RW) repeats (RW1-5) demonstrate antibacterial activity in plasma and platelets. Using WR strain of vaccinia virus (VV) as a model virus for enveloped virus in the present study, we tested the same nine synthetic peptides for their antiviral activity. A cell culture-based standard plaque reduction assay was utilized to estimate antiviral effectiveness of the peptides. Our analysis revealed that peptides PD3, PD4, and RW3 were virucidal against VV with PD3 demonstrating the highest antiviral activity of 100-fold reduction in viral titers, whereas, PD4 and RW3 peptide treatments resulted in 10–30-fold reduction. The EC50 values of PD3, PD4 and RW3 were found to be 40μg/ml, 50μg/ml and 6.5μM, respectively. In VV-spiked plasma samples, the virucidal activity of PD3, PD4 and RW3 was close to 100% (90–100-fold reduction). Overall, the present study constitutes a new proof-of-concept in developing peptide therapeutics for vaccinia virus infections in biothreat scenarios and as in vitro viral reduction agents.

A Peptide Derived from Phage Display Library Exhibits Antibacterial Activity against E. coli and Pseudomonas aeruginosa

Emergence of drug resistant strains to currently available antibiotics has resulted in the quest for novel antimicrobial agents. Antimicrobial peptides (AMPs) are receiving attention as alternatives to antibiotics. In this study, we used phage-display random peptide library to identify peptides binding to the cell surface of E. coli. The peptide with sequence RLLFRKIRRLKR (EC5) bound to the cell surface of E. coli and exhibited certain features common to AMPs and was rich in Arginine and Lysine residues. Antimicrobial activity of the peptide was tested in vitro by growth inhibition assays and the bacterial membrane permeabilization assay. The peptide was highly active against Gram-negative organisms and showed significant bactericidal activity against E. coli and P. aeruginosa resulting in a reduction of 5 log10 CFU/ml. In homologous plasma and platelets, incubation of EC5 with the bacteria resulted in significant reduction of E. coli and P. aeruginosa, compared to the peptide-free controls. The peptide was non-hemolytic and non-cytotoxic when tested on eukaryotic cells in culture. EC5 was able to permeabilize the outer membrane of E. coli and P. aeruginosa causing rapid depolarization of cytoplasmic membrane resulting in killing of the cells at 5 minutes of exposure. The secondary structure of the peptide showed a α-helical conformation in the presence of aqueous environment. The bacterial lipid interaction with the peptide was also investigated using Molecular Dynamic Simulations. Thus this study demonstrates that peptides identified to bind to bacterial cell surface through phage-display screening may additionally aid in identifying and developing novel antimicrobial peptides.

Identification and evaluation of a novel peptide binding to the cell surface of Staphylococcus aureus.

Identification of short peptides that serve as specific ligands to biological materials such as microbial cell surfaces has major implications in better understanding the molecular recognition of cell surfaces. In this study we screened a commercially available random phage-display library against Staphylococcus aureus cells and identified peptides specifically binding to the bacteria. A synthetic peptide (SA5-1) representing the consensus sequence (VPHNPGLISLQG) of the bacteria-binding peptide was evaluated for its binding potential against S. aureus. Dot-blot, immunoblot assay and ELISA results revealed the SA5-1 peptide to be highly specific to S. aureus. The SA5-1 peptide binding was optimal between pH 6.0 and 8.0. Nanogold Transmission Electron Microscopy demonstrated that the SA5-1 binds to the outer membrane surface of S. aureus. Diagnostic potential of the SA5-1 peptide was evaluated in human platelet samples spiked with S. aureus and specific detection of the bacteria by biotinylated-SA5-1 and streptavidin-conjugated fluorescent quantum dots. Fluorometry results indicated that the peptide was able to detect ∼100 organisms per ml in a spiked biological sample providing a proof-of-concept towards potential of this peptide as a S. aureus diagnostic tool that can be of use in different detection platforms.

Evaluation of antimicrobial peptides as novel bactericidal agents for room temperature–stored platelets

BACKGROUND: A single cost‐effective pathogen inactivation approach would help to improve the safety of our nations blood supply. Several methods and technologies are currently being studied to help reduce bacterial contamination of blood components. There is clearly need for simple and easy‐to‐use pathogen inactivation techniques specific to plasma, platelets (PLTs), and red blood cells.

Rubella virus P90 associates with the cytokinesis regulatory protein Citron-K kinase and the viral infection and constitutive expression of P90 protein both induce cell cycle arrest following S phase in cell culture

Summary.In utero infection of developing fetus by Rubella virus (RV) causes cell division inhibition of critical precursor cells in organogenesis, CNS-associated birth defects and induction of apoptosis in cell culture. The underlying mechanisms of RV-induced congenital abnormalities are not known. Here, we identified a novel interaction between RV replicase P90 protein and a cytokinesis-regulatory protein, the Citron-K kinase (CK), in a yeast two-hybrid cDNA library screen. Aberrations in cytokinesis and subsequent apoptosis do occur in specific cell types when the CK gene is knocked out or, its regulatory function is perturbed. Our analysis found that full-length P90 binds CK and in RV-infected cells P90 colocalizes with CK in the cytoplasm. Furthermore, during RV infection as well as cellular expression of P90 alone, we identified a discrete subpopulation of cells containing 4N DNA content, indicating that these cells are arrested in the cell cycle following S phase, suggesting that cellular expression of viral P90 during RV infection perturbs cytokinesis. Previous reports by others established that RV infection leads to apoptosis in cell culture. These observations together taken to the fetal organogenesis level, favor the idea that RV P90, by binding to cellular CK, invokes cell cycle aberrations resulting in the cell- and organ-specific growth inhibition and programmed cell death during RV infection in utero, which commonly is referred to as RV-induced teratogenesis.

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne.

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10(2) colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.

A Human Vaccine Strain of Lamb Rotavirus (Chinese) NSP4 Gene: Complete Nucleotide Sequence and Phylogenetic Analyses

A lamb strain of rotavirus has recently been licensed for use in China as a live vaccine to prevent rotavirus diarrhea in children. As rotavirus NSP4, especially the cytotoxic domain alone is considered to be associated with diarrhea, we sequenced gene segment 10, which encodes NSP4, of lamb rotavirus. Comparative analyses was performed to identify differences from human rotavirus strains, that might be associated with attenuation, and to ascertain whether the lamb rotavirus gene fits among the NSP4 of other sequenced rotavirus strains. Our comparative nucleotide sequence analysis suggests its close identity (91.17% homology) with that of group-A equine rotavirus (strain HI23). Multiple alignment of the deduced amino acid sequence of lamb NSP4 with that of other group A rotaviruses demonstrated homology ranging from 63.42% with that of porcine YM strain to 93.71% with equine HI23 strain of rotavirus. A group A-specific NSP4 monoclonal antibody recognized the glycosylated and unglycosylated forms of the protein from virus-infected lysates, suggesting a well-conserved group-specificity of the lamb NSP4. Phylogenetic analysis of the lamb rotavirus gene, with 60 other NSP4 gene sequences of human and animal rotavirus strains, demonstrated that the lamb rotavirus strain belongs to genotype A. Comparative analysis also revealed that although it is a vaccine strain, the NSP4 cytotoxic domain of lamb strain demonstrated an overall amino acid conservation similar to that of other strains, whose NSP4 alone causes diarrhea in animal models. These results taken together with our previous observations clearly reaffirm the idea that the attenuation phenotype of rotaviruses does not involve NSP4 cytotoxic domain, perhaps due to the suppression of NSP4 cytotoxic activity by other rotaviral proteins.