Enhanced antimicrobial activity of peptide-cocktails against common bacterial contaminants of ex vivo stored platelets.

Bacterial contamination of blood components such as ex vivo-stored platelets is a major safety risk in transfusion medicine. We have recently shown that synthetic antimicrobial peptides named PD1-PD4 derived from the thrombin-induced human platelet-derived antimicrobial proteins, and repeats of Arg-Trp (RW1-RW5) demonstrate microbicidal activity against selected bacteria and viruses. In the present study, we selected PD3, PD4, RW2, RW3 and RW4 and evaluated each individual peptide and their various combinations to see whether the cocktail regimen enhances the antimicrobial activity above and over the individual peptides. Stored platelet or plasma samples spiked with known titres of Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Bacillus cereus were treated with either individual peptides or with peptides in various combinations. Analyses revealed that individual peptides show moderate microbicidal activity (10- to 100-fold reduction) against the tested bacteria relative to their combined regimen. The peptide combinations (RW2 + RW4, RW2 + RW3 + RW4 and PD4 + RW3 + RW4) on the other hand enhanced the microbicidal activity (c.10 000-fold reduction) and revealed a minimal inhibitory concentration of 5 µM. Time-kill kinetics indicated that these three peptide combinations exhibited enhanced antimicrobial activity bringing about a 100-fold reduction of bacterial titres within 20 min of incubation. The present study therefore demonstrates the synergistic effect of antimicrobial peptides when used in combinations and provides a proof-of-concept of its potential application as a molecular tool towards pathogen reduction and further extends the possibility of using peptide combinatorial therapeutics as broad-spectrum antibiotics or as alternatives to combat drug-resistant bacteria.

Comparative molecular characterization of gene segment 11-derived NSP6 from lamb rotavirus LLR strain used as a human vaccine in China.

Sequence-length polymorphism is known for rotavirus genetic segment 11 (encodes non-structural protein, NSP6). With the exception of 11 strains that have the coding potential for a 98-residue NSP6, majority of the strains have the potential for a 92-residue NSP6. In nine strains, the coding potential for this protein is even shorter. This report focuses on the NSP6 gene nucleotide sequence of Lanzhou Lamb Rotavirus (LLR) strain and its comparative molecular characterization. The LLR strain is a G10 P12 type, which is in use as a licensed human vaccine in China. The LLR NSP6 was compared with 56 other rotaviral NSP6 sequences including a rhesus strain (RRV) available in the database. Analyses indicate that while RRV-NSP6 belongs to the majority (92-residue) group, the LLR NSP6 belongs to the 98-residue group. When the rotavirus NSP6 protein was expressed in cells as GFP fusion protein from human, simian and the LLR strains, they all demonstrated punctate cytoplasmic distribution and, contrary to the computer-aided prediction, the NSP6 did not undergo phosphorylation, which in itself is a novel observation for the rotavirus NSP6.

Rubella virus and birth defects: molecular insights into the viral teratogenesis at the cellular level.

BACKGROUND: In utero rubella virus (RV) infection of a fetus can result in birth defects that are often collectively referred to as congenital rubella syndrome (CRS). In extreme cases, fetal death can occur. In spite of the availability of a safe and effective vaccine against rubella, recent worldwide estimates are that more than 100,000 infants are born with CRS annually. RECENT PROGRESS: Recently, several significant findings in the field of cell biology, as well as in the RV replication and virus-cell interactions, have originated from the authors' laboratory, and other researchers have provided insights into RV teratogenesis. It has been shown that 1) an RV protein induces cell-cycle arrest by generating a subpopulation of tetraploid nuclei (i.e., 4N DNA) cells, perhaps representative of the tetraploid state following S phase in the cell cycle, due to its interaction with citron-K kinase (CK); 2) RV infection induces apoptosis in cell culture, and 3) CK functional perturbations lead to tetraploidy, followed by apoptosis, in specific cell types. CONCLUSIONS: Based on several similarities between known RV-associated fetal and cellular manifestations and CK deficiency-associated phenotypes, it is reasonable to postulate that P90-CK interaction in RV-infected cells interferes with CK function and induces cell-cycle arrest following S phase in a subpopulation, perhaps representative of tetraploid stage, which could lead to subsequent apoptosis in RV infection. Taking all these observations to the fetal organogenesis level, it is plausible that P90-CK interaction could perhaps be one of the initial steps in RV infection-induced apoptosis-associated fetal birth defects in utero.

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.

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.

The N- and C-terminal regions of rotavirus NSP5 are the critical determinants for the formation of viroplasm-like structures independent of NSP2.

Molecular events and the interdependence of the two rotavirus nonstructural proteins, NSP5 and NSP2, in producing viroplasm-like structures (VLS) were previously evaluated by using transient cellular coexpression of the genes for the two proteins, and VLS domains as well as the NSP2-binding region of NSP5 were mapped in the context of NSP2. Review of the previous studies led us to postulate that NSP2 binding of NSP5 may block the N terminus of NSP5 or render it inaccessible and that any similar N-terminal blockage may render NSP5 alone capable of producing VLS independent of NSP2. This possibility was addressed in this report by using two forms of NSP5-green fluorescent protein (GFP) chimeras wherein GFP is fused at either the N or the C terminus of NSP5 (GFP-NSP5 and NSP5-GFP) and evaluating their VLS-forming capability (by light and electron microscopy) and phosphorylation and multimerization potential independent of NSP2. Our results demonstrate that NSP5 alone can form VLS when the N terminus is blocked by fusion with a nonrotavirus protein (GFP-NSP5) but the C terminus is unmodified. Only GFP-NSP5 was able to undergo hyperphosphorylation and multimerization with the native form of NSP5, emphasizing the importance of an unmodified C terminus for these events. Deletion analysis of NSP5 mapped the essential signals for VLS formation to the C terminus and clearly suggested that hyperphosphorylation of NSP5 is not required for VLS formation. The present study emphasizes in general that when fusion proteins are used for functional studies, constructs that represent fusions at both the N and the C termini of the protein should be evaluated.

Identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle.

Peroxisomes are unimembrane, respiratory organelles of the cell. Transport of cellular proteins to the peroxisomal matrix requires a type 1 peroxisomal targeting signal (PTS1) which essentially constitutes a tripeptide from the consensus sequence S/T/A/G/C/N-K/R/H-L/I/V/M/A/F/Y. Although PTS-containing proteins have been identified in eukaryotes, prokaryotes, and parasites, viral proteins with such signals have not been identified so far. We report here the first instance of a virus, the rotavirus, which causes infantile diarrhea worldwide, containing a functional C-terminal PTS1 in one of its proteins (VP4). Analysis of 153 rotavirus VP4-deduced amino acid sequences identified five groups of conserved C-terminal PTS1 tripeptide sequences (SKL, CKL, GKL, CRL, and CRI), of which CRL is represented in approximately 62% of the sequences. Infection of cells by a CRL-containing representative rotavirus (SA11 strain) and confocal immunofluorescence analysis revealed colocalization of VP4 with peroxisomal markers and morphological changes of peroxisomes. Further, transient cellular expression of green fluorescent protein (GFP)-fused VP4CRL resulted in transport of VP4 to peroxisomes, whereas the chimera lacking the PTS1 signal, GFP-VP4DeltaCRL, resulted in diffuse cytoplasmic staining, suggesting a CRL-dependent targeting of the protein. The present study therefore demonstrates hitherto unreported organelle involvement, specifically of the peroxisomes, in rotaviral infections as demonstrated by using the SA11 strain of rotavirus and opens a new line of investigation toward understanding viral pathogenesis and disease mechanisms.

Correlation of autoimmune reactivity with hepatitis B and C virus (HBV and HCV) infection in histologically proven chronic liver diseases.

PURPOSE: To comprehensively study the possibility of autoimmune reactivity by hepatitis viruses B and C (HBV &HCV) in Indian chronic liver disease (CLD) patients. METHODS: One hundred and sixty histopathologically proven CLD cases and 100 matched controls were analysed for viral serology for HBV and HCV and autoimmune serology for antinuclear antibody (ANA), anti smooth muscle antibody (ASMA) and Liver kidney microsomal antibody (LKM) using standard immunofluorescence technique. RESULTS: 43.7% of cases were chronic hepatitis B while 16.2% were positive for HCV. CLD-B cases showed ANA positivity in 27.1% and ASMA positivity in 25.7%. CLD-C cases revealed 26.9%, 46.1% and 11.1% positivity for ANA, ASMA and LKM antibodies respectively. These rates and titres of autoantibodies were statistically significant (p= or < 0.02) when compared with that of controls. CONCLUSIONS: Based on the pattern of autoantibody positivity, it could be concluded that chronic HBV infection may induce autoimmune hepatitis (AIH) type I and chronic HCV infection might trigger AIH - Type II in Indian CLD cases.