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.

Gamma-phage lysin PlyG sequence-based synthetic peptides coupled with Qdot-nanocrystals are useful for developing detection methods for Bacillus anthracis by using its surrogates, B. anthracis-Sterne and B. cereus-4342.

BACKGROUND: Previous reports of site-directed deletion analysis on gamma (gamma)-phage lysin protein (PlyG) have demonstrated that removal of a short amino acid sequence in the C-terminal region encompassing a 10-amino acid motif (190LKMTADFILQ199) abrogates its binding activity specific to the cell wall of Bacillus anthracis. Whether short synthetic peptides representing the10-amino acid PlyG putative binding motif flanked by surrounding N- and C-terminal residues also selectively bind to the bacterial cell wall has not been evaluated. If such peptides do demonstrate selective binding to the cell wall, they could serve as bio-probes towards developing detection technologies for B. anthracis. Furthermore, by using B. anthracis (Sterne, 34F2), an animal vaccine and B. cereus-4342, a gamma-phage susceptible rare strain as surrogates of B. anthracis, development of proof-of-concepts for B. anthracis are feasible. RESULTS: Using four different methods, we evaluated six synthetic peptides representing the putative binding motif including flanking sequences (PlyG-P1 through P6) for the bacterial cell wall binding capacity. Our analysis identified PlyG-P1, PlyG-P3 and PlyG-P5 to have binding capability to both B. anthracis (Sterne, 34F2) and B. cereus-4342. The peptides however did not bind to B. cereus-11778, B. thuringiensis, and B. cereus-10876 suggesting their specificity for B. anthracis-Sterne and B. cereus-4342. PlyG-P3 in combination with fluorescent light microscopy detected even a single bacterium in plasma spiked with the bacteria. CONCLUSION: Overall, these studies illustrate that the short 10-amino acid sequence 'LKMTADFILQ' in fact is a stand-alone bacterial cell wall-binding motif of PlyG. In principle, synthetic peptides PlyG-P1, PlyG-P3 and PlyG-P5, especially PlyG-P3 coupled with Qdot-nanocrystals are useful as high-sensitivity bio-probes in developing detection technologies for B. anthracis.

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. STUDY DESIGN AND METHODS: In this report room temperature-stored PLTs of Days 0, 2, and 9 were analyzed by differential profiling for 52 apoptosis-associated human miRNAs. After total RNA extraction from the samples, a membrane array-based miRNA analysis was carried out. Prediction of target genes was performed by bioinformatics-based approaches. RESULTS: Our analysis revealed that during storage, Let-7a, -7c, -7e, -7f, -7g, and -7i miRNA profiles of the PLTs were barely detectable, while levels of miR-150, -151, -152, -184, -188, -196a, -197, and -202 remained at high levels in PLTs. The rest of the miRNA levels were in between. However, two miRNAs, Let-7b and miR-16, distinctly demonstrated an increasing trend while miR-7 and miR-145 showed a decreasing profile during PLT storage. For these four miRNAs, we also identified their potential target mRNAs. CONCLUSIONS: Overall, these results confirm the fact that miRNAs do exist in PLTs, and among 52 apoptosis-specific miRNAs studied, only a few selected miRNAs did perturb during PLT storage. Future experimental evaluation of these miRNA-target mRNA interactions will provide new insights into the molecular mechanisms of PLT storage-associated lesions.

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.

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.

The N-terminal conserved domain of rubella virus capsid interacts with the C-terminal region of cellular p32 and overexpression of p32 enhances the viral infectivity.

Cellular 'defense collagens' are produced to launch virus-specific responses to clear the invading viruses. Cellular p32, the C1q binding protein is one such protein. In this report, we identified the interaction of p32 derived from a human lung diploid cell line (WI-38) with rubella virus capsid (RVCP from Therien strain) N-terminal 28-amino acid domain, which is conserved among several RV strains including the vaccine strains. We further identified that the C-terminal 69 aa of the mature p32 is sufficient to interact with the CP. In addition, we observed that in three independent Vero 76-derived cell lines constitutively overexpressing p32, the RV infectivity was enhanced. Our results suggest that RV has evolved a strategy whereby one of its proteins is recruited to interact with, and exploit the cellular defense machinery to its advantage.