Biology subjects

Villafane, R.

Publications and source records attributed to Villafane, R..

4 recordsLinked to original sources

bioRxiv2022

Proteolytic Analysis of Epsilon 34 Phage Tailspike protein indicates Partial Sensitivity to Proteinase K

Purified bacteriophage {varepsilon}34 tailspike protein ({varepsilon}34 TSP) can bind to Salmonella newington (S. newington) via the binding site of the protein, which is the O antigens of the LPS of the bacterium. We demonstrated else-where that purified {varepsilon}34 TSP possessed bacteria lytic property on S. newington. The {varepsilon}34 TSP has been shown via computational prediction to consist of parallel {beta}-helices like that of P22 TSP. These protein moieties are among the simplest repetitive structural elements in proteins. There exist extensive research on the folding behavior of {beta}-helix proteins, which also provides insight on how amyloid fibrils are generated since these proteins consist of similar parallel {beta}-helix motifs. One of the most significantly studied system for investigating protein folding is the from the Salmonella bacteriophage P22. The major component of this protein is a right-handed parallel {beta}-helix with 13 rungs. Initial in silico analysis of the {varepsilon}34 phage TSP indicates similar structural similarity to the P22 TSP. Our previous studies indicated that despite the similarities of the two proteins, P22 TSP shows higher resistance to proteases (e.g. trypsin) and heat compared to {varepsilon}34 TSP. In this study we further proof that {varepsilon}34 TSP is partially sensitive to proteinase K, whereas P22 TSP is completely resistant to this protein. Detailed analysis indicates that specific structural motifs of {varepsilon}34 TSP is insensitive to the protease, whereas other regions of the protein showed susceptibility to it.

microbiology↗

bioRxiv2022

Capsaicin potently blocks Salmonella typhimurium invasion of Vero cells

As at 2021, the center for disease control (CDC) reported that Salmonella causes 1.2 million illness in the United States each year, with a mortality rate approaching 500 deaths per annum. Infants, the elderly, and persons with compromised immunity are the population with higher risk of mortality from this infection. At present there is no commercially available, safe and efficacious vaccine for the control and management of Salmonella typhimurium (S. typhimurium). More so, S. typhimurium has been shown to develop resistance against most antibiotics used for treatment of the infection. Capsaicin, a bioactive compound from Capsicum chinense (C. chinenses) is undoubtedly one of the most widely used spice in the world. This heat producing compound is not only been used as food additive but have been demonstrated to possess unique properties that have pharmacological, physiological, and antimicrobial applications. In this work, the antimicrobial property of pure capsaicin or capsaicin extract against S. typhimurium is tested to determine the compounds effectiveness in S. typhimurium inhibition. Capsaicin extract showed potent inhibition of S. typhimurium growth at concentrations as low as 100 ng/ml, whereas pure capsaicin comparatively showed poorer inhibition of the bacteria. Furthermore, both capsaicin extract and pure capsaicin potently blocked S. typhimurium invasion of an animal cell line in vitro. Taken together, this work revealed that capsaicin might work synergistically with dihydrocapsaicin or the other capsaicinoids to inhibit S. typhimurium growth, whereas individually, capsaicin or dihydrocapsaicin could potently block the bacteria entry and invasion of Vero cells.

microbiology↗

bioRxiv2021

Single Amino Acid Change Mutation in the Hydrophobic Core of the N-terminal Domain of P22 TSP affects the Proteins Stability.

The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly shifted the attention of researchers to critically investigate most viruses to understand specific characteristics that impart their virulence. For instance, the SARS-CoV-2 has undergone several mutations, with some variants classified as "variants of concern", e.g., the Omicron and Delta variant of SARS-CoV-2 are known for their rapid transmission and antigenicity due to mutation in the Spike protein. P22 bacteriophage is a bacterial virus that has a tailspike protein (TSP) that performs similar functions as the Spike protein of SARS-COV-2. We previously carried out a site-directed mutagenesis of the P22 TSP to bear disruptive mutations in the hydrophobic core of the N-terminal Domain (NTD), then partially characterized the properties of the mutant TSPs. In this process, the valine patch (triple valine residues that formed a hydrophobic core) was replaced with charged amino acids (Asp or lysine) or hydrophobic amino acids (Leucine or isoleucine). Some of the mutant TSPs characterized showed significant differences in migration in both native and SDS-PAGE. Mutants with such disruptive mutation are known to show non-native properties, and as expected, most of these mutants obtained showed significantly different properties from the WT P22 TSP. In this work, we further characterized these mutant species by computational and in vitro assays to demonstrate the validity of our previous inference that the valine patch is a critical player in the stability of the N-terminal domain of the P22 TSP.

microbiology↗

bioRxiv2021

The E34 Phage Tailspike Protein: An in vitro characterization, Structure Prediction, Potential Interaction with S. newington LPS and Cytotoxicity Assessment to Animal Cell Line.

The E34 phage is a member of the podoviridae family of phages, (short non-contractile tailed bacteriophages) that uses Salmonella newington as its host. This phage initiates the infection of its host via a specific interaction between its tailspike protein (TSP) and the lipopolysaccharides (LPS) of the bacterial. The E34 TSP is structurally similar and functionally equivalent to the P22 phage whose TSP has been well characterized and electron micrographs of both phages appear indistinguishable. The crystal structure of P22 phage TSP in complex with the O-antigen of S. typhimurium has been determined; and the active site of the TSP demonstrated to be the residues Asp392, Asp395 and Glu359 of the receptor binding domain. In another phage called E15, a phylogenetic relative of E34 phage, a short polysaccharide consisting of repeating units is responsible for the interaction between the E15 phage and Salmonella anatums LPS leading to the adsorption of the phage to the bacteria. Studies on E34 phage shows that it interacts with Salmonella newingtons O antigen polysaccharide component of the LPS, this polysaccharide consists of mannosyl-rhamnosyl-galactose repeating units joined together by {beta}-galactosyl linkages. However, no data exist regarding the specific residues of E34 TSP that are responsible for LPS binding and hydrolysis. In this study, the tailspike gene was cloned onto vector pET30a-LIC and expressed as a fusion protein termed the extended E34 TSP (EE34 TSP). We characterized the protein based on resistance to heat, SDS, and proteases; showing that the protein is heat resistant, shows aberrant electrophoretic mobility in the presence of SDS gradient, and actively binds to P22 phage heads to form hybrid phages that cannot infect P22 host. We also demonstrate via in silico study that the E34 TSP binds to and hydrolyses the O-antigen of its host via the ALA250, SER279 and ASP280 residues. Finally, testing E34 phage ability to protect Vero cells from Salmonella infection shows highly encouraging results, implying that E34 phage can be used in therapeutic/preventive medicine.

microbiology↗