Cloning, Amplified Expression and Bioinformatics Analysis of a Putative Nucleobase Cation Symporter-1 (NCS-1) Protein From Rhodococcus erythropolis

The Rhodococcus erythropolis gene DYC18_RS18060 (1437 bp) putatively codes for a secondary transporter of the Nucleobase Cation Symporter-1 (NCS-1) protein family (478 amino acids). The DYC18_RS18060 gene was successfully cloned from R. erythropolis genomic DNA with addition of EcoRI and PstI restriction sites at the 5′ and 3′ ends, respectively, using PCR technology. The amplified gene was introduced into IPTG-inducible plasmid pTTQ18 immediately upstream of the sequence coding for a His6-tag. The construct was transformed into Escherichia coli BL21(DE3), then amplified expression of the DYC18_RS18060-His6 protein was achieved with detection by SDS-PAGE and western blotting. Computational methods predicted that DYC18_RS18060 has a molecular weight of 51.1 kDa and isoelectric point of 6.58. The protein was predicted to be hydrophobic in nature (aliphatic index 113.24, grand average of hydropathicity 0.728) and to form twelve transmembrane spanning α-helices with both N- and C-terminal ends at the cytoplasmic side of the membrane. Whilst database sequence similarity searches and phylogenetic analysis suggested that the substrate of DYC18_RS18060 could be cytosine, this was not certain based on comparisons of residues involved in substrate binding in experimentally characterised NCS-1 proteins. This study has laid foundations for further structural and functional studies of DYC18_RS18060 and other NCS-1 proteins. Copyright(c) The Authors

Annotation of Potential Vaccine Targets and Design of a Multi-Epitope Subunit Vaccine against Yersinia pestis through Reverse Vaccinology and Validation through an Agent-Based Modeling Approach

Yersinia pestis is responsible for plague and major pandemics in Asia and Europe. This bacterium has shown resistance to an array of drugs commonly used for the treatment of plague. Therefore, effective therapeutics measurements, such as designing a vaccine that can effectively and safely prevent Y. pestis infection, are of high interest. To fast-track vaccine development against Yersinia pestis, herein, proteome-wide vaccine target annotation was performed, and structural vaccinology-assisted epitopes were predicted. Among the total 3909 proteins, only 5 (rstB, YPO2385, hmuR, flaA1a, and psaB) were shortlisted as essential vaccine targets. These targets were then subjected to multi-epitope vaccine design using different linkers. EAAK, AAY, and GPGPG as linkers were used to link CTL, HTL, and B-cell epitopes, and an adjuvant (beta defensin) was also added at the N-terminal of the MEVC. Physiochemical characterization, such as determination of the instability index, theoretical pI, half-life, aliphatic index, stability profiling, antigenicity, allergenicity, and hydropathy of the ensemble, showed that the vaccine is highly stable, antigenic, and non-allergenic and produces multiple interactions with immune receptors upon docking. In addition, molecular dynamics simulation confirmed the stable binding and good dynamic properties of the vaccine–TLR complex. Furthermore, in silico and immune simulation of the developed MEVC for Y. pestis showed that the vaccine triggered strong immune response after several doses at different intervals. Neutralization of the antigen was observed at the third day of injection. Conclusively, the vaccine designed here for Y. pestis produces an immune response; however, further immunological testing is needed to unveil its real efficacy.

Antifungal Activity of ToAP2D Peptide Against Sporothrix globosa

Improving clinical efficacy and reducing treatment time have been the focus of sporotrichosis therapy. Antimicrobial peptides ToAP2A, ToAP2C, and ToAP2D were synthesized on the basis of ToAP2 (AP02759), a peptide derived from the antimicrobial peptide database by the database filtering technology, and their physicochemical characteristics were analyzed. Compared with template peptide ToAP2, the modified peptides had much shorter length, lower molecular weight but significantly greater stability, which in return resulted in increases in the aliphatic index, hydrophilicity, and protein binding ability. Here, we show that the three derived peptides inhibit the growth of Sporothrix globosa, among which ToAP2D had the strongest anti-fungal activity. ToAP2D showed good serum stability without acute toxicity. The ToAP2D treatment inhibited the growth of S. globosa and enhanced apoptosis, which was evidenced by the upregulation of apoptosis-related protein caspase-3. The scanning electron microscopy analysis revealed deformation and rupture of S. globosa. The levels of mitochondrial membrane potential were decreased and that of the reactive oxygen species (ROS) were increased in S. globosa upon ToAP2D treatment. Moreover, ToAP2D activated metacaspase. In the in vivo study, we further demonstrated that ToAP2D inhibited the S. globosa infection of mice footpads, and its efficiency was nearly comparable to itraconazole. In summary, our results suggest that antimicrobial peptide ToAP2D has the potential for sporotrichosis therapy.

Designing of a novel multisubunit vaccine against Nipah virus structural proteins: A reverse vaccinology approach

Background The significant public health risk posed by NiV zoonosis and the lack of effective countermeasures against the intermittent outbreaks of the disease in the South and Southeast Asia region have entailed an imperative search for a protective vaccine to prevent or mitigate its epidemic potentiality. This is an endeavor to design an effective, safe multisubunit vaccine using an in silico reverse vaccinology approach. Methods The epitopes used for the construction of the candidate vaccine were meticulously predicted from five viral structural proteins (G, F, M, N, P) using several immunoinformatics tools to assess different epitope characteristics, namely, VaxiJen server for antigenicity, IEDB immunogenicity tool for immunogenicity, AlgPred server for allergenicity, ToxinPred for toxigenicity, IFNepitope server for interferon-gamma induction, Protparam server for physicochemical properties, GROMACS for simulation and simulation dynamics analysis, and finally, SnapGene tool for molecular cloning. Results The proposed vaccine molecule consisted of 501 amino acids, encompassing 7 B cell epitopes, 14 CTL epitopes, and 4 HTL epitopes. The physiochemical parameters of the vaccine construct showed a molecular weight of 54.6 kDa, an acidic stable molecule with an instability index of 38.3, aliphatic index of 62.89, and grand average of hydropathicity of -0.476. Moreover, the docking results and simulation dynamics of the vaccine molecule and TLR-3 showed global energy of 1.58 Kcal/mol, atomic contact energy of 2.98 Kcal/mol, and RMSD of 0.65 nm. The radius gyration showed a relatively steady value throughout the simulation period. a suggestive result of a stable compact structure and a promisingly effective vaccine construct. Conclusion In summary, the overall results of the multi-subunit vaccine molecule are suggestive of a promisingly effective vaccine against NiV infection in humans with a relatively stable compact structure, however, further experimental validation and assessment of pathogenic priming and autoimmunity induction are recommended.

Phospholipase A2 (PLA2) Sequences in Rattus norvegicus Genome

Phospholipase A2 is enzyme that hydrolyses phospholipids at sn-2 position. This class of enzymes are significant due to their ability to cleave membrane phospholipids and hence causing inflammation. The PLA2 enzymes present in Rattus norvegicus is extensively studied to predict its properties. The Protparam analysis was performed to predict the physical properties like number of amino acids, Theoretical pH, stability index value, aliphatic index value and GRAVY value. The SOPMA analysis predicted its structural properties like the number of alpha-helices and beta-strands. Hence the focus of the present study was to perform a preliminary in silico analysis to identify the PLA2 protein sequences in the genome of Rattus norvegicus.

Sequence-function Relationships in Phage-encoded Bacterial Cell Wall Lytic Enzymes and their Implications for Phage-derived Products Design

Phage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a landscape view of lysins’ structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G−) (higher net charge, amphipathic helices) are responsible for improved interaction with the G− envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecules design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G− surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages’ host bacteria, and sequence-predicted physicochemical properties. Based on such classifiers, an investigation of the differential appearance of certain features was conducted. Such analyses revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. Particularly, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment, and aliphatic index) were associated with G− phage lysins, appearing specifically at their C-terminal end. Evidence on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts has been provided, specifically supporting the nowadays common hypothesis that lysins from G− usually contain AMP-like regions. IMPORTANCE Phage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The potential of lysins as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance, but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysins diversity landscape by examining a set of phage lysin genes. This way, we have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures, and, thus, also the reach of their specialization regarding cell wall structures. These results provide clarity and evidence to sustain some of the common hypotheses in current literature, as well as make available an updated and characterized database of lysins sequences for further developments.

Vaccinomic approach for novel multi epitopes vaccine against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)

Background The spread of a novel coronavirus termed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in China and other countries is of great concern worldwide with no effective vaccine. This study aimed to design a novel vaccine construct against SARS-CoV-2 from the spike S protein and orf1ab polyprotein using immunoinformatics tools. The vaccine was designed from conserved epitopes interacted against B and T lymphocytes by the combination of highly immunogenic epitopes with suitable adjuvant and linkers. Results The proposed vaccine composed of 526 amino acids and was shown to be antigenic in Vaxigen server (0.6194) and nonallergenic in Allertop server. The physiochemical properties of the vaccine showed isoelectric point of 10.19. The instability index (II) was 31.25 classifying the vaccine as stable. Aliphatic index was 84.39 and the grand average of hydropathicity (GRAVY) was − 0.049 classifying the vaccine as hydrophilic. Vaccine tertiary structure was predicted, refined and validated to assess the stability of the vaccine via Ramachandran plot and ProSA-web servers. Moreover, solubility of the vaccine construct was greater than the average solubility provided by protein sol and SOLpro servers indicating the solubility of the vaccine construct. Disulfide engineering was performed to reduce the high mobile regions in the vaccine to enhance stability. Docking of the vaccine construct with TLR4 demonstrated efficient binding energy with attractive binding energy of − 338.68 kcal/mol and − 346.89 kcal/mol for TLR4 chain A and chain B respectively. Immune simulation significantly provided high levels of immunoglobulins, T-helper cells, T-cytotoxic cells and INF-γ. Upon cloning, the vaccine protein was reverse transcribed into DNA sequence and cloned into pET28a(+) vector to ensure translational potency and microbial expression. Conclusion A unique vaccine construct from spike S protein and orf1ab polyprotein against B and T lymphocytes was generated with potential protection against the pandemic. The present study might assist in developing a suitable therapeutics protocol to combat SARSCoV-2 infection.

Identification of the conserved domains of ADP-Glucose Pyrophosphorylase (AGPase) protein in sweetpotato (Ipomoea batatas (L.) Lam.) and its two wild relatives

The conserved domains are defined as recurring units in molecular evolution, which are commonly used to interpret the molecular function and biochemical structure of proteins. The AGPase amino acid sequences of three species from the Ipomoea genus were identified to investigate their physicochemical and biochemical characteristics. The molecular weights (MW), isoelectric point (pI), instability index (II), and grand average of hydropathy (GRAVY) showed considerable differences in each plant. The aliphatic index (AI) values of sweetpotato AGPase proteins were higher in the small subunit than in the large subunit. The AGPase proteins from sweetpotato contain an LbH_G1P_AT_C domain in the C-terminal region and various domains (NTP_transferase, ADP_Glucose_PP, or Glyco_tranf_GTA) in the N-terminal region. On the other hand, most of its two relatives (I. trifida and I. triloba) only contain the NTP_transferase domain in the N-terminal region. These findings suggested that these conserved domains were species specificity and related to the subunit types of AGPase proteins. The study may enable research on the AGPase-related specific characteristics of sweetpotatoes, which do not exist in the other two species, such as starch metabolism and tuberization mechanism.

Sequence-function Relationships in Phage-encoded Bacterial Cell Wall Lytic Enzymes and their Implications for Phage-derived Products Design

ABSTRACTPhage (endo)lysins are thought to be a viable alternative to usual antibiotic chemotherapy to fight resistant bacterial infections. However, a landscape view of lysins’ structure and properties regarding their function, with an applied focus, is somewhat lacking. Current literature suggests that specific features typical of lysins from phages infecting Gram-negative bacteria (G−) (higher net charge, amphipathic helices) are responsible for an improved interaction with G− envelope. Such antimicrobial peptide (AMP)-like elements are also of interest for antimicrobial molecules design. Thus, this study aims to provide an updated view on the primary structural landscape of phage lysins to clarify the evolutionary importance of several sequence-predicted properties, particularly for the interaction with the G− surface. A database of 2,182 lysin sequences was compiled, containing relevant information such as domain architectures, data on the phages’ host bacteria and sequence-predicted physicochemical properties. Based on such classifiers, an investigation on the differential appearance of certain features was conducted. Such analyses revealed different lysin architectural variants that are preferably found in phages infecting certain bacterial hosts. Particularly, some physicochemical properties (higher net charge, hydrophobicity, hydrophobic moment and aliphatic index) were associated to G− phage lysins, appearing specifically at their C-terminal end. Evidences on the remarkable genetic specialization of lysins regarding the features of the bacterial hosts have been provided, specifically supporting the nowadays common hypothesis that lysins from G− usually contain AMP-like regions.IMPORTANCEPhage-encoded lytic enzymes, also called lysins, are one of the most promising alternatives to common antibiotics. The lysins potential as novel antimicrobials to tackle antibiotic-resistant bacteria not only arises from features such as a lower chance to provoke resistance, but also from their versatility as synthetic biology parts. Functional modules derived from lysins are currently being used for the design of novel antimicrobials with desired properties. This study provides a view of the lysins diversity landscape by examining a set of phage lysin genes. This way, we have uncovered the fundamental differences between the lysins from phages that infect bacteria with different superficial architectures, and, thus, also the reach of their specialization regarding cell wall structures. These results provide clarity and evidences to sustain some of the common hypothesis in current literature, as well as make available an updated and characterized database of lysins sequences for further developments.

Bioinformatics analysis of calcium-dependent protein kinase 4 (CDPK4) as Toxoplasma gondii vaccine target

Objectives Toxoplasma gondii (T. gondii), an obligate intracellular apicomplexan parasite, could affect numerous warm-blooded animals, such as humans. Calcium-dependent protein kinases (CDPKs) are essential Ca2+ signaling mediators and participate in parasite host cell egress, outer membrane motility, invasion, and cell division. Results Several bioinformatics online servers were employed to analyze and predict the important properties of CDPK4 protein. The findings revealed that CDPK4 peptide has 1158 amino acid residues with average molecular weight (MW) of 126.331 KDa. The aliphatic index and GRAVY for this protein were estimated at 66.82 and – 0.650, respectively. The findings revealed that the CDPK4 protein comprised 30.14% and 34.97% alpha-helix, 59.84% and 53.54% random coils, and 10.02% and 11.49% extended strand with SOPMA and GOR4 tools, respectively. Ramachandran plot output showed 87.87%, 8.40%, and 3.73% of amino acid residues in the favored, allowed, and outlier regions, respectively. Also, several potential B and T-cell epitopes were predicted for CDPK4 protein through different bioinformatics tools. Also, antigenicity and allergenicity evaluation demonstrated that this protein has immunogenic and non-allergenic nature. This paper presents a basis for further studies, thereby provides a fundamental basis for the development of an effective vaccine against T. gondii infection.