Molecular characterisation of the GdhA- derivative of Pasteurella multocida B:2

Pasteurella multocida B:2 is an important veterinary pathogen causing fatal and acute haemorrhagic septicaemia (HS) in bovine. A live vaccine candidate, P. multocida B:2 GDH7 was reported to enable protection in cattle and buffaloes via intranasal (i. n.) administration. This potential vaccine was also reported to be self-transmitted from the vaccinated animal to the free-ranging animals allowing wider vaccination coverage. Prior to commercialisation, this potential vaccine requires further characterisation in accordance with the authoritative guidelines from the World Organisation for Animal Health (OIE). Hence, in this study, the potential vaccine strain, P. multocida B:2 GDH7 and the virulent parent strain were characterised through genomic and proteomic profiling. A crucial first step was to develop a sensitive yet simple and robust identification test to differentiate both strains which has been achieved by the development of a precise yet straightforward PCR method. In genomic profiling, Repetitive Extragenic Palindromic sequence-PCR (REP-PCR) was manipulated and both strains have a different display of genomic DNA band patterns. Some of the major OMPs were observed and prominent immunogens of P. multocida, OmpA and OmpH were observed to be expressed differently between these strains through SDS-PAGE analysis. In conclusion, a reproducible PCR detection method has enabled differentiation of both strains. Further characterisation of these strains shows a significantly different profile through genomic and proteomic profiling

Making the most of its short reads: a bioinformatics workflow for analysing the short-read-only data of Leishmania orientalis (formerly named Leishmania siamensis) isolate PCM2 in Thailand

Background: Leishmania orientalis (formerly named Leishmania siamensis) has been neglected for years in Thailand. The genomic study of L. orientalis has gained much attention recently after the release of the first high-quality reference genome of the isolate LSCM4. The integrative approach of multiple sequencing platforms for whole-genome sequencing has proven effective at the expense of considerably expensive costs. This study presents a preliminary bioinformatic workflow including the use of multi-step de novo assembly coupled with the reference-based assembly method to produce high-quality genomic drafts from the short-read Illumina sequence data of L. orientalis isolate PCM2. Results: The integrating multi-step de novo assembly by MEGAHIT and SPAdes with the reference-based method using the L. enriettii genome and salvaging the unmapped reads resulted in the 30.27 Mb genomic draft of L. orientalis isolate PCM2 with 3367 contigs and 8887 predicted genes. The results from the integrated approach showed the best integrity, coverage, and contig alignment when compared to the genome of L. orientalis isolate LSCM4 collected from the northern province of Thailand. Similar patterns of gene ratios and frequency were observed from the GO biological process annotation. Fifty GO terms were assigned to the assembled genomes, and 23 of these (accounting for 61.6% of the annotated genes) showed higher gene counts and ratios when results from our workflow were compared to those of the LSCM4 isolate. Conclusions: These results indicated that our proposed bioinformatic workflow produced an acceptable-quality genome of L. orientalis strain PCM2 for functional genomic analysis, maximising the usage of the short-read data. This workflow would give extensive information required for identifying strain-specific markers and virulence-associated genes useful for drug and vaccine development before a more exhaustive and expensive investigation

Identification of a conserved maxicircle and unique minicircles as part of the mitochondrial genome of Leishmania martiniquensis strain PCM3 in Thailand

Background: The mitochondrial DNA of trypanosomatids, including Leishmania, is known as kinetoplast DNAs (kDNAs). The kDNAs form networks of hundreds of DNA circles that are evidently interlocked and require complex RNA editing. Previous studies showed that kDNA played a role in drug resistance, adaptation, and survival of Leishmania. Leishmania martiniquensis is one of the most frequently observed species in Thailand, and its kDNAs have not been illustrated. Methods: This study aimed to extract the kDNA sequences from Illumina short-read and PacBio long-read whole-genome sequence data of L. martiniquensis strain PCM3 priorly isolated from the southern province of Thailand. A circular maxicircle DNA was reconstructed by de novo assembly using the SPAdes program, while the minicircle sequences were retrieved and assembled by the rKOMIC tool. The kDNA contigs were confirmed by blasting to the NCBI database, followed by comparative genomic and phylogenetic analysis. Results: We successfully constructed the complete circular sequence of the maxicircle (19,008 bp) and 214 classes of the minicircles from L. martiniquensis strain PCM3. The genome comparison and annotation showed that the maxicircle structure of L. martiniquensis strain PCM3 was similar to those of L. enriettii strain LEM3045 (84.29%), L. arabica strain LEM1108 (82.79%), and L. tarentolae (79.2%). Phylogenetic analysis also showed unique evolution of the minicircles of L. martiniquensis strain PCM3 from other examined Leishmania species. Conclusions: This was the first report of the complete maxicircle and 214 minicircles of L. martiniquensis strain PCM3 using integrated whole-genome sequencing data. The information will be helpful for further improvement of diagnosis methods and monitoring genetic diversity changes of this parasite

Amplification and bioinformatics analysis of conserved FAD-binding region of L-amino acid oxidase (LAAO) genes in gastropods compared to other organisms

This study aimed to investigate the conserved FAD-binding region of the L-amino acid oxidase (LAAO) genes in twelve gastropod genera commonly found in Thailand compared to those in other organisms using molecular cloning, nucleotide sequencing and bioinformatics analysis. Genomic DNA of gastropods and other invertebrates was extracted and screened using primers specific to the conserved FAD-binding region of LAAO. The amplified 143-bp fragments were cloned and sequenced. The obtained nucleotide sequences of 21 samples were aligned and phylogenetically compared to the LAAO-conserved FAD-binding regions of 210 other organisms from the NCBI database. Translated amino acid sequences of these samples were used in phylogenetics and pattern analyses. The phylogenetic trees showed clear separation of the conserved regions in fungi, invertebrates, and vertebrates. Alignment of the conserved 47-amino-acid FAD-binding region of the LAAOs showed 150 unique sequences among the 231 samples and these patterns were different from those of other flavoproteins in the amine oxidase family. An amino acid pattern analysis of five sub-regions (bFAD, FAD, FAD-GG, GG, and aGG) within the FAD-binding sequence showed high variation at the FAD-GG sub-region. Pattern analysis of secondary structures indicated the aGG sub-region as having the highest structural variation. Cluster analysis of these patterns revealed two major clusters representing the mollusc clade and the vertebrate clade. Thus, molecular phylogenetics and pattern analyses of sequence and structural variations could reflect evolutionary relatedness and possible structural conservation to maintain specific function within the FAD-binding region of the LAAOs in gastropods compared to other organisms. Keywords: L-amino acid oxidase (LAAO), FAD-binding region, Gastropods, Pattern analysis, Clusterin

Comparison of Hyaluronic Acid Biosynthetic Genes From Different Strains of

Pasteurella multocida produces a capsule composed of different polysaccharides according to the capsular serotype (A, B, D, E, and F). Hyaluronic acid (HA) is a component of certain capsular types of this bacterium, especially capsular type A. Previously, 2 HA biosynthetic genes from a capsular type A strain were studied for the industrial-scale improvement of HA production. Molecular comparison of these genes across different capsular serotypes of P multocida has not been reported. This study aimed to compare 8 HA biosynthetic genes ( pgi, pgm, galU, hyaC, glmS, glmM, glmU , and hyaD ) of 22 P multocida strains (A:B:D:F = 6:6:6:4) with those of other organisms using sequence and structural bioinformatics analyses. These 8 genes showed a high level of within-species similarity (98%-99%) compared with other organisms. Only the last gene of 4 strains with capsular type F (HN07, PM70, HNF01, and HNF02) significantly differed from those of other strains (82%). Analysis of amino acid patterns together with phylogenetic results showed that the HA biosynthetic genes of the type A were closely related within the group. The genes in the capsular type F strain were notably similar to those of the capsular type A strain. Protein structural analysis supported structural similarities of the encoded enzymes between the strains of capsular types A, B, D, and F, except for the Pgm, GlmS, GlmU, and HyaD proteins. Our bioinformatics analytic workflow proposed that variations observed within these genes could be useful for genetic engineering–based improvement of hyaluronic acid–producing enzymes

Understanding Snail Mucus Biosynthesis and Shell Biomineralisation through Genomic Data Mining of the Reconstructed Carbohydrate and Glycan Metabolic Pathways of the Giant African Snail (<i>Achatina fulica</i>)

The giant African snail (Order Stylommatophora: Family Achatinidae), Achatina fulica (Bowdich, 1822), is the most significant and invasive land snail pest. The ecological adaptability of this snail involves high growth rate, reproductive capacity, and shell and mucus production, driven by several biochemical processes and metabolism. The available genomic information for A. fulica provides excellent opportunities to hinder the underlying processes of adaptation, mainly carbohydrate and glycan metabolic pathways toward the shell and mucus formation. The authors analysed the 1.78 Gb draft genomic contigs of A. fulica to identify enzyme-coding genes and reconstruct biochemical pathways related to the carbohydrate and glycan metabolism using a designed bioinformatic workflow. Three hundred and seventy-seven enzymes involved in the carbohydrate and glycan metabolic pathways were identified based on the KEGG pathway reference in combination with protein sequence comparison, structural analysis, and manual curation. Fourteen complete pathways of carbohydrate metabolism and seven complete pathways of glycan metabolism supported the nutrient acquisition and production of the mucus proteoglycans. Increased copy numbers of amylases, cellulases, and chitinases highlighted the snail advantage in food consumption and fast growth rate. The ascorbate biosynthesis pathway identified from the carbohydrate metabolic pathways of A. fulica was involved in the shell biomineralisation process in association with the collagen protein network, carbonic anhydrases, tyrosinases, and several ion transporters. Thus, our bioinformatic workflow was able to reconstruct carbohydrate metabolism, mucus biosynthesis, and shell biomineralisation pathways from the A. fulica genome and transcriptome data. These findings could reveal several evolutionary advantages of the A. fulica snail, and will benefit the discovery of valuable enzymes for industrial and medical applications

Prediction of Antibacterial Peptides against Propionibacterium acnes from the Peptidomes of Achatina fulica Mucus Fractions

Acne vulgaris is a common skin disease mainly caused by the Gram-positive pathogenic bacterium, Propionibacterium acnes. This bacterium stimulates the inflammation process in human sebaceous glands. The giant African snail (Achatina fulica) is an alien species that rapidly reproduces and seriously damages agricultural products in Thailand. There were several research reports on the medical and pharmaceutical benefits of these snail mucus peptides and proteins. This study aimed to in silico predict multifunctional bioactive peptides from A. fulica mucus peptidome using bioinformatic tools for the determination of antimicrobial (iAMPpred), anti-biofilm (dPABBs), cytotoxic (ToxinPred) and cell-membrane-penetrating (CPPpred) peptides. Three candidate peptides with the highest predictive score were selected and re-designed/modified to improve the required activities. Structural and physicochemical properties of six anti-P. acnes (APA) peptide candidates were performed using the PEP&ndash;FOLD3 program and the four previous tools. All candidates had a random coiled structure and were named APAP-1 ori, APAP-2 ori, APAP-3 ori, APAP-1 mod, APAP-2 mod, and APAP-3 mod. To validate the APA activity, these peptide candidates were synthesized and tested against six isolates of P. acnes. The modified APA peptides showed high APA activity on three isolates. Therefore, our biomimetic mucus peptides could be useful for preventing acne vulgaris and further examined on other activities important to medical and pharmaceutical applications

Iron-associated protein interaction networks reveal the key functional modules related to survival and virulence of Pasteurella multocida

Pasteurella multocida causes respiratory infectious diseases in a multitude of birds and mammals. A number of virulence-associated genes were reported across different strains of P. multocida, including those involved in the iron transport and metabolism. Comparative iron-associated genes of P. multocida among different animal hosts towards their interaction networks have not been fully revealed. Therefore, this study aimed to identify the iron-associated genes from core- and pan-genomes of fourteen P. multocida strains and to construct iron-associated protein interaction networks using genome-scale network analysis which might be associated with the virulence. Results showed that these fourteen strains had 1587 genes in the core-genome and 3400 genes constituting their pan-genome. Out of these, 2651 genes associated with iron transport and metabolism were selected to construct the protein interaction networks and 361 genes were incorporated into the iron-associated protein interaction network (iPIN) consisting of nine different iron-associated functional modules. After comparing with the virulence factor database (VFDB), 21 virulence-associated proteins were determined and 11 of these belonged to the heme biosynthesis module. From this study, the core heme biosynthesis module and the core outer membrane hemoglobin receptor HgbA were proposed as candidate targets to design novel antibiotics and vaccines for preventing pasteurellosis across the serotypes or animal hosts for enhanced precision agriculture to ensure sustainability in food security

Prediction of anticancer peptides against MCF-7 breast cancer cells from the peptidomes of Achatina fulica mucus fractions

Several reports have shown antimicrobial and anticancer activities of mucous glycoproteins extracted from the giant African snail Achatina fulica. Anticancer properties of the snail mucous peptides remain incompletely revealed. The aim of this study was to predict anticancer peptides from A. fulica mucus. Two of HPLC-separated mucous fractions (F2 and F5) showed in vitro cytotoxicity against the breast cancer cell line (MCF-7) and normal epithelium cell line (Vero). According to the mass spectrometric analysis, 404 and 424 peptides from the F2 and F5 fractions were identified. Our comprehensive bioinformatics workflow predicted 16 putative cationic and amphipathic anticancer peptides with diverse structures from these two peptidome data. These peptides would be promising molecules for new anti-breast cancer drug development

Properties of Protein Hydrolysates and Bioinformatics Prediction of Peptides Derived from Thermal and Enzymatic Process of Skipjack Tuna (<i>Katsuwonus pelamis)</i> Roe

Currently, the use of skipjack tuna (Katsuwonus pelamis) roe to produce hydrolysate is limited, although it is a potentially valuable resource. This study aimed to investigate the physical and chemical characteristics of protein hydrolysates from tuna roe using autoclave and enzymes (alcalase and trypsin at 0.5 and 1.0% w/v). Bioinformatics was also applied to analyze the identified peptides. The hydrolysates were determined for amino acid composition, peptide profile patterns, antioxidant activity, solubility and foaming properties. The proteins were separated by SDS-PAGE before tryptic digestion and peptide identification by nano LC-ESI-MS/MS. The putative bioactivities of the identified peptides were predicted using bioinformatics prediction tools. The main amino acids found in all hydrolysates were cysteine, glycine and arginine (16.26–20.65, 10.67–13.61 and 10.87–12.08 g/100 g protein, respectively). The hydrolysates obtained from autoclaving showed lower molecular weights than those by the enzymatic method. The 0.1 g/mL concentration of hydrolysates provided higher antioxidant activities compared to the others. All hydrolysates had high solubility and exhibited foaming capacity and foam stability. Putative anti-hypertensive, anti-virus and anti-parasite activities were highly abundant within the obtained peptides. Moreover, predicted muti-bioactivity was indicated for seven novel peptides. In the future work, these peptides should be experimentally validated for further applications