In silico analysis of Naegleria fowleri cathepsin B paralogs: important drug targets.

Naegleria fowleri is a deadly human pathogen that causes primary amoebic meningoencephalitis (PAM). In this study, in silico investigations of two important N. fowleri cathepsin B paralogs, i.e., copies of genes resulting from a gene duplication event, were carried out using comparative modeling and molecular dynamics (MD) simulations. Comparative models of both paralogs showed significant architectural similarity with their template, i.e., rat cathepsin B. However, in N. fowleri cathepsin B (UniProt ID: X5D761) and putative cathepsin B (UniProt ID: M1HE19) enzymes, eleven and fifteen residues in the occluding loop regions were deleted, respectively, suggesting that these enzymes have a short occluding loop. Thus, it is concluded that N. fowleri cathepsin B and putative cathepsin B enzymes lack exopeptidase activity but possess enhanced endopeptidase activity and an affinity for macromolecular inhibitors. MD simulations further confirmed that prosegments (macromolecular inhibitors) bond more tightly with both enzymes than with wild-type cathepsin B. Additionally, a mutation was identified at an important N-glycosylation site; this mutation is believed to affect cathepsin B targeting inside the cell and make cathepsin B available in the extracellular environment. Due to this important N-glycosylation site mutation, these enzymes are secreted in the extracellular environment via an alternative, still unknown, posttranslational processing strategy. The present study is the first to predict the three-dimensional folds of N. fowleri cathepsin B paralogous enzymes, including a detailed description of the active site architecture and information about propeptide binding mode. This information can contribute to the discovery of novel and selective treatments that are effective against N. fowleri.

Draft genome sequence of a novel Bacillus glycinifermentans strain having antifungal and antibacterial properties.

OBJECTIVES: Bacillus spp. have been used as biocontrol agents against soilborne pathogens because they produce secondary metabolites that exhibit a wide range of antibacterial or antifungal properties. In this study, a novel strain of Bacillus glycinifermentans sp. (JRCGR-1) was identified and its genome was sequenced and annotated. The genome was explored for putative genes involved in antimicrobial activity. METHODS: Whole-genome sequencing was performed on an Illumina NextSeq 500 platform. Read quality was checked by FastQC, paired-end reads were trimmed using Sickle, and de novo assembly was performed using SPAdes v.3.11.11. QUAST 5.02 was used to assess the quality of contigs and scaffolds. Finally, the assembled scaffolds were annotated by Prokka v.1.13. Genes involved in antimicrobial metabolite biosynthesis were predicted using antiSMASH. Virulence and antimicrobial resistance genes were predicted using BacWGSTdb and the Comprehensive Antibiotic Resistance Database (CARD), respectively. RESULTS: The genome of B. glycinifermentans JRCGR-1 was 4 700 692 bp in size with a G + C content of 45.52%. Final assembly of the genome resulted into 84 contigs and 83 scaffolds (>500 bp length). Overall, the genome comprises 5174 genes, 32 tRNAs, 4 rRNAs, 1 tmRNA and 92 misc_RNAs. Eleven putative gene clusters responsible for antimicrobial metabolite biosynthesis were identified, including genes for biosynthesis of non-ribosomal lipopeptides and polyketides. Virulence and antimicrobial resistance genes were also identified in the genome. CONCLUSION: The presence of antimicrobial resistance genes in the genome of B. glycinifermentans JRCGR-1 makes it a potential biocontrol agent against soilborne pathogens.

Draft genome of a macrolide resistant XDR Salmonella enterica serovar Paratyphi A strain using a shotgun sequencing approach.

OBJECTIVES: Salmonella enterica serovar Paratyphi A, the causative pathogen of enteric fever, is a major public-health concern affecting millions of people around the world. We conducted whole-genome sequencing and analysis of a novel macrolide-resistant Salmonella Paratyphi A strain isolated from Karachi, Pakistan. METHODS: Genomic DNA of Salmonella Paratyphi A strain JRCGR-AK14 was sequenced on a MiSeq platform. Read quality was evaluated and paired-end reads were assembled into contigs and scaffolds. The quality of contigs and scaffolds was evaluated and assembled contigs were annotated. Virulence genes, antimicrobial resistance genes (ARGs), tRNAs, rRNAs, coding sequences and clustered regularly interspaced short palindromic repeats (CRISPRs) were identified. ARGs and mutations in quinolone-resistance determining regions (QRDRs) were identified by Antimicrobial Resistance Identification By Assembly (ARIBA) and ResFinder. Known and unknow mutations in the QRDRs were predicted. RESULTS: The genome of Salmonella Paratyphi A was calculated at 4529866 bp with 4381 genes and 1088 hypothetical proteins. Several putative genes coding for multidrug efflux pumps were identified. In addition, gene mutations conferring resistance to nitrofurantoin (e.g. marA, mdsC, Escherichia coli soxS), pulvomycin (e.g. H-NS, cpxA, E. coli EF-Tu) and fosfomycin (CRP, kdpE, E. coli glpT) were also identified. Several ARGs along with the mobile genetic element transposon Tn10 were also identified. It is evident from the results that diverse redundant mechanisms are involved in regulation of drug resistance in this strain. CONCLUSION: The current findings provide valuable data for understanding the multidrug resistance and pathogenic characteristics of clinical Salmonella Paratyphi A isolates.

Comparative genomics of an endophytic Pseudomonas putida isolated from mango orchard.

We analyzed the genome sequence of an endophytic bacterial strain Pseudomonas putida TJI51 isolated from mango bark tissues. Next generation DNA sequencing and short read de novo assembly generated the 5,805,096 bp draft genome of P. putida TJI51. Out of 6,036 protein coding genes in P. putida TJI51 sequences, 4,367 (72%) were annotated with functional specifications, while the remaining encoded hypothetical proteins. Comparative genome sequence analysis revealed that the P. putida TJI51genome contains several regions, not identified in so far sequenced P. putida genomes. Some of these regions were predicted to encode enzymes, including acetylornithine deacetylase, betaine aldehyde dehydrogenase, aldehyde dehydrogenase, benzoylformate decarboxylase, hydroxyacylglutathione hydrolase, and uroporphyrinogen decarboxylase. The genome of P. putida TJI51 contained three nonribosomal peptide synthetase gene clusters. Genome sequence analysis of P. putidaTJI51 identified this bacterium as an endophytic resident. The endophytic fitness might be linked with alginate, which facilitates bacterial colonization in plant tissues. Genome sequence analysis shed light on the presence of a diverse spectrum of metabolic activities and adaptation of this isolate to various niches.