In vitro and in silico analysis unravelled clinically desirable attributes of Bacillus altitudinis L-asparaginase.

AIMS: To identify a marine L-asparaginase with clinically desirable attributes and characterize the shortlisted candidate through in silico tools. METHODS AND RESULTS: Marine bacterial strains (number = 105) isolated from marine crabs were evaluated through a stepwise strategy incorporating the crucial attributes for therapeutic safety. The results demonstrated the potential of eight bacterial species for extracellular L-asparaginase production. However, only one isolate (Bacillus altitudinis CMFRI/Bal-2) showed clinically desirable attributes, viz. extracellular production, type-II nature, lack of concurrent L-glutaminase and urease activities, and presence of ansZ (functional gene for clinical type). The enzyme production was 22.55 ± 0.5 µM/mg protein/min within 24 h without optimization. The enzyme also showed good activity and stability in pH 7-8 and temperature 37°C, predicting the functioning inside the human body. The Michealis-Menten constant (Km) was 14.75 µM. Detailed in silico analysis based on functional gene authenticating the results of in vitro characterization and predicted the nonallergenic characteristic of the candidate. Docking results proved the higher affinity of the shortlisted candidate to L-asparagine than L-glutamine and urea. CONCLUSION: Comprehensively, the study highlighted B. altitudinis type II asparaginase as a competent candidate for further research on clinically safe asparaginases.

Characterization of novel L-asparaginases having clinically safe profiles from bacteria inhabiting the hemolymph of the crab, Scylla serrata (Forskål, 1775).

L-asparaginase (ASNase) is the principal chemotherapeutic agent against different blood cancers. The risks associated with current clinical preparations demand screening for novel ASNases. Accordingly, the study was conducted to shortlist ASNases having clinically safer profiles from a novel niche, namely, microbes in the gut and hemolymph of apparently healthy Scylla serrata. A four-step strategic approach incorporating the essential requirements for clinically safer profiles was followed. The initial step through plate assay showed five (9.61%) potential ASNase producers. The relative prevalence of ASNase producers was higher in hemolymph (13.33%) than gut (4.5%). The positive isolates were identified as Priestia aryabhattai, Priestia megaterium, Bacillus altitudinis, Shewanella decolorationis, and Chryseomicrobium amylolyticum. Quantitative profiles revealed high ASNase production (114.29 to 287.36 U/mL) without any optimization, with an added advantage of the extracellular production. The second step for substrate specificity studies revealed the absence of L-glutaminase and urease activities in ASNases from C. amylolyticum and P. megaterium, the most desirable properties for safe clinical applications. This is the first report of glutaminase and urease-free ASNase from these two bacteria. The third step ensured type II nature of selected ASNases, the targeted form in clinical applications. The fourth step confirmed the activity and stability in human physiological conditions. Altogether, the results revealed two potential ASNases with clinically compatible profiles.

A novel ferritin subunit gene from Asian green mussel, Perna viridis (Linnaeus, 1758).

Iron sequestration through ferritin forms a major part of innate immune response in molluscs and detailed understanding of ferritin gene and its functions can be directly applied in infection and disease management studies. Accordingly, identification and detailed molecular characterization of a ferritin subunit gene from a commercially significant marine mussel Perna viridis was targeted. Molecular screening using degenerate primers in total mantle RNA resulted in the amplification of a novel ferritin gene fragment having <87% identity to the reported ferritin gene sequences. Rapid amplification of cDNA ends-PCR was followed to generate complete cDNA sequence of P.viridis ferritin (PvFer). The complete cDNA was found to be 798 bp, containing an open reading frame of 522 bp, 5' untranslated region (UTR) of 112 bp and 3' UTR of 165 bp. The 5' UTR and 3' UTR were shown to contain an iron response element (IRE) and a polyadenylation signal (767AATAAA772) with poly (A) tail, respectively. Prediction of stem loop structure revealed that, PvFer-IRE can be folded into a typical secondary stem loop structure, having 5-CAGUGA-3' loop, proximal stem of five paired bases followed by a bulged cysteine, and six nucleotide bottom stem, indicating that expression of PvFer is regulated by iron at the translational level. ORF was found to encode 175 amino acid protein with calculated molecular mass of 19.97 kDa and isoelectric point of 4.97. Examination for signal peptide and phylogenetic analysis confirmed that PvFer belonged to cytosolic ferritins of molluscs. Conserved domain analysis showed that PvFer contained both ferroxidase diiron center and ferrihydrite nucleation center, analogous to ferritin M subunit of bony fishes and amphibians. However, amino acid sequence and glycosylation site showed more homology to vertebrate ferritin H subunits. Predicted 3D models of PvFer resembled the typical spatial features of ferritin proteins. The study forms the first comprehensive identification of a ferritin subunit gene in a true/common mussel (Order: Mytilida). Further, the detailed molecular phylogeny conducted through the present study revealed certain thought provoking insights on ferritin genes of the phylum Mollusca.

Epizootics of epizootic ulcerative syndrome among estuarine fishes of Kerala, India, under post-flood conditions.

Epizootic ulcerative syndrome (EUS), primarily caused by the water mold Aphanomyces invadans, is an OIE-notifiable disease, having potential impacts on fisheries. We report EUS epizootics among estuarine fishes of Kerala, India, during 2018, under post-flood conditions 3 decades after its primary outbreak. Six fish species (Mugil cephalus, Platycephalus sp., Scatophagus argus, Arius sp., Planiliza macrolepis and Epinephelus malabaricus) were infected, including the first confirmed natural case in E. malabaricus and P. macrolepis. Salinity, surface temperature, dissolved oxygen and pH of resident water during the epizootic were <2 ppt, 25°C, 4.1 ppm and 7.0. The presence of zoonotic bacterial pathogens (Aeromonas veronii, Shewanella putrefaciens, Vibrio vulnificus and V. parahaemolyticus) in tissues of affected fish indicates that EUS-infected fish may pose a public health hazard if not handled properly. Lack of clinical evidence in the region during the last 3 decades, a high number of affected fishes, including 2 new fish species, the severity of skin lesions and very low water salinity (<2 ppt) during the outbreak in contrast to historical water salinity records suggest relatively recent invasion by A. invadans. Phylogenetic analysis based on the internal transcribed spacer region of the rRNA gene showed that the same clone of pathogen has spread across different continents regardless of fish species and ecotypes (fresh/estuarine environments). Altogether, the present study provides baseline data which can be applied in EUS management strategies within brackish-water ecosystems. We recommend strict surveillance and development of sound biosecurity measures against the disease.