Isolation, Identification, and Characterization of L-asparaginase-Producing Human Commensal Bacterial Strains: A Promising Next-Gen Probiotics.

L-asparaginase is an FDA-approved drug for treating blood cancer, but its inherent antigenicity and L-glutaminase activity are associated with hypersensitivity and organ toxicity. Extracellularly produced glutaminase-free L-asparaginase from human commensal bacteria may be a good alternative to reduce the side effects of therapeutic L-asparaginase. Here, we report the isolation and characterization of fourteen L-asparaginase-producing bacterial strains belonging to the genera Acinetobacter, Escherichia, Klebsiella, and Pseudomonas from human stool and saliva samples. To the best of our knowledge, this is the first report of L-asparaginase-producing human commensal bacterial strains isolated from healthy individuals. L-asparaginase produced by fecal and salivary isolates exhibited significantly higher activity (3.64 to 16.96 U/ml) toward L-asparagine than L-glutamine. Interestingly, L-asparaginase from fecal isolates, Escherichia coli strains 3F1 and 3F2 and salivary isolate Klebsiella pneumoniae 3S3, exhibited no L-glutaminase activity. These isolates were also sensitive to all tested antibiotics. Additionally, these three isolates demonstrated tolerance to pH 3.0 (≥ 88% survival) and 0.3% bile (≥ 95% survival), indicating their potential as probiotics. Among these isolates, L-asparaginase from the highest-producing K. pneumoniae 3S3 strain was found to be a homodimer, with native and subunit molecular weights of 110 kDa and 55 kDa, respectively. The purified enzyme can be further explored for its antitumor and immunomodulatory properties. Overall, future research can be expanded to include the use of a pool of human commensal bacteria as genuine and alternative sources of L-asparaginase for effective cancer treatments and cutting-edge next-generation probiotics.

Characterization of a novel 4-guanidinobutyrase from Candida parapsilosis.

Enzymes of the ureohydrolase superfamily are specific in recognizing their substrates. While looking to broaden the substrate specificity of 4-guanidinobutyrase (GBase), we isolated a yeast, typed as Candida parapsilosis (NCIM 3689), that efficiently utilized both 4-guanidinobutyrate (GB) and 3-guanidinopropionate (GP) as a sole source of nitrogen. A putative GBase sequence was identified from its genome upon pBLAST query using the GBase sequence from Aspergillus niger (AnGBase). The C. parapsilosis GBase (CpGBase) ORF was PCR amplified, cloned, and sequenced. Further, the functional CpGBase protein expressed in Saccharomyces cerevisiae functioned as GBase and 3-guanidinopropionase (GPase). S. cerevisiae cannot grow on GB or GP. However, the transformants expressing CpGBase acquired the ability to utilize and grow on both GB and GP. The expressed CpGBase protein was enriched and analyzed for substrate saturation and product inhibition by γ-aminobutyric acid and ß-alanine. In contrast to the well-characterized AnGBase, CpGBase from C. parapsilosis is a novel ureohydrolase and showed hyperbolic saturation for GB and GP with comparable efficiency (Vmax/KM values of 3.4 and 2.0, respectively). With the paucity of structural information and limited active site data available on ureohydrolases, CpGBase offers an excellent paradigm to explore this class of enzymes.

Structure-guided approach to modify the substrate specificity of the protein human deglycase-1 (hDJ-1).

Glycation is a non-enzymatic reaction wherein sugars or dicarbonyls such as methylglyoxal (MGO) and glyoxal (GO) react with proteins, leading to protein inactivation. The hydrolysing enzyme human deglycase-1 (hDJ-1) is reported to decrease glycative stress by deglycating the modified proteins, specifically at cysteine, lysine, and arginine sites. This specificity of hDJ-1 is thought to be regulated by its active site cysteine residue (Cys106). Structural analysis of hDJ-1 by molecular docking and simulation studies, however, indicates a possible role of glutamate (Glu18) in determining its substrate specificity. To elucidate this, Glu18 present at the catalytic site of hDJ-1 was modified to aspartate (Asp18) by SDM, and the resultant mutant was termed mutant DJ-1 (mDJ-1). Both hDJ-1 and mDJ-1 were heterologously expressed in Escherichia coli BL21 (DE3) strain and purified to homogeneity. The hDJ-1 showed kcat values of 1.45 × 103 s-1, 3.6 × 102 s-1, and 3.1 × 102 s-1, and Km values 0.181 mM, 18.18 mM, and 12.5 mM for N-acetylcysteine (NacCys), N-acetyllysine (NacLys), and N-acetylarginine (NacArg), respectively. The mDJ-1 showed altered kcat values (8 × 102 s-1, 3.8 × 102 s-1, 4.9 × 102 s-1) and Km values of 0.14 mM, 6.25 mM, 5.88 mM for NacCys, NacLys and NacArg, respectively. A single amino acid change (Glu18 to Asp18) improved the substrate specificity of mDJ-1 toward NacLys and NacArg. Understanding hDJ-1's structure and enhanced functionality will facilitate further exploration of its therapeutic potential for the treatment of glycation-induced diabetic complications.

NADP-dependent glutamate dehydrogenases in a dimorphic zygomycete Benjaminiella poitrasii: Purification, characterization and their evaluation as an antifungal drug target.

BACKGROUND: It has been reported that the genes coding for NADP-dependent glutamate dehydrogenases (NADP-GDHs) showed a cause-effect relationship with Yeast-Hypha (YH) reversible transition in a zygomycete Benjaminiella poitrasii. As YH transition is significant in human pathogenic fungi for their survival and proliferation in the host, the NADP-GDHs can be explored as antifungal drug targets. METHODS: The yeast-form specific BpNADPGDH I and hyphal-form specific BpNADPGDH II of B. poitrasii were purified by heterologous expression in E. coli BL-21 cells and characterized. The structural analogs of L-glutamate, dimethyl esters of isophthalic acid (DMIP) and its derivatives were designed, synthesized and screened for inhibition of NADP-GDH activity as well as YH transition in B. poitrasii, and also in human pathogenic Candida albicans strains. RESULTS: The BpNADPGDH I and BpNADPGDH II were found to be homo-hexameric proteins with native molecular mass of 282 kDa and 298 kDa, respectively and subunit molecular weights of 47 kDa and 49 kDa, respectively. Besides the distinct kinetic properties, BpNADPGDH I and BpNADPGDH II were found to be regulated by cAMP-dependent- and Calmodulin (CaM) dependent- protein kinases, respectively. The DMIP compounds showed a more pronounced effect on H-form specific BpNADPGDH II and inhibited YH transition as well as growth in B. poitrasii and C. albicans strains. CONCLUSION: The present study will be useful to design and develop antifungal drugs against dimorphic human pathogens using glutamate dehydrogenase as a target. SIGNIFICANCE: Glutamate dehydrogenases can be explored as a target against human pathogenic fungi.

Molecular studies of NAD- and NADP-glutamate dehydrogenases decipher the conundrum of yeast-hypha dimorphism in zygomycete Benjaminiella poitrasii.

Benjaminiella poitrasii, a zygomycete, shows glucose- and temperature-dependent yeast (Y)-hypha (H) dimorphic transition. Earlier, we reported the biochemical correlation of relative proportion of NAD- and NADP-glutamate dehydrogenases (GDHs) with Y-H transition. Further, we observed the presence of one NAD-GDH and two form-specific NADP-GDH isoenzymes in B. poitrasii. However, molecular studies are necessary to elucidate the explicit role of GDHs in regulating Y-H reversible transition. Here, we report the isolation and characterization of one NAD (BpNADGDH, 2.643 kb) and two separate genes, BpNADPGDH I (Y-form specific, 1.365 kb) and BpNADPGDH II (H-form specific, 1.368 kb) coding for NADP-GDH isoenzymes in B. poitrasii. The transcriptional profiling during Y-H transition showed higher BpNADPGDH I expression in Y cells while expression of BpNADPGDH II was higher in H cells. Moreover, the yeast-form monomorphic mutant (Y-5) did not show BpNADPGDH II expression under normal dimorphism triggering conditions. Transformation with H-form specific BpNADPGDH II induced the germ tube formation in Y-5, which confirmed the cause-effect relationship between BpNADPGDH genes and morphological outcome in B. poitrasii. Interestingly, expression of H-form specific BpNADPGDH II also induced germ tube formation in human pathogenic, non-dimorphic yeast Candida glabrata, which further corroborated our findings.

The puzzle of highly virulent Metarhizium anisopliae strains from Annona squamosa fields against Helicoverpa armigera.

In our search for indigenous virulent strains of the entomopathogenic fungi, we observed that Metarhizium isolates from soils associated with Annona squamosa (custard apple) have higher virulence (>90% mortality of Helicoverpa armigera larvae at 1/10th spore concentration) than strains isolated from Solanum lycopersicum (tomato) fields. Proteomic analysis revealed two insecticidal cyclopeptides of A. squamosa origin in the M. anisopliae strains that led to higher virulence against H. armigera. Transcriptomic and genomic data indicated that M. anisopliae strains and A. squamosa had more than 20 genes in common, including those for cyclic hexapeptide synthase, non-ribosomal peptide synthetase, and plant cyclotide genes, which are involved in the biosynthesis of insecticidal cyclopeptides. These genes were absent in M. anisopliae strains isolated from the S. lycopersicum fields. Further, these strains can establish an endophytic relationship with A. squamosa suggesting that these rhizospheric strains originally could be endophytes, which were eventually released into the soil. Further, Metarhizium strains associated with Capsicum annuum (chili), Azadirachta indica (neem), and Carica papaya (papaya) - plants with insecticidal properties - also had higher virulence against H. armigera. Thus exploration of rhizospheres of plants producing insecticidal metabolites to isolate entomopathogenic fungi, per se, could be a viable strategy in agricultural for crop protection.

Development of Metarhizium anisopliae as a Mycoinsecticide: From Isolation to Field Performance.

A major concern when developing commercial mycoinsecticides is the kill speed compared to that of chemical insecticides. Therefore, isolation and screening for the selection of a fast-acting, highly virulent entomopathogenic fungus are important steps. Entomopathogenic fungi, such as Metarhizium, Beauveria, and Nomurea, which act by contact, are better suited than Bacillus thuringiensis or nucleopolyhedrosis virus (NPV), which must be ingested by the insect pest. In the present work, we isolated 68 Metarhizium strains from infected insects using a soil dilution and bait method. The isolates were identified by the amplification and sequencing of the ITS1-5.8S-ITS2 and 26S rDNA region. The most virulent strain of Metarhizium anisopliae was selected based on the median lethal concentration (LC50) and time (LT50) obtained in insect bioassays against III-instar larvae of Helicoverpa armigera. The mass production of spores by the selected strain was carried out with solid-state fermentation (SSF) using rice as a substrate for 14 days. Spores were extracted from the sporulated biomass using 0.1% tween-80, and different formulations of the spores were prepared. Field trials of the formulations for the control of an H. armigera infestation in pigeon peas were carried out by randomized block design. The infestation control levels obtained with oil and aqueous formulations (78.0% and 70.9%, respectively) were better than the 63.4% obtained with chemical pesticide.

Selection of reference genes for quantitative real-time RT-PCR assays in different morphological forms of dimorphic zygomycetous fungus Benjaminiella poitrasii.

Benjaminiella poitrasii, a dimorphic non-pathogenic zygomycetous fungus, exhibits a morphological yeast (Y) to hypha (H) reversible transition in the vegetative phase, sporangiospores (S) in the asexual phase and zygospores (Z) in the sexual phase. To study the gene expression across these diverse morphological forms, suitable reference genes are required. In the present study, 13 genes viz. ACT, 18S rRNA, eEF1α, eEF-Tu,eIF-1A, Tub-α, Tub-b, Ubc, GAPDH, Try, WS-21, NADGDH and NADPGDH were evaluated for their potential as a reference, particularly for studying gene expression during the Y-H reversible transition and also for other asexual and sexual life stages of B. poitrasii. Analysis of RT-qPCR data using geNorm, normFinder and BestKeeper software revealed that genes such as Ubc, 18S rRNA and WS-21 were expressed at constant levels in each given subset of RNA samples from all the morphological phases of B. poitrasii. Therefore, these reference genes can be used to elucidate the role of morpho-genes in B. poitrasii. Further, use of the two most stably expressed genes (Ubc and WS-21) to normalize the expression of the ornithine decarboxylase gene (Bpodc) in different morphological forms of B. poitrasii, generated more reliable results, indicating that our selection of reference genes was appropriate.