Insights into horizontal acquisition patterns of dormancy and reactivation regulon genes in mycobacterial species using a partitioning-based framework

Horizontal Gene Transfer (HGT) events, initially thought to be rare in Mycobacterium tuberculosis, have recently been shown to be involved in the acquisition of virulence operons in M. tuberculosis. We have developed a new partitioning framework based HGT prediction algorithm, called Grid3M, and applied the same for the prediction of HGTs in Mycobacteria. Validation and testing using simulated and real microbial genomes indicated better performance of Grid3M as compared with other widely used HGT prediction methods. Specific analysis of the genes belonging to dormancy/reactivation regulons across 14 mycobacterial genomes indicated that horizontal acquisition is specifically restricted to important accessory proteins. The results also revealed Burkholderia species to be a probable source of HGT genes belonging to these regulons. The current study provides a basis for similar analyses investigating the functional/evolutionary aspects of HGT genes in other pathogens. A database of Grid3M predicted HGTs in completely sequenced genomes is available at https://metagenomics.atc.tcs.com/Grid3M/ .     

Chickpea glutaredoxin (CaGrx) gene mitigates drought and salinity stress by modulating the physiological performance and antioxidant defense mechanisms

Glutaredoxins (Grxs) are short, cysteine-rich glutathione (GSH)-mediated oxidoreductases. In this study, a chickpea (Cicer arietinum L.) glutaredoxin [LOC101493651 (CaGrx)] gene has been selected based on screening experiments with two contrasting varieties of chickpea, PUSA-362 (drought-tolerant) and ICC-1882 (drought-sensitive) under drought and salinity. The tolerant variety showed higher CaGrx gene expression, as compared to less in the sensitive variety, under both the stresses. The CaGrx gene was then over-expressed in Arabidopsis thaliana and were exposed to drought and salinity. The over-expression of CaGrx elevated the activity of glutaredoxin, which induced antioxidant enzymes (glutathione reductase; GR, glutathione peroxidase; GPX, catalase; CAT, ascorbate peroxidase; APX, glutathione-S-transferase; GST, superoxide dismutase; SOD, monodehydroascorbate reductase; MDHAR, and dehydroascorbate reductase; DHAR), antioxidants (GSH and ascorbate) and stress-responsive amino acids (cysteine and proline). Enhancement in the antioxidant defense system possibly administered tolerance in transgenics against both stresses. CaGrx reduced stress markers (H₂O₂, TBARS, and electrolyte leakage) and enhanced root growth, seed germination, and survival against both stresses. The physiological parameters (net photosynthesis; P_N, water use efficiency; WUE, stomatal conductance; g_s, transpiration; E, electron transport rate; ETR, and photochemical quenching; qP), chlorophylls and carotenoids, were improved in the transgenics during both stresses, that maintained the photosynthetic apparatus and protected the plants from damage. The enhanced activity of the cysteine biosynthesis enzyme, o-acetylserine (thiol) lyase (OAS-TL), increased the cysteine level in the transgenics, which elevated glutathione biosynthesis to maintain the ascorbate–glutathione cycle under both stresses. This investigation verified that the CaGrx gene provides tolerance against salinity and drought, maintaining physiological and morphological performances, and could be exploited for genetic engineering approaches to overcome both the stresses in various crops.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-00999-z.     

Biodegradable Nanoparticle-Entrapped Vaccine Induces Cross-Protective Immune Response against a Virulent Heterologous Respiratory Viral Infection in Pigs

Biodegradable nanoparticle-based vaccine development research is unexplored in large animals and humans. In this study, we illustrated the efficacy of nanoparticle-entrapped UV-killed virus vaccine against an economically important respiratory viral disease of pigs called porcine reproductive and respiratory syndrome virus (PRRSV). We entrapped PLGA [poly (lactide-co-glycolides)] nanoparticles with killed PRRSV antigens (Nano-KAg) and detected its phagocytosis by pig alveolar macrophages. Single doses of Nano-KAg vaccine administered intranasally to pigs upregulated innate and PRRSV specific adaptive responses. In a virulent heterologous PRRSV challenge study, Nano-KAg vaccine significantly reduced the lung pathology and viremia, and the viral load in the lungs. Immunologically, enhanced innate and adaptive immune cell population and associated cytokines with decreased secretion of immunosuppressive mediators were observed at both mucosal sites and blood. In summary, we demonstrated the benefits of intranasal delivery of nanoparticle-based viral vaccine in eliciting cross-protective immune response in pigs, a potential large animal model.     

A computationally tractable birth-death model that combines phylogenetic and epidemiological data

Inferring the dynamics of pathogen transmission during an outbreak is an important problem in infectious disease epidemiology. In mathematical epidemiology, estimates are often informed by time series of confirmed cases, while in phylodynamics genetic sequences of the pathogen, sampled through time, are the primary data source. Each type of data provides different, and potentially complementary, insight. Recent studies have recognised that combining data sources can improve estimates of the transmission rate and the number of infected individuals. However, inference methods are typically highly specialised and field-specific and are either computationally prohibitive or require intensive simulation, limiting their real-time utility. We present a novel birth-death phylogenetic model and derive a tractable analytic approximation of its likelihood, the computational complexity of which is linear in the size of the dataset. This approach combines epidemiological and phylodynamic data to produce estimates of key parameters of transmission dynamics and the unobserved prevalence. Using simulated data, we show (a) that the approximation agrees well with existing methods, (b) validate the claim of linear complexity and (c) explore robustness to model misspecification. This approximation facilitates inference on large datasets, which is increasingly important as large genomic sequence datasets become commonplace.     

Specificity in Arabidopsis thaliana recruitment of root fungal communities from soil and rhizosphere

Biotic and abiotic conditions in soil pose major constraints on growth and reproductive success of plants. Fungi are important agents in plant soil interactions but the belowground mycobiota associated with plants remains poorly understood. We grew one genotype each from Sweden and Italy of the widely-studied plant model Arabidopsis thaliana. Plants were grown under controlled conditions in organic topsoil local to the Swedish genotype, and harvested after ten weeks. Total DNA was extracted from three belowground compartments: endosphere (sonicated roots), rhizosphere and bulk soil, and fungal communities were characterized from each by amplification and sequencing of the fungal barcode region ITS2. Fungal species diversity was found to decrease from bulk soil to rhizosphere to endosphere. A significant effect of plant genotype on fungal community composition was detected only in the endosphere compartment. Despite A. thaliana being a non-mycorrhizal plant, it hosts a number of known mycorrhiza fungi in its endosphere compartment, which is also colonized by endophytic, pathogenic and saprotrophic fungi. Species in the Archaeorhizomycetes were most abundant in rhizosphere samples suggesting an adaptation to environments with high nutrient turnover for some of these species. We conclude that A. thaliana endosphere fungal communities represent a selected subset of fungi recruited from soil and that plant genotype has small but significant quantitative and qualitative effects on these communities.     

Taxonomic Profiling and Metagenome Analysis of a Microbial Community from a Habitat Contaminated with Industrial Discharges

Industrial units, manufacturing dyes, chemicals, solvents, and xenobiotic compounds, produce liquid and solid wastes, which upon conventional treatment are released in the nearby environment and thus are the major cause of pollution. Soil collected from contaminated Kharicut Canal bank (N 22°57.878′; E 072°38.478′), Ahmedabad, Gujarat, India was used for metagenomic DNA preparation to study the capabilities of intrinsic microbial community in dealing with xenobiotics. Sequencing of metagenomic DNA on the Genome Sequencer FLX System using titanium chemistry resulted in 409,782 reads accounting for 133,529,997 bases of sequence information. Taxonomic analyses and gene annotations were carried out using the bioinformatics platform Sequence Analysis and Management System for Metagenomic Datasets. Taxonomic profiling was carried out by three different complementary approaches: (a) 16S rDNA, (b) environmental gene tags, and (c) lowest common ancestor. The most abundant phylum and genus were found to be “Proteobacteria” and “Pseudomonas,” respectively. Metagenome reads were mapped on sequenced microbial genomes and the highest numbers of reads were allocated to Pseudomonas stutzeri A1501. Assignment of obtained metagenome reads to Gene Ontology terms, Clusters of Orthologous Groups of protein categories, protein family numbers, and Kyoto Encyclopedia of Genes and Genomes hits revealed genomic potential of indigenous microbial community. In total, 157,024 reads corresponded to 37,028 different KEGG hits, and amongst them, 11,574 reads corresponded to 131 different enzymes potentially involved in xenobiotic biodegradation. These enzymes were mapped on biodegradation pathways of xenobiotics to elucidate their roles in possible catalytic reactions. Consequently, information obtained from the present study will act as a baseline which, subsequently along with other “-omic” studies, will help in designing future bioremediation strategies in effluent treatment plants and environmental clean-up projects.     

Stressful environments can indirectly select for increased longevity

Longevity is modulated by a range of conserved genes in eukaryotes, but it is unclear how variation in these genes contributes to the evolution of longevity in nature. Mutations that increase life span in model organisms typically induce trade‐offs which lead to a net reduction in fitness, suggesting that such mutations are unlikely to become established in natural populations. However, the fitness consequences of manipulating longevity have rarely been assessed in heterogeneous environments, in which stressful conditions are encountered. Using laboratory selection experiments, we demonstrate that long‐lived, stress‐resistant Caenorhabditis elegans age‐1(hx546) mutants have higher fitness than the wild‐type genotype if mixed genotype populations are periodically exposed to high temperatures when food is not limited. We further establish, using stochastic population projection models, that the age‐1(hx546) mutant allele can confer a selective advantage if temperature stress is encountered when food availability also varies over time. Our results indicate that heterogeneity in environmental stress may lead to altered allele frequencies over ecological timescales and indirectly drive the evolution of longevity. This has important implications for understanding the evolution of life‐history strategies.     

Generation and Characterization of ATP Analog-specific Protein Kinase Cδ

To better study the role of PKCδ in normal function and disease, we developed an ATP analog-specific (AS) PKCδ that is sensitive to specific kinase inhibitors and can be used to identify PKCδ substrates. AS PKCδ showed nearly 200 times higher affinity (K_m) and 150 times higher efficiency (k_cat/K_m) than wild type (WT) PKCδ toward N⁶-(benzyl)-ATP. AS PKCδ was uniquely inhibited by 1-(tert-butyl)-3-(1-naphthyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (1NA-PP1) and 1-(tert-butyl)-3-(2-methylbenzyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (2MB-PP1) but not by other 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1) analogs tested, whereas WT PKCδ was insensitive to all PP1 analogs. To understand the mechanisms for specificity and affinity of these analogs, we created in silico WT and AS PKCδ homology models based on the crystal structure of PKCι. N⁶-(Benzyl)-ATP and ATP showed similar positioning within the purine binding pocket of AS PKCδ, whereas N⁶-(benzyl)-ATP was displaced from the pocket of WT PKCδ and was unable to interact with the glycine-rich loop that is required for phosphoryl transfer. The adenine rings of 1NA-PP1 and 2MB-PP1 matched the adenine ring of ATP when docked in AS PKCδ, and this interaction prevented the potential interaction of ATP with Lys-378, Glu-428, Leu-430, and Phe-633 residues. 1NA-PP1 failed to effectively dock within WT PKCδ. Other PP1 analogs failed to interact with either AS PKCδ or WT PKCδ. These results provide a structural basis for the ability of AS PKCδ to efficiently and specifically utilize N⁶-(benzyl)-ATP as a phosphate donor and for its selective inhibition by 1NA-PP1 and 2MB-PP1. Such homology modeling could prove useful in designing molecules to target PKCδ and other kinases to understand their function in cell signaling and to identify unique substrates.     

Fetal DNA Methylation Associates with Early Spontaneous Preterm Birth and Gestational Age

Spontaneous preterm birth (PTB, <37 weeks gestation) is a major public health concern, and children born preterm have a higher risk of morbidity and mortality throughout their lives. Recent studies suggest that fetal DNA methylation of several genes varies across a range of gestational ages (GA), but it is not yet clear if fetal epigenetic changes associate with PTB. The objective of this study is to interrogate methylation patterns across the genome in fetal leukocyte DNA from African Americans with early PTB (24^1/7–34^0/7 weeks; N = 22) or term births (39^0/7–40^6/7weeks; N = 28) and to evaluate the association of each CpG site with PTB and GA. DNA methylation was assessed across the genome with the HumanMethylation450 BeadChip. For each individual sample and CpG site, the proportion of DNA methylation was estimated. The associations between methylation and PTB or GA were evaluated by fitting a separate linear model for each CpG site, adjusting for relevant covariates. Overall, 29 CpG sites associated with PTB (FDR<.05; 5.7×10⁻¹⁰<p<2.9×10⁻⁶) independent of GA. Also, 9637 sites associated with GA (FDR<.05; 9.5×10⁻¹⁶<p<1.0×10⁻³), with 61.8% decreasing in methylation with shorter GA. GA-associated CpG sites were depleted in the CpG islands of their respective genes (p<2.2×10⁻¹⁶). Gene set enrichment analysis (GSEA) supported enrichment of GA-associated CpG sites in genes that play a role in embryonic development as well as the extracellular matrix. Additionally, this study replicated the association of several CpG sites associated with gestational age in other studies (CRHBP, PIK3CD and AVP). Dramatic differences in fetal DNA methylation are evident in fetuses born preterm versus at term, and the patterns established at birth may provide insight into the long-term consequences associated with PTB.