Biology subjects

Das, B. K.

Publications and source records attributed to Das, B. K..

11 recordsLinked to original sources

bioRxiv2025

Promoter evolution in HIV-1C establishes latent reservoirs highly resistant to reversal

Latent viral reservoirs remain a major barrier to curing HIV-1, with the long-terminal repeat (LTR) and Tat playing crucial roles in regulating viral transcription. Subtype-specific transcription factor binding site (TFBS) variations within the LTR significantly influence latency and reservoir stability. In earlier work, we identified HIV-1C LTR variants with duplicated TFBS motifs, including NF-{kappa}B, AP1, RBEIII, and TCF-1/LEF-1. Using five cell models, including Jurkat and primary CD4 T cells, we compared canonical R-LTR and variant R2-LTR strains. Across sub-genomic reporters, single-round infections, and full-length viral vectors, we found that the balance between RBEIII and NF-{kappa}B motifs governs stability of latency. The two-viruses-one-cell system that normalized confounding environmental factors further revealed that latency is primarily controlled by intrinsic transcriptional circuits rather than external stimuli. In longitudinal studies of HIV-1 individuals from acute and chronic infection phases, we observed dominant R strains during early infection and the spontaneous emergence of R2 strains in nearly half of chronic-phase subjects, a process accelerated by ART. Upon CD4 T cell activation, R strains preferentially rebounded, while R2 strains showed strong resistance to reversal, even in subjects harbouring a co-infection. Together, these findings establish the clinical significance of LTR variation in latency regulation and identify the R2 phenotype as a critical determinant of reservoir persistence. These results underscore the importance of addressing reservoir heterogeneity in cure strategies, particularly in HIV-1C-prevalent regions.

microbiology↗

bioRxiv2024

Bradyrhizobium and the soybean rhizosphere: Species level bacterial population dynamics in established soybean fields, rhizosphere and nodules.

Bradyrhizobium fixes nitrogen symbiotically with soybean and is an agriculturally significant bacterium. Much is known about the Bradyrhizobium species that nodulate soybeans. Conversely, prevalence of Bradyrhizobium in soil and the rhizosphere is known only to the genus level as culture independent approaches have provided only partial 16S rRNA gene sequences, so that nodulating and non-nodulating species could not be distinguished. To track which species in bulk soil proliferate in the rhizosphere, and then nodulate, we sought to study population dynamics of Bradyrhizobium in soybean fields and rhizosphere at the species level. Recent advances in Oxford Nanopore Technologies provided us with higher fidelity and increased number of reads which enabled us to track Bradyrhizobium populations at the species level. We found evidence for 74 species of Bradyrhizobium within a community of 10,855 bacterial species in bulk soil and rhizosphere from three different soybean fields in South Dakota. The most predominant species in bulk soil and rhizosphere included B. liaoningense, B. americanum, and B. diversitatus, however none of these were isolated from nodules. Isolates from nodules included B. japonicum, B. elkanii and B. diazoefficiens. These nodulators also maintained populations in bulk soil and rhizosphere, although they were not the most prevalent Bradyrhizobium. Our findings reveal the rich diversity and community dynamics of Bradyrhizobium species in soybean field soil as well as in the rhizosphere. Our results showed that many species of the genus maintain populations in soybean field soil, even in the long-term absence of potential nodulating partners.

microbiology↗

bioRxiv2021

Transferrin receptor 1-mediated iron uptake regulates bone mass in mice via osteoclast mitochondria and cytoskeleton

Increased intracellular iron spurs mitochondrial biogenesis and respiration to satisfy high-energy demand during osteoclast differentiation and bone-resorbing activities. Transferrin receptor 1 (TFR1) mediates cellular iron uptake through endocytosis of iron-loaded transferrin and its expression increases during osteoclast differentiation. Nonetheless, the precise functions of TFR1 and TFR1-mediated iron uptake in osteoclast biology and skeletal homeostasis remain incompletely understood. To investigate the role of TFR1 in osteoclast lineage cells, we conditionally deleted Tfr1 gene in myeloid precursors or mature osteoclasts by crossing Tfr1- floxed mice with LysM-Cre and Ctsk-Cre mice, respectively. Skeletal phenotyping by {micro}CT and histology unveiled that loss of Tfr1 in osteoclast progenitor cells resulted in a three-fold increase in trabecular bone mass in the long bones of 10-week old female but not male mice. Although high trabecular bone volume in long bones was seen in both male and female mice with deletion of Tfr1 in mature osteoclasts, this phenotype was more pronounced in female knockout mice. Mechanistically, disruption of Tfr1 expression attenuated mitochondrial metabolism and cytoskeletal organization in mature osteoclasts, leading to decreased bone resorption with no impact on osteoclastogenesis. These results indicate that Tfr1-mediated iron uptake is specifically required for osteoclast function and is indispensable for bone remodeling.

cell biology↗

bioRxiv2021

Dual avatars of E. coli grxB encoded Glutaredoxin 2 perform ascorbate recycling and ion channel activities

Glutaredoxins (Grxs) are single-domain redox enzymes of the thioredoxin superfamily, and primarily function as glutathione (GSH) dependent disulphide reductases. Whereas, the E. coli Glutaredoxin 2 (EcGrx2) encoded by grxB has two conserved GST-fold domains, it still lacks a classical Grx-like functions. In this study, we show for the first time, that EcGrx2 exists in both soluble and membrane integrated forms. The soluble form associates with a previously unidentified GSH dependent dehydroascrobate (DHA) reductase, and the membrane integrated form possesses ion channel activities. Using enzyme kinetic data and structural data we unequivocally demonstrate that EcGrx2 recycles ascorbate (AsA) from DHA. This ability to recycle AsA is inhibited by Zinc (Zn2+). We also show that both wildtype and the E. coli grxB deletion mutant can be rescued from H2O2-induced oxidative stress using ascorbate as an antioxidant, which otherwise is only known as a carbon source in bacteria. Moreover, the grxB- mutant is susceptible to intracellular killing by ROS producing macrophages. We further discovered that EcGrx2 integrates into the native E. coli membrane and show that the purified soluble protein readily inserts into artificial lipid bilayer membrane and conducts ions in vitro. Our data demonstrates a highly conserved functional similarity among EcGrx2-orthologs and highlights that the utilization and subsequent recycling of ascorbate as an antioxidant by grxB harbouring gram-negative bacteria, including human pathogens, may provide a survival advantage under hostile oxidative environments.

biochemistry↗

bioRxiv2021

Comparative kinetic analysis of ascorbate (Vitamin-C) recycling dehydroascorbate reductases from plant and human.

Ascorbate, a primary antioxidant, gets readily oxidized to dehydroascorbate (DHA). Hence, recycling by dehydroascorbate reductase (DHAR) enzymes is vital for protection from cellular oxidative stress in eukaryotes. However, a detailed kinetic analysis of plant DHARs and their human orthologs; chloride intracellular channels (HsCLICs) is lacking. We demonstrate that DHAR from stress adapted pearl millet Pennisetum glaucum (PgDHAR) shows the highest turnover rate whereas HsCLIC1, 3, and 4 reduce DHA, albeit at lower rates. We further show that the catalytic cysteine is susceptible to varying levels of oxidation, supported by crystal structures and mass-spectrometry analysis. The differences in kinetic parameters among plant and human DHA reductases corroborate with the levels of reactive oxygen species H2O2 encountered in their respective intracellular environment. Our findings may have broader implications in crop improvement using pearl millet DHAR, and anti-cancer therapeutics targeting Vitamin-C recycling capability of human CLICs.

biochemistry↗

bioRxiv2021

The evolution of regulatory elements in the emerging promoter variant strains of HIV-1

In a multicentric, observational, investigator-blinded, and longitudinal clinical study of 764 ART-naive subjects, we identified nine different promoter-variant strains of HIV-1 subtype C (HIV-1C) emerging in the Indian population, with some of these variants being reported for the first time. Unlike several previous studies, our work here focuses on the evolving viral regulatory elements, not coding sequences. The emerging viral strains contain additional copies of the existing transcription factor binding sites (TFBS), including TCF-1/LEF-1, RBEIII, AP-1, and NF-{kappa}B, created by sequence duplication. The additional TFBS are genetically diverse and may blur the distinction between the modulatory region of the promoter and the viral enhancer. In a follow-up analysis, we found trends, but not significant associations between any specific variant promoter and prognostic markers, probably because the emerging viral strains might not have established mono infections yet. Illumina sequencing of four clinical samples containing a co-infection indicated the domination of one strain over the other and establishing a stable ratio with the second strain at the follow-up time-points. Since a single promoter regulates viral gene expression and constitutes the master regulatory circuit with Tat, the acquisition of additional and variant copies of the TFBS may significantly impact viral latency and latent reservoir characteristics. Further studies are urgently warranted to understand how the diverse TFBS profiles of the viral promoter may modulate the characteristics of the latent reservoir, especially following the initiation of antiretroviral therapy. Significance StatementA unique conglomeration of TFBS enables the HIV-1 promoter to accomplish two diametrically opposite functions - transcriptional activation and transcriptional silencing. The various phases of viral latency -establishment, maintenance, and reversal -collectively determine the replication fitness of individual viral strains. A profound variation in the TFBS composition of the viral promoter may significantly alter the viral latency properties and the latent reservoir characteristics. Although the duplication of certain TFBS remains a quality unique to HIV-1C, the high-level genetic recombination of HIV-1 may promote the transfer of such molecular properties to the other HIV-1 subtypes. The emergence of several promoter-variant viral strains may make the task of a functional cure more challenging in HIV-1C.

microbiology↗

bioRxiv2020

Plasma neutralizing antibodies in an infant with interclade HIV-1 superinfection preferentially neutralize superinfecting HIV-1 strains

HIV-1 superinfection is defined as infection by an unrelated second strain of HIV-1 after seroconversion due to primary infecting strain and has been associated with development of breadth in the neutralizing antibody (nAb) response, altered disease progression and efficacy of antiretrovirals; though conflicting observations have also been reported. Superinfection has been reported in HIV-1 infected adults. Recently we observed that multivariant infection in infants was associated with early induction of plasma broadly neutralizing antibodies (bnAbs) targeting diverse autologous viruses, however, there is paucity of information on infants with HIV-1 superinfection. Furthermore, the mechanisms by which superinfection in an infant, after priming by an initial infection, potentiate the evolution of a bnAb response have not been evaluated. Herein, we performed a longitudinal analysis and observed evolution of nAb responses in an antiretroviral naive perinatally HIV-1 infected infant, with interclade superinfection (clade C followed by a unique A1C recombinant). The nAb responses broadened rapidly after superinfection targeted an undefined glycan-dependent epitope on the superinfecting variant, while no enrichment of nAb response against the primary infecting strain occurred. Defining virological features in infants with sequential infection with highly divergent circulating viruses that improve nAb responses will contribute information that could be leveraged for optimization of multicomponent candidate vaccines. ImportanceHIV-1 infected infants develop bnAbs rapidly suggesting factors governing bnAb induction in infants are distinct from adults. HIV-1 superinfection is more common in adults whereas the stringent genetic bottleneck for transmission in infants often leads to infection by a single transmitted/founder HIV-1 strain. Longitudinal studies in infants with HIV-1 superinfection can provide key information on the viral factors that induce a bnAb response towards development of a polyvalent vaccine. Herein, we show that in infant who was sequentially infected with two HIV-1 strains from different clades, antibody responses were primarily generated against the superinfecting, second strain of HIV-1. These antibody responses were dependent on glycans, and targeted an undefined epitope in the C3V4 region of HIV-1 Env. A better understanding of how neutralizing antibody responses develop during natural HIV-1 superinfection in infants will provide information relevant to HIV Env vaccine development and evaluation.

immunology↗

bioRxiv2020

A rare mutation in an infant derived HIV-1 envelope glycoprotein alters interprotomer stability and susceptibility to broadly neutralizing antibodies targeting the trimer apex

The envelope glycoprotein (Env) of human immunodeficiency virus-1 (HIV-1) is the sole target of broadly neutralizing antibodies (bnAbs). Several mechanisms, such as acquisition of mutations due to the error prone reverse transcriptase, variability of loop length and alterations in glycan pattern are employed by the virus to shield neutralizing epitopes on the env, to sustain survival and infectivity within the host. Identification of mutations that can lead to viral evasion from host immune response is essential for optimization and engineering of Env based trimeric immunogens. Herein, we report a rare leucine to phenylalanine escape mutation (L184F) at the base of hypervariable loop 2 (population frequency of 0.0045%) in a nine-month-old perinatally HIV-1 infected infant broad neutralizer. The L184F mutation disrupted the intramolecular interaction, stabilizing the trimer apex thereby leading to viral escape from autologous plasma bnAbs and known bnAbs, targeting exclusively the N160 glycan at trimer apex and not any other known epitope. The L184F amino acid change led to acquisition of a relatively open trimeric configuration, often associated with tier 1 HIV-1 isolates and an increased susceptibility to neutralization by polyclonal plasma antibodies of weak neutralizers. While there was no impact of the L184F mutation on free virus transmission, a reduction in cell-to-cell transmission was observed. In conclusion, we report a viral escape mutation that plausibly destabilized the trimer apex and favoured evasion from broadly neutralizing antibodies. Such mutations, though rare, should be taken into consideration while designing an immunogen, based on a stable correctly-folded HIV-1 Env trimer. ImportanceDesign of HIV-1 envelope-based immunogens, capable of eliciting broadly neutralizing antibodies (bnAbs), are currently under active research. Some of the most potent bnAbs target the quaternary epitope at the V2 apex of HIV-1 Env trimer. By studying naturally circulating viruses from an HIV-1 perinatally infected infant, with plasma neutralizing antibodies targeted to the V2-apex, we identified a rare leucine to phenylalanine substitution in two out of six functional viral clones, that destabilized the trimer apex. This single amino acid alteration impaired the interprotomeric interactions that stabilize the trimer apex, resulting in an open trimer conformation, and escape from broadly neutralizing autologous plasma antibodies and known V2-apex directed bnAbs, thereby favouring viral evasion of the early bnAb response of the infected host. Defining the mechanisms by which viral mutations influence the sensitivity of HIV-1 to bnAbs is crucial for the development of effective vaccines against HIV-1 infection.

microbiology↗

bioRxiv2019

Multivariant HIV-1 infection in infants is associated with the induction of broadly neutralizing plasma antibodies.

Due to the extensive antigenic diversity of human immunodeficiency virus-1 (HIV-1), broadly neutralizing antibodies (bnAbs) develop in a subset of infected individuals over 2-3 years of infection. Interestingly, infected infants have been shown to develop plasma bnAbs frequently and as early as one-year post-infection, with features atypical than adult bnAbs, suggesting that the factors governing bnAb induction in infants are distinct from that in adults. Understanding the viral characteristics in infected infants with early bnAb responses will provide key information on the antigenic triggers driving B cell maturation pathways towards the induction of bnAbs. Herein, we evaluated the presence of plasma bnAbs in a cohort of 51 HIV-1 clade C perinatally infected infants of Indian origin and identified viral factors associated with early bnAb responses. Plasma bnAbs targeting V2-apex on the env were predominant in infant elite and broad neutralizers. Circulating viral variants in infant elite neutralizers were susceptible to known bnAbs against V2-apex while varied resistance profile to other bnAb classes was observed. In infant elite neutralizers, multivariant infection was associated with plasma bnAbs targeting diverse autologous viruses. Our data provides information supportive of polyvalent vaccination approaches capable of inducing V2-apex bnAbs against HIV-1.

immunology↗

bioRxiv2019

Immunosuppression, rather than inflammation, is a salientfeature of sepsis in an Indian cohort

1Sepsis remains a lethal ailment with imprecise treatment and ill-understood biology. A clinical transcriptomic analysis of sepsis patients was performed for the first time in India and revealed large-scale change in blood gene expression in patients of severe sepsis and septic shock admitted to ICU. Three biological processes were quantified using scores derived from the corresponding transcriptional modules. Comparison of the module scores revealed that genes associated with immune response were more suppressed compared to the inflammation-associated genes. These findings will have great implication in the treatment and prognosis of severe sepsis/septic shock if it can be translated into a bedside tool.

genomics↗

bioRxiv2018

Viral characteristics associated with sustenance of elite neutralizing activity in chronically HIV-1C infected monozygotic pediatric twins

Broad and potent neutralizing antibodies (bnAbs) with multiple epitope specificities evolve in HIV-1 infected children. Herein we studied two antiretroviral naive chronically HIV-1C in-fected monozygotic pediatric twins AIIMS_329 and AIIMS_330 with potent plasma bnAbs. Elite plasma neutralizing activity was observed since initial sampling at 78 months in AIIMS_330 and persisted throughout, while in AIIMS_329 it was seen at 90-months of age after which potency decreased overtime. We evaluated potential viral characteristics associated with the varied immune profile by generating single genome amplified pseudoviruses. The AIIMS_329 viruses generated from 90-month time point sampling were neutralization sensitive to second generation bnAbs and contemporaneous autologous plasma antibodies, while viruses from 112-months and 117-month timepoints were resistant to most bnAbs and autologous contemporaneous plasma. AIIMS_329 viruses developed resistance to plasma nAbs plausibly by N160-glycan loss, V1- and V4-loop lengthening. The viruses generated from AIIMS_330 at 90-month and 117-month timepoint showed varied susceptibility to bnAbs and autologous contemporaneous plasma antibodies while the viruses of 112-month timepoint, at which plasma nAb specificities mapped to the V2-glycan, V3-glycan and CD4bs, were resistant to autologous contemporaneous plasma antibodies as well as most bnAbs. We observed evolution of a viral pool in AIIMS_330 donor, comprising of plasma antibody neutralization sensitive or resistant diverse autologous viruses that in turn may have contributed to development and maintenance of elite neutralizing activity. The findings of this study provide information towards understanding factors involved in generation and maintenance of potent plasma nAbs.\n\nImportanceChronically HIV-1 infected children that develop elite neutralizing activity are suitable candidates to understand the mechanisms that lead to the co-evolution of virus and antibody response. Here, we evaluated the alterations in virus and antibody responses over time in chronically HIV-1C infected monozygotic pediatric twins, AIIMS_329 and AIIMS_330, who had acquired the infection by vertical transmission. AIIMS_330 retained the elite plasma neutralizing activity throughout, while in AIIMS_329, the potency decreased post 90 months of age. The corresponding viral pool from post 90-month samples in AIIMS_330 showed varied susceptibility, while that in AIIMS_329, developed resistance to bnAbs and autologous plasma antibodies. The findings of this study, conducted in twin children of same genetic make-up and infected at birth with a single source of HIV-1C, suggest that a viral pool with varied susceptibility to antibodies could have been one of the factors responsible for sustained elite neutralizing activity in AIIMS_330.

immunology↗