Biology subjects

Chakraborty, R.

Publications and source records attributed to Chakraborty, R..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Distinct immune responses to HIV and CMV in Hofbauer cells across gestation highlight evolving placental immune dynamics

Placental immune responses to Human Immunodeficiency Virus (HIV) and Human Cytomegalovirus (CMV) vary across gestational stages and may influence postnatal outcomes. This study investigates the innate immunity of Hofbauer cells from placentae obtained at early/mid-gestation (18-21.6 weeks) and term (>37 weeks). RNA sequencing and cytokine profiling reveal that early/mid-gestation HCs exhibit heightened differential gene expression responses compared to term HCs, indicating a distinct transcriptional activity in early pregnancy. Significant overlap in gene expression profiles of early/mid-gestation cells in response to CMV and HIV suggest similar innate immune responses, while term cells exhibit distinct patterns, reflecting the temporal evolution of placental immunity. Integration with Human Protein Atlas database reveals more placental-specific differentially expressed genes in early/mid-gestation HCs exposed to HIV and CMV compared to term cells. Functional analysis reveals downregulation of pathways related to oxygen stress, estrogen response, and KRAS signaling pathway in early/mid-gestation HCs, with HIV uniquely upregulating reactive oxygen species and CMV uniquely disrupting WNT {beta}-Catenin signaling. In term HCs, CMV exposure upregulates antiviral interferon (IFN) signaling and inflammatory pathways. Co-expression analysis highlights distinct molecular pathway enrichments across gestation, particularly with upregulation of IFN signaling and disruption of lipid metabolism in term CMV-exposed HCs. Cytokine profiling shows enhanced expression of GM-CSF, IFN-{gamma}, and Th2-associated cytokines in early/mid-gestation HCs, indicating heightened immune responsiveness. These findings reveal the dynamic nature of placental immunity and underscore the need for targeted interventions to address unique immune and metabolic disruptions caused by viral infections at distinct stages of pregnancy to improve fetal and infant health outcomes.

pathology↗

bioRxiv2024

SUV39H1 mediated regulation of KLF4 and KDM4A coordinate smooth muscle cell phenotypic plasticity

BackgroundReversible DNA methylation contributes to the phenotypic plasticity of vascular smooth muscle cells (VSMCs). This plasticity contributes to vascular growth remodeling, but also underlies cardiovascular pathologies, including intimal hyperplasia. We investigated the role of SUV39H1, a histone methyltransferase that generates the H3K9me3 repressive epigenetic mark, in VSMC plasticity MethodsWe applied knockdown, qPCR, western blotting, chromatin immunoprecipitation (ChIP) assays, and RNA-Seq in human coronary artery SMCs (hCASMCs), and murine carotid ligation to determine the role of SUV39H1 in VSMC plasticity. ResultsExpression of SUV39H1 and the H3K9me3 mark it generates increase, whereas the cognate H3K9me3 demethylase KDM4A decreases, over time during the progression of murine intimal hyperplasia following carotid artery ligation, with marked elevation of SUV39H1 and H3K9me in the neointima. SUV39H1 knockdown induced contractile genes and contractility while decreasing migration and proliferation in hCASMCs. Transcriptomic analysis confirmed that SUV39H1 promotes SMC dedifferentiation. SUV39H1 knockdown revealed that SUV39H1 promotes KLF4 upregulation by increasing KLF4 mRNA stability. PDGF-BB induced SUV39H1 expression and SUV39H1-dependent H3K9me3 modification of contractile gene promoters in hCASMC. SUV39H1 knockdown reduced the repressive H3K9me3 and 5mC marks but increased the activating H3K27Ac mark at these promoters. SUV39H1 knockdown also increased expression of KDM4A and its binding to contractile promoters, suggesting an opposing regulatory relationship between the writer and eraser of H3K9me3. ConclusionsWe identify SUV39H1 as an epigenetic regulator that promotes VSMC dedifferentiation by stabilizing KLF4 expression and by altering chromatin state. We report SUV39H1-dependent dynamic regulation of the repressive H3K9me3 mark at contractile gene promoters, and opposing regulation of the enzymes that write (SUV39H1) and erase (KDM4A) these marks during VSMC phenotypic switching. These studies suggest that coordinate regulation of both histone and DNA methylation contribute to VSMC phenotypic plasticity. HighlightsO_LIThe histone methyl transferase SUV39H1 is differentially regulated in VSMC phenotypic switching in culture and in vascular remodeling. SUV39H1 and the H3K9me3 mark it generates increase, whereas the cognate H3K9me3 eraser KDM4A decreases, during the progression of murine intimal hyperplasia. C_LIO_LISUV39H1 promotes PDGF-induced VSMC de-differentiation, stabilizing KLF4 mRNA. SUV39H1 opposes VSMC contractility and promotes proliferation and migration. C_LIO_LIPDGF-BB induces SUV39H1-dependent H3K9me3 marks at VSMC contractile gene promoters, and SUV39H1 loss of function alters multiple marks that govern chromatin accessibility at these promoters, with opposing effects on the repressive H3K9me3 and 5mC and activating H3K27Ac marks. C_LIO_LISUV39H1 generates H3K9me3 marks that are associated with silenced heterochromatin. We note dynamic regulation of this mark at VSMC contractile gene promoters, and opposing regulation of the enzymes that write (SUV39H1) and erase (KDM4A) these marks during VSMC phenotypic switching. C_LI

cell biology↗

bioRxiv2024

Synaptic vesicle endocytosis deficits underlie GBA-linked cognitive dysfunction in Parkinson's disease and Dementia with Lewy bodies

GBA is the major risk gene for Parkinsons disease (PD) and Dementia with Lewy Bodies (DLB), two common -synucleinopathies with cognitive deficits. We investigated the role of mutant GBA in cognitive decline by utilizing Gba (L444P) mutant, SNCA transgenic (tg), and Gba-SNCA double mutant mice. Notably, Gba mutant mice showed early cognitive deficits but lacked PD-like motor deficits or -synuclein pathology. Conversely, SNCA tg mice displayed age-related motor deficits, without cognitive abnormalities. Gba-SNCA mice exhibited both cognitive decline and exacerbated motor deficits, accompanied by greater cortical phospho--synuclein pathology, especially in layer 5 neurons. Single-nucleus RNA sequencing of the cortex uncovered synaptic vesicle (SV) endocytosis defects in excitatory neurons of Gba mutant and Gba-SNCA mice, via robust downregulation of genes regulating SV cycle and synapse assembly. Immunohistochemistry and electron microscopy validated these findings. Our results indicate that Gba mutations, while exacerbating pre-existing -synuclein aggregation and PD-like motor deficits, contribute to cognitive deficits through -synuclein-independent mechanisms, involving dysfunction in SV endocytosis.

neuroscience↗

bioRxiv2024

Multi-laboratory Study Establishes Reproducible Methods for Plant-Microbiome Research in Fabricated Ecosystems

Inter-laboratory replicability is crucial yet challenging in microbiome research. Leveraging microbiomes to promote soil health and plant growth requires understanding underlying molecular mechanisms using reproducible experimental systems. In a global collaborative effort involving five laboratories, we aimed to help advance reproducibility in microbiome studies by testing our ability to replicate synthetic community assembly experiments. Our study compared fabricated ecosystems constructed using two different synthetic bacterial communities, the model grass Brachypodium distachyon, and sterile EcoFAB 2.0 devices. All participating laboratories observed consistent inoculum-dependent changes in plant phenotype, root exudate composition, and final bacterial community structure where Paraburkholderia sp. OAS925 could dramatically shift microbiome composition. Comparative genomics and exudate utilization linked the pH-dependent colonization ability of Paraburkholderia, which was further confirmed with motility assays. The study provides detailed protocols, benchmarking datasets, and best practices to help advance replicable science and inform future multi-laboratory reproducibility studies.

microbiology↗

bioRxiv2024

A stable 15-member bacterial SynCom promotes Brachypodium growth under drought stress

Rhizosphere microbiomes are known to drive soil nutrient cycling and influence plant fitness during adverse environmental conditions. Field-derived robust Synthetic Communities (SynComs) of microbes that mimic the diversity of rhizosphere microbiomes can greatly advance a deeper understanding of such processes. However, assembling stable, genetically tractable, reproducible, and scalable SynComs remains challenging. Here, we present a systematic approach using a combination of network analysis and cultivation-guided methods to construct a 15-member SynCom from the rhizobiome of Brachypodium distachyon. This SynCom incorporates diverse strains from five bacterial phyla and demonstrates strong stability both in vitro and in planta. Genomic analysis of the individual strains revealed that they encode multiple plant growth-promoting traits, some of which were validated by laboratory phenotypic assays. Additionally, most strains encoded genes both for the synthesis of osmoprotectants (trehalose and betaine) and Na+/K+ transporters. These traits likely enabled the resilience of Brachypodium to drought stress where plants amended with SynCom recovered better than without. We further observed preferential colonization of SynCom strains around root tips under stress, likely due to active interactions between plant root metabolites and bacteria. Our results represent significant progress towards building and testing stable model SynComs for a better understanding of plant-microbe interactions.

microbiology↗

bioRxiv2024

Cyclophilin A Facilitates HIV-1 DNA Integration

Cyclophilin A (CypA) promotes HIV-1 infection by facilitating reverse transcription, nuclear entry and by countering the antiviral activity of TRIM5. These multifunctional roles of CypA are driven by its binding to the viral capsid. Interestingly, recent studies suggest that the HIV-1 capsid lattice enters the nucleus of an infected cell and uncoats just before integration. Therefore, we tested whether CypA-capsid interaction regulates post-nuclear entry steps of infection, particularly integration. First, we challenged CypA-expressing (CypA+/+) and CypA-depleted (CypA-/-) cells with HIV-1 particles and quantified the resulting levels of provirus. Surprisingly, CypA-depletion significantly reduced integration, an effect that was independent of CypAs effect on reverse transcription, nuclear entry, and the presence or absence of TRIM5. Additionally, cyclosporin A, an inhibitor that disrupts CypA-capsid binding, inhibited HIV-1 integration in CypA+/+ cells but not in CypA-/- cells. Accordingly, HIV-1 capsid mutants (G89V and P90A) deficient in CypA binding were also blocked at integration in CypA+/+ cells but not in CypA-/- cells. Then, to understand the mechanism, we assessed the integration activity of HIV-1 preintegration complexes (PICs) extracted from infected cells. The PICs from CypA-/- cells had lower activity in vitro compared to those from CypA+/+ cells. PICs from cells depleted for CypA and TRIM5 also had lower activity, suggesting that CypAs effect on PIC activity is independent of TRIM5. Finally, addition of CypA protein significantly stimulated the integration activity of PICs extracted from both CypA+/+ and CypA-/-cells. Collectively, these results suggest that CypA promotes HIV-1 integration, a previously unknown role of this host factor. ImportanceHIV-1 capsid interaction with host cellular factors is essential for establishing a productive infection. However, the molecular details of such virus-host interactions are not fully understood. Cyclophilin A (CypA) is the first host protein identified to specifically bind to the HIV-1 capsid. Now it is established that CypA promotes reverse transcription and nuclear entry steps of HIV-1 infection. In this report, we show that CypA promotes HIV-1 integration by binding to the viral capsid. Specifically, our results demonstrate that CypA promotes HIV-1 integration by stimulating the activity of the viral preintegration complex and identifies a novel role of CypA during HIV-1 infection. This new knowledge is important because recent reports suggest that an operationally intact HIV-1 capsid enters the nucleus of an infected cell.

microbiology↗

bioRxiv2024

High-throughput protein characterization by complementation using DNA barcoded fragment libraries

Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we use complementation of auxotrophs and DNA barcode sequencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. Although assay effectiveness ranged with respect to source genome, with 41% of expected enzymes recovered, even distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron showed success. Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein on an amino acid basis. Among unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seqs utility is demonstrated, with future applications in ecology, health, and engineering.

genomics↗

bioRxiv2024

Differential alpha-Synuclein-induced Autophagy Dysfunction in Neuronal and Microglial Cells drives Tunneling Nanotubes and Aggregate Spread to Microglia

Tunneling nanotubes (TNTs) play a crucial role in intercellular communication, enabling a dynamic network for the transfer of molecular cargo over long distances between connected cells. Previous studies have demonstrated efficient, directional transfer of -Synuclein (-Syn) aggregates from neurons to microglia, with endosomal trafficking and lysosomal processing identified as the primary events following -Syn internalization. Using human neuronal and microglial cell lines, we found that microglia exhibit higher lysosomal turnover, particularly through lysophagy, whereas neuronal lysosomes display compromised degradative capacity and impaired autophagic flux. This deficiency results in less efficient degradation of aggregates in neurons. Moreover, perturbation of autophagy enhances TNT-mediated transfer of aggregate from neuronal cells to microglia. In contrast, microglia co-cultured with -Syn-containing neurons upregulate autophagy flux, enabling efficient degradation of the transferred aggregates. These findings were further validated using human induced pluripotent stem cells (hiPSC)-derived neurons and microglia. Overall, our study highlights the distinct responses of neurons and microglia to -Syn aggregates and identifies dysfunctional autophagy in neurons as a key driver of the preferential and directional transfer of aggregates to microglia.

cell biology↗

bioRxiv2024

Epigenetic Remodeling in Human Coronary Artery Smooth Muscle Cell Phenotypic Switching

BackgroundSmooth muscle cell (SMC) dedifferentiation contributes to repair and remodeling, but also cardiovascular pathologies. To understand this plasticity, the epigenetic landscape in SMC phenotypic switching was profiled. MethodsGenome-wide analyses of histone modifications (ChIP-seq), chromatin architecture (ATAC-seq), and transcriptomes (RNA-seq) were performed on human coronary artery SMCs (CASMC) treated with rapamycin (contractile phenotype) and PDGF-BB (synthetic phenotype). ResultsAnalyses of differentially acetylated promoter regions identified ZEB and ZBT7A as novel enriched regulatory motifs. There were more changes in the enhancer epigenome than in promoters in CASMC phenotypic switching. Rapamycin-activated enhancers were associated with differentiation and TGF-{beta} signaling pathways and were most enriched in TEAD, SRF and SMAD motifs, whereas PDGF-induced enhancers were associated with ERK signaling and migration pathways, and were most enriched in ETV4, SOX5, and FOS motifs. GATA, TEAD, and SMCA1 motifs were enriched in CASMC enhancer open chromatin compared to other cell types. Candidate enhancers with single nucleotide polymorphisms linked to cardiovascular disease were markedly enriched in active enhancers and super enhancers and showed significant activity in reporter assays. In CASMC promoters and enhancers, common regulatory motifs were often enriched in both the differentiated and dedifferentiated phenotypes, suggesting that differential cofactor binding, as occurs with SRF at CArG elements, may be a more widespread mechanism underlying phenotypic switching. ConclusionsThese data identify novel regulatory elements engaged in SMC phenotypic switching and provide a comprehensive profile of SMC promoters, enhancers, super enhancers, and chromatin accessibility as a significant resource for studies of CASMC phenotype. Research PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIThis work identifies key regulatory elements and widespread changes in chromatin accessibility engaged in SMC phenotypic switching, including novel motifs enriched in promoters and enhancers. In CASMC promoters and enhancers, common regulatory motifs were often enriched in both the differentiated and dedifferentiated phenotypes, suggesting that differential cofactor binding, as occurs with SRF at CArG elements, may be a more widespread mechanism underlying phenotypic switching. C_LIO_LIThis work identifies distinct enhancer profiles: enhancers activated by rapamycin were associated with TGF-{beta} signaling and differentiation, while PDGF-induced enhancers were associated with ERK signaling and migration. C_LIO_LIEnhancer elements containing single nucleotide polymorphisms (SNPs) associated with cardiovascular disease from genome wide association studies (GWAS) showed notable enrichment across active enhancers and super enhancers. Select enhancers demonstrated statistically significant activity in reporter gene assays. C_LI What Question Should Be Addressed Next?O_LIThe cis regulatory elements identified in this work suggest new transcription factors that can be tested to determine whether and how they may influence SMC phenotypic modulation. C_LIO_LIThese studies could be extended to other stimuli to identify epigenomic signatures associated with CASMC transitions to other phenotypes including macrophages and chondrocytes. C_LI

genomics↗

bioRxiv2024

Genomic and environmental controls on Castellaniella biogeography in an anthropogenically disturbed subsurface

Castellaniella species have been isolated from a variety of mixed-waste environments including the nitrate and multiple metal contaminated subsurface at the Oak Ridge Reservation (ORR). Previous studies examining microbial community composition and nitrate removal at ORR during biostimulation efforts reported increased abundances of members of the Castellaniella genus concurrent to increased denitrification rates. Thus, we asked how genomic and abiotic factors control the Castellaniella biogeography at the site to understand how these factors may influence nitrate transformation in an anthropogenically impacted setting. ORR Castellaniella strains showed a higher degree of genetic diversification than those originating from non-ORR sites, which we attribute to the multitude of extreme stressors faced in the ORR subsurface. We report the isolation and characterization of several Castellaniella strains from the ORR subsurface. Five of these isolates match at 100% identity (at the 16S rRNA gene V4 region) to two Castellaniella amplicon sequence variants (ASVs), ASV1 and ASV2, that have persisted in the ORR subsurface for at least two decades. However, ASV2 has consistently higher relative abundance in samples taken from the site and was also the dominant blooming denitrifier population during a prior biostimulation effort. We found that the ASV2 representative strain has greater resistance to mixed metal stress than the ASV1 representative strains. We attribute this resistance, in part, to the large number of unique heavy metal resistance genes identified on a genomic island in the ASV2 representative genome. Additionally, we suggest that the relatively lower fitness of ASV1 may be connected to the loss of the nitrous oxide reductase (nos) operon (and associated nitrous oxide reductase activity) due to the insertion at this genomic locus of a mobile genetic element carrying copper resistance genes. This study demonstrates the value of integrating genomic, environmental, and phenotypic data to characterize the biogeography of key microorganisms in contaminated sites.

microbiology↗

bioRxiv2024

VEGF counteracts shear stress-determined arterial fate specification during capillary remodeling.

Postnatal vascular morphogenesis is a dynamic process that initially involves angiogenesis to form the capillary bed, followed by its partial remodeling into arteries. Vascular endothelial growth factor A (VEGF hereafter), the primary inducer of angiogenesis, is also implicated in arterial specification in developmental contexts. However, it is unclear why arterial patterning is spatiotemporally segregated from angiogenesis, while postnatal arterial specification in animal models with blocked VEGF signaling remains unstudied. Here, we report that VEGF does not induce arterial fate in the capillaries, instead serving as a physiological brake to attenuate fluid shear stress (FSS)-driven capillary-to-arterial cell fate transition. Mouse models with disrupted VEGF signaling reveal impaired angiogenesis but intact and ectopic arterialization. Mechanistically, mechanosensitive transcription factor Sox17 determines the FSS-arterial program, while VEGF suppresses Sox17 transcription activity at arterial promoters and enhancers sites. Angiogenic signaling, while essential for the initial capillary morphogenesis, inhibits arterial specification, thereby protecting the capillary bed from premature arterialization driven by flow. These findings establish a new paradigm in which precise spatiotemporal coordination of environmental stimuli orchestrates angiogenesis, arterial patterning and capillary maintenance during vascular morphogenesis.

developmental biology↗

bioRxiv2024

Gamma protocadherins in vascular endothelial cells inhibit Klf2/4 to promote atherosclerosis

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of mortality worldwide1. Laminar shear stress (LSS) from blood flow in straight regions of arteries protects against ASCVD by upregulating the Klf2/4 anti-inflammatory program in endothelial cells (ECs)2-8. Conversely, disturbed shear stress (DSS) at curves or branches predisposes these regions to plaque formation9,10. We previously reported a whole genome CRISPR knockout screen11 that identified novel inducers of Klf2/4. Here we report suppressors of Klf2/4 and characterize one candidate, protocadherin gamma A9 (Pcdhga9), a member of the clustered protocadherin gene family12. Pcdhg deletion increases Klf2/4 levels in vitro and in vivo and suppresses inflammatory activation of ECs. Pcdhg suppresses Klf2/4 by inhibiting the Notch pathway via physical interaction of cleaved Notch1 intracellular domain (NICD Val1744) with nuclear Pcdhg C-terminal constant domain (CCD). Pcdhg inhibition by EC knockout (KO) or blocking antibody protects from atherosclerosis. Pcdhg is elevated in the arteries of human atherosclerosis. This study identifies a novel fundamental mechanism of EC resilience and therapeutic target for treating inflammatory vascular disease.

cell biology↗

bioRxiv2023

Biomimetic Lipid-polymer composite membrane model to study Permeability through Cornea

The excised animal cornea is the gold standard for testing and evaluating drug permeability through a cornea. However, it has a concise shelf life and encounters ethical concerns. Also, ex-vivo models, which are incomplete replicas of the human cornea, may provide faulty results in the pre-clinical studies. To circumvent these problems, we have proposed an in-vitro biomimetic lipid-polymer composite membrane (BLCM) model as an artificial cornea to study drug permeability. We designed and fabricated a free-standing, electro-spun polystyrene (PS) nanofibrous membrane and impregnated its pores with phosphatidylcholine (PC). SEM, FTIR, and goniometer characterized the BLCM. Permeation data of the drug ganciclovir through the BLCM model in a Franz-diffusion cell corroborates with the excised goat corneal system. Also, owing to the simple and scalable fabrication method, BLCM can be used as an alternative to animal models for initial drug permeability screening and studies and accelerate drug development.

bioengineering↗

bioRxiv2023

Fermented polyherbal formulation restored ricinoleic acid-induced diarrhea in Sprague Dawley rats and exhibited in vitro antibacterial effect on multiple antibiotics resistant gastric pathogens

The involvement of multiple antibiotics resistant gastric pathogens in diarrhea aggravates the disease condition uncontrollably. Current study was aimed at finding and developing a suitable formulation utilizing multiple natural components from known plant sources to augment current therapeutic outcomes. Hydro-ethanolic extraction method was applied through boiling and fermentation, on ancient observation based efficacious plant parts for developing antidiarrheal polyherbal formulation AP-01. Animal study model of diarrhea was used to evaluate safety and efficacy of the formulation. The formulation was tested in vitro on four different multiple antibiotics resistant gastric pathogens collected from national repository. The formulation depicted no cytotoxicity on normal gut cells and was efficacious at 10 ml/Kg single dose in relieving symptoms of diarrhea by 79.71%, in comparison with standard drug showing reduction of symptoms by 83.01%. AP-01 exhibited delaying the gastric motility. Symptoms of diarrhea ceased to occur within 321 minutes with AP-01, where the standard drug took 308 minutes. AP-01 was found successful at a viable dosage regimen of 75 to 100 microliter per ml, in inhibiting growth of different pathogens from Enterobacteriaceae family possessing resistance against several classes of antibiotics, in culture media. Chemical analysis revealed different alkaloids, flavonoids and polyphenols those probably work in unison through multiplex modes of action to arrest diarrhea and inhibit pathogens at the same time. These promising results shown by AP-01 should definitely evoke an effort to dive deep into research and development for better therapeutic formulations for infectious diarrhea by harvesting the arsenal of nature.

microbiology↗

bioRxiv2023

Fermented polyherbal formulation ameliorates the severity of acute multiple - antibiotics - resistant Pseudomonas aeruginosa infected burn wound in rat burn model

Pseudomonas aeruginosa, a Gram-negative opportunistic bacterium has emerged as a cause of life-threatening infections in topical burn wounds. Current therapeutic approaches thru wound dressings and systemic medicines are far from satisfactory; multiple-antibiotic-resistance shown by pathogens contribute to failures of therapy causing mortality. This animal study was conducted to check efficacy of one Ayurveda based fermented polyherbal preparation (AP 01) against multiple antibiotic resistant Pseudomonas aeruginosa HW01 infected rat burn wounds. AP-01 was applied on artificially inflected burn wound on rat model infected with Multiple Antibiotics Resistant Pseudomonas aeruginosa to register the healing effects in terms of reduction in residual wound area percentage, presence of C-reactive protein in blood, presence of viable bacteria colony while keeping conventional antibiotics as positive control. The polyherbal preparation had reduced the infected residual burn wound area at 40.63% {+/-} 0.69 from the initial burn wound area within two weeks after a single intervention; whereas residual burn wound area remained much higher in case of animals left untreated and in case of the animals treated with control drug. Restoration to normalcy of serum C-reactive protein level were also achieved earlier in case of polyherbal AP-01 treated groups than other groups. Fermented formulation using components of AP-01 singly or in different combinations were never been tested earlier for topical application in infected burn-wound. The formulation AP-01 was found superior in terms of rate of healing and control of infection by multiple-antibiotic-resistant P. aeruginosa strains in burn wounds in rat model.

microbiology↗

bioRxiv2023

High diversity of Proteobacteria amid the dominance of a few Aquificae species in the vent-water of a Trans-Himalayan sulfur-borax hot spring

Trans-Himalayan hot spring waters rich in boron, chlorine, sodium and sulfur (but poor in calcium and silicon) are known based on PCR-amplified 16S rRNA gene sequence data to harbor high diversities of infiltrating bacterial mesophiles. Yet, little is known about the community structure and functions, primary productivity, mutual interactions, and thermal adaptations of the microorganisms present in the steaming waters discharged by these geochemically peculiar spring systems. We revealed these aspects of a bacteria-dominated microbiome (microbial cell density [~]8.5 x 104 mL-1; live:dead cell ratio 1.7) thriving in the boiling (85{degrees}C) fluid vented by a sulfur-borax spring called Lotus Pond, situated at 4436 m above the mean sea-level, in the Puga valley of eastern Ladakh, on the Changthang plateau. Assembly, annotation, and population-binning of >15-GB metagenomic sequence illuminated the numeral predominance of Aquificae. While members of this phylum accounted for 80% of all 16S rRNA-encoding reads within the metagenomic dataset, 14% of such reads were attributed to Proteobacteria. Post assembly, only 25% of all protein-coding genes identified were attributable to Aquificae, whereas 41% was ascribed to Proteobacteria. Annotation of metagenomic reads encoding 16S rRNAs, and/or PCR-amplified 16S rRNA genes, identified 163 bacterial genera, out of which 66 had been detected in past investigations of Lotus Ponds vent-water via 16S amplicon sequencing. Among these 66, Fervidobacterium, Halomonas, Hydrogenobacter, Paracoccus, Sulfurihydrogenibium, Tepidimonas, Thermus and Thiofaba (or their close phylogenomic relatives) were presently detected as metagenome-assembled genomes (MAGs). Remarkably, the Hydrogenobacter related MAG alone accounted for [~]56% of the entire metagenome, even though only 15 out of the 66 genera consistently present in Lotus Ponds vent-water have strains growing in the laboratory at >45{degrees}C, reflecting the continued existence of the mesophiles in the ecosystem. Furthermore, the metagenome was replete with genes crucial for thermal adaptation in the context of Lotus Ponds geochemistry and topography. In terms of sequence similarity, a majority of those genes were attributable to phylogenetic relatives of mesophilic bacteria, while functionally they rendered functions such as encoding heat shock proteins, molecular chaperones, and chaperonin complexes; proteins controlling/modulating/inhibiting DNA gyrase; universal stress proteins; methionine sulfoxide reductases; fatty acid desaturases; different toxin-antitoxin systems; enzymes protecting against oxidative damage; proteins conferring flagellar structure/function, chemotaxis, cell adhesion/aggregation, biofilm formation, and quorum sensing. The Lotus Pond Aquificae not only dominated the microbiome numerically but also acted potentially as the main primary producers of the ecosystem, with chemolithotrophic sulfur oxidation (Sox) being the fundamental bioenergetic mechanism, and reductive tricarboxylic acid (rTCA) cycle the predominant carbon fixation pathway. The Lotus Pond metagenome contained several genes directly or indirectly related to virulence functions, biosynthesis of secondary metabolites including antibiotics, antibiotic resistance, and multi-drug efflux pumping. A large proportion of these genes being attributable to Aquificae, and Proteobacteria (very few were ascribed to Archaea), it could be worth exploring in the future whether antibiosis helped the Aquificae overcome niche overlap with other thermophiles (especially those belonging to Archaea), besides exacerbating the bioenergetic costs of thermal endurance for the mesophilic intruders of the ecosystem.

genomics↗

bioRxiv2023

Decoding Root Biogeography: Building Reduced Complexity Functional Rhizosphere Microbial Consortia

The rhizosphere microbiome plays a crucial role in supporting plant productivity and contributes to ecosystem functioning by regulating nutrient cycling, soil integrity, and carbon storage. However, characterizing their functional attributes and microbial relationships remains challenging due to their complex taxonomic and functional compositions. To enable such studies, the development of reduced complexity microbial consortia derived from the rhizosphere microbiome of the natural ecosystem is highly desirable. Designing and assembling reduced complexity consortia that mimic natural communities with consistent, stable, predictable features are highly sought after but is challenging to deliver. Here we present our systematic controlled design towards successful assembly of several such rhizosphere derived reduced complexity consortia. From Brachypodium grown in natural soil under controlled lab conditions, we enriched the root-associated microbes, utilizing carbon compounds prevalent in Brachypodium root exudates. By transferring the enrichments every 3 or 7 days for 9 generations, we developed both fast and slow-growing microbial communities. 16S rRNA amplicon analysis revealed that both inoculum and carbon substrates significantly influence microbial community composition. For example, 1/10 R2A preferentially enriched Amplicon Sequence Variants (ASVs) from slow growing taxa vital to plant including Acidobacteria and Verrucomicrobia. Network analysis revealed that although fast and slow growing microbial consortia have distinct key taxa, the key hubs (keystone taxa) for both belong to genera with plant growth promoting (PGP) traits. This suggests that PGP bacteria might play a central role in controlling the microbial networks among rhizospheric microbiomes. Based on the stability and richness results from different transfers, most carbon substrates lead to microbial consortia with reduced complexity and high stability after a few transfers. The stability tests of the derived microbial consortia also showed high stability, reproducibility, and revivability of the constructed microbial consortia. Our study represents a significant step towards understanding and harnessing the potential of rhizosphere microbiomes, with implications for sustainable agriculture and environmental management.

microbiology↗

bioRxiv2023

Iodidimonas, a bacterium unable to degrade hydrocarbons, thrives in a bioreactor treating oil and gas produced water

Iodidimonas is a genus recently described in bioreactors treating oil and gas produced water and in iodide rich brines. Besides the ability to oxidize iodine, little is known about the metabolic capabilities that enable Iodidimonas sp. to occupy this unique ecological niche. We isolated, characterized, and sequenced three strains belonging to the Iodidimonas genus from the sludge of a membrane bioreactor treating produced water. We describe the genomic features of these isolates and compare them with the only other four isolate genomes reported from this genus, as well as a metagenome-assembled genome from the source bioreactor. To survive in the produced water, Iodidimonas isolates had several genes associated with mitigating salinity, heavy metal and organic compound stress. While the isolates could utilize a wide variety of carbon substrates, they failed to degrade aliphatic or aromatic hydrocarbons, consistent with the lack of genes associated with common hydrocarbon degradation pathways in their genomes. We hypothesize these microbes may lead a scavenging lifestyle in the bioreactor and similar iodide-rich brines. ImportanceOccupying a niche habitat and having few representative isolates, genus Iodidimonas is a relatively understudied Alphaproteobacterial group. This genus has garnered attention due to its ability to corrode pipes in iodine production facilities and generate iodinated organic compounds during treatment of oil and gas produced water. The iodinated organic compounds are likely to be carcinogenic and may pose issues with recycling the treated water. Hence, detailed characterization of the metabolic potential of these isolates is not only of economic importance, but also sheds light on adaptation of this microbe to its environmental niche.

ecology↗