Biology subjects

Kulkarni, R.

Publications and source records attributed to Kulkarni, R..

30 records · Page 2Linked to original sources

bioRxiv2022

FAM21 is critical for TLR2-CLEC4E-mediated dendritic cell function against Candida albicans

FAM21 (family with sequence similarity 21) is a component of the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) protein complex that mediates actin polymerization at endosomal membranes to facilitate sorting of cargo-containing vesicles out of endosomes. To study the function of FAM21 in vivo, we generated conditional knockout (cKO) mice in the C57BL/6 background in which FAM21 was specifically knocked out of CD11c-positive dendritic cells. Bone marrow-derived dendritic cells (BMDC) from those mice displayed enlarged early endosomes, and altered cell migration and morphology relative to wildtype (WT) cells. FAM21-cKO cells were less competent in phagocytosis and antigen processing in vitro, though antigen presentation was not affected. More importantly, we identified the TLR2/CLEC4E signaling pathway as being downregulated in FAM21-cKO BMDCs when challenged with its specific ligand Candida albicans. Moreover, FAM21-cKO mice were more susceptible to C. albicans infection than WT mice. Reconstitution of WT BMDCs in FAM21-cKO mice rescued them from lethal C. albicans infection. Thus, our study highlights the importance of FAM21 in a host immune response against a significant pathogen.

cell biology↗

bioRxiv2021

Glycosuria alters uropathogenic Escherichia coli global gene expression and virulence

Uropathogenic Escherichia coli (UPEC) is the principal etiology of more than half of urinary tract infections (UTI) in humans with diabetes mellitus. Epidemiological data and studies in mouse model of ascending UTI have elucidated various host factors responsible for increasing the susceptibility of diabetic hosts to UPEC-UTI. In contrast, the nature of alterations in UPEC physiology mediated by diabetic urinary microenvironment and the contributions of altered UPEC physiology in shaping UPEC-UTI pathogenesis in diabetes have not been examined. Our central hypothesis is that glycosuria directly induces urinary virulence of UPEC. We compared virulence characteristics and gene expression in human UPEC strains UTI89 (cystitis) and CFT073 (pyelonephritis) exposed for 2h, in vitro to human urine either in the presence or absence of glycosuria (600mg/dl glucose). Compared to control UPEC exposed to nutrient-rich culture medium LB, glycosuria-exposed UPEC exhibited significant increase in biofilm formation and reduction in the hemagglutination of Guinea pig erythrocytes (a surrogate measure of type 1 piliation). In addition, analysis of UTI89 transcriptome by RNA sequencing revealed that 2h-long, in vitro exposure to glycosuria also significantly alters expression of virulence and metabolic genes central to urinary virulence of UPEC. In summary, our results provide novel insights into how glycosuria-mediated early changes in UPEC fitness may facilitate UTI pathogenesis in the diabetic urinary microenvironment. IMPORTANCEUropathogenic Escherichia coli (UPEC) is an important causative agent of urinary tract infections in diabetic humans. We examined the effects of in vitro exposure to glycosuria (presence of glucose in urine) on the virulence and gene expression by UPEC. Our results show that glycosuria rapidly (in 2h) alters UPEC gene expression, induces biofilm formation, and suppresses hemagglutination. These results offer a novel insight into the pathogenesis of UPEC in the urinary tract.

microbiology↗

bioRxiv2021

Amplification and attenuation of noisy expression by export processes

Inside mammalian cells, single genes are known to be transcribed in stochastic bursts leading to the synthesis of nuclear RNAs that are subsequently exported to the cytoplasm to create mRNAs. We systematically characterize the role of export processes in shaping the extent of random fluctuations (i.e. noise) in the mRNA level of a given gene. Using the method of Partitioning of Poisson arrivals, we derive an exact analytical expression for the noise in mRNA level assuming that the nuclear retention time of each RNA is an independent and identically distributed random variable following an arbitrary distribution. These results confirm recent experimental/theoretical findings that decreasing the nuclear export rate buffers the noise in mRNA level, and counterintuitively, decreasing the noise in the nuclear retention time enhances the noise in the mRNA level. Next, we further generalize the model to consider a dynamic extrinsic disturbance that affects the nuclear-to-cytoplasm export. Our results show that noise in the mRNA level varies non-monotonically with the disturbance timescale. More specifically, high- and low-frequency external disturbances have little impact on the mRNA noise level, while noise is amplified at intermediate frequencies. In summary, our results systematically uncover how the coupling of bursty transcription with nuclear export can both attenuate or amplify noise in mRNA levels depending on the nuclear retention time distribution and the presence of extrinsic fluctuations.

systems biology↗

bioRxiv2021

Vaccinia virus-based vaccines confer protective immunity against SARS-CoV-2 virus in Syrian hamsters

COVID-19 in humans is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) that belongs to the beta family of coronaviruses. SARS-CoV-2 causes severe respiratory illness in 10-15% of infected individuals and mortality in 2-3%. Vaccines are urgently needed to prevent infection and to contain viral spread. Although several mRNA- and adenovirus-based vaccines are highly effective, their dependence on the "cold chain" transportation makes global vaccination a difficult task. In this context, a stable lyophilized vaccine may present certain advantages. Accordingly, establishing additional vaccine platforms remains vital to tackle SARS- CoV-2 and any future variants that may arise. Vaccinia virus (VACV) has been used to eradicate smallpox disease, and several attenuated viral strains with enhanced safety for human applications have been developed. We have generated two candidate SARS-CoV-2 vaccines based on two vaccinia viral strains, MVA and v-NY, that express full-length SARS-CoV-2 spike protein. Whereas MVA is growth-restricted in mammalian cells, the v-NY strain is replication-competent. We demonstrate that both candidate recombinant vaccines induce high titers of neutralizing antibodies in C57BL/6 mice vaccinated according to prime-boost regimens. Furthermore, our vaccination regimens generated TH1-biased immune responses in mice. Most importantly, prime-boost vaccination of a Syrian hamster infection model with MVA-S and v-NY-S protected the hamsters against SARS-CoV-2 infection, supporting that these two vaccines are promising candidates for future development. Finally, our vaccination regimens generated neutralizing antibodies that partially cross-neutralized SARS-CoV-2 variants of concern.

immunology↗

bioRxiv2021

Human urine alters methicillin-resistant Staphylococcus aureus virulence and transcriptome

Gram-positive methicillin-resistant Staphylococcus aureus (MRSA) is an emerging cause of hospital-associated urinary tract infections, especially in catheterized individuals. Despite being rare, MRSA-UTI are prone to potentially life-threatening exacerbations such as bacteremia that can be refractory to routine antibiotic therapy. Hence, MRSA-UTI is an important of research topic. To delineate molecular mechanisms governing MRSA urinary pathogenesis, we exposed three S. aureus strains, of which two were MRSA, to human urine and analyzed virulence characteristics and gene expression. We also analyzed MRSA-1369 transcriptome following cultivation in human urine for 2h. Our results reveal that human urine induces global changes in MRSA transcriptome, marked by changes in genes encoding proteins involved in metabolic pathways, virulence, and transcriptional regulators. In addition, in vitro assays also showed that human urine alters, in a strain-specific manner, adherence to human bladder epithelial cells and fibronectin, hemolysis of sheep RBCs, and surface hydrophobicity. In summary, our results provide first important insights into how the urine may specifically alter MRSA physiology in turn facilitating MRSA survival in the nutrient-limiting and hostile urinary microenvironment. ImportanceMethicillin-resistant Staphylococcus aureus (MRSA) is an uncommon cause of urinary tract infections (UTI) in the general population. However, it is important to understand MRSA pathophysiology in the urinary tract because isolation of MRSA in urine samples is often secondary to potentially life-threatening MRSA bacteremia. In this report, we describe that cultivation in human urine alters MRSA global gene expression and virulence. We hypothesize that these alterations may aid MRSA adapt to the nutrient-limiting, immunologically hostile conditions within the urinary tract.

microbiology↗

bioRxiv2020

Plasmids encode niche-specific traits in Lactobacillaceae

The species of family Lactobacillaceae are found in highly diverse environments and play an important role in fermented foods and probiotic products. Many of these species have been individually reported to harbor plasmids that encode important genes. In this study, we performed comparative genomic analysis of the publically available data of 512 plasmids from 282 strains represented by 51 species of this family and correlated the genomic features of plasmids with the ecological niches in which these species are found. Two-third of the species had at least one plasmid-harboring strain. Plasmid abundance and GC content were significantly lower in the vertebrate-adapted species as compared to the nomadic and free-living species. Hierarchical clustering (HCL) highlighted the distinct nature of plasmids from the nomadic and free-living species than those from the vertebrate-adapted species. EggNOG assisted functional annotation revealed that genes associated with transposition, conjugation, DNA repair and recombination, exopolysaccharide production, metal ion transport, toxin-antitoxin system, and stress tolerance were significantly enriched on the plasmids of the nomadic and in some cases nomadic and free-living species. On the other hand, genes related to anaerobic metabolism, ABC transporters, and major facilitator superfamily were found to be overrepresented on the plasmids of the vertebrate-adapted species. These genomic signatures are correlated to the comparatively nutrient-depleted, stressful and dynamic environments of nomadic and free-living species and nutrient-rich and anaerobic environments of the vertebrate-adapted species. Thus, these results indicate the contribution of the plasmids in the adaptation of lactobacilli to the respective habitats. This study also underlines the potential application of these plasmids in improving the technological and probiotic properties of lactic acid bacteria. Impact statementThe bacteria of the family Lactobacillaceae are present in the wide range of habitats and play an important role in human health, fermented foods and chemical industries. A few studies have demonstrated the presence of plasmids in the individual strains of Lactobacillaceae species encoding various traits. Extensive data of genome sequences of the lactobacilli are becoming available; however, no comprehensive analysis of the plasmid-encoded genes and determining their biological relevance across lactobacilli has been undertaken at a larger scale. In this study, we explored the genomic content of 512 plasmids of Lactobacillaceae species and correlated it to the three types of these species according to their ecological niches - vertebrate-adapted, free-living and nomadic. Comparatively lower plasmid abundance and GC content in the vertebrate-adapted species could be correlated to the presence of these species in the nutrient-rich environment. The genomic content of the plasmids was consistent with the respective lifestyle adopted by lactobacilli suggesting that the plasmids might enhance the niche-specific fitness of the strains. The plethora of important genes present on the plasmids can also make them a highly useful tool in improving the probiotic, technological and food-related properties of lactobacilli. Data summaryNucleotide sequences of plasmids of Lactobacillus strains for which complete genome sequences were available were retrieved from the NCBI genome [https://www.ncbi.nlm.nih.gov/genome] and PATRIC 3.5.41 databases on 31st March 2019. The dataset includes 512 nucleotide sequences of plasmids of 282 strains belonging to genus Lactobacillus before its reclassification into several genera (1). Details of the plasmids have been given in Table S1.

genomics↗

bioRxiv2020

Direct Quantification of in vivo Mutagenesis Using Duplex Sequencing

The ability to accurately measure mutations is critical for basic research and identification of potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and dont fully represent other species such as humans. Next generation sequencing (NGS) is a conceptually attractive alternative for mutation detection in the DNA of any organism, however, the limit of resolution for standard NGS is poor. Technical error rates (~1x10-3) of NGS obscure the true abundance of somatic mutations, which can exist at pernucleotide frequencies [≤]1x10-7. Using Duplex Sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by 3 carcinogens in 5 tissues of 2 strains of mice within 31 days following exposure. We observed a strong correlation between mutation induction measured by Duplex Sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified the primordial signs of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex Sequencing can be broadly applied to chemical safety testing, basic mutational research, and related clinical uses. SIGNIFICANCE STATEMENTError-corrected next generation sequencing (ecNGS) can be used to rapidly detect and quantify the in vivo mutagenic impact of environmental exposures or endogenous processes in any tissue, from any species, at any genomic location. The greater speed, higher scalability, richer data outputs, as well as cross-species and cross-locus applicability of ecNGS compared to existing methods make it a powerful new tool for mutational research, regulatory safety testing, and emerging clinical applications.

genomics↗

bioRxiv2020

Unique roles of ATAC and SAGA KAT2A complexes in normal and malignant hematopoiesis

Epigenetic histone modifiers are key players in cell fate decisions. Significant research has focused on their enzymatic activity, but less is known about the contextual role of the complexes they integrate. We focus on KAT2A, a histone acetyltransferase we recently associated with leukemia stem cell maintenance, and which participates in ATAC and SAGA complexes. We show that ATAC is uniquely required for maintenance of normal and leukemia stem and progenitor cells, while SAGA more specifically contributes to cell identity. This dichotomy sets a paradigm for investigating epigenetic activities in their macromolecular context and informs epigenetic regulator targeting for translational purposes.

cell biology↗

bioRxiv2020

Genotypic characterization of the U.S. peanut core collection

Cultivated peanut (Arachis hypogaea) is an important oil, food, and feed crop worldwide. The USDA peanut germplasm collection currently contains 8,982 accessions. In the 1990s, 812 accessions were selected as a core collection on the basis of phenotype and country of origin. The present study reports genotyping results for the entire available core collection. Each accession was genotyped with the Arachis_Axiom2 SNP array, yielding 14,430 high-quality, informative SNPs across the collection. Additionally, a subset of 253 accessions was replicated, using between two and five seeds per accession, to assess heterogeneity within these accessions. the genotypic diversity of the core is mostly captured in five genotypic clusters, which have some correspondence with botanical variety and market type. There is little genetic clustering by country of origin, reflecting peanuts rapid global dispersion in the 18th and 19th centuries. A genetic cluster associated with the hypogaea/aequatoriana/peruviana varieties, with accessions coming primarily from Bolivia, Peru, and Ecuador, is consistent with these having been the earliest landraces. The genetics, phenotypic characteristics, and biogeography are all consistent with previous reports of tetraploid peanut originating in Southeast Bolivia. Analysis of the genotype data indicates an early genetic radiation, followed by regional distribution of major genetic classes through South America, and then a global dissemination that retains much of the early genetic diversity in peanut. Comparison of the genotypic data relative to alleles from the diploid progenitors also indicates that subgenome exchanges, both large and small, have been major contributors to the genetic diversity in peanut. All data is available at the National Ag Library: https://doi.org/10.15482/USDA.ADC/1518508 and at PeanutBase: https://peanutbase.org/data/public/Arachis_hypogaea/mixed.esm.KNWV

genomics↗

bioRxiv2019

The effects of e-cigarette vapor exposure on the transcriptome and virulence of Streptococcus pneumoniae

The effects of e-cigarette vapor (EV) exposure on the physiology of respiratory microflora are not fully defined. We analyzed the effects of exposure to vapor from nicotine-containing and nicotine-free e-liquid formulations on virulence and transcriptome of Streptococcus pneumoniae strain TIGR4, a pathogen that asymptomatically colonizes human nasopharyngeal mucosa. TIGR4 was pre-exposed for 2h to nicotine-containing EV extract (EVE+NIC), nicotine-free EV extract (EVE-NIC), cigarette smoke extract (CSE), or nutrient-rich TS broth (control). The differences in the treatment and control TIGR4 were explored using transcriptome sequencing, in vitro virulence assays, and in vivo mouse model of acute pneumonia. The analysis of RNASeq profiles revealed modest changes in the expression of 14 genes involved in sugar transport and metabolism in EVE-NIC pre-exposed TIGR4 compared to the control. While, EVE+NIC or CSE exposure altered expression of 264 and 982 genes, respectively, most of which were involved in metabolism and stress response. Infection in a mouse model of acute pneumonia with control TIGR4 or with TIGR4 pre-exposed to EVE+NIC, EVE-NIC, or CSE did not show significant differences in disease parameters, such as bacterial organ burden and respiratory cytokine response. Interestingly, TIGR4 exposed to CSE or EVE+NIC (but not EVE-NIC) exhibited moderate induction of biofilm formation. However, none of the treatment groups showed significant alterations in pneumococcal hydrophobicity or epithelial cell adherence. In summary, our study reports that exposure to EV significantly alters the S. pneumoniae transcriptome in a nicotine-dependent manner without affecting pneumococcal virulence.\n\nImportanceWith the increasing popularity of e-cigarettes amongst cigarette smoking and non-smoking adults and children, and the recent reports of vaping related lung illnesses and deaths, further analysis of the adverse health effects of e-cigarette vapor (EV) exposure is warranted. Since pathogenic bacteria such as Streptococcus pneumoniae can colonize the human nasopharynx as commensals, they may be affected by the exposure to bioactive chemicals in EV. Hence in this study we examined the effects of EV exposure on the physiology of S. pneumoniae strain TIGR4. In order to differentiate between the effects of nicotine and non-nicotine components, we specifically compared RNASeq profiles and virulence of TIGR4 exposed to vapor from nicotine-containing and nicotine-free e-liquid formulations. We observed that nicotine-containing EV augmented TIGR4 biofilms and altered expression of TIGR4 genes predominantly involved in metabolism and stress response. However, neither nicotine-containing nor nicotine-free EV affected TIGR4 virulence in a mouse model.

microbiology↗

bioRxiv2018

Loss Of Kat2a Enhances Transcriptional Noise And Depletes Acute Myeloid Leukemia Stem-Like Cells

Acute Myeloid Leukemia (AML) is an aggressive hematological malignancy with abnormal progenitor self-renewal and defective myelo-monocytic differentiation. Its pathogenesis comprises subversion of transcriptional regulation, through mutation and by hijacking normal chromatin regulation. Kat2a is a histone acetyltransferase central to promoter activity that we recently associated with stability of pluripotency networks, and identified as a genetic vulnerability in AML. Through combined chromatin profiling and single-cell transcriptomics, we demonstrate that Kat2a contributes to leukemia propagation through homogeneity of transcriptional programs and preservation of leukemia stem-like cells. Kat2a loss reduces transcriptional bursting frequency in a subset of gene promoters, generating enhanced variability of transcript levels but minimal effects on mean gene expression. Destabilization of target programs shifts cellular equilibrium out of self-renewal towards differentiation. We propose that control of transcriptional variability is central to leukemia stem-like cell propagation, and establish a paradigm exploitable in different tumors and at distinct stages of cancer evolution.

cancer biology↗

bioRxiv2018

Histone Acetyltransferase Kat2a Stabilises Pluripotency with Control of Transcriptional Heterogeneity

Cell fate transitions in mammalian stem cell systems have often been associated with transcriptional heterogeneity, however existing data have failed to establish a functional or mechanistic link between the two phenomena. Experiments in unicellular organisms support the notion that transcriptional heterogeneity can be used to facilitate adaptability to environmental changes and have identified conserved chromatin-associated factors that modulate levels of transcriptional noise. Herein, we show destabilisation of pluripotency-associated gene regulatory networks through increased transcriptional heterogeneity of mouse embryonic stem cells in which paradigmatic histone acetyl-transferase, and candidate noise modulator, Kat2a (yeast orthologue Gcn5) has been inhibited. Functionally, network destabilisation associates with reduced pluripotency and accelerated mesendodermal differentiation, with increased probability of transitions into lineage commitment. Thus, we functionally link transcriptional heterogeneity to cell fate transitions through manipulation of the histone acetylation landscape of mouse embryonic stem cells and establish a general paradigm that could be exploited in other normal and malignant stem cell fate transitions.

developmental biology↗