Biology subjects

Mehra, A.

Publications and source records attributed to Mehra, A..

9 recordsLinked to original sources

bioRxiv2026

Temperate Phage Shape Honey Bee Gut Microbiome Structure and Response to Antibiotic Treatment

Bacteriophage (phage) are hypothesized to play a significant role in modulating gut microbiomes. Yet, in vivo research examining the role of phage in gut ecosystems remains sparse, largely due to a lack of tractable model systems. Here, we use the honey bee (Apis mellifera) gut as a model to test the hypotheses that phage-bacteria interactions in the gut are temporally variable and that stress, in the form of antibiotics, can enhance phage killing of their bacterial hosts. First, we isolated and characterized a novel temperate phage which infects a bee-specific strain of Bifidobacterium. We then mono-colonized adult honey bees with this Bifidobacterium strain and tested how phage treatment impacts bacterial abundance over time. Next, using a series of in vitro and in vivo experiments, we examined how phage-bacteria interactions change in response to treatment with tetracycline, an antibiotic commonly used in commercial beekeeping. Finally, to probe the biological mechanisms underlying different phage-antibiotic synergies, we assayed how different classes of antibiotics impacted bacterial growth with and without phage infection. Our results indicate that a single temperate phage can both promote the ability of its Bifidobacterium host to colonize the gut, while also increasing host sensitivity to antibiotic treatment. Together, these findings demonstrate that environmental stressors can shift phage-bacteria interactions from mutualism to antagonism, highlighting the significance of phage in shaping how gut microbiomes respond to antibiotic or xenobiotic perturbation.

microbiology↗

bioRxiv2025

Designing a robust whole-cell biosensor platform for detection of toxic metals using intein splicing inhibition of Mycobacterium tuberculosis SufB protein

Disruption of the natural geochemical cycle by human activities has led to bioaccumulation of metals, posing a global health threat. Hence, there is a pressing need for simple, sensitive, yet eco-friendly biosensor setups to monitor metal contamination in the environment. Existing biosensors are limited by poor efficiency, stability issues, and complex instrumentation requiring skilled operators. To address these caveats, current study explores how intein-mediated protein splicing, a spontaneous post-translational process, can be adapted for metal-biosensing by coupling metal-dependent splicing inhibition to viability loss of native microbial cells. Toxic metal ions like Cd2 and Hg2 attenuated the splicing activity of Mtb SufB precursor protein over a concentration range of 25 {micro}M to 2 mM, while Pb2 and Cr3 failed to do so. An innovative biosensor platform was designed for colorimetric detection of metal ions via simple Alamar Blue assay, where attenuated Mtb strain (H37Ra) served as the indicator cells. Metal-induced SufB splicing inhibition led to loss of viability of H37Ra cells, while addition of metal-specific chelators reversed the effect. Multiplexing ability was evaluated by including known splicing inhibitors like Cu2, Zn2, and Pt4 over various concentration range alongside Cd2 and Hg2. The simple 96-well plate format enables multiplexed qualitative metal detection, while colorimetric absorbance measurement ensures metal quantification. The designed biosensor offers low-cost, user-friendly, and sensitive assay for high-throughput metal detection, utilizing whole-cell native organisms carrying metal-sensing precursor protein. Thus, this approach can be implemented in standard biological laboratories for robust metal screening process in environmental and industrial effluents.

microbiology↗

bioRxiv2025

Phase variation of Clostridioides difficile colony morphology occurs via modulation of cell division

Phase variation of C. difficile colony morphology occurs via modulation of transcription cmrRST, which encodes a three-protein signal transduction system. Response regulators CmrR and CmrT promote rough colony development, cell elongation and chaining, surface motility, and disease in the hamster model of infection, while impairing swimming motility and biofilm formation. Using RNA-Seq, we identified the CmrR and CmrT-dependent transcriptional differences in rough and smooth colonies. Further analysis showed that CmrT, but not CmrR, is required for differential expression of most of the genes. Two CmrT-regulated genes, herein named mrpA and mrpB, were together sufficient for restoring all CmrT-dependent in vitro phenotypes in a cmrT mutant and alleviating selection of cmr phase ON cells during growth on an agar surface. MrpA and MrpB are uncharacterized proteins with no known function but are highly conserved in C. difficile. Using immunoprecipitation and mass spectrometry to identify interacting partners, we found that MrpA interacts with the septum-site determining protein MinD and several other proteins involved in cell division and cell shape determination. Ectopic expression of mrpAB resulted in atypical cell division, consistent with MrpAB interference with MinD function. Our findings reveal a potential mechanism by which phase variation of CmrRST modulates colony morphology and motility: in cmr phase ON cells, CmrT-mediated expression of mrpAB interferes with normal cell division resulting elongated cells that enable expansion of the population across a surface while limiting swimming motility. AUTHOR SUMMARYC. difficile can reversibly switch between two colony morphology variants that differ in multiple additional phenotypes. Previous work determined that the phenotypic switch occurs through phase variation of the CmrRST signal transduction system, however the CmrRST-regulated genes that mediate these phenotypes were unknown. Here, we identified the CmrR- and CmrT-regulated genes that are differentially expressed between rough and smooth colonies and identified two genes, mrpA and mrpB, that together are sufficient to confer phenotypes associated with cmrRST expression. Protein interaction studies revealed that MrpA interacts with MinD, a protein that helps ensure symmetric cell division in bacteria. Based on these findings, we propose that MrpA disrupts MinD function leading to aberrant cell division, resulting in elongated cells that form rough colonies. This study reveals previously uncharacterized proteins that affect C. difficile cell division and broadly impact the physiology of this pathogen.

microbiology↗

bioRxiv2025

Enhancing RNA 3D Folding Prediction via Transformer and Equivariant Architectures under Resource Constraints

Predicting the three-dimensional structure of RNA molecules is a fundamental problem in computational biology, with applications in understanding gene regulation, viral function, and drug design. Existing RNA structure prediction methods often rely on large models or heavy computation, as exemplified by protein folding solutions like AlphaFold2 [2], which makes them impractical under tight resource constraints. In this work, we present an efficient neural architecture that integrates a Transformer encoder enhanced with Rotary Positional Embeddings (RoPE) and a small residual MLP block, along with an optional E(n)-equivariant refinement module. We train our model on the Stanford Ribonanza RNA 3D dataset [11] using BF16 precision on a single NVIDIA H100 (80 GB) GPU. Our model achieves lower RMSD than simpler baselines, demonstrating that modern Transformer and equivariant techniques can be effectively applied to RNA folding under practical compute constraints.

bioinformatics↗

bioRxiv2025

Bactericidal activity of ZnO nanoparticles-anti TB drugs combination towards H37Rv strain and multidrug-resistant isolates of Mycobacterium tuberculosis via SufB splicing inhibition

Tuberculosis (TB) remains a significant global health threat, claiming millions of lives annually. Despite advancements in treatment, the emergence of drug-resistant strains has hindered effective TB control. The current management for TB is prolonged with severe side effects, leading to poor patient compliance. Metal-based nanoparticles are shown to manage drug-sensitive TB when combined with anti-TB drugs. However, mycobactericidal potential of nanoparticles towards drug-resistant TB is not confirmed yet. This work explores the bactericidal potential of Zinc Oxide Nanoparticles (ZnONPs, 40 nm) in managing both drug-sensitive and drug-resistant TB in combination with anti-TB drugs. It was found that ZnONPs inhibit generation of active SufB protein via splicing inhibition, an essential event for Mycobacterium tuberculosis (Mtb) survival. While TEM and UV-visible spectroscopy identified NPs[~]protein interaction, SEM visualised extensive membrane damage in H37Rv and multidrug-resistant (MDR) Mtb cells. Alamar blue assay and spread plate method detected minimum inhibitory concentration and minimum bactericidal concentration of ZnONPs towards H37Rv strain and MDR Mtb isolates. In vitro studies identified a combination with ZnONPs that reduced effective doses for anti-TB drugs towards H37Rv and MDR Mtb isolates. A correlation to splicing inhibition was made by performing Alamar blue assay in SufB intein-less microbe, Mycobacterium smegmatis. A similar drug combination, attenuated the mycobacterial load, inflammation in the spleen & lungs, and protected against Mtb induced splenomegaly in infected mice. Thus, ZnONPs can be used as potent additive in anti-TB regimen to manage drug-susceptible and drug-resistant TB, addressing challenges such as prolonged therapy, drug toxicity and poor patient compliance.

microbiology↗

bioRxiv2024

flt1 inactivation promotes zebrafish cardiac regeneration by enhancing endothelial activity and limiting the fibrotic response

Summary statementflt1 inactivation promotes zebrafish cardiac regeneration by enhancing coronary revascularization and endocardial expansion, while limiting myofibroblast differentiation. VEGFA administration has been explored as a pro-angiogenic therapy for cardiovascular diseases including heart failure for several years, but with little success. Here we investigate a different approach to augment VEGFA bioavailability: by deleting the VEGFA decoy receptor VEGFR1/FLT1, one can achieve more physiological VEGFA concentrations. We find that following cryoinjury, zebrafish flt1 mutant hearts display enhanced coronary revascularization and endocardial expansion, increased cardiomyocyte dedifferentiation and proliferation, and decreased scarring. Suppressing Vegfa signaling in flt1 mutants abrogates these beneficial effects of flt1 deletion. Transcriptomic analyses of cryoinjured flt1 mutant hearts reveal enhanced endothelial MAPK/ERK signaling and downregulation of the transcription factor gene egr3. Using newly generated genetic tools, we observe egr3 upregulation in the regenerating endocardium, and find that Egr3 promotes myofibroblast differentiation. These data indicate that with enhanced Vegfa bioavailability, the endocardium limits myofibroblast differentiation via egr3 downregulation, thereby providing a more permissive microenvironment for cardiomyocyte replenishment after injury.

molecular biology↗

bioRxiv2024

Unique growth and morphology properties of Clade 5 Clostridioides difficile strains revealed by single-cell time-lapse microscopy

Clostridioides difficile is a gastrointestinal pathogen of both humans and agricultural animals and thus a major One Health threat. The C. difficile species consists of five main clades, with Clade 5 currently undergoing speciation from Clades 1-4. Clade 5 strains are highly prevalent in agricultural animals and can cause zoonotic infections, suggesting that these strains have evolved phenotypes that distinguish them from Clade 1-4 strains. Here, we compare the growth properties of Clade 5 strains to those of Clade 1-4 strains using anaerobic time-lapse microscopy coupled with automated image analysis. Our analyses indicate that Clade 5 strains grow faster and are more likely to form long chains of cells than Clade 1-4 strains. Using comparative genomic and CRISPRi analyses, we show that the chaining phenotype of Clade 5 strains is driven by the orientation of the invertible cmr switch sequence, with chaining strains exhibiting a bias to the cmr-ON state. Interestingly, Clade 5 strains with a bias towards the cmr-ON state shifted to a largely cmr-OFF state during murine infection, suggesting that the cmr-OFF state is under positive selection during infection. Collectively, our data reveal that Clade 5 strains have distinct growth properties, which may allow them to inhabit diverse ecological niches. Author SummaryThe Clade 5 strains of the Clostridioides difficile species are so phylogenetically divergent that they almost meet the threshold of being a distinct species. Although these strains are ubiquitously isolated from agricultural and environmental settings and an important source of zoonotic and community-acquired infections, it is unclear whether they have distinct phenotypic properties that allow them to colonize diverse hosts or persist in the environment. By combining a novel anaerobic time-lapse microscopy method with automated image analysis, we discovered that Clade 5 strains grow faster than strains from other C. difficile clades and that they frequently form long chains. These chaining properties are driven by the environmentally responsive expression of a non-canonical signal transduction system, which our analyses suggest is selected against during murine infection. Collectively, our analyses reveal that Clade 5 strains have distinct growth properties that may promote their persistence in the environment.

microbiology↗

bioRxiv2021

Multiple regulatory mechanisms control the production of CmrRST, an atypical signal transduction system in Clostridioides difficile

Clostridioides difficile, an intestinal pathogen and leading cause of nosocomial infection, exhibits extensive phenotypic heterogeneity through phase variation by site-specific recombination. The signal transduction system CmrRST, which encodes two response regulators (CmrR and CmrT) and a sensor kinase (CmrS), impacts C. difficile cell and colony morphology, surface and swimming motility, biofilm formation, and virulence in an animal model. CmrRST is subject to phase variation through site-specific recombination and reversible inversion of the cmr switch, and expression of cmrRST is also regulated by c-di-GMP through a riboswitch. The goal of this study was to determine how the cmr switch and c-di-GMP work together to regulate cmrRST expression. We generated "phase locked" strains by mutating key residues in the right inverted repeat flanking the cmr switch. Phenotypic characterization of these phase locked cmr-ON and -OFF strains demonstrates that they cannot switch between rough and smooth colony morphologies, respectively, or other CmrRST-associated phenotypes. Manipulation of c-di-GMP levels in these mutants showed that c-di-GMP promotes cmrRST expression and associated phenotypes independent of cmr switch orientation. We identified multiple promoters controlling cmrRST transcription, including one within the ON orientation of cmr switch and another that is positively autoregulated by CmrR. Overall, this work reveals a complex regulatory network that governs cmrRST expression and a unique intersection of phase variation and c-di-GMP signaling. These findings suggest that multiple environmental signals impact the production of this signaling transduction system. IMPORTANCEClostridioides difficile is a leading cause of hospital-acquired intestinal infections in the U.S. The CmrRST signal transduction system controls numerous physiological traits and processes in C. difficile, including cell and colony morphology, motility, biofilm formation, and virulence. Here we define the complex, multi-level regulation of cmrRST expression, including stochastic control through phase variation, modulation by the second messenger c-di-GMP, and positive autoregulation by CmrR. The results of this study suggest that multiple, distinct environmental stimuli and selective pressures must be integrated to appropriately control cmrRST expression.

microbiology↗

bioRxiv2020

Targeted delivery of acid alpha-glucosidase corrects skeletal muscle phenotypes in Pompe disease mice

Lysosomal diseases are a class of genetic disorders predominantly caused by loss of lysosomal hydrolases, leading to lysosomal and cellular dysfunction. Enzyme Replacement Therapy (ERT), where recombinant enzyme is given intravenously, internalized by cells, and trafficked to the lysosome, has been applied to treat several lysosomal diseases. However, current ERT regimens do not correct disease phenotypes in all affected organs because the biodistribution of enzyme uptake does not match that of the affected cells and tissues that require the enzyme. We present here targeted ERT, an approach that utilizes antibody-enzyme fusion proteins to target the enzyme to specific tissues. The antibody moiety recognizes transmembrane proteins involved in lysosomal trafficking and that are also preferentially expressed in those cells most affected in disease. Using Pompe disease (PD) as an example, we show that targeted ERT is superior to ERT in treating the skeletal muscle phenotypes of PD mice.

bioengineering↗