Biology subjects

Wadhwa, N.

Publications and source records attributed to Wadhwa, N..

13 recordsLinked to original sources

bioRxiv2026

Increased flagellation enhances bacterial migration against and across flow

Rheotaxis enables bacteria to swim against fluid flow, influencing their dispersal and colonization of surfaces. Although flagella generate the propulsion required for swimming, whether having more flagella improves rheotaxis remains unclear. Here, we varied flagellation using an Escherichia coli strain with tunable flagellar expression and tracked near-surface swimming under controlled microfluidic flow. Greater flagellation increased swimming speed without flow, reduced downstream transport under flow, and increased the fraction of tracks moving upstream. More flagellated cells also swam upstream for longer before switching direction. Greater flagellation also enhanced directed migration across the flow, suggesting that flagellar number alters the balance between propulsion, upstream alignment, and chiral turning near surfaces. A near-surface swimming model, in which flagellar number affects the swimming speed and the tilt of the cell toward the surface reproduced the trends in downstream transport, upstream swimming, and lateral migration. These results identify flagellar number as a determinant of how bacteria resist downstream transport and migrate across near-surface flows.

biophysics↗

bioRxiv2026

Stator pool size shapes the mechanosensitivity of the bacterial flagellar motor

The bacterial flagellar motor adapts to mechanical load by adding or removing stator units, which can either be inactive in the inner membrane (the stator pool) or attached to the motor in a torque-generating configuration. This adaptation is well understood, but what sets the load range over which the motor responds remains unclear. Building on a previous two-state model of stator binding, we derive how the on-rate depends on the diffusing stator pool and find that it increases in proportion to pool size. The model predicts that pool size governs motor occupancy at low load but not at high load, where the motor saturates regardless of how many stators are available. We confirmed this prediction in Escherichia coli: raising stator expression increased swimming speed and single-motor rotation rate at low load. This dependence on expression weakened as load rose and was undetectable at high load. The model further predicts that larger pools make the motor mechanosensitive at lower loads, which our measurements of load-induced occupancy change support. Mechanosensitivity is therefore not fixed by motor architecture alone, but also determined by stator abundance, which cells can set through expression to position their mechanosensory range. SignificanceUsing a statistical mechanics model of stator binding, we predict how flagellar motor output depends on mechanical load and on the number of stators a cell expresses. Combining the model with measurements of swimming cells and single motors, we find that the size of the stator pool sets the range of load over which the motor is mechanosensitive. Mechanosensitivity is therefore a property the cell can tune through stator expression, not a fixed feature of the motor, a principle that may extend to other force-responsive molecular complexes assembled from a limited pool of components.

biophysics↗

bioRxiv2025

Long-range coupling regulates stator dynamics in the bacterial flagellar motor

The bacterial flagellar motor generates torque through MotAB stator complexes which couple ion flux to rotation. Stators anchor in the peptidoglycan cell wall and dynamically remodel in response to changes in external conditions such as the mechanical load, yet how stator anchoring is regulated remains unknown. Here, we show that long-range allosteric interactions within the MotB periplasmic domain tune stator binding in Escherichia coli. Using coarse-grained elastic-network modeling and co-evolutionary analyses, we identified residues mechanically coupled to peptidoglycan-interacting loops of MotB. Targeted mutagenesis at these coupled sites produced distinct motility phenotypes in some mutants, exhibiting altered swimming speeds compared to wild-type and characteristic expression-dependent swimming trends, indicating mutation-specific effects on stator dynamics or torque. Single-motor measurements distinguished mutants with altered torque from those with altered stator dynamics. Molecular dynamics simulations revealed that mutations at distal positions reshape loop flexibility in ways that quantitatively correlate with swimming speeds. These results demonstrate that allosteric communication within MotB propagates across length scales to modulate the performance of the entire motor, revealing how local molecular changes can tune large-scale bacterial motion.

biophysics↗

bioRxiv2025

Osmotic stress triggers fast and reversible PMF collapse in Escherichia coli

Environmental stressors routinely impact bacteria and affect their physiology. Among the most important physiological parameters is the proton motive force (PMF), that powers vital cellular processes and molecular machines. Measuring how PMF responds to environmental stress in real time requires tools that capture rapid physiological changes with high temporal resolution. Here, we use the bacterial flagella motor as an in vivo voltmeter to probe PMF dynamics during hyperosmotic shock. Because motor rotation frequency scales linearly with PMF, this approach enables single-cell electrophysiology with high temporal resolution. We find that hyperosmotic stress causes a rapid and reversible loss of PMF in Escherichia coli, independent of the osmolyte used or the presence of potassium ions. We corroborate these findings by using the Nernstian dye, tetramethyl rhodamine methyl ester (TMRM), showing that hyperosmotic shock leads to membrane depolarization. Together, our results highlight the efficacy of the flagellar motor as powerful tool for probing bacterial electrophysiology and reveal that hyperosmotic stress directly disrupts cellular energetics in addition to its mechanical effects.

biophysics↗

bioRxiv2024

Generation of a Transgenic Mouse Model for Investigating Mitochondria in Sperm

Mitochondria play a crucial role in the sperm development; however, the mechanisms regulating their function in sperm remain poorly understood. Developing a method to regulate the expression of a target gene within the mitochondria of sperm is a vital step in this area of research. In this study, we aimed to create a system for expressing a transgene in the mitochondria of sperm. As a proof of concept, we generated transgenic mice that express green fluorescent protein (GFP) fused with a mitochondrial localization signal (MLS) driven by the phosphoglycerate kinase 2 (PGK2) promoter, which facilitates the transgene expression in the sperm. Although the PGK2 promoter has previously shown to drive gene expression in spermatocytes and spermatids, the novelty of our approach lies in the combination of PGK2-driven MLS-GFP expression to study mitochondria in vivo. We established two founder lines of transgenic mice through pronuclear microinjection, and MLS-GFP expression was confirmed in the mitochondria of sperm cells using fluorescence microscopy and flow cytometry. Consequently, we provide a novel platform for investigating mitochondrial function in sperm, where GFP can be substituted with other genes of interest to examine their effects on mitochondria. This system specifically targets sperm mitochondria, offering an innovative approach for studying mitochondrial function in vivo.

cell biology↗

bioRxiv2024

Surfing Motility: A Novel Propulsion-Independent Mechanism for Surface Migration in Salmonella and E. coli

Bacterial motility over surfaces is crucial for colonization, biofilm formation, and pathogenicity. Surface motility in Escherichia coli and Salmonella enterica is traditionally believed to rely on flagellar propulsion. Here, we report a novel mode of motility, termed "swashing," where these bacteria migrate on agar surfaces without functional flagella. Mutants lacking flagellar filaments and motility proteins exhibit rapid surface migration comparable to wild-type strains. Unlike previously described sliding motility, swashing is inhibited by surfactants and requires fermentable sugars. We propose that the fermentation of sugars at the colony edge produces osmolytes, creating local osmotic gradients that draw water from the agar, forming a fluid bulge that propels the colony forward. Our findings challenge the established view that flagellar propulsion is required for surface motility in E. coli and Salmonella, and highlight the role of a fermentation in facilitating bacterial spreading. This discovery expands our understanding of bacterial motility, offering new insights into bacterial adaptive strategies in diverse environments. Significance StatementBacteria move on surfaces using a variety of mechanisms, with important implications for their growth and survival in both the clinical setting (such as on the surface of medical devices) and in the wild. Surface motility in the medically important model species S. enterica and E. coli has been extensively studied and is thought to require flagellar propulsion. Here, we show surface expansion in these species even in the absence of propulsion by the flagella. Instead, movement is tied to fermentation and surface tension: As cells ferment sugars, they create local osmolarity gradients, which generate a wave of fluid on which the cells "swash."

microbiology↗

bioRxiv2023

SARS-CoV-2 antibodies cross-react and enhance dengue infection

The presence of non-neutralizing antibodies of any dengue serotype, increase the severity of subsequent infection by other dengue serotypes. During SARS-CoV-2 pandemic, the number of symptomatic dengue cases increased in India. We describe that antibodies isolated from convalescent plasma from COVID-19 patients enhances DENV2 infection in vitro. CR3022, one antibody against SARS-CoV-2 spike protein, also showed elevated DENV2 infection in vitro. In silico protein-protein interactions between the spike antibodies and the DENV2 E-protein revealed significant interactions. Likewise, few monoclonal/polyclonal antibodies against SARS-CoV-2 showed increased dengue infection in vitro. Importantly, the AG129 mice infected with SARS-CoV-2 three-weeks prior to DENV2 infection, showed elevated dengue pathogenesis. Thus, highlighting the possibilities of elevated infection and symptomatic dengue disease in COVID-19 survivors.

immunology↗

bioRxiv2022

Mechanisms of ion selectivity and rotor coupling in the bacterial flagellar sodium-driven stator unit

Bacteria swim using a flagellar motor that is powered by stator units. These stator units are energized by an ionic gradient across the membrane, typically proton or sodium. The presumed monodirectional rotation of the stator units allows the bidirectional rotation of the flagellar motor. However, how ion selectivity is attained, how ion transport triggers the directional rotation of the stator unit, and how the stator unit is incorporated into the motor remain largely unclear. Here we have determined by cryo-electron microscopy the structure of the Na+-driven type stator unit PomAB from the gram-negative bacterium Vibrio alginolyticus in both lipidic and detergent environments, at a resolution up to 2.5 [A]. The structure is in a plugged, auto-inhibited state consisting of five PomA subunits surrounding two PomB subunits. The electrostatic potential map uncovers sodium ion binding sites within the transmembrane domain, which together with functional experiments and explicit solvent molecular dynamics simulations, suggest a mechanism for ion translocation and selectivity. Resolved conformational isomers of bulky hydrophobic residues from PomA, in the vicinity of key determinant residues for sodium ion coupling of PomB, prime PomA for clockwise rotation. The rotation is tightly blocked by the trans-mode organization of the PomB plug motifs. The structure also reveals a conformationally dynamic helical motif at the C-terminus of PomA, which we propose regulates the distance between PomA subunit cytoplasmic domains and is involved in stator unit-rotor interaction, concomitant stator unit activation, and torque transmission. Together, our studies provide mechanistic insight for understanding flagellar stator unit ion selectivity and incorporation of the stator units into the motor.

microbiology↗

bioRxiv2022

Ancestral SARS-CoV-2 driven antibody repertoire diversity in an unvaccinated individual correlates with expanded neutralization breadth

Understanding the quality of immune repertoire triggered during natural infection can provide vital clues that form the basis for development of humoral immune response in some individuals capable of broadly neutralizing pan SARS-CoV-2 variants. We assessed the diversity of neutralizing antibody responses developed in an unvaccinated individual infected with ancestral SARS-CoV-2 by examining the ability of the distinct B cell germline-derived monoclonal antibodies (mAbs) in neutralizing known and currently circulating Omicron variants by pseudovirus and authentic virus neutralization assays. The ability of the antibodies developed post vaccination in neutralizing Omicron variants was compared to that obtained at baseline of the same individual and to those obtained from Omicron breakthrough infected individuals by pseudovirus neutralization assay. Broadly SARS-CoV-2 neutralizing mAbs representing unique B cell lineages with non-overlapping epitope specificities isolated from a single donor varied in their ability to neutralize Omicron variants. Plasma antibodies developed post vaccination from this individual demonstrated neutralization of Omicron BA.1, BA.2 and BA.4 with increased magnitude and found to be comparable with those obtained from other vaccinated individuals who were infected with ancestral SARS-CoV-2. Development of B cell repertoire capable of producing antibodies with distinct affinity and specificities for the antigen immediately after infection capable of eliciting broadly neutralizing antibodies offers highest probability in protecting against evolving SARS-CoV-2 variants. ImportanceDevelopment of robust neutralizing antibodies in SARS-CoV-2 convalescent individuals is known, however varies at population level. We isolated monoclonal antibodies from an individual infected with ancestral SARS-CoV-2 in early 2020 that not only varied in their B cell lineage origin but also varied in their capability and potency to neutralize all the known VOC and currently circulating Omicron variants. This indicated establishment of unique lineages that contributed in forming B cell repertoire in this particular individual immediately following infection giving rise to diverse antibody responses that could compensate each other in providing broadly neutralizing polyclonal antibody response. Individuals who were able to produce such potent polyclonal antibody responses after infection have a higher chance of being protected from evolving SARS-CoV-2 variants.

immunology↗

bioRxiv2021

A multi-state dynamical process underpins mechano-adaptation in the bacterial flagellar motor

Adaptation is a defining feature of living systems. The bacterial flagellar motor adapts to changes in the external mechanical environment by adding or removing torque-generating stator units. However, the molecular mechanism for mechanosensitive motor remodeling remains unclear. Here, we induced stator disassembly using electrorotation, followed by the time-dependent assembly of the individual stator units into the motor after electrorotation was terminated. From these experiments, we extracted detailed statistics of the dwell times that comprise the stochastic dynamics of the binding and unbinding of stator units. Dwell times reveal multiple timescales, indicating the existence of multiple binding states of the stator units. Based on these results, we propose a minimal model in which the stator unit can occupy four different states - two bound states with very different rates of unbinding, a diffusive unbound state, and a transiently detached state. Our minimal model quantitatively explains multiple features of the experimental data and allows us to determine the transition rates among all four states. Our experiments and modeling suggest a mechanism of mechano-adaptive remodeling of the bacterial flagellar motor in which torque generated by bound stator units controls their effective unbinding rate by modulating the transition between the two bound states. Furthermore, the binding rate of stator units with the motor has a non-monotonic dependence on the number of bound units, likely because of two counteracting effects of motor rotation on the binding process.

biophysics↗

bioRxiv2021

Mechanosensitive remodeling of the bacterial flagellar motor is independent of direction of rotation

Motility is critical for the survival and dispersal of bacteria, and it plays an important role during infection. How bacteria regulate motility is thus a question of broad interest. Regulation of bacterial motility by chemical stimuli is well studied, but recent work has added a new dimension to the problem of motility control. The bidirectional flagellar motor of the bacterium Escherichia coli recruits or releases torque-generating units (stator units) in response to changes in load. Here, we show that this mechanosensitive remodeling of the flagellar motor is independent of direction of rotation. Remodeling rate constants in clockwise rotating motors and in counterclockwise rotating motors, measured previously, fall on the same curve if plotted against torque. Increased torque decreases the off rate of stator units from the motor, thereby increasing the number of active stator units at steady state. A simple mathematical model based on observed dynamics provides quantitative insight into the underlying molecular interactions. The torque-dependent remodeling mechanism represents a robust strategy to quickly regulate output (torque) in response to changes in demand (load). SignificanceMacromolecular machines carry out most of the biological functions in living organisms. Despite their significance, we do not yet understand the rules that govern the self-assembly of large multi-protein complexes. The bacterial flagellar motor tunes the assembly of its torque-generating stator complex with changes in external load. Here, we report that clockwise and counterclockwise rotating motors have identical remodeling response to changes in the external load, suggesting a purely mechanical mechanism for this regulation. Autonomous control of self-assembly may be a general strategy for tuning the functional output of protein complexes. The flagellar motor is a prime example of a macromolecular machine in which the functional regulation of assembly can be rigorously studied.

biophysics↗

bioRxiv2020

Structure and function of stator units of the bacterial flagellar motor

Many bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how these changes power rotation of the flagellum, remain unknown. Here we present ~3 [A]-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational direction. One Sentence SummaryStructural basis of torque generation in the bidirectional bacterial flagellar motor

biophysics↗

bioRxiv2018

Cell-intrinsic genetic regulation of peripheral memory-phenotype T cell frequencies

Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while recently activated -B and -CD4 T cell frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets, suggesting genetic regulation of memory lymphocyte frequencies. To explore this possibility further, we screened effector memory (EM)-phenotype T cell frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4EM and/or CD8EM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.

immunology↗