Biology subjects

Sukumar, S.

Publications and source records attributed to Sukumar, S..

10 recordsLinked to original sources

bioRxiv2026

Extracellular vesicle-bound bacterial toxin pneumolysin triggers membrane engagement and damage beyond canonical pore formation

Pneumolysin (PLY) is a cholesterol-dependent pore-forming toxin and a key virulence factor of Streptococcus pneumoniae, the leading cause of pneumonia worldwide in children aged below 5 years. At sublytic toxin doses, host cells shed PLY-laden extracellular vesicles (EVs) during membrane repair response. However, it remains unclear how these toxin-bearing EVs engage and damage target cell membranes. Here, we combine molecular dynamics simulations, liposome fusion assays, and cell-based experiments to elucidate the membrane interaction potential of vesicle-bound PLY. Simulations indicate that EV-embedded PLY uses an exposed helix to bind target cell membranes, inducing pronounced curvature, bilayer thinning, and water influx. Liposome fusion assays demonstrate that both PLY and membrane cholesterol promote vesicle-membrane interactions. Characterization of vesicle subpopulations released from PLY-challenged monocytes by western blotting and immunogold electron microscopy demonstrated that plasma membrane-derived microvesicles are preferentially enriched in membrane-bound PLY compared to small extracellular vesicles. Consistent with these findings, MVs purified from wild-type PLY-challenged monocytes, but not the toxoid mutant PLYW433F fuse with human immune cells, delivering toxin and causing membrane damage. Together our results reveal a noncanonical, pore-independent mode of toxin dissemination, in which, vesicle-bound pneumolysin fuses and destabilize target cell membranes, representing a new proposal for EV-mediated toxin activity and a potential target for therapeutic intervention.

microbiology↗

bioRxiv2024

Reward invigorates isometric gripping actions

Individuals exhibit a propensity to move faster toward more rewarding stimuli. While this phenomenon has been observed in movements, the effect of reward on implicit control of isometric actions, like gripping or grasping, is relatively unknown. How reward-related invigoration generalizes to other effortful actions is an important question. Reward invigorates reaching movements and saccades, supporting the idea that reward pays the additional effort cost of moving faster. Effort in isometric force generation is less understood, so here we ask whether and how reward-related invigoration generalizes to isometric force gripping. And if so, what implicit characteristics of gripping change when there is a prospect of reward? Participants (N=19) gripped a force transducer and the force applied was mapped to radial position of an onscreen cursor. Each trial, a target appeared in one of four locations; increasing grip force moved the cursor toward the target. The gripping action was interchangeable for all target positions. In each block of 100 trials, one target was consistently rewarded, while the other targets were not. When gripping to acquire the rewarded target, participants reacted faster, generated force more rapidly and to a greater extent, while intriguingly maintaining the same accuracy and integral of force over time. These findings support the generalization of reward-related invigoration in isometric force tasks, and that the brain exquisitely trades-off reward and effort costs to obtain reward more rapidly without compromising accuracy or more effort costs than necessary. NEW & NOTEWORTHYGripping actions are important for day-to-day tasks, for medical diagnostics like strength and force control, and for choice selection in decision-making experiments. Comparing isometric gripping responses to reward and nonreward cues, we observed reward-based invigoration mediated by selective increases in effort. These findings can be leveraged to provide additional insight into the decision making process, and better understand the effect of reward on movement vigor and the implicit control of accuracy.

neuroscience↗

bioRxiv2024

The cold-inducible RNA-binding protein RBM3 stabilises viral mRNA at the cooler temperatures of the upper respiratory tract.

Temperature is a critical determinant of host-pathogen interactions in the respiratory tract, where influenza A virus (IAV) has adapted to the cooler environment of the upper respiratory tract (URT) to enable efficient replication and transmission. The cold-inducible RNA-binding protein RBM3 is highly expressed in nasopharyngeal tissue and is known to stabilise mRNAs under hypothermic conditions; however, its role during viral infection has not been defined. Here, we identify RBM3 as a key host factor that facilitates IAV replication at sub-physiological temperatures. siRNA-mediated knockdown of RBM3 significantly impaired viral replication, while its constitutive overexpression at 37{degrees}C restored replication to levels typically observed at 33{degrees}C. Mechanistically, RBM3 binds directly to viral nucleoprotein (NP) mRNA, prolonging mRNA half-life and inevitably leading to increased viral production. This effect was abolished in an RNA-binding-deficient RBM3 mutant, confirming the requirement for direct RNA interaction, visualised by smiFISH-PLA. Crucially, this positive regulation of NP mRNA and interaction was validated in well-differentiated primary nasal epithelial cells, highlighting the physiological relevance of RBM3 in the human URT. These results reveal a temperature-sensitive host-virus interaction that promotes IAV replication in the cooler URT, a key site for viral shedding and transmission. By linking environmental temperature, host RNA-binding proteins, and viral mRNA stability, this study uncovers a novel mechanism of respiratory virus adaptation and identifies RBM3 as a potential therapeutic target for limiting early-stage viral replication and transmission. Author SummaryTo establish productive infection and ensure transmission, respiratory viruses such as influenza A virus (IAV) must adapt to the diverse environments of the human respiratory tract. One key challenge is the temperature gradient that exists between the upper and lower respiratory tract. The upper respiratory tract (URT), the main site of viral entry and transmission, maintains a lower physiological temperature ([~]33{degrees}C) than the lower respiratory tract ([~]37{degrees}C, LRT), creating a distinct cellular environment. In this study, we investigated how this cooler URT temperature alters the landscape of RNA-binding proteins (RBPs), which play a central role in regulating gene expression after transcription. Using mass spectrometry-based profiling and molecular virology approaches, we identified a cold-inducible RBP, RBM3, as significantly enriched at 33{degrees}C and acting as a key proviral host factor during IAV infection. We show that RBM3 binds directly to viral nucleoprotein (NP) mRNA, mainly in the cytoplasm, in both immortalised cell models and primary nasal epithelial cells. This interaction promotes the stability of NP mRNA thereby enhancing the overall production of infectious virions. Importantly, disrupting RBM3s RNA-binding ability abolished this proviral effect. These findings reveal that RBM3, elevated in the cooler URT environment, directly supports IAV replication. This work highlights how subtle physiological differences in host tissue can reshape post-transcriptional regulation and influence the outcome of respiratory virus infection.

microbiology↗

bioRxiv2024

Myoferlin is an essential component of late-stage vRNP trafficking vesicles for enveloped RNA viruses.

The Rab11a endosomal recycling pathway is exploited by important respiratory RNA viruses such as IAV and RSV, aiding viral egress from the apical surface of polarized epithelial cells. Late in infection, Rab11a-containing vesicles specifically transport viral ribonucleoprotein (vRNP) complexes towards the cell surface before packaging and budding. Rather than employing traditional Rab11a-positive recycling endosomes, virus-infected cells generate remodelled Rab11a-containing vesicles, as observed during IAV infection. Besides Rab11a, no other conserved host co-factors have been identified among these various vRNP trafficking vesicles. Here we discovered and confirmed myoferlins association with IAV vRNPs in the cytoplasm and colocalisation with Rab11a during late stages of infection. We also found that this role was conserved in late-stage vRNP trafficking of other viruses, including RSV and SeV. Myoferlin likely recruits the EHD family of proteins, which are involved in endosomal biogenesis, to these unique vRNP trafficking endosomes, highlighting myoferlins pivotal role in viral replication.

microbiology↗

bioRxiv2023

Identifying cellular RNA-binding proteins during infection uncovers a role for MKRN2 in influenza mRNA trafficking

Utilisation of RNA-binding proteins (RBPs) is an important aspect of post-transcriptional regulation of viral RNA. Viruses such as influenza A viruses (IAV) interact with RBPs to regulate processes including splicing, nuclear export and trafficking, while also encoding RBPs within their genomes, such as NP and NS1. But with almost 1000 RBPs encoded within the human genome it is still unclear what role, if any, many of these proteins play during viral replication. Using the RNA interactome capture (RIC) technique, we isolated RBPs from IAV infected cells to unravel the RBPome of mRNAs from IAV infected human cells. This led to the identification of one particular RBP, MKRN2, that associates with and positively regulates IAV mRNA. Through further validation, we determined that MKRN2 is involved in the nuclear-cytoplasmic trafficking of IAV mRNA likely through an association with the RNA export mediator GLE1. In the absence of MKRN2, IAV mRNAs accumulate in the nucleus of infected cells, which we suspect leads to their degradation by the nuclear RNA exosome complex. MKRN2, therefore, appears to be required for the efficient nuclear export of IAV mRNAs in human cells.

microbiology↗

bioRxiv2023

Disentangling the effects of metabolic cost and accuracy on movement vigor

AO_SCPLOWBSTRACTC_SCPLOWOn any given day, we make countless reaching movements to objects around us. While such ubiquity may suggest uniformity, each movement is unique in the speed with which it is made. Some movements are slow, while others are fast. These variations in reach speed have long been known to be influenced by accuracy constraints; we slow down when accuracy demands are high. However, in other forms of movement like walking, metabolic cost is the primary determinant of movement speed. Here we ask, how do metabolic cost and accuracy interact to determine speed of reaching movements? First we systematically measure the effect of increasing mass on the metabolic cost of reaching across a range of movement speeds. Next, in a sequence of three experiments, we examine how added mass affects preferred movement speeds in a simple reaching task with increasing accuracy requirements. We find that mass consistently increased metabolic cost and led to slower movements. Yet, intriguingly, preferred reach speeds were slower than metabolically optimal. We then demonstrate how a single model that, critically, considers both accuracy and metabolic cost can explain preferred movement speeds across the range of accuracy and effort requirements tested. Together, these findings provide a unifying framework to explain the combined effects of metabolic cost and accuracy on movement speed, and also highlight the integral role metabolic cost plays in determining reach speed.

neuroscience↗

bioRxiv2021

Conserved induction of distinct antiviral signalling kinetics by primate interferon lambda 4 proteins

Interferon lambdas (IFN{lambda}) (also known as type III IFNs) are critical cytokines that combat infection predominantly at barrier tissues, such as the lung, liver and gastrointestinal tract. Humans have four IFN{lambda}s (1-4) where IFN{lambda}1-3 show [~]80-95% homology and IFN{lambda}4 is the most divergent displaying only [~]30% sequence identity. Variants in IFN{lambda}4 in humans are associated with the outcome of infection, such as with hepatitis C virus. However, how IFN{lambda}4 variants impact cytokine signalling in other tissues and how well this is conserved is largely unknown. In this study we address whether differences in antiviral signalling exist between IFN{lambda}4 variants in human hepatocyte and intestinal cells, comparing them to IFN{lambda}3. We demonstrate that compared to IFN{lambda}3, wild-type human IFN{lambda}4 induces a signalling response with distinct magnitudes and kinetics, which is modified by naturally-occurring variants P70S and K154E in both cell types. IFN{lambda}4s distinct antiviral response was more rapid yet transient compared to IFN{lambda}1 and 3. Additionally, divergent antiviral kinetics were also observed using non-human primate IFN{lambda}s and cell lines. Furthermore, an IFN{lambda}4-like receptor-interacting interface failed to alter IFN{lambda}1s kinetics. Together our data provide further evidence that major functional differences exist within the IFN{lambda} gene family. These results highlight the possible tissue specialisation of IFN{lambda}s and encourage further investigation of the divergent, non-redundant activities of IFN{lambda}4 and other IFN{lambda}s. Contribution to the FieldViral infections remain major causes of death and disease in humans and other animals. Interferons (IFNs) are a diverse group of host signalling proteins that can induce a potent antiviral state in cells and are intimately involved in the outcome of infection. Genetic variants within one IFN (interferon lambda 4, IFN{lambda}4) are associated with the outcome of hepatitis C infection in humans. However, how IFN{lambda}4 functions - and how natural variants affect its activity - remains poorly understood. Comparing how the antiviral activity changes over time following stimulation with different IFN{lambda}s, we identified that IFN{lambda}4 induces a more rapid antiviral state compared to other IFN{lambda}s in liver and intestinal cells. Importantly, this response was conserved within human variants and between humans and non-human primates (chimpanzee and Rhesus macaque). Our results shed light on the unique functions of the divergent IFN{lambda}4 protein.

immunology↗

bioRxiv2021

Effects of reward history on decision-making and movement vigor

During foraging, animals explore a site and harvest reward, and then abandon that site and travel to the next opportunity. One aspect of this behavior involves decision-making, while the other involves movement control. We recently proposed that control of decision-making and movements may be linked via a desire to maximize a single normative utility: the sum of all rewards acquired, minus all efforts expended, divided by time. If this is the case, then the history of rewards, and not just its immediate availability, should dictate how long one decides to stay and harvest reward, and how fast one travels to the next opportunity. We tested this theory in a series of experiments in which humans used their hand to harvest tokens at a reward patch, and then used their arm to reach toward a subsequent opportunity. Experiencing a history of poor rewards not only led people to forage longer, but they also reached slower toward the next reward site. Thus, reward history had a consistent effect on both the decision-making process regarding when to abandon a reward site, and the motor control process regarding how fast to move to the next opportunity.

neuroscience↗

bioRxiv2018

Validation of a low-cost, carbon dioxide-based cryoablation system for percutaneous tumor ablation

Breast cancer rates are rising in low- and middle-income countries (LMICs), yet there is a lack of accessible and cost-effective treatment. As a result, the cancer burden and death rates are highest in LMICs. In an effort to meet this need, our work presents the design and feasibility of a low-cost cryoablation system using widely-available carbon dioxide as the only consumable. This system uses an 8-gauge outer-diameter needle and Joule-Thomson expansion to percutaneously necrose tissue with cryoablation. Bench top experiments characterized temperature dynamics in ultrasound gel demonstrated that isotherms greater than 2 cm were formed. Further, this system was applied to mammary tumors in an in vivo rat model and necrosis was verified by histopathology. Finally, freezing capacity under a large heat load was assessed with an in vivo porcine study, where volumes of necrosis greater than 1.5 cm in diameter confirmed by histopathology were induced in a highly perfused liver after two 7-minute freeze cycles. These results demonstrate the feasibility of a carbon-dioxide based cryoablation system for improving solid tumor treatment options in resource-constrained environments.

bioengineering↗

bioRxiv2017

A Polymorphic Residue That Attenuates Interferon Lambda 4 Activity in Hominid Lineages

As antimicrobial signalling molecules, type III or lambda interferons (IFN{lambda}s) are critical for defence against infection by diverse pathogens. Counter-intuitively, expression of one member of the family, IFN{lambda}4, is associated with decreased clearance of hepatitis C virus (HCV) in the human population; by contrast, a natural in-frame nucleotide insertion that abrogates IFN{lambda}4 production improves viral clearance. To further understand how genetic variation between and within species affects IFN{lambda}4 function, we screened a panel of extant coding variants of human IFN{lambda}4 and identified three variants that substantially affect antiviral activity (P70S, L79F and K154E). The most notable variant was K154E, which enhanced in vitro activity in a range of antiviral and interferon stimulated gene (ISG) assays. This more active E154 variant of IFN{lambda}4 was found only in African Congo rainforest Pygmy hunter-gatherers. Remarkably, E154 was highly conserved as the ancestral residue in mammalian IFN{lambda}4s yet K154 is the dominant variant throughout evolution of the hominid genus Homo. Compared to chimpanzee IFN{lambda}4, the human orthologue had reduced activity due to amino acid substitution of glutamic acid with lysine at position 154. Meta-analysis of published gene expression data from humans and chimpanzees showed that this difference in activity between K154 and E154 in IFN{lambda}4 is consistent with differences in antiviral gene expression in vivo during HCV infection. Mechanistically, our data suggest that human-specific K154 likely affects IFN{lambda}4 activity by reducing secretion and potency. We postulate that evolution of an IFN{lambda}4 with attenuated activity in humans (K154) likely contributes to distinct host-specific responses to and outcomes of infection, such as HCV.

immunology↗