Biology subjects

Kaushansky, A.

Publications and source records attributed to Kaushansky, A..

18 recordsLinked to original sources

bioRxiv2026

Plasmodium blood-stage induces trained immunity in hepatocytes

Plasmodium parasites, the causative agents of malaria, are transmitted at high levels in endemic areas and sequential infections are common. Using a mouse model of infection we discovered liver burden was suppressed in blood-stage experienced, compared to naive, animals, independently of adaptive responses and inflammation. We observed greater chromatin accessibility of a subset of interferon stimulated genes in blood-stage experienced animals, and a rapid increase in transcription of these genes upon sporozoite challenge. Ex vivo stimulation of hepatocytes from blood-stage experienced mice also led to a rapid and elevated response upon treatment with an unrelated antigen, ctDNA. Taken together, these data are consistent with a model in which hepatocytes are reprogramed by blood stage Plasmodium infection to exhibit trained memory akin to what has been described in innate immune cells. The consequences of training of hepatocytes could be wide-reaching and might alter responses to diverse pathogens and other stimuli.

immunology↗

bioRxiv2026

Modulation of Ferroptosis During Early Mycobacterium tuberculosis Infection Contributes to Beijing Lineage Strain SA161 Virulence

Although spread of internalized Mtb from the initial infected alveolar macrophages (AMs) is a crucial determinant of infection outcomes, the role of cell death in facilitating this spread, and how it is regulated by Mtb remains poorly understood. Ferroptosis, a lipid peroxidation-mediated form of necrotic cell death, contributes to pathology during later stages of infection. However, the role of ferroptosis in early AM cell death remains inadequately defined, and its induction during infection has been primarily studied with the laboratory strain, H37Rv. Using gene set variation analysis of single cell RNAseq data profiling Mtb-infected murine lungs, we found that the hypervirulent Beijing sublineage clinical strain SA161 is associated with an elevated pro-ferroptotic transcriptional response in AMs compared to H37Rv by 17 days post infection. Consistent with these transcriptional profiles, we found that SA161 induced increased lipid peroxidation in comparison to H37Rv during infection in vitro and in vivo. Administration of the lipid peroxidation inhibitor, ferrostatin-1 (Fer-1), reduces this Mtb-induced lipid peroxidation. Notably, we found that administration of Fer-1 to Mtb-infected mice significantly reduced bacterial burden for SA161 at 14 dpi while having no effect on H37Rv. Microscopic analysis of SA161-infected lung lesions at 14 dpi suggests that inhibition of ferroptosis-driving lipid peroxidation results in a greater proportion of AMs amongst infected cells and decreased neutrophil-associated IFN signaling. Collectively, these findings reveal that ferroptosis plays an important role during early infection with virulent clinical strains of Mtb by influencing bacterial spread and signaling of immune cell responders, potentially informing host-directed intervention strategies.

immunology↗

bioRxiv2025

A Machine Learning- 3D Microvessel Platform Identifies Kinase Targets Restoring Blood-Brain-Barrier Endothelial Integrity.

Disruption of the brain endothelial barrier is a hallmark of traumatic brain injury (TBI), and contributes to cerebral edema, coagulopathy, and delayed neurological deficits, yet there are no mechanism-guided therapies that directly stabilize human brain vessels. Here we used a three-dimensional (3D) human brain endothelial vessel platform with quantifiable macromolecular barrier function to assess responses to thrombin and plasma from patients with TBI. This endothelial-focused 3D system isolates the central cellular regulator of blood-brain barrier function and enables mechanistic analysis of endothelial signaling and drug responses as a foundation for personalized vascular therapeutics. By integrating this platform with machine learning-guided kinase analysis, we identified kinase pathways associated with barrier stabilization versus disruption and nominated inhibitors that reversed thrombin- and TBI plasma-induced leakage as well as compounds that exacerbated endothelial loss. This work establishes an important first step toward precision vascular therapeutics in TBI, by linking patient-derived plasma phenotypes to specific endothelial barrier responses and pharmacologically tractable kinase signaling, and it provides a generalizable framework for mechanistic drug discovery across neurovascular disorders. SIGNIFICANCE STATEMENTBlood-brain barrier (BBB) disruption drives cerebral edema, coagulopathy, ischemia, and neurological deficits after TBI. Targeting kinases as therapeutics is promising but challenged by complex inhibitor profiles and patient variability. We present a machine learning-guided platform built on 3D perfusable human brain endothelial-centric microvessels to systematically screen and select kinase inhibitors that reverse TBI-induced endothelial barrier breakdown. By pinpointing key kinase and gene regulators and demonstrating restoration of endothelial barrier integrity in patient-derived plasma models, our approach provides a transformative strategy for stabilizing the endothelial barrier after TBI and offers a versatile framework for therapeutic discovery across neurovascular disorders.

bioengineering↗

bioRxiv2025

Unlocking new understanding of Plasmodium sporozoite biology with expansion microscopy

Transmission of malaria relies on the formation of sporozoites in the mosquito midgut and their subsequent invasion of the salivary gland. Despite their importance, our understanding of the cell biology of sporozoite formation and salivary gland invasion is limited. Here, we apply a technique called Mosquito Tissue Ultrastructure Expansion Microscopy (MoTissU-ExM), which physically expands infected mosquito tissues while preserving both host and parasite ultrastructure. Using MoTissU-ExM we are able to observe a range of parasite structures and organelles including features previously seen only by electron microscopy as well as structure not observed before. We leverage MoTissU-ExM to investigate a number of cell biology events during sporozoite formation and salivary gland invasion. In particular we focus on the rhoptries, a secretory organelle important for host cell invasion. We establish a timeline for sporozoite rhoptry biogenesis, show that two rhoptries are used up during salivary gland invasion, and provide the first evidence that rhoptry pairs are specialized for different invasion events. Building on this new knowledge, we characterize the rhoptry protein RON11 and identify it as the first protein involved in sporozoite rhoptry biogenesis. Disruption of RON11 led to the production of sporozoites that specifically fail to invade the salivary gland epithelial cell, thereby blocking transmission of these parasites.

microbiology↗

bioRxiv2024

Systems-level reconstruction of kinase phosphosignaling networks regulating endothelial barrier integrity using temporal data

Phosphosignaling networks control cellular processes. We built kinase-mediated regulatory networks elicited by thrombin stimulation of brain endothelial cells using two computational strategies: Temporal Pathway Synthesizer (TPS), which uses phosphoproteomics data as input, and Temporally REsolved KInase Network Generation (TREKING), which uses kinase inhibitor screens. TPS and TREKING predicted overlapping barrier-regulatory kinases connected with unique network topology. Each strategy effectively describes regulatory signaling networks and is broadly applicable across biological systems.

systems biology↗

bioRxiv2024

Using machine learning to dissect host kinases required for Leishmania internalization and development

The Leishmania life cycle alternates between promastigotes, found in the sandfly, and amastigotes, found in mammals. When an infected sandfly bites a host, promastigotes are engulfed by phagocytes (i.e., neutrophils, dendritic cells, and macrophages) to establish infection. When these phagocytes die or break down, amastigotes must be re-internalized to survive within the acidic phagolysosome and establish disease. To define host kinase regulators of Leishmania promastigote and amastigote uptake and survival within macrophages, we performed an image-based kinase regression screen using a panel of 38 kinase inhibitors with unique yet overlapping kinase targets. We also targeted inert beads to complement receptor 3 (CR3) or Fc{gamma} receptors (FcR) as controls by coating them with complement/C3bi or IgG respectively. Through this approach, we identified several putative host kinases that regulate receptor-mediated phagocytosis and/or the uptake of L. amazonensis. Findings included kinases previously implicated in Leishmania uptake (such as Src family kinases (SFK), Abl family kinases (ABL1/c-Abl, ABL2/Arg), and spleen tyrosine kinase (SYK)), but we also uncovered many novel kinases. Our methods also predicted host kinases necessary for promastigotes to convert to amastigotes or for amastigotes to survive within macrophages. Overall, our results suggest that the concerted action of multiple interconnected networks of host kinases are needed over the course of Leishmania infection, and that the kinases required for the parasites life cycle may differ substantially depending on which receptors are bound and the life cycle stage that is internalized. In addition, using our screen, we identified kinases that appear to preferentially regulate the uptake of parasites over beads, indicating that the methods required for Leishmania to be internalized by macrophages may differ significantly from generalized phagocytic mechanisms. Our findings are intended to be used as a hypothesis generation resource for the broader scientific community studying the roles of kinases in host-pathogen interactions.

microbiology↗

bioRxiv2023

Host kinase regulation of Plasmodium vivax dormant and replicating liver stages

Upon transmission to the liver, Plasmodium vivax parasites form replicating schizonts, which continue to initiate blood-stage infection, or dormant hypnozoites that reactivate weeks to months after initial infection. P. vivax phenotypes in the field vary significantly, including the ratio of schizonts to hypnozoites formed and the frequency and timing of relapse. Evidence suggests that both parasite genetics and environmental factors underly this heterogeneity. We previously demonstrated that data on the effect of a panel of kinase inhibitors with overlapping targets on Plasmodium liver stage infection, in combination with a computational approach called kinase regression (KiR), can be used to uncover novel host regulators of infection. Here, we applied KiR to evaluate the extent to which P. vivax liver-stage parasites are susceptible to changes in host kinase activity. We identified a role for a subset of host kinases in regulating schizont and hypnozoite infection and schizont size and characterized overlap as well as variability in host phosphosignaling dependencies between parasite forms and across multiple patient isolates. Striking, our data point to variability in host dependencies across P. vivax isolates, suggesting one possible origin of the heterogeneity observed across P. vivax in the field.

microbiology↗

bioRxiv2023

Multiple Receptor Tyrosine Kinases Regulate Dengue Infection of Hepatocytes

Dengue is an arboviral disease causing severe illness in over 500,000 people each year. Currently, there is no way to constrain dengue in the clinic. Host kinase regulators of dengue virus (DENV) infection have the potential to be disrupted by existing therapeutics to prevent infection and/or disease progression. To evaluate kinase regulation of DENV infection, we performed kinase regression (KiR), a machine learning approach that predicts kinase regulators of infection using existing drug-target information and a small drug screen. We infected hepatocytes with DENV in vitro in the presence of a panel of 38 kinase inhibitors then quantified the effect of each inhibitor on infection rate. We employed elastic net regularization on these data to obtain predictions of which of 300 kinases are regulating DENV infection. Thirty-six kinases were predicted to have a functional role. Intriguingly, seven of the predicted kinases - EPH receptor A4 (EPHA4), EPH receptor B3 (EPHB3), EPH receptor B4 (EPHB4), erb-b2 receptor tyrosine kinase 2 (ERBB2), fibroblast growth factor receptor 2 (FGFR2), Insulin like growth factor 1 receptor (IGF1R), and ret proto-oncogene (RET) - belong to the receptor tyrosine kinase (RTK) family, which are already therapeutic targets in the clinic. We demonstrate that predicted RTKs are expressed at higher levels in DENV infected cells. Knockdown of ERBB2, FGFR2 and IGF1R reduces DENV infection in hepatocytes. Finally, we observe differential temporal induction of ERBB2 and IGF1R following DENV infection, highlighting their unique roles in regulating DENV. Collectively, our findings underscore the significance of multiple RTKs in DENV infection and advocate further exploration of RTK-oriented interventions against dengue.

systems biology↗

bioRxiv2023

Identification of potent and selective N-myristoyltransferase inhibitors of Plasmodium vivax liver stage hypnozoites and schizonts

New drugs targeting multiple stages of the malaria-causing parasite, Plasmodium, are needed to reduce and eliminate malaria worldwide. N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme, and a validated chemically tractable drug target for malaria. Previous efforts have failed to target NMT owing to the low selectivity for the Plasmodium enzyme compared with human NMTs. Herein, we applied a structure-guided approach using previously reported NMT inhibitors as scaffolds to develop a new generation of Plasmodium vivax NMT (PvNMT) targeting compounds. We report a series of compounds with IC50 values in the nM range and an order of magnitude improved selectivity to Plasmodium NMT over human NMT (HsNMT). X-ray co-crystallization of PvNMT with a representative lead compound, 12b, supported the prevailing hypothesis that a conformational difference in a key tyrosine residue of PvNMT and HsNMT drives the selectivity between these enzymes. The compounds were triaged based on their selectivity for PvNMT. They significantly decreased P. falciparum blood-stage parasite load, with IC50 values ranging from 0.36 M to 1.25 M. The compounds exhibited a dose-dependent inhibition of P. vivax liver stage schizont and hypnozoite infection, consistently, in three different P. vivax isolates with IC50 values ranging from 2.2 M to 6 M and from 1.2 M to 12 M. Our data provide evidence that NMT inhibitors could be multistage antimalarials, targeting both dormant and developing liver stage parasites, which is essential for malaria elimination. One Sentence SummaryPotent and selective N-myristoyltransferase inhibitors of Plasmodium vivax hypnozoites and schizonts were synthesized and tested.

pharmacology and toxicology↗

bioRxiv2022

Temporally resolved kinase regulatory networks control endothelial barrier integrity

Vascular leak is a common disease complication, yet the signaling networks regulating barrier integrity are incompletely understood. We developed a novel methodology, Temporally REsolved KInase Network Generation (TREKING), which combines a 28-kinase inhibitor screen with machine learning and network reconstruction to build time-resolved, functional phosphosignaling networks. We demonstrated the utility of TREKING for identifying pathways mediating barrier integrity following thrombin stimulation with or without tumor necrosis factor (TNF) activation in brain endothelial cells. TREKING assigned distinct barrier phenotypes to mitogen-activated protein kinase (MAPK) pathways and revealed a condition-specific MAPKAPK2/MK2 switch kinase pathway with early barrier-disruptive activity in both inflammatory conditions, but late barrier-restorative activity exclusively in the absence of TNF pre-conditioning. MAPKAPK2/MK2 was activated with expected distinct kinetics under the two inflammatory conditions and late activation was linked to a MAP3K20/ZAK-MAPK14/p38-MAPKAPK2/MK2 pathway. Beyond MAPKs, TREKING predicts extensive interconnected networks that control barrier integrity and is a tool for dissecting complex temporal phosphosignaling networks across biological systems.

systems biology↗

bioRxiv2021

Elucidating spatially-resolved changes in host signaling during Plasmodium liver-stage infection

Upon transmission to the human host, Plasmodium sporozoites exit the skin, are taken up by the blood stream, and then travel to the liver where they infect and significantly modify a single hepatocyte. Low infection rates within the liver have made proteomic studies of infected hepatocytes challenging, particularly in vivo, and existing studies have been largely unable to consider how protein and phosphoprotein differences are altered at different spatial locations within the heterogeneous liver. Using digital spatial profiling, we characterized changes in host signaling during Plasmodium yoelii infection in vivo without disrupting the liver tissue, and measured variation between infected cells. Moreover, we measured alterations in protein expression around infected hepatocytes and identified a subset of CD163+ Kupffer cells that migrate towards infected cells during infection. These data offer the first insight into the heterogeneity of the infected hepatocyte in situ and provide insights into how the parasite may alter the local microenvironment to influence its survival and modulate immunity.

microbiology↗

bioRxiv2021

Germinal center activity and B cell maturation promote protective antibody responses against Plasmodium pre-erythrocytic infection

Blocking Plasmodium, the causative agent of malaria, at the asymptomatic pre-erythrocytic stage would abrogate disease pathology and prevent transmission. Rodent-infectious species of Plasmodium such as P. yoelii (Py) serve as key tools to study vaccine efficacy and disease biology in immune-competent experimental animals. Here we evaluated the differences in vaccine-elicited humoral immunity in two widely used, and vastly diverged, inbred mouse strains, BALB/cJ and C57BL/6J, and identified immunological factors associated with protection. We vaccinated with Py circumsporozoite protein (CSP), the major surface antigen on the sporozoite, and evaluated protective efficacy after mosquito bite challenge. Vaccination achieved 60% sterile protection and otherwise delayed blood stage patency in BALB/cJ mice, whereas; all C57BL/6J mice were infected similar to controls. Interestingly, protection was mediated by antibodies, and could be passively transferred from immunized BALB/cJ mice into naive C57BL/6J. Dissection of the underlying immunological features of protection revealed early deficits in antibody titers and polyclonal avidity in C57BL/6J mice. Additionally, PyCSP-vaccination in BALB/cJ induced a significantly higher proportion of antigen-specific B-cells and class-switched memory B-cell (MBCs) populations than in C57BL/6J mice. Strikingly, C57BL/6J mice also had markedly fewer germinal center experienced, CSP-specific class-switched MBCs compared to BALB/cJ mice. Analysis of the IgG {gamma} chain repertoires by next generation sequencing in PyCSP-specific memory B-cell repertoires also revealed higher somatic hypermutation rates in BALB/cJ mice than in C57BL/6J mice. These findings indicate that BALB/cJ mice achieved higher levels of B cell maturation in response to vaccination with PyCSP, which likely enabled the development of protective antibody responses. Overall, our study indicates that germinal center activity and B cell maturation are key processes in the development of vaccine-elicited protective antibodies against CSP.

immunology↗

bioRxiv2020

Dengue activates mTORC2 signaling to counteract apoptosis and maximize viral replication

The mechanistic target of rapamycin (mTOR) functions in at least two distinct complexes: mTORC1, which regulates cellular anabolic-catabolic homeostasis, and mTORC2, which is an important regulator of cell survival and cytoskeletal maintenance. mTORC1 has been implicated in the pathogenesis of flaviviruses including dengue, where it contributes to the establishment of a pro-viral autophagic state. In contrast, the role of mTORC2 in viral pathogenesis is unknown. In this study, we explore the consequences of a physical protein-protein interaction between dengue non-structural protein 5 (NS5) and host cell mTOR proteins during infection. Using shRNA to differentially target mTORC1 and mTORC2 complexes, we show that mTORC2 is required for optimal dengue replication. Furthermore, we show that mTORC2 is activated during viral replication, and that mTORC2 counteracts virus-induced apoptosis, promoting the survival of infected cells. This work reveals a novel mechanism by which the dengue flavivirus can promote cell survival to maximize viral replication.

microbiology↗

bioRxiv2020

Antibody interference by a non-neutralizing antibody abrogates humoral protection against Plasmodium liver stage

Both subunit and attenuated whole sporozoite vaccination strategies against Plasmodium infection have shown promising initial results in malaria-naive westerners but exhibited less efficacy in malaria-exposed individuals in endemic areas. It has been hypothesized that preexisting immunity to malaria represents a significant roadblock to the development of a protective vaccine. Here, we demonstrate proof-of-concept that non-neutralizing antibodies (nNAb) can directly interfere with protective anti-PyCSP humoral responses. We developed and characterized a novel monoclonal antibody, RAM1, against the P. yoelii sporozoite major surface antigen, circumsporozoite protein (CSP). Unlike the canonical PyCSP repeat domain binding and neutralizing antibody (NAb) 2F6, RAM1 does not inhibit sporozoite traversal or entry of hepatocytes in vitro. Though 2F6 and RAM1 bind non-overlapping regions of the CSP-repeat domain, pretreatment with RAM1 abrogated 2F6s capacity to block sporozoite traversal and invasion in vitro. Importantly, RAM1 reduced the efficacy of the polyclonal humoral response against CSP in vivo, paralleling the observed reduced efficacy of RTS,S in malaria-exposed populations. Taken together, our data demonstrate the interference of non-neutralizing antibodies with the efficacy of NAbs and may impact the efficacy of anti-CSP vaccines in malaria-exposed individuals.

immunology↗

bioRxiv2020

A Genome-wide CRISPR-Cas9 Screen Identifies Host Factors Essential for Optimal Plasmodium Liver Stage Development.

Prior to initiating symptomatic malaria, a single Plasmodium sporozoite infects a hepatocyte and develops into thousands of merozoites, in part by scavenging host resources. We show that host microtubules dynamically reorganize around the developing liver stage (LS) parasite. Using a genome-wide CRISPR-Cas9 screen, we identified host regulators of cytoskeleton organization, vesicle trafficking, ER/Golgi stress and lipid biogenesis that regulate Plasmodium LS development. These novel regulators of infection, including Centromere Protein J (CENPJ), led us to interrogate how microtubule organizing centers (MTOCs) are regulated during infection. Foci of {gamma}-tubulin localized to the parasite periphery; depletion of CENPJ exacerbated this re-localization and increased infection. Further, we show that the Golgi acts as a non-centrosomal MTOC by organizing {gamma}-tubulin and stimulating microtubule nucleation at the parasite periphery. Collectively, we show that the Plasmodium LS recruits the host Golgi to form MT mediated conduits along which host organelles are recruited to the PVM, to support liver stage development. Our findings suggest many host-targeted pharmacological inhibitors may inhibit LS infection.

cell biology↗

bioRxiv2020

A system-level gene regulatory network model for Plasmodium falciparum

Many of the gene regulatory processes of Plasmodium falciparum, the deadliest malaria parasite, remain poorly understood. To develop a comprehensive guide for exploring this organisms gene regulatory network, we generated a system-level model of Plasmodium falciparum gene regulation using a well-validated, machine-learning approach for predicting interactions between transcription regulators and their targets. The resulting network accurately predicts expression levels of transcriptionally coherent gene regulatory programs in independent transcriptomic data sets from parasites collected by different research groups in diverse laboratory and field settings. Thus, our results indicate that our gene regulatory model has predictive power and utility as a hypothesis-generating tool for illuminating clinically relevant gene regulatory mechanisms within Plasmodium falciparum. Using the set of regulatory programs we identified, we also investigated correlates of artemisinin resistance based on gene expression coherence. We report that resistance is associated with incoherent expression across many regulatory programs, including those controlling genes associated with erythrocyte-host engagement. These results suggest that parasite populations with reduced artemisinin sensitivity are more transcriptionally heterogenous. This pattern is consistent with a model where the parasite utilizes bet-hedging strategies to diversify the population, rendering a subpopulation more able to navigate drug treatment.

systems biology↗

bioRxiv2019

Plasmodium secretion induces hepatocyte lysosome exocytosis and promotes parasite entry

The invasion of a suitable host hepatocyte by Plasmodium sporozoites is an essential step in malaria infection. We demonstrate that in infected hepatocytes, lysosomes are redistributed away from the nucleus, and surface exposure of lysosomal-associated membrane protein (LAMP1) is increased. Lysosome exocytosis in infected cells occurs independently of sporozoite traversal. Instead, a sporozoite-secreted factor is sufficient for the process. Knockdown of the SNARE proteins involved in lysosome-plasma membrane fusion reduces lysosome exocytosis and Plasmodium infection. In contrast, promoting fusion between the lysosome and plasma membrane dramatically increases infection. Our work demonstrates new parallels between Plasmodium sporozoite entry of hepatocytes and infection by the excavate pathogen, Trypanosoma cruzi and raises the question of whether convergent evolution has shaped host cell invasion by divergent pathogens.

cell biology↗

bioRxiv2018

Ferroptosis-like signaling facilitates a potent innate defense against Plasmodium infection

The facets of host control during Plasmodium liver infection remain largely unknown and conventional innate regulatory pathways are only minimally effective at eliminating parasites1-3. Ferroptosis, a recently described form of iron-dependent cell death that drives accumulation of reactive oxygen species and lipid peroxides, but has not yet been shown to function as an innate immune response4,5. Inducing ferroptosis with pharmacologicals or by genetic perturbation of its negative regulators, GPX4 and SLC7a11, dramatically reduces survival of the Plasmodium Liver Stage. In contrast, knockdown or knockout of NOX1 or knockdown of TFR1, which are required for ferroptosis, increases the number of Liver Stage parasites. Moreover, we demonstrate that blocking ferroptosis renders parasite-infected hepatocytes resistant to P53-mediated hepatocyte death. Our work establishes that ferroptotic signaling serves to control Plasmodium infection in the liver and raises the possibility that ferroptosis operates as an axis of the innate immune system to defend against intracellular pathogens.

microbiology↗