Biology subjects

Aitchison, J. D.

Publications and source records attributed to Aitchison, J. D..

At least 19 recordsLinked to original sources

bioRxiv2026

Subunit vaccination enhances protection conferred by prior Mycobacterium tuberculosis exposure

Mycobacterium tuberculosis (Mtb) remains the leading cause of death from a single infectious agent. Although BCG protects against disseminated tuberculosis in infants, it has failed to curb transmission among adults. Animal models are used to prioritize vaccines entering clinical trials, but current models use Mtb-naive mice, limiting their relevance to humans in endemic settings, the primary targets of vaccine trials. Here we used a mouse model of Mtb exposure, CoMtb, where a cervical lymph node Mtb infection protects against aerosol challenge, consistent with historical observations in humans that prior Mtb exposure reduces disease risk. When we compared the impact of CoMtb on vaccine efficacy, BCG failed to provide added protection over CoMtb, while protein subunit vaccination further reduced lung burdens. This protection was associated with decreased KLRG1+ vascular CD4+ T cells and enhanced polyfunctional lung CD4+ T cells before and after aerosol challenge. Our findings were corroborated in an analysis of vaccinated humans stratified by QuantiFERON-TB status, which revealed a similar enrichment of polyfunctional CD4+ T cells in Mtb-exposed individuals. These data support the use of CoMtb to test vaccine efficacy in Mtb-exposed individuals and highlight the potential of subunit vaccines in endemic regions.

immunology↗

bioRxiv2026

A Chemical-Genetic Interaction Matrix Reveals Drug Mechanism and Genetic Architecture

To probe drug mechanism of action (MOA) and interrogate the genetic architecture of human cells, we carried out isogenic genome-wide CRISPR/Cas9 knockout screens against 310 diverse drugs, bioactive compounds, and stress conditions. Stringent statistical correction for gene knockout fitness defects yielded a large-scale matrix of >12,000 high confidence chemical-genetic interactions (CGIs). This dataset revealed many previously unappreciated off-target effects for well-characterized compounds and novel MOAs for uncharacterized compounds. The CGI matrix uncovered dense genetic modules that yielded new biological insights into phospholipidosis, mitotic regulation, metabolism, the DNA damage response, and mTOR signaling. The dataset allowed identification of multi-drug sensitization and resistance mechanisms, inference of gene function, elaboration of cross-process connectivity, evaluation of the cell type specificity of CGIs, prediction of chemical synergism, and extensive annotation of understudied genes. This resource provides a map of the genetic landscape in human cells and a framework to help guide drug discovery.

systems biology↗

bioRxiv2025

Host oxidative stress primes mycobacteria for rapid antibiotic resistance evolution

The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (Mtb) threatens global TB control, yet the mechanisms enabling rapid evolution of drug resistance in Mtb remain poorly understood. Here we reveal that pre-existing mutations in oxidative stress response genes create permissive genomic backgrounds that accelerate high-level isoniazid resistance (INHR) without fitness costs, challenging the paradigm that resistance mutations always precede their fitness compensatory adaptations. Using M. smegmatis mc2155 (Msm) as a model, we show that brief exposure to sublethal INH (2x IC50) enriches for "low-level resistance and tolerance" (LLRT) mutants in a single step. These LLRT mutants, particularly those with ohrR loss-of-function mutations, acquire high-level resistance (> 500x IC50) at 6-fold higher rates than wildtype, primarily through otherwise-deleterious mycothiol biosynthesis mutations that become tolerable in the oxidative stress-buffered background. Crucially, we demonstrate that sublethal oxidative stress alone, mimicking host immune pressure, nearly tripled the rate of INH resistance evolution in Msm. Bayesian analysis of 1,578 clinical Mtb isolates from Vietnam confirmed that mutations in oxidative stress response genes were significantly associated with the emergence of INHR strains (p-value = 1.09x10-7). Independently, reanalysis of genome-wide CRISPRi screens revealed that the OSR network and high Bayes probability genes are functionally associated with treatment escape and survival with multiple antibiotics, including isoniazid, rifampicin, ethambutol, bedaquiline, vancomycin, clarithromycin, linezolid, and streptomycin. Our findings that host-imposed oxidative stress and inadequate drug penetration may synergistically prime Mtb populations for rapid resistance evolution suggest that targeting pre-resistance mechanisms, such as oxidative stress defenses, could help slow the emergence of antibiotic resistance in tuberculosis.

microbiology↗

bioRxiv2025

Single-cell profiling reveals that dynamic lung immune responses distinguish protection from susceptibility to tuberculosis

The mechanisms that underlie protective immunity to Mycobacterium tuberculosis (Mtb) remain incompletely defined. To identify immune correlates associated with protection, we performed single-cell RNA sequencing of lung immune cells after aerosol Mtb infection of naive mice and mice with contained Mtb infection (CoMtb), a model of naturally acquired resistance, across multiple time points, mouse strains, and Mtb strains. Across conditions, protection was associated with distinct temporal patterns of immune activation, cell recruitment, and resolution. Early after challenge, CoMtb mice exhibited rapid, transient recruitment and activation of CD4 T cells, macrophages, NK, and NKT cells, accompanied by short-lived bursts of type I and II interferon signaling, increased oxidative phosphorylation, and enhanced chemokine-mediated cell-cell communication. In contrast, primary infection elicited delayed but sustained interferon and neutrophil responses and higher bacterial burdens. These data indicate that protection involves dynamically coordinated immune pathways rather than the magnitude of any single response. Transcriptional features of CoMtb overlapped with those observed in nonhuman primates following intravenous BCG vaccination, including enrichment of activated tissue-resident CD4 T cells and innate effector populations. Together, these findings support a model in which effective immunity to Mtb depends on the timing and coordination of immune activation, providing a framework for vaccine strategies that reproduce protective lung immune dynamics.

immunology↗

bioRxiv2025

A Conditional Cas9 System for Stage-Specific Gene Editing in P. falciparum

The malaria parasite has a complex lifecycle involving various host cell environments in both human and mosquito hosts. The parasite must tightly regulate gene expression at each stage in order to adapt to its current environment while continuing development. However, it is challenging to study gene function and regulation of essential genes across the parasites multi-host lifecycle. Thus, we adapted a recently developed a single-plasmid dimerizable Cre recombinase system for rapamycin-controllable expression of Cas9, allowing for conditional introduction of mutations. We explored rates of gene deletion using varying repair template lengths, showing functionality of donor templates under 250bp for homology-directed repair. As a proof of concept, we conditionally disrupted two uncharacterized genes in blood and gametocyte stages, identifying new stage-specific phenotypes. ImportanceAs progress towards eliminating malaria has stalled, there is a pressing need for new antimalarials and vaccines. Genes essential to multiple stages of development represent ideal candidates for both antimalarials and vaccines. However, much of the parasite genome remains uncharacterized. Conditional gene perturbation approaches are needed in order to study gene function across the lifecycle. Currently available tools are limited in their ability to perturb genes at the scale required for large screens. We describe a tool that allows for conditional introduction of desired mutations by controlling Cas9 with the DiCre-loxP system. We demonstrate the accessibility of this approach by designing gRNA-donor pairs that can be commercially synthesized. This toolkit provides a scalable system for identifying new drug and vaccine candidates targeting multiple stages of the parasite lifecycle

microbiology↗

bioRxiv2024

APOE Protects Against Severe Infection with Mycobacterium tuberculosis by Restraining Production of Neutrophil Extracellular Traps

While neutrophils are the predominant cell type in the lungs of humans with active tuberculosis (TB), they are relatively scarce in the lungs of most strains of mice that are used to study the disease. However, similar to humans, neutrophils account for approximately 45% of CD45+ cells in the lungs of Apoe-/- mice on a high-cholesterol (HC) diet following infection with Mycobacterium tuberculosis (Mtb). We hypothesized that the susceptibility of Apoe-/- HC mice might arise from an unrestrained feed-forward loop in which production of neutrophil extracellular traps (NETs) stimulates production of type I interferons by pDCs which in turn leads to the recruitment and activation of more neutrophils, and demonstrated that depleting neutrophils, depleting plasmacytoid dendritic cells (pDCs), or blocking type I interferon signaling, improved the outcome of infection. In concordance with these results, we found that Mtb-infected in Apoe-/-HC mice produce high levels of LTB4 and 12-HETE, two eicosanoids known to act as neutrophil chemoattractants and showed that blocking leukotriene B4 (LTB4) receptor signaling also improved the outcome of tuberculosis. While production of NETs has been associated with severe tuberculosis in other mouse models and in humans, a causative role for NETs in the pathology has not been directly established. We demonstrate that blocking the activation of peptidylarginine deiminase 4 (PAD4), an enzyme critical to NET formation, leads to fewer NETs in the lungs and, strikingly, completely reverses the hypersusceptibility of Apoe-/-HC mice to tuberculosis.

immunology↗

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

CD4-mediated immunity shapes neutrophil-driven tuberculous pathology

Pulmonary Mycobacterium tuberculosis (Mtb) infection results in highly heterogeneous lesions ranging from granulomas with central necrosis to those primarily comprised of alveolitis. While alveolitis has been associated with prior immunity in human post-mortem studies, the drivers of these distinct pathologic outcomes are poorly understood. Here, we show that these divergent lesion structures can be modeled in C3HeB/FeJ mice and are regulated by prior immunity. Using quantitative imaging, scRNAseq, and flow cytometry, we demonstrate that Mtb infection in the absence of prior immunity elicits dysregulated neutrophil recruitment and necrotic granulomas. In contrast, prior immunity induces rapid recruitment and activation of T cells, local macrophage activation, and diminished late neutrophil responses. Depletion studies at distinct infection stages demonstrated that neutrophils are required for early necrosis initiation and necrosis propagation at chronic stages, whereas early CD4 T cell responses prevent neutrophil feedforward circuits and necrosis. Together, these studies reveal fundamental determinants of tuberculosis lesion structure and pathogenesis, which have important implications for new strategies to prevent or treat tuberculosis.

immunology↗

bioRxiv2024

Automated, image-based quantification of peroxisome characteristics with perox-per-cell

perox-per-cell automates cumbersome, image-based data collection tasks often encountered in peroxisome research. The software processes microscopy images to quantify peroxisome features in yeast cells. It uses off-the-shelf image processing tools to automatically segment cells and peroxisomes and then outputs quantitative metrics including peroxisome counts per cell and spatial areas. In validation tests, we found that perox-per-cell output agrees well with manually-quantified peroxisomal counts and cell instances, thereby enabling high-throughput quantification of peroxisomal characteristics. The software is available at https://github.com/AitchisonLab/perox-per-cell

bioinformatics↗

bioRxiv2024

Deleteome-Tools: Utilizing a compendium of yeast deletion strain transcriptomes to identify co-functional genes

We introduce DeleteomeTools, an R package that leverages the Deleteome compendium of yeast single-gene deletion transcriptomes to predict gene function. Primarily, the package provides functions for identifying similarities between the transcriptomic signatures of deletion strains, thereby associating genes of interest with others that may be functionally related. We describe how our software predicted a novel relationship between the yeast nucleoporin Nup170 and the Ctf18-RFC complex, which was confirmed experimentally, revealing a previously unknown link between nuclear pore complexes and the DNA replication machinery. To assess the packages broader predictive capabilities, we performed a systematic evaluation that tested how well it predicted Gene Ontology (GO) annotations already applied to the subset of genes deleted in Deleteome strains. We show that our package predicted a majority of reported GO:biological process annotations with semantic similarities ranging from moderate to identical. We also discuss how our strategy for quantifying similarity between deletion strains, which relies on differential expression signatures, differs from other approaches that use global expression profiles and why it has the potential to identify functional relationships that might otherwise go undetected.

bioinformatics↗

bioRxiv2023

Predicting host-based, synthetic lethal antiviral targets from omics data

Traditional antiviral therapies often have limited effectiveness due to toxicity and development of drug resistance. Host-based antivirals, while an alternative, may lead to non-specific effects. Recent evidence shows that virus-infected cells can be selectively eliminated by targeting synthetic lethal (SL) partners of proteins disrupted by viral infection. Thus, we hypothesized that genes depleted in CRISPR KO screens of virus-infected cells may be enriched in SL partners of proteins altered by infection. To investigate this, we established a computational pipeline predicting SL drug targets of viral infections. First, we identified SARS-CoV-2-induced changes in gene products via a large compendium of omics data. Second, we identified SL partners for each altered gene product. Last, we screened CRISPR KO data for SL partners required for cell viability in infected cells. Despite differences in virus-induced alterations detected by various omics data, they share many predicted SL targets, with significant enrichment in CRISPR KO-depleted datasets. Comparing data from SARS-CoV-2 and influenza infections, we found possible broad-spectrum, host-based antiviral SL targets. This suggests that CRISPR KO data are replete with common antiviral targets due to their SL relationship with virus-altered states and that such targets can be revealed from analysis of omics datasets and SL predictions.

bioinformatics↗

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↗

bioRxiv2022

Distinct immune responses associated with vaccination status and protection outcomes after malaria challenge

Understanding immune mechanisms that mediate malaria protection is critical for improving vaccine development. Vaccination with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) induces high level of sterilizing immunity against malaria and serves as a valuable tool for the study of protective mechanisms. To identify vaccine-induced and protection-associated responses during malaria infection, we performed transcriptome profiling of whole blood and in-depth cellular profiling of PBMCs from volunteers who received either PfRAS or noninfectious mosquito bites, followed by controlled human malaria infection (CHMI) challenge. In-depth single-cell profiling of cell subsets that respond to CHMI in mock-vaccinated individuals showed a predominantly inflammatory transcriptome response. Whole blood transcriptome analysis revealed that gene sets associated with interferon responses and T and B cell signatures were increased and decreased, respectively, in protected vaccinees as early as one day following CHMI. In contrast, non-protected vaccinees and mock-vaccinated individuals exhibited shared transcriptome changes after CHMI characterized by decreased innate cell signatures and inflammatory responses. Additionally, immunophenotyping data showed different induction profiles of v{delta}2+{gamma}{delta} T cells, CD56+ CD8+ T effector memory (Tem) cells, and non-classical monocytes between protected vaccinees and individuals developing blood-stage parasitemia, following treatment and resolution of infection. Our data provide key insights in understanding immune mechanistic pathways of PfRAS-induced protection and infective CHMI. We demonstrate that protective immunity by PfRAS is associated with early changes in interferon and adaptive immune responses. Author summaryMalaria poses a significant global health threat, causing over half a million deaths annually. Effective vaccines are critically needed to prevent malaria disease. Our incomplete understanding of immune mechanisms that mediate malaria protection is hampering the development of effective vaccines. Irradiated sporozoite vaccines can induce highly sterilizing protection against malaria and are a valuable tool for the analysis of immune protection. Here, we aimed to characterize correlates of immune protection in individuals vaccinated with a suboptimal dose of irradiated sporozoite and subsequently challenged with live malaria parasite. Blood samples were taken before and after malaria challenge, and gene expression and cell type profiles were measured. We observed that the trajectories of immune response after malaria challenge is highly distinct between protected and non-protected vaccinees. We observed early perturbations in interferon response and adaptive immune cells in protected vaccinees whereas inflammatory and innate cell response were unique to non-protected vaccinees. We also observed that the immune profile after malaria challenge was distinctly similar between non-protected vaccinees and mock-vaccinated individuals. Our study sheds light on the dynamics of vaccine-induced immune responses that are associated with protection from malaria after CHMI.

immunology↗

bioRxiv2022

The nucleoporin Nup170 mediates subtelomeric gene silencing through the Ctf18-RFC complex and PCNA

The nuclear pore complex (NPC) physically interacts with chromatin and regulates gene expression. The inner ring nucleoporin Nup170 has been implicated in chromatin organization and the maintenance of gene silencing in subtelomeric regions. To gain insight into how Nup170 regulates this process, we used protein-protein interaction, genetic interaction, and transcriptome correlation analyses to identify the Ctf18-RFC complex, an alternative proliferating cell nuclear antigen (PCNA) loader, as a facilitator of the gene regulatory functions of Nup170. The Ctf18-RFC complex is recruited to a subpopulation of NPCs that lack the nuclear basket proteins Mlp1 and Mlp2. In the absence of Nup170, PCNA levels on DNA are reduced, resulting in the loss of silencing of subtelomeric genes. Increasing PCNA levels on DNA by removing Elg1, which is required for PCNA unloading, rescues subtelomeric silencing defects in nup170{Delta}. The NPC therefore mediates subtelomeric gene silencing by regulating PCNA levels on DNA.

cell biology↗

bioRxiv2021

Systems analysis of immune responses to attenuated P. falciparum malaria sporozoite vaccination reveals excessive inflammatory signatures correlating with impaired immunity

Immunization with radiation-attenuated sporozoites (RAS) can confer sterilizing protection against malaria, although the mechanisms behind this protection are incompletely understood. We performed a systems biology analysis of samples from the Immunization by Mosquito with Radiation Attenuated Sporozoites IMRAS) trial, which comprised P. falciparum RAS-immunized (PfRAS), malaria-naive participants whose protection from malaria infection was subsequently assessed by controlled human malaria infection (CHMI). Blood samples collected after initial PfRAS immunization were analyzed to compare immune responses between protected and non-protected volunteers leveraging integrative analysis of whole blood RNA-seq, high parameter flow cytometry, and single cell CITEseq of PBMCs. This analysis revealed differences in early innate immune responses indicating divergent paths associated with protection. In particular, elevated levels of inflammatory responses early after the initial immunization were detrimental for the development of protective adaptive immunity. Specifically, non-classical monocytes and early type I interferon responses induced within 1 day of PfRAS vaccination correlated with impaired immunity. Non-protected individuals also showed an increase in Th2 polarized T cell responses whereas we observed a trend towards increased Th1 and T-bet+ CD8 T cell responses in protected individuals. Temporal differences in genes associated with natural killer cells suggest an important role in immune regulation by these cells. These findings give insight into the immune responses that confer protection against malaria and may guide further malaria vaccine development.

immunology↗

bioRxiv2021

Nanobody Repertoires for Exposing Vulnerabilities of SARS-CoV-2

Despite the great promise of vaccines, the COVID-19 pandemic is ongoing and future serious outbreaks are highly likely, so that multi-pronged containment strategies will be required for many years. Nanobodies are the smallest naturally occurring single domain antigen binding proteins identified to date, possessing numerous properties advantageous to their production and use. We present a large repertoire of high affinity nanobodies against SARS-CoV-2 Spike protein with excellent kinetic and viral neutralization properties, which can be strongly enhanced with oligomerization. This repertoire samples the epitope landscape of the Spike ectodomain inside and outside the receptor binding domain, recognizing a multitude of distinct epitopes and revealing multiple neutralization targets of pseudoviruses and authentic SARS-CoV-2, including in primary human airway epithelial cells. Combinatorial nanobody mixtures show highly synergistic activities, and are resistant to mutational escape and emerging viral variants of concern. These nanobodies establish an exceptional resource for superior COVID-19 prophylactics and therapeutics.

biochemistry↗

bioRxiv2021

Autophagy as a mechanism for adaptive prediction-mediated emergence of drug resistance

Drug resistance is a major problem in treatment of microbial infections and cancers. There is growing evidence that a transient drug tolerant state may precede and potentiate the emergence of drug resistance. Therefore, understanding the mechanisms leading to tolerance is critical for combating drug resistance and for the development of effective therapeutic strategy. Through laboratory evolution of yeast, we recently demonstrated that adaptive prediction (AP), a strategy employed by organisms to anticipate and prepare for a future stressful environment, can emerge within 100 generations by linking the response triggered by a neutral cue (caffeine) to a mechanism of protection against a lethal agent (5-FOA). Here, we demonstrate that mutations selected across multiple laboratory evolved lines had linked the neutral cue response to core genes of autophagy. Across these evolved lines, conditional activation of autophagy through AP conferred tolerance, and potentiated subsequent selection of mutations in genes specific to overcoming the toxicity of 5-FOA. We propose a model to explain how extensive genome-wide genetic interactions of autophagy facilitates emergence of AP over short evolutionary timescales to potentiate selection of resistance-conferring mutations.

evolutionary biology↗

bioRxiv2020

A synthetic lethal interaction with a viral-induced hypomorph of GBF1

Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of cancer-specific mutations. Synthetic lethal interactions of viral-induced hypomorphs have the potential to be similarly targeted for the development of host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for synthetic lethal partners of GBF1 revealed ARF1 as the top hit, disruption of which, selectively killed cells that synthesize poliovirus 3A. Thus, viral protein interactions can induce hypomorphs that render host cells vulnerable to perturbations that leave uninfected cells intact. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2. SummaryUsing a viral-induced hypomorph of GBF1, Navare et al., demonstrate that the principle of synthetic lethality is a mechanism to selectively kill virus-infected cells.

molecular biology↗