Biology subjects

Forrester, S.

Publications and source records attributed to Forrester, S..

15 recordsLinked to original sources

bioRxiv2026

A pH-dependent protein kinase cascade regulates divergent differentiation of Leishmania in the sand fly.

Leishmania parasites must rapidly adapt to fluctuating environments to ensure survival and transmission. While acidic pH in the sand fly vector is a conserved developmental trigger, sensing mechanisms remain poorly understood. Using a barcoded protein kinase library, we screened for regulators of acid adaptation in Leishmania mexicana, identifying nine protein kinases influencing survival at low pH, including a haptomonad differentiation regulator protein kinase (HDRK1). We demonstrate that HDRK1 null mutants ({Delta}hdrk1) are predisposed to differentiate to haptomonad-like forms at low pH. While {Delta}hdrk1 mutants successfully infect the sand fly midgut, they fail to colonise the stomodeal valve, compromising transmission. Integrated transcriptomic and proteomic analyses revealed that at low pH, {Delta}hdrk1 mutants enter a low energy state reminiscent of AMPK-activated cells. We identified a second protein kinase, HDRK2, establishing a pH-dependent signalling pathway that governs the developmental fate of the parasite, directing differentiation towards either mammalian-infective metacyclic or vector-attached haptomonad stages. Finally, our screen revealed that phosphoinositide balance, regulated by the lipid kinases PI4K and PI4P5K, is vital for acid adaptation, and identified two STE transmembrane kinases as potential pH sensors. Together these findings provide a framework for how Leishmania detects and survives acid stress to coordinate its life cycle.

microbiology↗

bioRxiv2026

A network perspective on the role of c-di-GMP-associated protein complexes in biofilm formation.

1.The secondary messenger cyclic di-GMP is a ubiquitous bacterial signal that regulates the switch from a free-swimming to a sessile biofilm-forming lifestyle. Many biofilm-forming Pseudomonas species possess numerous c-di-GMP-binding proteins (CDGs) which regulate gene expression, protein activity, and protein complexes. However, the mechanisms by which numerous CDG effectors form a coherent signaling network to coordinate lifestyle changes remain poorly understood. We addressed this knowledge gap by focusing on ten CDG proteins involved in biofilm development in P. fluorescens SBW25. We used an integrated approach combining a protein interaction network from genome-wide yeast two-hybrid (Y2H) screens with large-scale biofilm and motility phenotype analyses via CRISPR interference (CRISPRi). Our network associated c-di-GMP signaling with processes such as signal transduction, solute transport, secretion, virulence, transcriptional regulation, DNA repair, and cell division. We discovered unknown functions of two CDG proteins in DNA repair and cell division, supporting the significance of our network. Notably, the phosphodiesterase DipA interacts with numerous CDG proteins through GGDEF domains. Phenotypic analyses revealed that CDG partners were highly correlated or strongly anticorrelated with DipA. These findings suggest that DipA is a central hub for CDG interactions that integrates opposing modules. These findings support the hub-based model of c-di-GMP signaling, which is crucial for localized control and rapid adaptation to environmental changes.

microbiology↗

bioRxiv2026

A Spatio-Temporal Analysis Framework for Characterizing Radiation-Induced Genomic Instability

Chronic low-dose ionizing radiation induces complex genomic instability encompassing both structural variants and point mutations, yet these alterations are typically analyzed as independent events, limiting detection of mechanistic coupling between rearrangement formation and localized mutagenesis at breakpoint junctions. This gap is particularly consequential given the widespread occupational and environmental exposure contexts such as nuclear energy, medical imaging, and environmental contamination, where coupled genomic alterations may contribute to cancer risk through mechanisms invisible to type-agnostic analyses. We developed an integrated analytical framework combining temporal pattern tracking, breakpoint-proximal mutation enrichment analysis, and systematic testing across all structural variant types to resolve these coupled dynamics across dose and time. Applying this framework to whole-genome sequencing data from primary human endothelial cells (HUVEC) exposed to chronic low-dose gamma radiation (0.20-2.62 mGy/hr) over three weeks, we identified inversion-specific mutagenic coupling; doublet base substitutions (DBS) were 7.13-fold enriched within 10bp of inversion breakpoints, a signal absent from other structural variant types, with sharp distance-dependent decay indicating localized mutagenesis at these junctions. Temporal analysis further revealed divergent fates of co-occurring alterations: inversions appeared transiently while DBS mutations showed greater persistence. These results illustrate how systematic integration of dose, time, and variant-type dimensions can uncover coupled mutagenic mechanisms that remain invisible in static or type-agnostic analyses. The framework is broadly applicable to longitudinal sequencing studies of genotoxic exposures, with applications to cancer genomics, radiation risk assessment, and mechanistic studies of DNA repair fidelity.

bioinformatics↗

bioRxiv2026

Interpretable transcriptome-to-phenotype modeling of cell-painting nuclear morphology features from RNA-seq under low-dose radiation exposure

With rapid advancements in high-throughput multi-modal profiling techniques across molecular, cellular to tissue scales, translating such multi-modal data into knowledge discovery for foundational understanding of cellular mechanisms is central in modern biomedical sciences. In this study, we focus on understanding how low-dose radiation exposure perturbs cellular morphology by linking high-dimensional transcriptomic responses to quantitative cell-phenotype readouts over time. We present a time-resolved inverse modeling framework that associates gene expression changes via RNA-sequencing with nuclear morphology features obtained from cell-painting imaging. Morphology responses were defined as treated-control differences for multiple nuclear features including size, shape, intensity, and textures, indexed by radiation dose and week. To capture time-dependent associations while maintaining interpretability, both RNA-sequencing and cell-painting data were stratified into four temporal phases (weeks 1-2, 3-4, 5-6, 7-9) and phase-dependent effects are encoded via gene-phase interaction predictors. To reduce confounding by dose trends and to evaluate generalization across time, we used a two-stage leave-one-week-out procedure: (i) a dose-only baseline model produced out-of-week residuals for each morphology feature, and (ii) elastic-net regression on phase-aware predictors modeled residual variation not explained by dose. Hyperparameters were selected via an exhaustive grid search scored by the correlation between observed residuals and out-of-week residual predictions, with additional sparsity diagnostics based on nonzero coefficient counts per fold. Stable predictors were identified by selection frequency and sign consistency across folds, then pruned further for multicollinearity and parsimony. Final reduced models were fit using ordinary least squares with heteroskedasticity-consistent standard errors to report effect estimates robust to non-constant variance. This workflow yields a transparent, time-stratified set of transcriptomic predictors associated with longitudinal nuclear morphology changes and provides a reproducible foundation for downstream biological interpretation and validation.

bioinformatics↗

bioRxiv2025

Anopheles (Kerteszia) cruzii, the main malaria vector in the Brazilian Atlantic Forest, is a complex of at least five cryptic species

Malaria, a tropical disease caused by Plasmodium and transmitted by Anopheles, remains a public health concern in Brazil. While most cases occur in the Amazon, transmission persists in the Atlantic Forest, where Anopheles mosquitoes of the Kerteszia subgenus are the primary vectors of human and simian malaria. Previous studies using cytogenetics, isoenzymes, and molecular markers have suggested cryptic species within Anopheles (Kerteszia) cruzii and Anopheles (Kerteszia) bellator. We sequenced 55 genomes: 35 An. cruzii s.l. (four with Nanopore and 31 with Illumina), 12 An. bellator s.l., and eight An. homunculus, the latter two with Illumina. Phylogenomic analysis revealed at least five cryptic species within An. cruzii s.l., labelled A-E, with evidence of sympatry in some locations. Anopheles bellator s.l. also forms a species complex, comprising at least three distinct lineages. These cryptic species showed high genetic differentiation (FST range: 0.4-0.7), typical of interspecific comparisons. In contrast, An. homunculus populations showed low differentiation (FST [~] 0.2), suggesting a single widespread species. Our analysis confirms cryptic speciation in An. cruzii and An. bellator, but not in An. homunculus. These findings are important for understanding malaria transmission in the Atlantic Forest, given that vector competence may differ among cryptic species.

evolutionary biology↗

bioRxiv2025

The Dirofilaria immitis unc-49 gene encodes a pharmacologically unique cys-loop GABA receptor

Nematode cys-loop GABA receptors play important roles in movement and locomotion. GABA receptors encoded by unc-49 appear to be widespread in nematode genomes including parasitic nematodes but are poorly characterized in filarial parasites. Dirofilaria immitis is a filarial parasite responsible for heartworm disease in dogs. Macrocyclic lactones are widely used to prevent heartworm infection. However, like many anthelmintics, resistance has emerged and new solutions are needed including the discovery of new anthelmintic drug targets. In this study, we report the isolation of two unc-49 subunit mRNAs (unc-49b and unc-49c) from the canine parasitic nematode D. immitis. When expressed in Xenopus oocytes, Dim-UNC-49B formed a functional homomeric GABA-gated channel, whereas Dim-UNC-49C did not. We further demonstrated that Dim-UNC-49B and Dim-UNC-49C gave rise to a functional heteromeric channel. Pharmacological characterization of these receptors and cross-species co-expression with UNC-49 subunits from the parasitic nematode Haemonchus contortus showed some unique properties compared to the same receptors characterized from other nematodes. These results revealed new properties of UNC-49 receptors in filarial worms.

pharmacology and toxicology↗

bioRxiv2025

Genomic reconstruction of Bacillus anthracis from complex environmental samples enables high throughput identification and lineage assignment in Pakistan

Bacillus anthracis, the causative agent of anthrax, is a highly virulent zoonotic pathogen primarily affecting domesticated and wild herbivores. Human exposure to B. anthracis is primarily through contact with infected animals or contaminated animal products. In Pakistan, where livestock vaccines are largely unavailable and infected carcasses are often disposed of improperly, the risk to humans, wildlife and livestock is significant. Currently, diagnosis of anthrax infections and outbreak tracing necessitates the isolation and culturing of B. anthracis, a process that requires BSL-3 facilities. In this study, we show that positive identification, genome reconstruction and lineage assignment can be accomplished using bioinformatic analysis of DNA extracted directly from environmental samples that would otherwise provide the starting material for isolation and culturing. This approach does not require laboratory target enrichment as is necessary for other pathogens, due in part to the extremely high bacterial load in the bloodstream in the deceased animals. Using these methods, we greatly expand the knowledge of endemic B. anthracis in Pakistan. We provide the first reference B. anthracis genomes from Pakistan since the 1970s and identify A.Br.014 Aust94 as a minor circulating sublineage alongside dominant A.Br.047 Vollum. Future work will focus on limits of detection and will determine if this bioinformatic method can be expanded more broadly for B. anthracis or other pathogens to replace typical culture-based methods.

bioinformatics↗

bioRxiv2023

Response of soil bacteria to PUREX chemicals suggests biomarker utility and bioremediation potential

Chemicals involved in plutonium uranium reduction extraction (PUREX) have the potential to be released from nuclear reprocessing facilities and accumulate in the environment. In order to understand how soil microbial communities respond to contamination by PUREX chemicals, we carried out a series of microcosm experiments, exposing chemically diverse soils to a range of concentrations of key chemicals used in the PUREX process. We tested 4 PUREX chemicals, and 5 soil types using 16S rRNA amplicon sequencing, determining that responses of microbial communities are dependent on the soil type in which they reside, and that tributyl phosphate exposure appears to generate the most reproducible and detectable shifts in microbial communities. We identified a number of key taxa that are consistently enriched in soils exposed to tributyl phosphate. These key taxa are either in the family Rhizobiaceae or genus Pseudomonas. The relative abundance of these key taxa is concentration dependent, and their abundance remains elevated at least 100 days post initial exposure. Using whole-shotgun metagenomic sequencing, we reconstructed the genomes of these key taxa and find a number of putative phosphotriesterase genes found only in Rhizobiaceae. We find the abundance of phosphotriesterase genes is significantly higher in samples exposed to tributyl phosphate. These phosphotriesterase genes, which degrade tributyl phosphate into dibutyl phosphate and butanol, may serve as effective biomarkers for tributyl phosphate contaminated soil, as well as a method for future bioremediation. ImportanceNuclear materials reprocessing facilities have the capacity to release toxic chemicals during normal operations or accidents. This study examines the ways in which chemicals involved with nuclear materials reprocessing impact microorganisms in the soil. Our intention was to understand the consequences of the release of these chemicals on ecosystems that may surround these reprocessing facilities. We find soil microbial communities change in response to some chemicals but not others, and that tributyl phosphate appears to generate the most reproducible and detectable shifts in microbial communities. Microorganisms in the family Rhizobiaceae increase in abundance in response to the addition of tributyl phosphate, and an examination of the genomes of these microbes suggest they may be able to break down tributyl phosphate to access the phosphosphate present in this chemical. Overall, this work demonstrates that changes in soil microbial communities in response to contamination with chemicals from nuclear materials reprocessing facilities may be predictable, and these responses could be leveraged to remediate contamination.

microbiology↗

bioRxiv2022

Parasite genotype is a major predictor of mortality from visceral leishmaniasis

Abstract/SummaryO_ST_ABSBackgroundC_ST_ABSVisceral leishmaniasis (VL) is a potentially fatal disease mainly caused by Leishmania infantum in South America and L. donovani in Asia and Africa. Disease outcomes have been associated with patient genotype, nutrition, age, sex, comorbidities, and co-infections. In this study, we examine the effects of parasite genetic variation on VL disease severity in Brazil. MethodsWe collected and sequenced the genomes of 109 L. infantum isolates from patients in northeast Brazil and retrieved matching patient clinical data from medical records, including mortality, sex, HIV co-infection and laboratory data (creatinine, haemoglobin, leukocyte and platelet counts). We identified genetic differences between parasite isolates, including single nucleotide polymorphisms (SNPs), small insertions/deletions (indels), and variations in genic, intergenic, and chromosome copy numbers (copy number variants, CNVs). To describe associations between the parasite genotypes and clinical outcomes, we applied quantitative genetics methods of heritability and genome-wide association studies (GWAS), treating clinical outcomes as traits that may be influenced by parasite genotype. FindingsMultiple aspects of the genetic analysis indicate that parasite genotype affects clinical outcomes. We estimate that parasite genotype explains 83% chance of mortality (narrow sense heritability, h2 = 0{middle dot}83{+/-}0{middle dot}17), and has a significant relationship with patient sex (h2 = 0{middle dot}60{+/-}0{middle dot}27). Impacts of parasite genotype on other clinical traits are lower (h2 [≤]0{middle dot}34). GWAS analysis identified multiple parasite genetic loci that were significantly associated with clinical outcomes; 17 CNVs that were significantly associated with mortality, two with creatinine and one with bacterial co-infection, jaundice and HIV co-infection; and two SNPs/indels and six CNVs that associate with age, jaundice, HIV and bacterial co-infections, creatinine, and/or bleeding sites. InterpretationParasite genotype is an important factor in VL disease severity in Brazil. Our analysis indicates that specific genetic differences between parasites act as virulence factors, enhancing risks of severe disease and mortality. More detailed understanding of these virulence factors could be exploited for novel therapies. Author SummaryMultiple factors contribute to the risk of mortality from visceral leishmaniasis (VL), including, patient genotype, comorbidities, and nutrition. Many of these factors will be influenced by socio-economic biases 1. Our work suggests that the virulence of the infecting parasite is an important risk factor for mortality. We pinpoint some specific genomic markers that are associated with mortality, which can lead to a greater understanding of the molecular mechanisms that cause severe VL disease, to genetic markers for virulent parasites and to the development of drug and vaccine therapies.

evolutionary biology↗

bioRxiv2022

Tissue specific dual RNA-seq defines host-parasite interplay in murine visceral leishmaniasis caused by Leishmania donovani and Leishmania infantum

Visceral leishmaniasis is associated with hepato-splenomegaly and altered immune and haematological parameters in both pre-clinical animal models and humans. We studied mouse experimental visceral leishmaniasis caused by Leishmania infantum and Leishmania donovani in BALB/c mice using dual RNA-seq to investigate the transcriptional response of host and parasite in liver and spleen. We identified only 4 species-specific parasite expressed genes (SSPEGs; log2FC >1, FDR <0.05) in the infected spleen, and none in the infected liver. For the host transcriptome, we found 789 differentially expressed genes (DEGs; log2FC >1, FDR <0.05) in the spleen that were common to both infections, with IFN{gamma} signaling and complement and coagulation cascade pathways highly enriched, and an additional 286 and 186 DEGs that were selective to L. donovani and L. infantum infection respectively. Among those, there were network interactions between genes of amino acid metabolism and PPAR signaling in L. donovani infection and increased IL1{beta} and positive regulation of fatty acid transport in L. infantum infection, although no pathway enrichment was observed. In the liver, there were 1939 DEGs in mice infected with either L. infantum or L. donovani in comparison to uninfected mice, and the most enriched pathways were IFN{gamma} signaling, neutrophil mediated immunity, complement and coagulation, cytokine-chemokine responses and hemostasis. Additionally, 221 DEGs were selective in L. donovani and 429 DEGs in L. infantum infections. These data show that the host response for these two visceral leishmaniasis infection models is broadly similar, and [~]10% of host DEGs vary in infections with either parasite species. ImportanceVisceral leishmaniasis (VL) is caused by two species of Leishmania parasites, L. donovani in the Old World and L. infantum in the New World and countries bordering the Mediterranean. Although cardinal features such as hepato-splenomegaly and alterations in blood and immune function are evident, clinical presentation may vary by geography, with for example severe bleeding often associated with VL in Brazil. Although animal models of both L. donovani and L. infantum have been widely used to study disease pathogenesis, a direct side-by-side comparison of how these parasites species impact the infected host and/or how they might respond to the stresses of mammalian infection has not been previously reported. Identifying common and distinct pathways to pathogenesis will be important to ensure that new therapeutic or prophylactic approaches will be applicable across all forms of VL.

microbiology↗

bioRxiv2021

Ipsilesional motor cortex activation with high-force unimanual handgrip contractions of the less-affected limb in participants with stroke

Stroke is a leading cause of severe disability that often presents with unilateral motor impairment. Conventional rehabilitation approaches focus on motor practice of the affected limb and aim to suppress brain activity in the contralesional hemisphere to facilitate ipsilesional hemispheric neuroplasticity subserving motor recovery. Previous research has also demonstrated that exercise of the less-affected limb can promote motor recovery of the affected limb through the interlimb transfer of the trained motor task, termed cross-education. One of the leading theories for cross-education proposes that the interlimb transfer manifests from ipsilateral cortical activity during unimanual motor tasks, and that this ipsilateral cortical activity results in motor related neuroplasticity giving rise to contralateral improvements in motor performance. Conversely, exercise of the less-affected limb promotes contralesional brain activity which is typically viewed as contraindicated in stroke recovery due to the interhemispheric inhibitory influence onto the ipsilesional hemisphere. High-force unimanual handgrip contractions are known to increase ipsilateral brain activation in control participants, but it remains to be determined if this would be observed in participants with stroke. Therefore, this study aimed to determine how parametric increases in handgrip force during repeated contractions with the less-affected limb impacts brain activity bilaterally in participants with stroke and in a cohort of neurologically intact controls. In this study, higher force contractions were found to increase brain activation in the ipsilesional/ipsilateral hemisphere in both groups (p = .002), but no between group differences were observed. These data suggest that high-force exercise with the less-affected limb may promote ipsilesional cortical plasticity to promote motor recovery of the affected-limb in participants with stroke.

neuroscience↗

bioRxiv2021

Multiple targets of balancing selection in Leishmania donovani complex parasites

The Leishmania donovani species complex are the causative agents of visceral leishmaniasis, which cause 20-40,000 fatalities a year. Here, we conduct a screen for balancing selection in this species complex. We used 387 publicly-available L. donovani and L. infantum genomes, and sequence 93 isolates of L. infantum from Brazil to describe the global diversity of this species complex. We identify five genetically-distinct populations that are sufficiently represented by genomic data to search for signatures of selection. We find that signals of balancing selection are generally not shared between populations, consistent with transient adaptive events, rather than long-term balancing selection. We then apply multiple diversity metrics to identify candidate genes with robust signatures of balancing selection, identifying a curated set of 19 genes with robust signatures. These include zeta toxin, nodulin-like and flagellum attachment proteins. This study highlights the extent of genetic divergence between L. donovani complex parasites and provides genes for further study.

evolutionary biology↗

bioRxiv2019

The genome of the zoonotic malaria parasite Plasmodium simium reveals adaptions to host-switching

BackgroundPlasmodium simium, a malaria parasite of non-human primates (NHP) was recently shown to cause zoonotic infections in humans in Brazil. We sequenced the P. simium genome to investigate its evolutionary history and to identify any genetic adaptions that may underlie the ability of this parasite to switch between host species. ResultsPhylogenetic analyses based on whole genome sequences of P. simium from humans and NHPs reveals that P. simium is monophyletic within the broader diversity of South American Plasmodium vivax, suggesting P. simium first infected NHPs as a result of a host-switch of P. vivax from humans. The P. simium isolates show the closest relationship to Mexican P. vivax isolates. Analysis of erythrocyte invasion genes reveals differences between P. vivax and P. simium, including large deletions in the Duffy Binding Protein 1 (DBP1) and Reticulocyte Binding Protein 2a genes of P. simium. Analysis of P. simium isolated from NHPs and humans revealed a deletion of 38 amino acids in DBP1 present in all human-derived isolates, whereas NHP isolates were multi-allelic. ConclusionsAnalysis of the P. simium genome confirmed a close phylogenetic relationship between P. simium and P. vivax, and suggests a very recent American origin for P. simium. The presence of the DBP1 deletion in all human-derived isolates tested suggests that this deletion, in combination with other genetic changes in P. simium, may facilitate the invasion of human red blood cells and may explain, at least in part, the basis of the recent zoonotic infections.

genomics↗

bioRxiv2019

The mRNA-bound proteome of Leishmania mexicana: novel genetic insight into an ancient parasite

Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Utilizing optimised crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1,400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, while the crosslinked RBPome is magnitudes more enriched the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L.mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.

molecular biology↗

bioRxiv2018

Interrogation of genes controlling biofilm formation using CRISPR interference in Pseudomonas fluorescens

Bacterial biofilm formation involves multigenic signaling and regulatory pathways that control the transition from motile to sessile lifestyle, production of extracellular polymeric matrix, and maturation of the biofilm complex 3D structure. Biofilms are extensively studied because of their importance in biomedical, ecological and industrial settings. Genetic approaches based on gene inactivation are powerful for mechanistic studies but often are labor intensive, limiting systematic gene surveys to the most tractable bacterial hosts. Here, we adapted the CRISPR interference (CRISPRi) system for use in P. fluorescens. We found that CRISPRi is applicable to three genetically and physiologically diverse species, SBW25, WH6 and Pf0-1 and affords extended periods of time to study complex phenotypes such as cell morphology, motility and biofilm formation. In SBW25, CRISPRi-mediated silencing of the GacA/S two-component system and genes regulated by cylic-di-GMP produced phenotypes similar to those previously described after gene inactivation in various Pseudomonas. Combined with detailed confocal microscopy of biofilms, our study also revealed novel phenotypes associated with biofilm architecture and extracellular matrix biosynthesis as well as the potent inhibition of SBW25 biofilm formation mediated by the PFLU1114 protein. Thus, CRISPRi is a reliable and scalable approach to interrogate gene networks in the diverse P. fluorescens group.

microbiology↗