Biology subjects

Holmes, M. J.

Publications and source records attributed to Holmes, M. J..

At least 19 recordsLinked to original sources

bioRxiv2026

HIV-exposure related disruptions in functional and structural connectivity in the central auditory system in adolescence

BackgroundChildren who are HIV-exposed but uninfected (CHEU) face elevated risks of hearing loss and language deficits compared to HIV-unexposed peers. The central auditory system (CAS) undergoes substantial maturational changes during adolescence, yet no neuroimaging study has examined its structural or functional integrity in CHEU. Prior work in this cohort identified white matter (WM) alterations in regions adjacent to the CAS at age 7, and reduced auditory working memory in CHEU relative to unexposed children (CHUU). AimTo characterise WM integrity and functional connectivity (FC) of the CAS and related regions in CHEU at age 11, to investigate structural and functional network topology, and to examine associations between imaging outcomes and neurocognitive function. MethodsForty-eight children aged 11-12 (20 CHEU, 28 CHUU) from an ongoing longitudinal neurodevelopmental cohort underwent 3T MRI including diffusion tensor imaging (DTI) and resting-state fMRI (RS-fMRI). CAS regions (cochlear nucleus/superior olivary complex, inferior colliculus [IC], medial geniculate nucleus [MGN], and primary auditory cortex [PAC]) were manually segmented and combined with an automated atlas. DTI probabilistic tractography was performed, extracting FA, MD, AD, RD, fractional number of tracts, and tract volume. FC was computed using Pearson correlations between regional time series. Graph theory measures (degree, strength, transitivity, nodal and local efficiency) were derived for structural and functional networks. RS-fMRI group comparisons used Bayesian multilevel modelling (matrix-based and region-based analyses), while DTI comparisons used linear models with FDR correction. Neurocognitive testing employed the KABC-II. ResultsNo significant group differences in DTI WM metrics (FA, MD, AD, RD) were observed after FDR correction. CHEU demonstrated higher structural nodal strength in the left IC (FDR-significant) and in the bilateral rostral middle frontal cortex (rMFC) and right cuneus. RS-fMRI revealed lower FC between the bilateral IC in CHEU, alongside reduced FC in the left caudate, left hippocampus CA3, left pericalcarine, and left lingual gyrus. CHEU showed higher FC between the left MGN and right precentral, left postcentral, and right rMFC; the right PAC also showed higher FC to the right rMFC and left postcentral gyrus. No significant group differences were observed in functional nodal measures. No significant associations were found between structural or functional imaging outcomes and neurocognitive scores after multiple comparison correction. DiscussionStructural and functional alterations within the CAS were most prominent in the IC, with increased nodal strength in CHEU potentially reflecting compensatory structural connectivity, and reduced interhemispheric FC between the bilateral IC suggesting disrupted auditory integration. Altered FC between the MGN/PAC and cortical regions, including the rMFC and sensorimotor cortices, may reflect differences in top-down auditory processing. The absence of imaging-cognition associations at age 11 suggests that these connectivity differences do not, at this stage, translate into measurable deficits in auditory or language-related neurocognitive performance. ConclusionThis is the first study to examine functional and structural connectivity of the CAS in CHEU children. HIV exposure is associated with subtle but discernible alterations in IC connectivity and in CAS links to cortical regions at age 11, without detectable neurocognitive correlates. Longitudinal follow-up and inclusion of audiological and ART exposure data are needed to clarify the developmental and functional consequences of these findings.

neuroscience↗

bioRxiv2025

SpoIIE drives asymmetric cell division in B. subtilis by sequential modulation of the cytokinesis machinery

To form a dormant spore, Bacillus subtilis and related endospore-forming bacteria divide asymmetrically to generate daughter cells of unequal size, the smaller of which becomes the spore. The transmembrane protein SpoIIE repositions the cell division machinery and controls cytokinesis during sporulation, but the molecular basis for the precise placement of the asymmetrical division site to the quarter cell point is unknown. Here, we applied live-cell fluorescence microscopy techniques to reveal that SpoIIE localizes with the treadmilling components of the cell division machinery. We found that SpoIIE opposes the inhibitory activity of the MinCD complex, which prevents assembly of Z-rings near the cell poles. Cells expressing a variant of SpoIIE with its transmembrane region replaced by an unrelated transmembrane anchor assembled condensed Z-rings that were unable to initiate constriction. This reveals a new function of SpoIIE and a possible checkpoint licensing cytokinesis downstream of Z-ring condensation. Potentially explaining the role of SpoIIE in cytokinesis, we demonstrated that SpoIIEs transmembrane region interacts with DivIB, an enigmatic structural component of the cell wall synthesis complex required for cytokinesis during sporulation. Finally, we found that FtsZ filaments are unusually short during sporulation, which requires the transmembrane domain of SpoIIE. Together, these results demonstrate that SpoIIE sequentially influences polar divisome assembly at distinct steps to drive asymmetric cell division.

microbiology↗

bioRxiv2025

Evidence of functional connectivity disruptions between auditory and non-auditory regions in adolescents living with HIV

Children living with perinatally acquired HIV (CPHIV) demonstrate hearing impairments and language processing delays even in the presence of combination antiretroviral therapy (cART). Even though efficient processing of sound relies on the proper function of both the peripheral and central auditory auditory systems (PAS; CAS), investigations on the effect of HIV on the auditory system have predominantly focused on the PAS. Additionally, language processing requires the efficient interaction between CAS brain regions and non-auditory regions. Investigating the functional connectivity (FC) within the CAS and between the CAS and non-auditory regions may reveal the influence of HIV on regions involved in auditory function. Within a Bayesian stastical framework, we used resting-state functional magnetic resonance imaging (RS-fMRI) to map FC in the CAS as well as between CAS regions and non-auditory regions of 11-year-old CPHIV. Graph theory was used to investigate the regional effects of HIV on functional brain network properties. Finally we explored the relationships between FC and neurocognitive outcomes relating to auditory and language processing. We hypothesized that CPHIV would show disruptions in FC between CAS regions as well as between CAS and non-auditroy regions. Secondly, we hypothesized that in CPHIV, regional brain network properties would be altered compared to their uninfected peers (CHUU). Finally we hypothesized that FC and functional network regional outcomes would be related to neurocognitive outcomes. Our investigation revealed lower FC of the primary auditory cortex (PAC) in CPHIV as well as disruptions in FC between CAS regions (including the PAC) and non-auditry regions including hippocampal sub-regions, the lingual gyri and basal ganglia. Functional network analysis reavealed lower nodal degree and efficiency in CAS regions including the cochlear nucleus/superior olivary complex (CN/SOC) and the inferior colliculus (IC). We also report associations between the nodal efficiency of middle temporal and superior frontal regions and delayed recall, a neurocognitive marker of working memory, present in CHUU but not in CPHIV. Our results demonstrate FC alterations in the PAC and between CAS regions and non-auditory regions involved in limbic, visual and motor processing, as well as disruptions to the regional properties of the CAS regions as part of the functional brain network. These results provide insight into the state of the CAS FC in the presence of HIV and its possible role in the hearing and language impairments seen in this population.

neuroscience↗

bioRxiv2025

GCN5a is a telomeric lysine acetyltransferase whose loss primes Toxoplasma gondii for latency

Toxoplasma gondii is a protozoan parasite that causes persistent infection in warm-blooded vertebrates by undergoing differentiation from a replicative stage (tachyzoites) to a latent encysted stage (bradyzoites). Stage differentiation is critical for transmission and pathogenesis and relies on gene regulation driven by a network of transcription and epigenetic factors. We previously found in non-cystogenic type I RH strain parasites that the lysine acetyltransferase (KAT), GCN5a, is dispensable in tachyzoites but required to upregulate stress-response genes, suggesting a link with bradyzoite conversion. To address this possibility, we generated endogenously tagged GCN5a parasites and a genetic knockout in cystogenic type II Pru strain. We show that GCN5a protein, but not mRNA, increases during differentiation and complexes with unique protein partners, most of which contain AP2 domains. Pru strain tachyzoites lacking GCN5a augment bradyzoite-specific gene expression in the absence of stress. Loss of GCN5a slowed tachyzoite replication and heightened sensitivity to bradyzoite conversion but resulted in smaller cyst sizes compared to wildtype. Using CUT&Tag, we delineated the chromosomal occupancy of GCN5a relative to the essential KAT, GCN5b. While GCN5b localizes to coding regions, GCN5a surprisingly localizes exclusively to telomeres. These findings suggest that the loss of GCN5a leads to telomere dysfunction, which slows replication and promotes the transition to latency. IMPORTANCEToxoplasma gondii is a single-celled parasite that persists in warm-blooded hosts, including humans, because it converts into latent tissue cysts. Switching from its replicating form into dormant cysts is a tightly regulated process that involves epigenetic factors such as lysine acetyltransferases GCN5a and GCN5b. This study is the first to examine the role of GCN5a in a cyst-forming Toxoplasma strain. We found that GCN5a protein, but not mRNA, increases during cyst development. Additionally, parasites lacking GCN5a replicate more slowly and are quicker to form cysts when stressed. We show that GCN5a and GCN5b work in different multi-protein complexes and localize to different areas of the genome; while GCN5b targets promoters of gene coding regions, GCN5a is exclusively found at telomeric regions. Our findings suggest a novel role for GCN5a in telomere biology that, when depleted, produces a fitness defect that favors development of latent stages.

microbiology↗

bioRxiv2024

Cap-independent translation directs stress-induced differentiation of the protozoan parasite Toxoplasma gondii

Translational control mechanisms modulate microbial latency of eukaryotic pathogens, enabling them to evade immunity and drug treatments. The protozoan parasite Toxoplasma gondii persists in hosts by differentiating from proliferative tachyzoites to latent bradyzoites, which are housed inside tissue cysts. Transcriptional changes facilitating bradyzoite conversion are mediated by a Myb domain transcription factor called BFD1, whose mRNA is present in tachyzoites but not translated into protein until stress is applied to induce differentiation. We addressed the mechanisms by which translational control drives BFD1 synthesis in response to stress-induced parasite differentiation. Using biochemical and molecular approaches, we show that the 5-leader of BFD1 mRNA is sufficient for preferential translation upon stress. The translational control of BFD1 mRNA is maintained when ribosome assembly near its 5-cap is impaired by insertion of a 5-proximal stem-loop and upon knockdown of the Toxoplasma cap-binding protein, eIF4E1. Moreover, we show that a trans-acting RNA-binding protein called BFD2/ROCY1 is necessary for cap-independent translation of BFD1 through its binding to the 5-leader. Translation of BFD2 mRNA is also suggested to be preferentially induced under stress, but by a cap-dependent mechanism. These results show that translational control and differentiation in Toxoplasma proceed through cap-independent mechanisms in addition to canonical cap-dependent translation. Our identification of cap-independent translation in protozoa underscores the antiquity of this mode of gene regulation in cellular evolution and its central role in stress-induced life-cycle events.

microbiology↗

bioRxiv2024

Using short-read 16S rRNA sequencing of multiple variable regions to generate high-quality results to a species level.

IntroductionShort-read amplicon sequencing studies have typically focused on 1-2 variable regions of the 16S rRNA gene. Species-level resolution is limited in these studies, as each variable region enables the characterisation of a different subsection of the microbiome. Although long-read sequencing techniques take advantage of all 9 variable regions by sequencing the entire 16S rRNA gene, they are substantially more expensive. This work assessed the feasibility of accurate species-level resolution and reproducibility using a relatively new sequencing kit and bioinformatics pipeline developed for short-read sequencing of multiple variable regions of the 16S rRNA gene. In addition, we evaluated the potential impact of different sample collection methods on our outcomes. MethodsUsing xGenTM 16S Amplicon Panel v2 kits, sequencing of all 9 variable regions of the 16S rRNA gene was carried out on an Illumina MiSeq platform. Mock cells and mock DNA for 8 bacterial species were included as extraction and sequencing controls respectively. Within-run and between-run replicate samples, and pairs of stool and rectal swabs collected at 0-5 weeks from the same participants, were incorporated. Observed relative abundances of each species were compared to theoretical abundances provided by ZymoBIOMICS. Paired Wilcoxon rank sum tests and distance-based intraclass correlation coefficients were used to statistically compare alpha and beta diversity measures, respectively, for pairs of replicates and stool/rectal swab sample pairs. ResultsUsing multiple variable regions of the 16S ribosomal Ribonucleic Acid (rRNA) gene, we found that we could accurately identify taxa to a species level and obtain highly reproducible results at a species level. Yet, the microbial profiles of stool and rectal swab sample pairs differed substantially despite being collected concurrently from the same infants. ConclusionThis protocol provides an effective means for studying infant gut microbial samples at a species level. However, sample collection approaches need to be accounted for in any downstream analysis.

microbiology↗

bioRxiv2023

Translation initiation factor eIF1.2 promotes Toxoplasma stage conversion by regulating levels of key differentiation factors

AbstractsThe parasite Toxoplasma gondii persists in its hosts by converting from replicating tachyzoites to latent bradyzoites housed in tissue cysts. The molecular mechanisms that mediate T. gondii differentiation remain poorly understood. Through a mutagenesis screen, we identified translation initiation factor eIF1.2 as a critical factor for T. gondii differentiation. A F97L mutation in eIF1.2 or the genetic ablation of eIF1.2 ({Delta}eif1.2) markedly impeded bradyzoite cyst formation in vitro and in vivo. We demonstrated, at single-molecule level, that the eIF1.2 F97L mutation impacts the scanning process of the ribosome preinitiation complex on a model mRNA. RNA sequencing and ribosome profiling experiments unveiled that {Delta}eif1.2 parasites are defective in upregulating bradyzoite induction factors BFD1 and BFD2 during stress-induced differentiation. Forced expression of BFD1 or BFD2 significantly restored differentiation in {Delta}eif1.2 parasites. Together, our findings suggest that eIF1.2 functions by regulating the translation of key differentiation factors necessary to establish chronic toxoplasmosis.

microbiology↗

bioRxiv2023

mRNA cap-binding protein eIF4E1 is a novel regulator of Toxoplasma gondii latency

The protozoan parasite Toxoplasma gondii causes serious opportunistic disease due to its ability to persist in patients as latent tissue cysts. The molecular mechanisms coordinating conversion between proliferative parasites (tachyzoites) and dormant cysts (bradyzoites) are not fully understood. We previously showed that phosphorylation of eIF2 accompanies bradyzoite formation, suggesting that this clinically relevant process involves regulation of mRNA translation. In this study, we investigated the composition and role of eIF4F multi-subunit complexes in translational control. Using CLIPseq, we find that the cap-binding subunit, eIF4E1, localizes to the 5-end of all tachyzoite mRNAs, many of which show evidence of stemming from heterogenous transcriptional start sites. We further show that eIF4E1 operates as the predominant cap-binding protein in two distinct eIF4F complexes. Using genetic and pharmacological approaches, we found that eIF4E1 deficiency triggers efficient spontaneous formation of bradyzoites without stress induction. Consistent with this result, we also show that stress-induced bradyzoites exhibit reduced eIF4E1 expression. Overall, our findings establish a novel role for eIF4F in translational control required for parasite latency and microbial persistence. SignificanceToxoplasma gondii is an opportunistic pathogen of importance to global human and animal health. There are currently no chemotherapies targeting the encysted form of the parasite. Consequently, a better understanding of the mechanisms controlling encystation are required. Here we show that the mRNA cap-binding protein, eIF4E1, is involved in regulating the encystation process. Encysted parasites reduce eIF4E1 levels and depletion of eIF4E1 decreases the translation of ribosome-associated machinery and drives Toxoplasma encystation. Together, these data reveal a new layer of mRNA translational control that regulates parasite encystation and latency.

microbiology↗

bioRxiv2023

Estimating the reproduction number, R0, from agent-based models of tree disease spread

Tree populations worldwide are facing an unprecedented threat from a variety of tree diseases and invasive pests. Their spread, exacerbated by increasing globalisation and climate change, has an enormous environmental, economic and social impact. Computational agent-based models are a popular tool for describing and forecasting the spread of tree diseases due to their flexibility and ability to reveal collective behaviours. In this paper we present a versatile agentbased model with a Gaussian infectivity kernel to describe the spread of a generic tree disease through a synthetic treescape. We then explore several methods of calculating the basic reproduction number R0, a characteristic measurement of disease infectivity, defining the expected number of new infections resulting from one newly infected individual throughout their infectious period. It is a useful comparative summary parameter of a disease and can be used to explore the threshold dynamics of epidemics through mathematical models. We demonstrate several methods of estimating R0 through the agent-based model, including contact tracing, inferring the Kermack-McKendrick SIR model parameters using the linear noise approximation, and an analytical approximation. As an illustrative example, we then use the model and each of the methods to calculate estimates of R0 for the ash dieback epidemic in the UK.

ecology↗

bioRxiv2023

Processive movement of Staphylococcus aureus essential septal peptidoglycan synthases is independent of FtsZ treadmilling and drives cell constriction

Bacterial cell division is mediated by the tubulin-homolog FtsZ, which recruits peptidoglycan (PG) synthesis enzymes to the division site. Septal PG synthases promote inward growth of the division septum, but the mechanisms governing the spatiotemporal regulation of these enzymes are poorly understood. Recent studies on various organisms have proposed different models for the relationship between the movement and activity of septum-specific PG synthases and FtsZ treadmilling. Here, we studied the movement dynamics of conserved cell division proteins relative to the rates of septum constriction and FtsZ treadmilling in the Gram-positive pathogen Staphylococcus aureus. The septal PG synthesis enzyme complex FtsW/PBP1 and its putative activator protein, DivIB, moved processively, around the division site, with the same velocity. Impairing FtsZ treadmilling did not affect FtsW and DivIB velocities or septum constriction rates. Contrarily, inhibition of PG synthesis slowed down or completely stopped both septum constriction and the directional movement of FtsW/PBP1 and DivIB. Our findings support a model for S. aureus in which a single population of processively moving FtsW/PBP1 remains associated with DivIB to drive cell constriction independently of treadmilling FtsZ filaments.

microbiology↗

bioRxiv2023

LRphase: an efficient method for assigning haplotype identity to long reads

MotivationUnderstanding the functional effects of sequence variation is among the primary goals of contemporary genomics. Individual human genomes contain millions of variants which are thought to contribute to phenotypic variability and differential disease risks at the population level. However, because variants rarely act in isolation, we cannot accurately predict functional effects without first considering the potential effects of other interacting variants on the same chromosome. This information can be obtained by phasing the read data from sequencing experiments. However, no standalone tools are available to simply phase reads based on known haplotypes. Here we present LRphase: a user-friendly utility for simple phasing of long sequencing reads. Availability and ImplementationLRphase is implemented in Python, and is freely available at https://github.com/Boyle-Lab/LRphase, under the MIT license. Version 1.1.0, described in this manuscript, is available through the pip and Bioconda repositories (e.g., "pip install lrphase==1.1.0"). Contactapboyle@umich.edu Supplementary InformationSupplementary methods are available as part of the online version of this publication.

bioinformatics↗

bioRxiv2022

AP2XII-2 coordinates transcriptional repression for Toxoplasma gondii sexual commitment

Toxoplasma gondii is a widespread protozoan parasite that has significant impact on human and veterinary health. The parasite undergoes a complex life cycle involving multiple hosts and developmental stages. How Toxoplasma transitions between life cycle stages is poorly understood, yet central to controlling transmission. Of particular neglect are the factors that contribute to its sexual development, which takes place exclusively in feline intestines. While epigenetic repressors have been shown to play an important role in silencing spurious gene expression of sexually committed parasites, the specific factors that recruit this generalized machinery to the appropriate genes remains largely unexplored. Here, we establish that a member of the AP2 transcription factor family, AP2XII-2, is targeted to genomic loci associated with sexually committed parasites along with the epigenetic regulators of transcriptional silencing, HDAC3 and MORC. Despite widespread association with gene promoters, AP2XII-2 is required for silencing of relatively few genes. Using CUT&Tag methodology, we identify two major genes associated with sexual development downstream of AP2XII-2 control, AP2X-10 and the amino acid hydroxylase AAH1. Our findings show that AP2XII-2 is a key contributor to the gene regulatory pathways modulating Toxoplasma sexual development. IMPORTANCEToxoplasma gondii is a parasite that undergoes its sexual stage exclusively in feline intestines, making cats a major source of transmission. A better understanding of the proteins controlling the parasites life cycle stage transitions is needed for the development of new therapies aimed to treat toxoplasmosis and transmission of the infection. Genes that regulate the sexual stages need to be turned on and off at the appropriate times, activities that are mediated by specific transcription factors that recruit general machinery to silence or activate gene expression. In this study, we identify a transcription factor called AP2XII-2 as being important for repression of a subset of sexual stage genes, including a sexual stage-specific AP2 factor (AP2X-10) and a protein (AAH1) required to construct the infectious oocysts expelled by infected cats.

microbiology↗

bioRxiv2021

m6A RNA methylation facilitates pre-mRNA 3-end formation and is essential for viability of Toxoplasma gondii

Toxoplasma gondii is an obligate intracellular parasite that can cause serious opportunistic disease in the immunocompromised or through congenital infection. To progress through its life cycle, Toxoplasma relies on multiple layers of gene regulation that includes an array of transcription and epigenetic factors. Over the last decade, the modification of mRNA has emerged as another important layer of gene regulation called epitranscriptomics. Here, we report that epitranscriptomics machinery exists in Toxoplasma, namely the methylation of adenosines (m6A) in mRNA transcripts. We identified novel components of the m6A methyltransferase complex and determined the distribution of m6A marks within the parasite transcriptome. m6A mapping revealed the modification to be preferentially located near the 3-boundary of mRNAs within the consensus sequence, YGCAUGCR. Knockdown of the m6A writer enzyme METTL3 resulted in diminished m6A marks, loss of a target transcript, and a complete arrest of parasite replication. Furthermore, we examined the two proteins in Toxoplasma that possess YTH domains, which bind m6A marks, and showed them to be integral members of the cleavage and polyadenylation machinery that catalyzes the 3-end processing of pre-mRNAs. Together, these findings establish that the m6A epitranscriptome is essential for parasite viability by contributing to the processing of mRNA 3-ends. Author SummaryToxoplasma gondii is a parasite of medical importance that causes disease upon immuno-suppression. Uncovering essential pathways that the parasite uses for its basic biological processes may reveal opportunities for new anti-parasitic drug therapies. Here, we describe the machinery that Toxoplasma uses to modify specific adenosine residues within its messenger RNAs (mRNA) by N6-adenosine methylation (m6A). We discovered that m6A mRNA methylation is prevalent in multiple stages of the parasite life cycle and is required for parasite replication. We also establish that m6A plays a major role in the proper maturation of mRNA. Two proteins that bind m6A modifications on mRNA associate with factors responsible for the cleavage and final processing steps of mRNA maturation. Since all of the machinery is conserved from plants to Toxoplasma and other related parasites, we propose that this system operates similarly in these organisms.

microbiology↗

bioRxiv2020

Dynamics of bacterial cell division: Z ring condensation is essential for cytokinesis

How proteins in the bacterial cell division complex (the divisome) coordinate to divide bacteria remains unknown. To explore how these proteins collectively function, we conducted a complete dynamic characterization of the proteins involved, and then examined the function of FtsZ binding proteins (ZBPs) and their role in cytokinesis. We find that the divisome consists of two dynamically distinct subcomplexes: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a directionally-moving complex that includes cell wall synthases. FtsZ filaments treadmill at steady state and the ZBPs have no effect on filament dynamics. Rather, ZBPs bundle FtsZ filaments, condensing them into Z rings. Z ring condensation increases the recruitment of cell wall synthesis enzymes to the division site, and this condensation is necessary for cytokinesis.

microbiology↗

bioRxiv2020

The inducible lac operator-repressor system is functional in zebrafish cells

BackgroundZebrafish are a foundational model organism for studying the spatio-temporal activity of genes and their regulatory sequences. A variety of approaches are currently available for editing genes and modifying gene expression in zebrafish, including RNAi, Cre/lox, and CRISPR-Cas9. However, the lac operator-repressor system, a component of the E. coli lac operon which has been adapted for use in many other species and is a valuable, flexible tool for studying the inducible modulation of gene expression, has not previously been tested in zebrafish. ResultsHere we demonstrate that the lac operator-repressor system robustly decreases expression of firefly luciferase in cultured zebrafish fibroblast cells. Our work establishes the lac operator-repressor system as a promising tool for the manipulation of gene expression in whole zebrafish. ConclusionsOur results lay the groundwork for the development of lac-based reporter assays in zebrafish, and adds to the tools available for investigating dynamic gene expression in embryogenesis. We believe that this work will catalyze the development of new reporter assay systems to investigate uncharacterized regulatory elements and their cell-type specific activities.

molecular biology↗

bioRxiv2019

The secreted acid phosphatase domain-containing GRA44 from Toxoplasma gondii is required for C-myc induction in infected cells

During host cell invasion, the eukaryotic pathogen Toxoplasma gondii forms a parsitophorous vacuole to safely reside within, while partitioned from host cell defense mechanisms. From within this safe niche parasites sabotage multiple host cell systems including gene expression, apoptosis and intracellular immune recognition by secreting a large arsenal of effector proteins. Many parasite proteins studied for active host cell manipulative interactions have been kinases. Translocation of effectors from the parasitophorous vacuole into the host cell is mediated by a putative translocon complex, which includes proteins MYR1, MYR2, and MYR3. Whether other proteins are involved in the structure or regulation of this putative translocon is not known. We have discovered that the secreted protein GRA44, which contains a putative acid phosphatase domain, interacts with members of this complex and is required for host cell effects downstream of effector secretion. We have determined GRA44 is processed in a region with homology to sequences targeted by protozoan proteases of the secretory pathway and that both major cleavage fragments are secreted into the parasitophorous vacuole. Immunoprecipitation experiments showed that GRA44 interacts with a large number of secreted proteins included MYR1. Importantly, conditional knockdown of GRA44 resulted in a lack of host cell cMyc upregulation, which mimics the phenotype seen when members of the translocon complex are genetically disrupted. Thus, the putative acid phosphatase GRA44 is crucial for host cell alterations during Toxoplasma infection and is associated with the translocon complex which Toxoplasma relies upon for success as an intracellular pathogen. IMPORTANCEApproximately one third of humans are infected with the parasite Toxoplasma gondii. Toxoplasma infections can lead to severe disease in those with a compromised or suppressed immune system. Additionally, infections during pregnancy present a significant health risk to the developing fetus. Drugs that target this parasite are limited, have significant side effects, and do not target all disease stages. Thus, a thorough understanding of how the parasite propagates within a host is critical in the discovery of novel therapeutic targets. To replicate Toxoplasma requires to enter the cells of the infected organism. In order to survive the environment inside a cell, Toxoplasma secretes a large repertoire of proteins, which hijack a number of important cellular functions. How these Toxoplasma proteins move from the parasite into the host cell is not well understood. Our work shows that the putative phosphatase GRA44 is part of a protein complex responsible for this process.

microbiology↗

bioRxiv2019

Simultaneous ribosome profiling of human host cells infected with Toxoplasma gondii

Toxoplasma gondii is a ubiquitous obligate intracellular parasite that infects the nucleated cells of warm-blooded animals. From within the parasitophorous vacuole in which they reside, Toxoplasma tachyzoites secrete an arsenal of effector proteins that can reprogram host gene expression to facilitate parasite survival and replication. Gaining a better understanding of how host gene expression is altered upon infection is central for understanding parasite strategies for host invasion and for developing new parasite therapies. Here, we applied ribosome profiling coupled with mRNA measurements to concurrently study gene expression in the parasite and in host human foreskin fibroblasts. By examining the parasite transcriptome and translatome, we identified potential upstream open reading frames that may permit the stress-induced preferential translation of parasite mRNAs. We also determined that tachyzoites reduce host death-associated pathways and increase survival, proliferation, and motility in both quiescent and proliferative host cell models of infection. Additionally, proliferative cells alter their gene expression in ways consistent with massive transcriptional rewiring while quiescent cells were best characterized by re-entry into the cell cycle. We also identified a translational control regimen consistent with mTOR activation in quiescent cells, and to a lesser degree in proliferative cells. This study illustrates the utility of the method for dissection of gene expression programs simultaneously in parasite and host.\n\nImportanceToxoplasma gondii is a single-celled parasite that has infected up to one-third of the worlds population. Significant overhauls in gene expression in both the parasite and the host cell accompany parasite invasion, and a better understanding of these changes may lead to the development of new therapeutic agents. In this study, we employed ribosome profiling to determine the changes that occur at the levels of transcription and translation in both the parasite and the infected host cell at the same time. We discovered features of Toxoplasma mRNAs that suggest a means for controlling parasite gene expression under stressful conditions. We also show that differences in host gene expression occur depending on whether they are confluent or not. Our findings demonstrate the feasibility of using ribosomal profiling to interrogate the host-parasite dynamic under a variety of conditions.

microbiology↗

bioRxiv2018

The convergent evolution of caste in ants and honey bees is based on a shared core of ancient reproductive genes and many plastic genes

Eusociality has convergently evolved multiple times, but the genomic basis of caste-based division of labor and degree to which independent origins of eusociality have utilized common genes remain largely unknown. Here we characterize caste-specific transcriptomic profiles across development and adult body segments from pharaoh ants (Monomorium pharaonis) and honey bees (Apis mellifera), representing two independent origins of eusociality. We identify a substantial shared core of genes upregulated in the abdomens of queen ants and honey bees that also tends to be upregulated in mated female flies, suggesting that these genes are part of a conserved insect reproductive groundplan. Outside of this shared groundplan, few genes are differentially expressed in common. Instead, the majority of the thousands of caste-associated genes are plastically-expressed, rapidly evolving, and relatively evolutionarily young. These results emphasize that the recruitment of both highly conserved and lineage-specific genes underlie the convergent evolution of novel traits such as eusociality.

evolutionary biology↗