Biology subjects

Keroack, C. D.

Publications and source records attributed to Keroack, C. D..

6 recordsLinked to original sources

bioRxiv2025

Ribosomal Architecture and rRNA Modification Landscape in the Tick-Borne Parasite Babesia divergens

Babesia is a tick-borne intracellular apicomplexan parasite responsible for diseases ranging from mild to fatal, with a broadening geographic distribution. Due to the complex life cycle of Babesia species, their survival depends on the precise control of gene expression, which is primarily regulated by epigenetic, transcriptional, and post-transcriptional mechanisms. High-resolution structural information on key components of the translation machinery, such as ribosomes, could aid in the development of antiparasitic drugs. Here, we report cryo-EM ribosome structures (2.6 [A]) from the tick-borne apicomplexan pathogen Babesia divergens, showing associated tRNAs, an mRNA fragment, and RACK1, a signaling scaffold crucial to translation regulation. Density map analysis displays ribosome regions at atomic resolution (1.7 [A]), which, when combined with nanopore sequencing, enabled the comprehensive identification of rRNA modifications, including modifications unreported in other organisms. The new rRNA modifications localize not only to the reduced Babesia rRNA expansion segments but also to functionally essential ribosomal sites, uncovering new avenues for therapeutic intervention against babesiosis.

microbiology↗

bioRxiv2023

Single cell expression and chromatin access of the Toxoplasma gondii lytic cycle identifies AP2XII-8 as an essential pivotal controller of a ribosome regulon

Sequential lytic cycles driven by cascading transcriptional waves underlie pathogenesis in the apicomplexan parasite Toxoplasma gondii. This parasites unique division by internal budding, short cell cycle, and jumbled up classically defined cell cycle stages have restrained in-depth transcriptional program analysis. Here, unbiased transcriptome and chromatin accessibility maps throughout the lytic cell cycle were established at the single cell level. Correlated pseudo-timeline assemblies of expression and chromatin profiles mapped transcriptional versus chromatin level transition points promoting the cell division cycle. Sequential clustering analysis identified putatively functionally related gene groups facilitating parasite division. Promoter DNA motif mapping revealed patterns of combinatorial regulation. Pseudo-time trajectory analysis revealed transcriptional bursts at different cell cycle points. The dominant burst in G1 was driven by transcription factor AP2XII-8, which engages TGCATGCG/A and TATAAGCCG motifs, and promoted the expression of a regulon encoding 40 ribosomal proteins. Overall, the study provides integrated, multi-level insights into apicomplexan transcriptional regulation.

microbiology↗

bioRxiv2023

Comparative chemical genomics in Babesia species identifies the alkaline phosphatase phoD as a novel determinant of resistance

Babesiosis is an emerging zoonosis and widely distributed veterinary infection caused by 100+ species of Babesia parasites. The diversity of Babesia parasites, coupled with the lack of potent inhibitors necessitates the discovery of novel conserved druggable targets for the generation of broadly effective antibabesials. Here, we describe a comparative chemogenomics (CCG) pipeline for the identification of novel and conserved targets. CCG relies on parallel in vitro evolution of resistance in independent populations of evolutionarily-related Babesia spp. (B. bovis and B. divergens). We identified a potent antibabesial inhibitor from the Malaria Box, MMV019266. We were able to select for resistance to this compound in two species of Babesia, achieving 10-fold or greater resistance after ten weeks of intermittent selection. After sequencing of multiple independently derived lines in the two species, we identified mutations in a single conserved gene in both species: a membrane-bound metallodependent phosphatase (putatively named PhoD). In both species, the mutations were found in the phoD-like phosphatase domain, proximal to the predicted ligand binding site. Using reverse genetics, we validated that mutations in PhoD confer resistance to MMV019266. We have also demonstrated that PhoD localizes to the endomembrane system and partially with the apicoplast. Finally, conditional knockdown and constitutive overexpression of PhoD alter the sensitivity to MMV019266 in the parasite: overexpression of PhoD results in increased sensitivity to the compound, while knockdown increases resistance, suggesting PhoD is a resistance mechanism. Together, we have generated a robust pipeline for identification of resistance loci, and identified PhoD as a novel determinant of resistance in Babesia species. HighlightsO_LIUse of two species for in vitro evolution identifies a high confidence locus associated with resistance C_LIO_LIResistance mutation in phoD was validated using reverse genetics in B. divergens C_LIO_LIPerturbation of phoD using function genetics results in changes in the level of resistance to MMV019266 C_LIO_LIEpitope tagging reveals localization to the ER/apicoplast, a conserved localization with a similar protein in diatoms C_LIO_LITogether, phoD is a novel resistance determinant in multiple Babesia spp. C_LI

molecular biology↗

bioRxiv2022

Babesia divergens egress from host cells is orchestrated by essential and druggable kinases and proteases

A unique aspect of apicomplexan biology is the requirement for egress from and invasion into host red blood cells (RBCs). The cellular mechanisms and molecular mediators of RBC egress and invasion remain poorly characterized in Babesia spp., a group of parasites of veterinary importance and emerging cause of zoonotic disease. Through the use of video microscopy, transcriptomics, and chemical genetics we have implicated signaling, proteases and gliding motility in egress and/or invasion by Babesia divergens. We developed CRISPR/Cas9 and two inducible knockdown systems to perform a genetic screen of putative mediators of egress. We found that proteases ASP2 and ASP3 are required for invasion, and the latter is also required for egress. Strikingly, parasites continue to replicate intracellularly in the absence of the protein kinases, PKG or CDPK4, indicating that they are required for exit from the replication cycle and egress. These essential molecules present druggable targets for Babesia spp. All together we have established a molecular framework for the spread of infection through host RBCs, with egress of B. divergens more closely resembling T. gondii than the more evolutionarily related Plasmodium spp. HighlightsO_LIEgress in Babesia divergens requires host cell lysis and parasite motility C_LIO_LITranscriptomics can be used to identify egress and invasion proteins C_LIO_LIKnockdown of the proteases, ASP2 and ASP3, inhibit egress and invasion C_LIO_LIInhibition of PKG or CDPK4 signaling results in continued intracellular replication C_LI

microbiology↗

bioRxiv2022

Comparative single-cell transcriptional atlases of Babesia species reveal conserved and species-specific expression profiles

Babesia is a genus of Apicomplexan parasites that infect red blood cells in vertebrate hosts. Pathology occurs during rapid replication cycles in the asexual blood-stage of infection. Current knowledge of Babesia replication cycle progression and regulation is limited and relies mostly on comparative studies with related parasites. Due to limitations in synchronizing Babesia parasites, fine-scale time-course transcriptomic resources are not readily available. Single-cell transcriptomics provides a powerful unbiased alternative for profiling asynchronous cell populations. Here, we applied single-cell RNA sequencing to three Babesia species (B. divergens, B. bovis, and B. bigemina). We used analytical approaches and algorithms to map the replication cycle and construct pseudo-synchronized time-course gene expression profiles. We identify clusters of co-expressed genes showing just-in-time expression profiles, with gradually cascading peaks throughout asexual development. Moreover, clustering analysis of reconstructed gene curves reveals coordinated timing of peak expression in epigenetic markers and transcription factors. Using a regularized Gaussian Graphical Model, we reconstructed co-expression networks and identified conserved and species-specific nodes. Motif analysis of a co-expression interactome of AP2 transcription factors identified specific motifs previously reported to play a role in DNA replication in Plasmodium species. Finally, we present an interactive web-application to visualize and interactively explore the datasets.

bioinformatics↗

bioRxiv2020

Fussing about fission: defining variety among mainstream and exotic apicomplexan cell division modes

Cellular reproduction defines life, yet our textbook-level understanding of cell division is limited to a small number of model organisms centered around humans. The horizon on cell division variants is expanded here by advancing insights on the fascinating cell division modes found in the Apicomplexa, a key group of protozoan parasites. The Apicomplexa display remarkable variation in offspring number, whether karyokinesis follows each S/M-phase or not, and whether daughter cells bud in the cytoplasm or bud from the cortex. We find that the terminology used to describe the various manifestations of asexual apicomplexan cell division emphasizes either the number of offspring or site of budding, which are not directly comparable features and has led to confusion in the literature. Division modes have been primarily studied in two human pathogenic Apicomplexa, malaria-causing Plasmodium spp. and Toxoplasma gondii, a major cause of opportunistic infections. Plasmodium spp. divide asexually by schizogony, producing multiple daughters per division round through a cortical budding process, though at several life-cycle nuclear amplifications are not followed by karyokinesis. T. gondii divides by endodyogeny producing two internally budding daughters per division round. Here we add to this diversity in replication mechanisms by considering the cattle parasite Babesia bigemina and the pig parasite Cystoisospora suis. B. bigemina produces two daughters per division round by a binary fission mechanism whereas C. suis produces daughters through both endodyogeny and multiple internal budding known as endopolygeny. In addition, we provide new data from the causative agent of equine protozoal myeloencephalitis (EPM), Sarcocystis neurona, which also undergoes endopolygeny but differs from C. suis by maintaining a single multiploid nucleus. Overall, we operationally define two principally different division modes: internal budding found in cyst-forming Coccidia (comprising endodyogeny and two forms of endopolygeny) and external budding found in the other parasites studied (comprising the two forms of schizogony, binary fission and multiple fission). Progressive insights into the principles defining the molecular and cellular requirements for internal versus external budding, as well as variations encountered in sexual stages are discussed. The evolutionary pressures and mechanisms underlying apicomplexan cell division diversification carries relevance across Eukaryota. Contribution to the FieldMechanisms of cell division vary dramatically across the Tree of Life, but the mechanistic basis has only been mapped for several model organisms. Here we present cell division strategies across Apicomplexa, a group of obligate intracellular parasites with significant impact on humans and domesticated animals. Asexual apicomplexan cell division is organized around assembly of daughter buds, but division forms differ in the cellular site of budding, number of offspring per division round, whether each S-phase follows karyokinesis and if mitotic rounds progress synchronously. This varies not just between parasites, but also between different life-cycle stages of a given species. We discuss the historical context of terminology describing division modes, which has led to confusion on how different modes relate to each other. Innovations in cell culture and genetics together with light microscopy advances have opened up cell biological studies that can shed light on this puzzle. We present new data for three division modes barely studied before. Together with existing data, we show how division modes are organized along phylogenetic lines and differentiate along external and internal budding mechanisms. We also discuss new insights into how the variations in division mode are regulated at the molecular level, and possess unique cell biological requirements.

microbiology↗