Donna Huber, Author at Center for Tropical and Emerging Global Diseases

Evaluating the Benefits and Limits of Multiple Displacement Amplification With Whole-Genome Oxford Nanopore Sequencing

Donna Huber on February 28, 2025

Circos plot illustrating a synteny comparison between the reference S. aureus ATCC-29213 genome sequence and pre- and post-amplification genome assemblies.

Multiple displacement amplification (MDA) outperforms conventional PCR in long fragment and whole-genome amplification, making it attractive to couple MDA with long-read sequencing of samples with limited quantities of DNA to obtain improved genome assemblies. Here, we explore the efficacy and limits of MDA for efficient low-cost genome sequence assembly using Oxford Nanopore Technologies (ONTs) rapid library preparations and minION sequencing. We successfully generated almost complete genome sequences for all organisms examined, including Gram-positive (Staphylococcus aureus, Enterococcus faecium) and Gram-negative (Escherichia coli) prokaryotes and one challenging eukaryotic pathogen (Cryptosporidium spp) representing a broad spectrum of critical infectious disease pathogens. High-quality data from those samples were generated starting with only 0.025 ng of total DNA. Controlled sheared DNA samples exhibited a distinct pattern of size increase after MDA, which may be associated with the amplification of long, low-abundance fragments present in the assay, as well as generating concatemeric sequences during amplification. To address concatemers, we developed a computational pipeline (CADECT: Concatemer Detection Tool) to identify and remove putative concatemeric sequences. This study highlights the efficacy of MDA in generating high-quality genome assemblies from limited amounts of input DNA. Also, the CADECT pipeline effectively mitigated the impact of concatemeric sequences, enabling the assembly of contiguous sequences even in cases where the input genomic DNA was degraded. These results have significant implications for the study of organisms that are challenging to culture in vitro, such as Cryptosporidium, and for expediting critical results in clinical settings with limited quantities of available genomic DNA.

Fiifi Agyabeng-Dadzie, Megan S Beaudry, Alex Deyanov, Haley Slanis, Minh Q Duong, Randi Turner, Asis Khan, Cesar A Arias, Jessica C Kissinger, Travis C Glenn, Rodrigo de Paula Baptista. Mol Ecol Resour. 2025 Feb 28:e14094. doi: 10.1111/1755-0998.14094.

A limitation lifted: A conditional knockdown system reveals essential roles for Polo-like kinase and Aurora kinase 1 in Trypanosoma cruzi cell division

Donna Huber on February 18, 2025

Figure 6 from https://doi.org/10.1073/pnas.2416009122 — PLK is essential for normal cytokinesis in amastigotes.

While advances in genome editing technologies have simplified gene disruption in many organisms, the study of essential genes requires development of conditional disruption or knockdown systems that are not available in most organisms. Such is the case for Trypanosoma cruzi, a parasite that causes Chagas disease, a severely neglected tropical disease endemic to Latin America that is often fatal. Our knowledge of the identity of essential genes and their functions in T. cruzi has been severely constrained by historical challenges in very basic genetic manipulation and the absence of RNA interference machinery. Here, we describe the development and use of self-cleaving RNA sequences to conditionally regulate essential gene expression in T. cruzi. Using these tools, we identified essential roles for Polo-like and Aurora kinases in T. cruzi cell division, mirroring their functions in Trypanosoma brucei. Importantly, we demonstrate conditional knockdown of essential genes in intracellular amastigotes, the disease-causing stage of the parasite in its human host. This conditional knockdown system enables the efficient and scalable functional characterization of essential genes in T. cruzi and provides a framework for the development of conditional gene knockdown systems for other nonmodel organisms.

J. Wiedeman, R. Harrison, & R.D. Etheridge, Proc. Natl. Acad. Sci. U.S.A. 122 (8) e2416009122, https://doi.org/10.1073/pnas.2416009122 (2025).

Combined fluorescent in situ hybridization and F- ara-EdU staining on whole mount Hymenolepis diminuta

Donna Huber on February 13, 2025

Co-localization of F-ara-EdU incorporation and cycling cell marker expression. Laser-scan confocal micrographs of H. diminuta stained by FISH for cycling cell markers mcm2/mcm7 followed by detection of S-phase marker, F-ara-EdU . Nuclei are labeled with DAPI.

Hymenolepis diminuta is a parasitic tapeworm that utilizes rats as hosts and offers advantages over human parasitic tapeworms and free-living flatworms as a model system to study the biology and pathology of helminth infections. H. diminuta is minimally infectious to humans, easy to maintain in the lab, demonstrates impressive growth, regeneration, and reproductive capabilities, and is amenable to loss-of-function manipulations. As an emerging model, tool development is critical to increasing the utility of this system. This study introduces a novel protocol for H. diminuta that combines fluorescent in situ hybridization (FISH) and 2′-Deoxy-2′-fluoro-5-ethynyluridine (F-ara-EdU) uptake and staining. Our protocol allows for the spatial detection of gene expression and simultaneous identification of proliferating cells. Dual labeling of F-ara-EdU and stem cell markers revealed a distinct expression pattern in different anatomical regions, especially in the head and neck. We demonstrate optimal labeling without permeabilization, streamlining the protocol. We also demonstrate generalizability using FISH for other tissue markers. The protocol was applied to perform bulk lineage tracing, revealing that stem cells can differentiate into neuronal and tegumental cells within 3 days. Our protocol provides an important tool in the arsenal for investigating gene expression and cell proliferation in H. diminuta, contributing valuable insights into the biology of parasitic tapeworms and potentially opening new avenues for the study of human parasitic tapeworms.

Mohamed Ishan, Isabell R Skipper, Tania Rozario. Biol Methods Protoc. 2025 Feb 13;10(1):bpaf011. doi: 10.1093/biomethods/bpaf011. eCollection 2025.

PfFBXO1 is essential for inner membrane complex formation in Plasmodium falciparum during both asexual and transmission stages

Donna Huber on February 7, 2025

PfFBXO1 localization by immunofluorescence stained with anti-V5 (PfFBXO1)

Plasmodium species replicate via schizogony, which involves asynchronous nuclear divisions followed by semi-synchronous segmentation and cytokinesis. Successful segmentation requires a double-membranous structure known as the inner membrane complex (IMC). Here we demonstrate that PfFBXO1 (PF3D7_0619700) is critical for both asexual segmentation and gametocyte maturation. In Toxoplasma gondii, the FBXO1 homolog, TgFBXO1, is essential for the development of the daughter cell scaffold and a component of the daughter cell IMC. We demonstrate PfFBXO1 forming a similar IMC initiation scaffold near the apical region of developing merozoites and unilaterally positioned in gametocytes of P. falciparum. While PfFBXO1 initially localizes to the apical region of dividing parasites, it displays an IMC-like localization as segmentation progresses. Similarly, PfFBXO1 localizes to the IMC region in gametocytes. Following inducible knockout of PfFBXO1, parasites undergo abnormal segmentation and karyokinesis, generating inviable daughters. PfFBXO1-deficient gametocytes are abnormally shaped and fail to fully mature. Proteomic analysis identified PfSKP1 as one of PfBXO1’s stable interacting partners, while other major proteins included multiple IMC pellicle and membrane proteins. We hypothesize that PfFBXO1 is necessary for IMC biogenesis, chromosomal maintenance, vesicular transport, and ubiquitin-mediated translational regulation of proteins in both sexual and asexual stages of P. falciparum.

Sreelakshmi K Sreenivasamurthy, Carlos Gustavo Baptista, Christopher M West, Ira J Blader, Jeffrey D Dvorin. Commun Biol. 2025 Feb 7;8(1):190. doi: 10.1038/s42003-025-07619-6.

mSphere of Influence: Lighting up organellar communication in protozoan parasites

Donna Huber on February 6, 2025

Diego Huet works in molecular parasitology, focusing on the organellar biology of Toxoplasma gondii. In this mSphere of Influence article, he reflects on how the article “Efficient proximity labeling in living cells and organisms with turboID” (Branon et al., 2018) impacted his research and the strategies used to dissect inter-organellar interactions in T. gondii.

Diego Huet. mSphere. 2025 Feb 6:e0057424. doi: 10.1128/msphere.00574-24.

Novel antibodies detect nucleocytoplasmic O-fucose in protist pathogens, cellular slime molds, and plants

Donna Huber on February 6, 2025

Cellular adaptations to change often involve post-translational modifications of nuclear and cytoplasmic proteins. An example found in protists and plants is the modification of serine and threonine residues of dozens to hundreds of nucleocytoplasmic proteins with a single fucose (O-fucose). A nucleocytoplasmic O-fucosyltransferase occurs in the pathogen Toxoplasma gondii, the social amoeba Dictyostelium, and higher plants, where it is called Spy because mutants have a spindly appearance. O-fucosylation, which is required for optimal proliferation of Toxoplasma and Dictyostelium, is paralogous to the O-GlcNAcylation of nucleocytoplasmic proteins of plants and animals that are involved in stress and nutritional responses. O-fucose was first discovered in Toxoplasma using Aleuria aurantia lectin, but its broad specificity for terminal fucose residues on N- and O-linked glycans in the secretory pathway limits its use. Here we present affinity-purified rabbit antisera that are selective for the detection and enrichment of proteins bearing fucose-O-Ser or fucose-O-Thr. These antibodies detect numerous nucleocytoplasmic proteins in Toxoplasma, Dictyostelium, and Arabidopsis, as well as O-fucose occurring on secretory proteins of Dictyostelium and mammalian cells except when blocked by further glycosylation. The antibodies label Toxoplasma, Acanthamoeba, and Dictyostelium in a pattern reminiscent of O-GlcNAc in animal cells including nuclear pores. The O-fucome of Dictyostelium is partially conserved with that of Toxoplasma and is highly induced during starvation-induced development. These antisera demonstrate the unique antigenicity of O-fucose, document the conservation of the O-fucome among unrelated protists, and enable the study of the O-fucomes of other organisms possessing O-fucosyltransferase-like genes.IMPORTANCEO-fucose (O-Fuc), a form of mono-glycosylation on serine and threonine residues of nuclear and cytoplasmic proteins of some parasites, other unicellular eukaryotes, and plants, is understudied because it is difficult to detect owing to its neutral charge and lability during mass spectrometry. Yet, the O-fucosyltransferase enzyme (OFT) is required for optimal growth of the agent for toxoplasmosis, Toxoplasma gondii, and an unrelated protist, the social amoeba Dictyostelium discoideum. Furthermore, O-fucosylation is closely related to the analogous process of O-GlcNAcylation of thousands of proteins of animal cells, where it plays a central role in stress and nutritional responses. O-Fuc is currently best detected using Aleuria aurantia lectin (AAL), but in most organisms, AAL also recognizes a multitude of proteins in the secretory pathway that are modified with fucose in different ways. By establishing the potential to induce highly specific rabbit antisera that discriminate O-Fuc from all other forms of protein fucosylation, this study expands knowledge about the protist O-fucome and opens a gateway to explore the potential occurrence and roles of this intriguing posttranslational modification in bacteria and other protist pathogens such as Acanthamoeba castellanii.

Megna Tiwari, Elisabet Gas-Pascual, Manish Goyal, Marla Popov, Kenjiroo Matsumoto, Marianne Grafe, Ralph Gräf, Robert S Haltiwanger, Neil Olszewski, Ron Orlando, John C Samuelson, Christopher M West. mSphere. 2025 Feb 6:e0094524. doi: 10.1128/msphere.00945-24.

Toxoplasma chitinase-like protein orchestrates cyst wall glycosylation to facilitate effector export and cyst turnover

Donna Huber on January 29, 2025

TgCLP1 is a microneme protein that is secreted and targeted to the cyst wall.

Toxoplasma bradyzoites reside in tissue cysts that undergo cycles of expansion, rupture, and release to foster chronic infection. The glycosylated cyst wall acts as a protective barrier, although the processes responsible for formation, remodeling, and turnover are not understood. Herein, we identify a noncanonical chitinase-like enzyme TgCLP1 that localizes to micronemes and is targeted to the cyst wall after secretion. Genetic deletion of TgCLP1 resulted in a thickened cyst wall that decreased cyst turnover, blocked the export of virulence effectors into host cells, and resulted in failure to persist during chronic infection. Genetic complementation with a series of mutants revealed that the GH19 glycosidase domain was crucial for regulating glycosylation of several glycoproteins in the cyst wall. Overall, our findings reveal that TgCLP1 is a multifunctional survival factor that modifies glycoproteins within the cyst wall to modulate export of virulence effectors and regulate turnover of tissue cysts.

Yong Fu, Tadakimi Tomita, Louis M Weiss, Christopher M West, L David Sibley. Proc Natl Acad Sci U S A. 2025 Feb 4;122(5):e2416870122. doi: 10.1073/pnas.2416870122.

Stereospecific Resistance to N2-Acyl Tetrahydro-β-carboline Antimalarials Is Mediated by a PfMDR1 Mutation That Confers Collateral Drug Sensitivity

Donna Huber on January 14, 2025

Half the world’s population is at risk of developing a malaria infection, which is caused by parasites of the genus Plasmodium. Currently, resistance has been identified to all clinically available antimalarials, highlighting an urgent need to develop novel compounds and better understand common mechanisms of resistance. We previously identified a novel tetrahydro-β-carboline compound, PRC1590, which potently kills the malaria parasite. To better understand its mechanism of action, we selected for and characterized resistance to PRC1590 in Plasmodium falciparum. Through in vitro selection of resistance to PRC1590, we have identified that a single-nucleotide polymorphism on the parasite’s multidrug resistance protein 1 (PfMDR1 G293V) mediates resistance to PRC1590. This mutation results in stereospecific resistance and sensitizes parasites to other antimalarials, such as mefloquine, quinine, and MMV019017. Intraerythrocytic asexual stage specificity assays have revealed that PRC1590 is most potent during the trophozoite stage when the parasite forms a single digestive vacuole (DV) and actively digests hemoglobin. Moreover, fluorescence microscopy revealed that PRC1590 disrupts the function of the DV, indicating a potential molecular target associated with this organelle. Our findings mark a significant step in understanding the mechanism of resistance and the mode of action of this emerging class of antimalarials. In addition, our results suggest a potential link between resistance mediated by PfMDR1 and PRC1590’s molecular target. This research underscores the pressing need for future research aimed at investigating the intricate relationship between a compound’s chemical scaffold, molecular target, and resistance mutations associated with PfMDR1.

Emily K Bremers, Joshua H Butler, Leticia S Do Amaral, Emilio F Merino, Hanan Almolhim, Bo Zhou, Rodrigo P Baptista, Maxim Totrov, Paul R Carlier, Maria Belen Cassera. ACS Infect Dis. 2025 Jan 14. doi: 10.1021/acsinfecdis.4c01001.

The Life & Times of the SchistoKid – People, Parasites, and Plagues podcast

Donna Huber on January 2, 2025

Professor Emeritus Daniel Colley is the featured guest on the People, Parasites, and Plagues podcast. Learn more about his time spent with the CDC, becoming the director of the Center for Tropical and Emerging Global Diseases at UGA, and the research abroad that sparked his passion for schistosomes in this episode.

Mono-allelic epigenetic regulation of polycistronic transcription initiation by RNA polymerase II in Trypanosoma brucei

Donna Huber on December 20, 2024

TLF resistance correlates with HpHbR allelic expression

Unique for a eukaryote, protein-coding genes in trypanosomes are arranged in polycistronic transcription units (PTUs). This genome arrangement has led to a model where Pol II transcription of PTUs is unregulated and changes in gene expression are entirely post-transcriptional. Trypanosoma brucei brucei is unable to infect humans because of its susceptibility to an innate immune complex, trypanosome lytic factor (TLF) in the circulation of humans. The initial step in TLF-mediated lysis of T.b.brucei requires high affinity haptoglobin/hemoglobin receptor (HpHbR) binding. Here, we demonstrate that by in vitro selection with TLF, resistance is obtained in a stepwise process correlating with loss of HpHbR expression at an allelic level. RNA-seq, Pol II ChIP, and run-on analysis indicate HpHbR silencing is at the transcriptional level, where loss of Pol II binding at the promoter region specifically shuts down transcription of the HpHbR-containing gene cluster and the adjacent opposing gene cluster. Reversible transcriptional silencing of the divergent PTUs correlates with DNA base J modification of the shared promoter region. Base J function in establishing transcriptional silencing, rather than maintenance, is suggested by the maintenance of PTU silencing following the inhibition of J-biosynthesis and subsequent loss of the modified DNA base. Therefore, we show that epigenetic mechanisms exist to regulate gene expression via Pol II transcription initiation of gene clusters in a mono-allelic fashion. These findings suggest epigenetic chromatin-based regulation of gene expression is deeply conserved among eukaryotes, including early divergent eukaryotes that rely on polycistronic transcription.IMPORTANCEThe single-cell parasite Trypanosoma brucei causes lethal diseases in both humans and livestock. T. brucei undergoes multiple developmental changes to adapt in different environments during its digenetic life cycle. With protein-coding genes organized as polycistronic transcription and apparent absence of promoter-mediated regulation of transcription initiation, it is believed that developmental gene regulation in trypanosomes is essentially post-transcriptional. In this study, we found reversible Pol II transcriptional silencing of two adjacent polycistronic gene arrays that correlate with the novel DNA base J modification of the shared promoter region. Our findings support epigenetic regulation of Pol II transcription initiation as a viable mechanism of gene expression control in T. brucei. This has implications for our understanding how trypanosomes utilize polycistronic genome organization to regulate gene expression during its life cycle.

Rudo Kieft, Laura Cliffe, Haidong Yan, Robert J Schmitz, Stephen L Hajduk, Robert Sabatini. mBio. 2024 Dec 20:e0232824. doi: 10.1128/mbio.02328-24.

Author: Donna Huber