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Author: Donna Huber

The Human Milk Oligosaccharide Lacto-N-Fucopentaose III Conjugated to Dextran Inhibits HIV Replication in Primary Human Macrophages

Donna Huber on March 12, 2025

LNFPIII inhibits HIV replication in primary human macrophages: Macrophages were infected with HIV at MOI 0.01 for 5 h, followed by washing with PBS to remove unbound virions. Fresh medium was added.

Background/Objectives: Individuals with HIV on combined antiretroviral therapy (ART) with virologic suppression exhibit chronic immune activation and immune dysfunction. Numerous studies have shown that human milk oligosaccharide (HMO) controls the postnatal transmission of HIV-1, but its effect on adult HIV-1 infection is not known. The purpose of this study was to investigate the anti-HIV activity of Lacto-N-fucopentaose III (LNFPIII) in adult blood-borne macrophages. Methods: Primary human monocyte-derived macrophages from the blood of HIV-seronegative individuals were infected with HIV and treated with or without dextran-conjugated LNFPIII (P3DEX). HIV replication was measured by quantifying the accumulation of HIV Gag p24 in the culture supernatants by ELISA. The quantities of chemokines MIP-1α, MIP-1β, and CCL5 in the culture supernatant were also measured by ELISA. The expression of IL-1β, IL-18, TNFα, IL-10, BECN1, and housekeeping gene HuPO in the macrophages was determined by qRT PCR. The expression of NF-kB, LC3, p62, and β-actin was measured by immunoblotting. Results: We found that P3DEX controls HIV replication without affecting HIV binding and/or internalization by human macrophages. The treatment of HIV-infected macrophages with P3DEX increased the quantity of beta (β)-chemokines MIP-1α, CCL5, and MIP-1β, which are known to have anti-HIV activity. Furthermore, the treatment of HIV-infected macrophages with P3DEX increased autophagic flux in a TLR8-dependent manner and ameliorated the expression of proinflammatory cytokines. These results suggest that P3DEX is a prominent milk-derived sugar that simultaneously augments anti-viral mechanisms and controls immune activation. These findings prudently justify the use and clinical development of P3DEX as a host-directed therapeutic option for people living with HIV.

Tablow Shwan Media, Medhini Ramesh, Olivia Isa Lee, Lucy Njideka Ubaka, Donald A Harn, Thomas Norberg, Frederick Quinn, Ankita Garg. Nutrients. 2025 Mar 2;17(5):890. doi: 10.3390/nu17050890.

Stable colonization of the model kissing bug Rhodnius prolixus by Trypanosoma cruzi Y strain

Donna Huber on March 12, 2025

The Y strain of Trypanosoma cruzi stably infects the vector Rhodnius prolixus.

Trypanosoma cruzi is a single-celled eukaryotic parasite responsible for Chagas disease, a major cause of morbidity and mortality in Central and South America. While the host-pathogen interactions of T. cruzi have been extensively studied in vertebrate models, investigations into its interactions within its insect host remain limited. To address this gap and establish a genetically tractable system for studying parasite-vector dynamics, we conducted quantitative kinetic infection studies using the Y strain of T. cruzi and the model vector Rhodnius prolixus. We began by comparing parasite infection kinetics from two genetically diverse strains of T. cruzi, Brazil and Y, and demonstrated that ingested parasites from both strains transiently expand in the anterior regions of the insect digestive tract with stable colonization occurring in the hindgut over the long term. Notably, we demonstrated that the clonal Y strain, contrary to previous reports, can effectively infect and persist across multiple developmental stages of R. prolixus. Additionally, comparison of movement of parasites versus inert fluorescent microspheres introduced into artificial blood meals suggests that T. cruzi colonization of the R. prolixus gut occurs passively through peristaltic movement during digestion, rather than through active parasite-mediated chemotaxis. These findings highlight the T. cruzi Y strain – R. prolixus model system as a promising tool for the in-depth molecular characterization of parasite-vector interactions, potentially offering new insights into the biology of this neglected and deadly human pathogen.

Ruby E Harrison, Kevin J Vogel, Ronald Drew Etheridge. PLoS Negl Trop Dis. 2025 Mar 12;19(3):e0012906. doi: 10.1371/journal.pntd.0012906.

Jessica Kissinger named 2025 University Professor

Donna Huber on March 4, 2025

photo of Jessica KissingerCTEGD faculty member Jessica Kissinger earned the distinction of University Professor, a title bestowed on those who have made a significant impact on the university in addition to fulfilling their regular academic responsibilities.

An integral voice on the university’s 2020 and 2025 strategic planning committees, Kissinger has championed initiatives designed to move UGA into the ranks of the world’s elite research universities. The recommendations developed by Kissinger and her colleagues helped lay the groundwork for strategic faculty hiring initiatives that have attracted leading researchers and scholars to UGA and new programs that have increased graduate student enrollment and support for graduate students.

“In a nutshell, I am a strategist and problem solver with a vision, who has worked hard to make UGA better for all,” she said.

Kissinger’s impact on the university includes her service as a member of the university’s Goldwater Selection Committee since 2015, and as a standing member of the Committee for Fellowships and Awards in the Franklin College of Arts and Sciences. She has also provided valuable guidance to senior leadership as a member of the Provost’s Working Group on Centers and Institutes and the Digital Infrastructure Development Committee.

Kissinger is a founding member of UGA’s Institute of Bioinformatics, a group dedicated to facilitating interdisciplinary research in bioinformatics and computational biology and its applications. Under her leadership as director from 2011-2019, the institute grew to include faculty from four colleges and more than 45 graduate students. The institute unifies the exploration of genomics and bioinformatics on campus and provides graduate training in a setting that melds the two disciplines unlike many bioinformatics programs in the U.S.

The success of the Georgia Advanced Computing Resource Center, a high-performance computing and networking infrastructure for UGA researchers, can also be linked to Kissinger’s leadership. She was part of a team that established UGA’s first centralized high-performance computing cluster, and she was a tireless advocate for expanding these resources for researchers across campus. Additionally, she encouraged the GACRC to provide centralized storage and she supported the early adoption of graphical processing units, or GPUs, the driving computational power behind artificial intelligence computing.

Kissinger has been recognized many times for research and leadership. She is a recipient of the Creative Research Medal, the Lamar Dodd Creative Research Award, the Faculty Excellence in Diversity Leadership Award and the Richard F. Reiff Internationalization Award, all presented by UGA. She is a fellow of the American Association for the Advancement of Science and the American Society for Tropical Medicine and Hygiene. Most recently, she was awarded a Fulbright U.S. Scholar award to teach and conduct research at Makerere University in Uganda.

University Professors receive a permanent salary increase of $10,000 and a yearly academic support of $5,000. Nominations from the deans of UGA’s schools and colleges are reviewed by a committee, which makes a recommendation to the provost.

Full story is available at https://news.uga.edu/carmichael-kissinger-named-2025-university-professors/

The contribution of the Golgi and the endoplasmic reticulum to calcium and pH homeostasis in Toxoplasma gondii

Donna Huber on March 3, 2025

TgCAXL1 localizes to the Golgi apparatus and the endoplasmic reticulum.

The cytosolic Ca2+ concentration of all cells is highly regulated demanding the coordinated operation of Ca2+ pumps, channels, exchangers and binding proteins. In the protozoan parasite Toxoplasma gondii calcium homeostasis, essential for signaling, governs critical virulence traits. However, the identity of most molecular players involved in signaling and homeostasis in T. gondii are unknown or poorly characterized. In this work we studied a putative calcium proton exchanger, TgGT1_319550 (TgCAXL1), which belongs to a family of Ca2+/proton exchangers that localize to the Golgi apparatus. We localized TgCAXL1 to the Golgi and the endoplasmic reticulum (ER) of T. gondii and validated its role as a Ca2+/proton exchanger by yeast complementation. Characterization of a knock-out mutant for TgCAXL1 (Δcaxl) underscored the role of TgCAXL1 in Ca2+ storage by the ER and acidic stores, most likely the Golgi. Most interestingly, TgCAXL1 function is linked to the Ca2+ pumping activity of the Sarcoplasmic Reticulum Ca2+-ATPase (TgSERCA). TgCAXL1 functions in cytosolic pH regulation and recovery from acidic stress. Our data showed for the first time the role of the Golgi in storing and modulating Ca2+ signaling in T. gondii and the potential link between pH regulation and TgSERCA activity, which is essential for filling intracellular stores with Ca2+.

Abigail Calixto, Katherine Moen, Silvia Nj Moreno. J Biol Chem.. 2025 Mar 3:108372. doi: 10.1016/j.jbc.2025.108372.

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
Diego Huet, assistant professor in the College of Pharmacy and the Center for Tropical & Emerging Global Diseases, studies parasites that cause disease in both humans and animals. His lab has ramped up a project to better understand the biology of Toxoplasma gondii , an organism carried by cats that is related to the parasite that causes malaria. (Photo by Lauren Corcino)

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

Fig 1 Anti-fucopeptide antisera.

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.