publications Archives - Center for Tropical and Emerging Global Diseases

Seeing the unseen: illuminating Toxoplasma gondii’s metabolic manipulation

Donna Huber on July 12, 2024

Intracellular infection by a pathogen induces significant rewiring of host cell signaling and biological processes. Understanding how an intracellular pathogen such as Toxoplasma gondii modulates host cell metabolism with single-cell resolution has been challenged by the variability of infection within cultures and difficulties in separating host and parasite metabolic processes. A new study from Gallego-Lopez and colleagues (G. M. Gallego-López, E. C. Guzman, D. E. Desa, L. J. Knoll, M. C. Skala, mBio e00727-24, 2024, https://doi.org/10.1128/mbio.00727-24) applies a quantitative imaging approach to evaluate the host cell metabolism during intracellular infection with Toxoplasma. This study provides important insights into host metabolic responses to Toxoplasma infection and offers a valuable tool to dissect the mechanisms underlying parasite infection and pathophysiology.

Diego Huet, Victoria Jeffers. mBio. 2024 Jul 12:e0121124. doi: 10.1128/mbio.01211-24.

Domestic Dog Infection with Trypanosoma cruzi from Northern and Southern Regions of Mexico

Donna Huber on July 1, 2024

Reactive StatPak immunochromatography test results from dogs sampled between December 2018 and October 2019 in Reynosa, Tamaulipas and Tuxtla Gutierrez, Chiapas, Mexico.

Background: Chagas disease or American trypanosomiasis, caused by Trypanosoma cruzi and vectored by triatomines, affects millions of people worldwide. In endemic countries including Mexico, infections in domestic animals, such as dogs, may affect the risk of human disease when they serve as a source of infection to vectors that subsequently infect humans. Materials and Methods: We conducted a cross-sectional study of 296 dogs from two cities near the northern and southern borders of Mexico: Reynosa, Tamaulipas, and Tuxtla Gutierrez, Chiapas. Infection was measured based on testing of blood using T. cruzi quantitative PCR (qPCR) and up to three antibody detection assays. The StatPak immunochromatographic assay was used to screen samples and the indirect fluorescent antibody (IFA) and multiplex microsphere immunoassay (MIA) tests were used as secondary tests on all samples that screened positive and a subset of negatives. Serologic positivity was defined based on reactivity on at least two independent tests. Results: Of the 280 samples tested for parasite DNA, two (0.7%) were positive, one of which (0.4%) was confirmed as T. cruzi discrete typing unit TcIV. Overall, 72 (24.3%) samples were reactive for T. cruzi antibodies via StatPak of which 8 were also positive using MIA and 2 were also positive using IFA (including one of the PCR-positive dogs). Overall, nine dogs (3.4%) met study criteria of positivity based on either/both serology or PCR tests. Positive dogs were found in both regions of Mexico; five (2.7%) from Reynosa and four (3.6%) from Tuxtla Gutierrez. We found no association between infection status and state of origin, sex, age group, breed group, neighborhood, and whether other pets lived in the home. Conclusion: Our results re-emphasize dogs’ utility as sentinels for T. cruzi in Mexico and underscore the need for improved veterinary diagnostic tests and parasite surveillance at the household level in endemic countries.

Edward Davila, Nadia A Fernandez-Santos, José Guillermo Estrada-Franco, Lihua Wei, Doireyner Daniel Velázquez-Ramírez, Rosario García-Miranda, Cesar Irecta Nájera, Raúl Cruz-Cadena, Carlos Guichard-Romero, Carlos Rodriguez, Rick Tarleton, Mario A Rodríguez-Pérez, Héctor Ochoa-Díaz-López, Gabriel L Hamer, Sarah A Hamer. Vector Borne Zoonotic Dis. 2024 Jul 1. doi: 10.1089/vbz.2023.0110

Reciprocal interactions between neuropeptide F and RYamide regulate host attraction in the mosquito Aedes aegypti

Donna Huber on July 1, 2024

NPF accumulates in EECs of the posterior midgut after adult emergence but rapidly depletes after blood feeding.

Female mosquitoes produce eggs in gonadotrophic cycles that are divided between a previtellogenic and vitellogenic phase. Previtellogenic females consume water and sugar sources like nectar while also being attracted to hosts for blood feeding. Consumption of a blood meal activates the vitellogenic phase, which produces mature eggs and suppresses host attraction. In this study, we tested the hypothesis that neuropeptide Y-like hormones differentially modulate host attraction behavior in the mosquito Aedes aegypti. A series of experiments collectively indicated that enteroendocrine cells (EECs) in the posterior midgut produce and release neuropeptide F (NPF) into the hemolymph during the previtellogenic phase which stimulates attraction to humans and biting behavior. Consumption of a blood meal, which primarily consists of protein by dry weight, down-regulated NPF in EECs until mature eggs developed, which was associated with a decline in hemolymph titer. NPF depletion depended on protein digestion but was not associated with EEC loss. Other experiments showed that neurons in the terminal ganglion extend axons to the posterior midgut and produce RYamide, which showed evidence of increased secretion into circulation after a blood meal. Injection of RYamide-1 and -2 into previtellogenic females suppressed host attraction, while coinjection of RYamides with or without short NPF-2 also inhibited the host attraction activity of NPF. Overall, our results identify NPF and RYamide as gut-associated hormones in A. aegypti that link host attraction behavior to shifts in diet during sequential gonadotrophic cycles.

Xiaoyi Dou, Kangkang Chen, Mark R Brown, Michael R Strand. Proc Natl Acad Sci U S A. 2024 Jul 9;121(28):e2408072121. doi: 10.1073/pnas.2408072121.

An Overview of Management Considerations for Mongolian Gerbils (Meriones unguiculatus), Cats (Felis catus), and Dogs (Canis familiaris) as Hosts for Brugia Infection

Donna Huber on June 26, 2024

Lymphatic filariasis is a mosquito-borne parasitic infection affecting an estimated 51.4 million people. Brugia malayi and Brugia pahangi are used in research because common nonprimate research species such as Mongolian gerbils (Meriones unguiculatus), cats (Felis catus), and dogs (Canis familiaris) can maintain the life cycle of these species of filarial nematodes. Although overall care and management of animals infected with Brugia spp. is relatively straightforward, there are some unique challenges and special considerations that must be addressed when managing a research colony infected with these parasites. In this review, we discuss our experience, share insight into biosafety and clinical management, and describe the expected clinical signs associated with Brugia infection in gerbils, cats, and dogs.

Catherine A Chambers, Christopher C Evans, Gianni A Campellone, Mary A McCrackin, Andrew R Moorhead, Leanne C Alworth. Comp Med. 2024 Jun 26. doi: 10.30802/AALAS-CM-24-034.

Optimization of diastereomeric dihydropyridines as antimalarials

Donna Huber on June 18, 2024

The increase in research funding for the development of antimalarials since 2000 has led to a surge of new chemotypes with potent antimalarial activity. High-throughput screens have delivered several thousand new active compounds in several hundred series, including the 4,7-diphenyl-1,4,5,6,7,8-hexahydroquinolines, hereafter termed dihydropyridines (DHPs). We optimized the DHPs for antimalarial activity. Structure-activity relationship studies focusing on the 2-, 3-, 4-, 6-, and 7-positions of the DHP core led to the identification of compounds potent (EC₅₀ < 10 nM) against all strains of P. falciparum tested, including the drug-resistant parasite strains K1, W2, and TM90-C2B. Evaluation of efficacy of several compounds in vivo identified two compounds that reduced parasitemia by >75 % in mice 6 days post-exposure following a single 50 mg/kg oral dose. Resistance acquisition experiments with a selected dihydropyridine led to the identification of a single mutation conveying resistance in the gene encoding for Plasmodium falciparum multi-drug resistance protein 1 (PfMDR1). The same dihydropyridine possessed transmission blocking activity. The DHPs have the potential for the development of novel antimalarial drug candidates.

Kurt S Van Horn, Yingzhao Zhao, Prakash T Parvatkar, Julie Maier, Tina Mutka, Alexis Lacrue, Fabian Brockmeier, Daniel Ebert, Wesley Wu, Debora R Casandra, Niranjan Namelikonda, Jeanine Yacoub, Martina Sigal, Spencer Knapp, David Floyd, David Waterson, Jeremy N Burrows, James Duffy, Joseph L DeRisi, Dennis E Kyle, R Kiplin Guy, Roman Manetsch. Eur J Med Chem. 2024 Jun 18:275:116599. doi: 10.1016/j.ejmech.2024.116599.

Evaluation of diagnostic techniques for early detection of heartworm in experimentally infected dogs: identification of Dirofilaria immitis-derived microRNA in the initial 28 weeks post-inoculation

Donna Huber on June 13, 2024

Background: Dirofilaria immitis, commonly known as heartworm (HW), is a parasitic nematode transmitted by various mosquito species, leading to heartworm disease (HWD) in dogs. Diagnosis of HW typically involves antigen or microfilariae detection, or visualization of adult worms through imaging or post mortem examination. Polymerase chain reaction (PCR) and micro RNA (miRNA) detection have been explored for HW diagnosis.

Methods: Three dogs, previously experimentally infected with HW, underwent blood sampling every 4 weeks for 7 months. Samples were assessed for antigen presence after heat treatment, PCR amplification, and microfilaria examination using Giemsa-stained thick smears. Additionally, whole blood aliquots underwent miRNA deep sequencing and bioinformatic analysis.

Results: Heartworm antigen was detectable after heat treatment at 20 weeks post-inoculation and via PCR at 24 weeks, with microfilariae observed in peripheral blood smears at 28 weeks. However, deep miRNA sequencing revealed that the miRNA candidate sequences are not consistently expressed before 28 weeks of infection.

Conclusions: While ancillary molecular methods such as PCR and miRNA sequencing may be less effective than antigen detection for detecting immature larval stages in an early stage of infection, our experimental findings demonstrate that circulating miRNAs can still be detected in 28 weeks post-infection.

Daniel Felipe Barrantes Murillo, Elyssa J Campbell, Andrew R Moorhead, Chengming Wang. Parasit Vectors. 2024 Jun 13;17(1):258. doi: 10.1186/s13071-024-06337-y.

The Toxoplasma oxygen-sensing protein, TgPhyA, is required for resistance to interferon gamma-mediated nutritional immunity in mice

Donna Huber on June 10, 2024

All FiguresNextPrevious
Fig 1. TgPHYa knockout in type II strain parasites. — Fig 1. TgPHYa knockout in type II strain parasites.

As Toxoplasma gondii disseminates through its host, the parasite must sense and adapt to its environment and scavenge nutrients. Oxygen (O2) is one such environmental factor and cytoplasmic prolyl 4-hydroxylases (PHDs) are evolutionarily conserved O2 cellular sensing proteins that regulate responses to changes in O2 availability. Toxoplasma expresses 2 PHDs. One of them, TgPHYa hydroxylates SKP1, a subunit of the SCF-E3 ubiquitin ligase complex. In vitro, TgPHYa is important for growth at low O2 levels. However, studies have yet to examine the role that TgPHYa or any other pathogen-encoded PHD plays in virulence and disease. Using a type II ME49 Toxoplasma TgPHYa knockout, we report that TgPHYa is important for Toxoplasma virulence and brain cyst formation in mice. We further find that while TgPHYa mutant parasites can establish an infection in the gut, they are unable to efficiently disseminate to peripheral tissues because the mutant parasites are unable to survive within recruited immune cells. Since this phenotype was abrogated in IFNγ knockout mice, we studied how TgPHYa mediates survival in IFNγ-treated cells. We find that TgPHYa is not required for release of parasite-encoded effectors into host cells that neutralize anti-parasitic processes induced by IFNγ. In contrast, we find that TgPHYa is required for the parasite to scavenge tryptophan, which is an amino acid whose levels are decreased after IFNγ up-regulates the tryptophan-catabolizing enzyme, indoleamine dioxygenase (IDO). We further find, relative to wild-type mice, that IDO knockout mice display increased morbidity when infected with TgPHYa knockout parasites. Together, these data identify the first parasite mechanism for evading IFNγ-induced nutritional immunity and highlight a novel role that oxygen-sensing proteins play in pathogen growth and virulence.

Charlotte Cordonnier, Msano Mandalasi, Jason Gigley, Elizabeth A Wohlfert, Christopher M West, Ira J Blader. PLoS Biol. 2024 Jun 10;22(6):e3002690. doi: 10.1371/journal.pbio.3002690.

The first Cryptosporidium meeting: a concerted effort to fight cryptosporidiosis

Donna Huber on June 7, 2024

The first biennial Cryptosporidium meeting was held on 10–12 March 2024 in Philadelphia, PA, USA. The organizers, Dr Boris Striepen and Dr Christopher Hunter, welcomed more than 130 attendees to the University of Pennsylvania School of Veterinary Medicine. The meeting opened with a panel discussion featuring a diverse group of researchers, clinicians, non-profit and industry partners who offered unique insights into the problems of cryptosporidiosis. Seven research themed sessions (‘Impact of cryptosporidiosis’, ‘Population genetics’, ‘Genomics and new tools for research and translation’, ‘Parasite cell and developmental biology’, ‘Host–parasite interaction and immunity’, ‘Cryptosporidium metabolism and emerging targets’, and ‘Immunity to Cryptosporidium and vaccines’), as well as two poster sessions completed the meeting. A farewell dinner in the domed Asia gallery of the Penn Museum was organized for all the attendees. The meeting was graciously supported by the Bill and Melinda Gates Foundation, the Burroughs Wellcome Fund, Novartis, Zoetis, and several centers and departments of the University of Pennsylvania. In this TrendsTalk, we invited the session chairs to highlight the innovative research and discoveries presented during the inaugural Cryptosporidium meeting.

Wes van Voorhis, Joyce Siwila, Jessica C Kissinger, Natalia Bayona Vásquez, Guy Robinson, Rodrigo Baptista, Asis Khan, Amandine Guérin, Yi-Wei Chang, Zannatun Noor, N Bishara Marzook, Sumiti Vinayak, Sam Arnold, Chelsea Marie, Robert K M Choy, Mattie C Pawlowic, Rajiv S Jumani. Trends Parasitol. 2024 Jun;40(6):431-438. doi: 10.1016/j.pt.2024.04.005

Cryptosporidium Genomics – Current Understanding, Advances, and Applications

Donna Huber on May 31, 2024

Genome assembly impacts annotation quality, gene family member estimates, and genetic variation analyses.

Purpose of review: Here we highlight the significant contribution that genomics-based approaches have had on the field of Cryptosporidium research and the insights these approaches have generated into Cryptosporidium biology and transmission.

Recent findings: There are advances in genomics, genetic manipulation, gene expression, and single-cell technologies. New and better genome sequences have revealed variable sub-telomeric gene families and genes under selection. RNA expression data now include single-cell and post-infection time points. These data have provided insights into the Cryptosporidium life cycle and host-pathogen interactions. Antisense and ncRNA transcripts are abundant. The critical role of the dsRNA virus is becoming apparent.

Summary: The community’s ability to identify genomic targets in the abundant, yet still lacking, collection of genomic data, combined with their increased ability to assess function via gene knock-out, is revolutionizing the field. Advances in the detection of virulence genes, surveillance, population genomics, recombination studies, and epigenetics are upon us.

Fiifi Agyabeng-Dadzie, Rui Xiao, Jessica C Kissinger. Curr Trop Med Rep. 2024;11(2):92-103. doi: 10.1007/s40475-024-00318-y.

The Toxoplasma gondii F-Box Protein L2 Functions as a Repressor of Stage Specific Gene Expression

Donna Huber on May 30, 2024

Fig 5. TgFBXL2 localizes to a perinucleolar compartment.

Toxoplasma gondii is a foodborne pathogen that can cause severe and life-threatening infections in fetuses and immunocompromised patients. Felids are its only definitive hosts, and a wide range of animals, including humans, serve as intermediate hosts. When the transmissible bradyzoite stage is orally ingested by felids, they transform into merozoites that expand asexually, ultimately generating millions of gametes for the parasite sexual cycle. However, bradyzoites in intermediate hosts differentiate exclusively to disease-causing tachyzoites, which rapidly disseminate throughout the host. Though tachyzoites are well-studied, the molecular mechanisms governing transitioning between developmental stages are poorly understood. Each parasite stage can be distinguished by a characteristic transcriptional signature, with one signature being repressed during the other stages. Switching between stages require substantial changes in the proteome, which is achieved in part by ubiquitination. F-box proteins mediate protein poly-ubiquitination by recruiting substrates to SKP1, Cullin-1, F-Box protein E3 ubiquitin ligase (SCF-E3) complexes. We have identified an F-box protein named Toxoplasma gondii F-Box Protein L2 (TgFBXL2), which localizes to distinct perinucleolar sites. TgFBXL2 is stably engaged in an SCF-E3 complex that is surprisingly also associated with a COP9 signalosome complex that negatively regulates SCF-E3 function. At the cellular level, TgFBXL2-depleted parasites are severely defective in centrosome replication and daughter cell development. Most remarkable, RNAseq data show that TgFBXL2 conditional depletion induces the expression of stage-specific genes including a a large cohort of genes necessary for sexual commitment. Together, these data suggest that TgFBXL2 is a latent guardian of stage specific gene expression in Toxoplasma and poised to remove conflicting proteins in response to an unknown trigger of development.

Carlos G Baptista, Sarah Hosking, Elisabet Gas-Pascual, Loic Ciampossine, Steven Abel, Mohamed-Ali Hakimi, Victoria Jeffers, Karine Le Roch, Christopher M West, Ira J Blader. PLoS Pathog. 2024 May 30;20(5):e1012269. doi: 10.1371/journal.ppat.1012269.

Category: publications