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Activity of Antibacterial/Antifungal Compounds against the Protozoan Parasite, Toxoplasma gondii

Donna Huber on August 28, 2025

We investigated the antiparasitic activity of several antimicrobial drug leads against Toxoplasma gondii tachyzoites and, in one case, bradyzoites. Carbazole and phenylthiazole aminoguanidine anti-infectives, originally developed as antibacterial and antifungal agents, showed potent activity, with IC₅₀ values as low as 2 μM. This potency was comparable to that observed with the tuberculosis drug candidate SQ109 and a series of its analogs. Notably, SQ109 also significantly reduced the viability of in vivo-derived bradyzoites. All compounds acted, at least in part, as protonophore uncouplers by collapsing the ΔpH component of the proton motive force. Furthermore, SQ109 and the tetrahydrocarbazole (THCz) compounds disrupted the mitochondrial membrane potential in T. gondii tachyzoites. While SQ109 is known to activate macrophages to an M1 phenotype, we observed no significant difference in its activity against T. gondii grown in fibroblasts versus macrophages, likely due to the parasite’s residence within the protective parasitophorous vacuole. We also examined correlations between compound activity against the yeast Saccharomyces cerevisiae, and the bacterium Mycobacterium smegmatis, finding significant correlations between the collapse of the proton motive force and antiproliferative activity. Taken together, our findings underscore the potential of these antimicrobial agents as promising leads for the development of new antiparasitic therapies against T. gondii.

Davinder Singh, Melissa A Sleda, Satish R Malwal, Akanksha M Pandey, Yiyuan Chen, Ruijie Zhou, Feyisola Adewole, Katie Sadowska, Oluseye K Onajole, Silvia N J Moreno, Eric Oldfield. ACS Infect Dis. 2025 Aug 28. doi: 10.1021/acsinfecdis.5c00609.

Benzo-ring modification on Malaria Box hit MMV008138: effects on antimalarial potency and microsomal stability

Donna Huber on August 15, 2025

Tetrahydro-β-carboline 1 (MMV008138) controls growth of asexual blood-stage Plasmodium falciparum by inhibiting IspD, an enzyme in the MEP pathway for synthesis of a critical metabolite, isopentenyl pyrophosphate (IPP). We have previously investigated the structure activity relationship (SAR) of three of its four rings (B, C, and D). In this report we investigate the SAR of the benzo- (i.e. A-ring) of 1, with the goal of increasing its in vitro antimalarial potency and metabolic stability. As in our previous studies of the B- and C-ring substitution, extreme sensitivity to substitution was also seen in the benzo-ring. In total, 19 benzo-ring substitution variants of 1 were prepared. When tested against multidrug-resistant (Dd2 strain) P. falciparum, only three derivatives (20a, c, d) possessed asexual blood stage (ABS) activity with EC50 values within 3-fold of the parent. As hoped, one analog (20c) showed a marked improvement in microsomal stability. However, this improvement unfortunately did not improve plasma exposure relative to 1, and did not lead to oral efficacy in a mouse model of malaria.

Maryam Ghavami, Haibo Li, Lixuan Liu, Joshua H Butler, Sha Ding, Grant J Butschek, Reagan S Haney, R McAlister Council-Troche, R Justin Grams, Emilio F Merino, Jennifer M Davis, Maxim Totrov, Maria B Cassera, Paul R Carlier. RSC Med Chem. 2025 Aug 15. doi: 10.1039/d5md00439j.

Antimalarial spirooxindole alkaloids with a rare 6/5/5/6/6 polycyclic skeleton from the fungus Penicillium citrinum YSC-1 isolated from a medicinal plant

Donna Huber on August 5, 2025

OSMAC (one strain many compounds) provides a convenient strategy to produce chemically diverse and novel natural products. In the study, the application of an OSMAC approach on a fungus Penicillium citrinum YSC-1 isolated from a medicinal plant Chloranthus japonicus, using different culture media, led to the isolation and identification of seven new spirooxindole alkaloids penicitrimicins A-G (1-7) with a rare 6/5/5/6/6 polycyclic skeleton, along with two known compounds (8-9). The new structures were characterized based on the comprehensive spectroscopic analyses, including 1D, 2D NMR and HRESIMS data. The absolute configurations of compounds 1-7 were determined by modified Mosher ester methodology, PGME derivatization, X-ray crystallographic analysis, and quantum chemical calculations. Biological evaluation revealed that these spirooxindole alkaloids exhibited good biocompatibility (<5 % hemolysis and > 80 % cell viability) while displaying obvious antimalarial activity against Plasmodium falciparum Dd2 strain, with EC50 values spanning 0.9-2.4 μM. Furthermore, stage-specific assays revealed that compound 5 displayed significant inhibitory effects on the developmental transition of asexual blood-stage parasites, effectively blocking their progression to subsequent lifecycle stages.

Pei-Qian Wu, Jun-Su Zhou, Leticia S Do Amaral, Maria B Cassera, Jian-Min Yue, Bin Zhou. Bioorg Chem. 2025 Aug 5:164:108825. doi: 10.1016/j.bioorg.2025.108825.

Gene regulation in Cryptosporidium: New insights and unanswered questions

Donna Huber on July 15, 2025

Parasites of the genus Cryptosporidium have evolved to have a highly compact genome of ∼9.1 Mb. The mechanisms that regulate gene expression in Cryptosporidium spp. remain incompletely understood at all levels, including chromatin accessibility, transcription factor activation and repression and RNA processing. This review discusses possible mechanisms of gene regulation in Cryptosporidium spp., including histone modifications, cis regulatory elements, transcription factors and non-coding RNAs. Cryptosporidium spp. are among the most basal branching apicomplexans and existing evidence suggests that they diverge from other members of their phylum via retention of the E2F/DP1 transcription factor family, and the recent discovery that C. parvum produces polycistronic transcripts. Most of what we know about gene regulation in the genus Cryptosporidium is based on sequence conservation and homology with other members of the phylum Apicomplexa, and in some cases, more distant eukaryotes. Very few putative gene regulatory components identified in Cryptosporidium spp. are supported by experimental confirmation. This review summarizes what we know about gene regulation in Cryptosporidium spp. and identifies gaps in our current understanding.

Samantha Gunasekera, Jessica C Kissinger. Curr Res Parasitol Vector Borne Dis. 2025 Jun 17:8:100280. doi: 10.1016/j.crpvbd.2025.100280. eCollection 2025.

New T2T assembly of Cryptosporidium parvum IOWA II annotated with Legacy-Compatible Gene identifiers

Donna Huber on June 19, 2025

FIgure 1 Amino acid alignment between GP60 sequences from all four C. parvum IOWA II genome assemblies analyzed. — Amino acid alignment between GP60 sequences from all four C. parvum IOWA II genome assemblies analyzed.

Cryptosporidium parvum is a significant pathogen causing gastrointestinal infections in humans and animals. It is spread through ingesting contaminated food and water. Despite its global health significance, generating a C. parvum genome sequence has been challenging for many reasons including cloning and challenging subtelomeric regions. A new, gapless, hybrid, telomere-to-telomere genome assembly was created for C. parvum IOWA II, here termed CpBGF. It reveals 8 chromosomes, a genome size of 9,259,183 bp, and resolves complex subtelomeric regions. To facilitate ease of use and consistency with the literature, the chromosomes have been oriented, and genes in this annotation have been given similar gene IDs as those used in the 2004, C. parvum IOWA II reference genome sequence. The new annotation utilized considerable RNA expression evidence including single-molecule Iso-Seq data; thus, untranslated regions, long noncoding RNAs, and antisense RNAs are annotated. The CpBGF genome assembly serves as a valuable resource for understanding the biology, pathogenesis, and transmission of C. parvum, and it facilitates the development of diagnostics, drugs, and vaccines against cryptosporidiosis.

Rodrigo de Paula Baptista, Rui Xiao, Yiran Li, Travis C Glenn, Jessica C Kissinger. Sci Data. 2025 Jun 19;12(1):1039. doi: 10.1038/s41597-025-05364-3.

Highly cross-reactive and competent effectors dominate the CD8+ T cell response in Trypanosoma cruzi infection

Donna Huber on June 16, 2025

T. cruzi-specific CD8+ T cells cross-react with multiple flagellar peptides.

CD8+ T cells are key effectors in immune control of Trypanosoma cruzi infection. Within C57BL/6 mice, the T. cruzi-specific CD8+ T cell response is largely comprised of T cells recognizing trans-sialidase (TS)- and mucin-encoded epitopes. Despite their immunodominance, these epitope-specific CD8+ T cells are entirely dispensable for immune control. In a screen for epitopes inducing “protective” CD8+ T cells, we uncovered a high level of cross-reactivity within the TSKb20-specific CD8+ T cell response. This cross-reactivity was driven by the TSKb20 epitope itself and not the infection. TCR sequencing defined key characteristics of the TSKb20 repertoire including biased TRBV12-1/12-2 and TRBJ2-1/2-7 gene usage and a highly dominant CDR3β motif. The dispensability of the TSKb20 response in the control of T. cruzi infection along with the broad reactivity of this T cell population prompted us to assess the relative effector capacity of TSKb20 T cells at the site of reinfection. Similar to other activated CD8+ T cells at the site, TSKb20-specific CD8+ T cells expressed transcriptional patterns associated with effector function, suggesting that TSKb20 T cells are capable and likely participants in parasite control. These results indicate that broad TCR reactivity does not compromise the ability of TSKb20-specific T cells to develop into phenotypically functional effectors. Additionally, the failure to identify individual parasite epitopes capable of driving a protective CD8+ T cell response challenges the paradigm that individual T. cruzi epitopes, including highly immunodominant ones, are critical to or exploitable for the potent recognition of T. cruzi-infected host cells and infection control.

Molly E Bunkofske, Pradyot Dash, Walid Awad, Paul G Thomas, Rick L Tarleton. J Immunol. 2025 Jun 16:vkaf107. doi: 10.1093/jimmun/vkaf107.

From Target Product Profiles (TPPs) to Target Specimen Profiles (TSPs): A New Concept in Infectious Disease Biobanking for Diagnostic Applications

Donna Huber on June 13, 2025

A target specimen profile (TSP) corresponds to the required characteristics of the specimen panels needed to demonstrate that a diagnostic kit meets the target product profile (TPP). TSPs can guide biobanks in the prospective collection of sample panels to support the development and validation of diagnostics.

Fay Betsou, Warren Fransman, Patrick Lammie. Diagnostics (Basel). 2025 Jun 13;15(12):1503. doi: 10.3390/diagnostics15121503.

Protocol for laboratory rearing and infection tracking of Rhodnius prolixus using 3D-printable designs

Donna Huber on June 13, 2025

Human infections by Trypanosoma cruzi propagate via its blood-feeding triatomine vector. Investigating parasite-vector interactions depends upon robust techniques to rear insects and analyze infections. Here, we present a protocol for laboratory rearing and infection tracking of Rhodnius prolixus. We describe steps for housing, feeding, and sorting strategies using 3D-printable designs. We also detail procedures for gut dissection, fecal collection, and parasite re-isolation. This protocol describes techniques that support efforts to understand and mitigate vector-mediated Chagas disease transmission.

Ruby E Harrison, Ronald Drew Etheridge. STAR Protoc. 2025 Jun 13;6(3):103894. doi: 10.1016/j.xpro.2025.103894.

The importance of persistence and dormancy in Trypanosoma cruzi infection and Chagas disease

Donna Huber on June 5, 2025

Dormant and metabolically active T. cruzi amastigotes.

Trypanosoma cruzi typically establishes a life-long infection in its mammalian hosts, causing the destruction of muscle tissues and ultimately resulting in potentially fatal Chagas disease. In this review, we consider the array of avoidance mechanisms that allow for T. cruzi persistence, many of which are unconventional among protozoan pathogens but which collectively are highly effective in the face of otherwise potent host immune responses. We also reflect on the phenomenon of dormancy in T. cruzi amastigotes, which is likely not involved in the long-term persistence of infection. Lastly, we consider how these phenomena of persistence and dormancy complicate the effectiveness of potential therapeutic interventions to prevent Chagas disease.

Molly E Bunkofske, Fernando J Sanchez-Valdez, Rick L Tarleton. Curr Opin Microbiol. 2025 Jun 5:86:102615. doi: 10.1016/j.mib.2025.102615.

Chemical and Genetic Validation of an Essential Calcium Entry Channel of Trypanosoma brucei as a Therapeutic Target

Donna Huber on June 3, 2025

The Trypanosoma brucei group of parasites causes Nagana in cattle and human African trypanosomiasis, or sleeping sickness, in humans. Current drugs against these parasites have severe toxicity, vaccines are not available, and development of drug resistance makes finding new chemotherapeutic targets imperative. Ion channels, which are involved in several biological processes, are targets of many therapeutically useful agents, and they remain significantly underexplored as therapeutic targets in parasites. Here, we report the presence of a voltage gated Ca²⁺ channel (VGCC, TbCa_v), which is localized in the flagellar plasma membrane (PM) of T. brucei and is essential for proliferation of both bloodstream (BSF) and procyclic forms (PCF) of the parasite. TbCa_V is a single subunit channel capable of transporting Ca²⁺ when expressed in mutant yeast lacking PM Ca²⁺ channels or in HEK293T cells. Through the virtual screening of a commercial chemical library using dynamic ensembles of various conformations of TbCa_v and associated docking analyses, several inhibitors of TbCa_v were discovered. As pharmacological validation of the essential roles of TbCa_v, these compounds were shown to inhibit T. brucei growth with the most potent agent, N-(7-nitro-2,1,3-benzoxadiazol-4-yl) acetamide (NBD-A), exhibiting an EC₅₀ of 25 ± 3 nM and no cytotoxicity in Vero cells possessing related channels. Thus, such studies constitute pharmacological validation of TbCa_v as a viable therapeutic target of T. brucei.

Guozhong Huang, Harmanpreet Singh, Priti Singh, Rohit Kumar Varshnaya, Donald Hamelberg, Binghe Wang, Roberto Docampo. ACS Infect Dis. 2025 Jun 3. doi: 10.1021/acsinfecdis.5c00329.

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