Pub Date : 2025-12-01Epub Date: 2025-09-19DOI: 10.1016/j.ijpddr.2025.100615
Yu Yu , Guo Huilin , Liujia Li , Guiquan Guan , Qiping Zhao , Shunhai Zhu , Jinwen Wang , Fanghe Zhao , Hui Dong , Hongyu Han
Coccidiosis, which is primarily caused by Eimeria spp., poses a persistent challenge to poultry health and production worldwide. The emergence and spread of drug-resistant strains have significantly compromised the efficacy of anticoccidial therapies. We previously used RNA-seq to demonstrate that ribosomal protein L27 is differentially expressed in drug-sensitive and maduramicin-resistant strains of E. tenella (EtRPL27). In the present study, an RT–qPCR analysis showed that its expression is stage-specific, with the highest levels in sporozoites and second-generation merozoites. Immunofluorescence revealed both the cytoplasmic and partial surface localization of EtRPL27. Notably, EtRPL27 transcript levels were significantly elevated in a maduramicin-resistant strain relative to the drug-sensitive strain. Functional assays showed that anti-EtRPL27 antibodies inhibited the invasion of sporozoites, whereas the transgenic overexpression of EtRPL27 enhanced both the invasion efficiency and pathogenicity of E. tenella in chickens. Importantly, EtRPL27 overexpression reduced its sensitivity to maduramicin, evident as increased oocyst output and a higher anticoccidial index. Resistance classification based on standard indices confirmed moderate-to-high resistance levels in the EtRPL27-overexpressing strain. These findings demonstrate that EtRPL27 is closely associated with both virulence and maduramicin resistance in E. tenella, indicating its potential utility as a molecular marker and therapeutic target.
{"title":"Ribosomal protein L27 contributes to virulence and maduramicin resistance in Eimeria tenella","authors":"Yu Yu , Guo Huilin , Liujia Li , Guiquan Guan , Qiping Zhao , Shunhai Zhu , Jinwen Wang , Fanghe Zhao , Hui Dong , Hongyu Han","doi":"10.1016/j.ijpddr.2025.100615","DOIUrl":"10.1016/j.ijpddr.2025.100615","url":null,"abstract":"<div><div>Coccidiosis, which is primarily caused by <em>Eimeria</em> spp., poses a persistent challenge to poultry health and production worldwide. The emergence and spread of drug-resistant strains have significantly compromised the efficacy of anticoccidial therapies. We previously used RNA-seq to demonstrate that ribosomal protein L27 is differentially expressed in drug-sensitive and maduramicin-resistant strains of <em>E. tenella</em> (<em>Et</em>RPL27). In the present study, an RT–qPCR analysis showed that its expression is stage-specific, with the highest levels in sporozoites and second-generation merozoites. Immunofluorescence revealed both the cytoplasmic and partial surface localization of <em>Et</em>RPL27. Notably, <em>Et</em>RPL27 transcript levels were significantly elevated in a maduramicin-resistant strain relative to the drug-sensitive strain. Functional assays showed that anti-<em>Et</em>RPL27 antibodies inhibited the invasion of sporozoites, whereas the transgenic overexpression of <em>Et</em>RPL27 enhanced both the invasion efficiency and pathogenicity of <em>E. tenella</em> in chickens. Importantly, <em>Et</em>RPL27 overexpression reduced its sensitivity to maduramicin, evident as increased oocyst output and a higher anticoccidial index. Resistance classification based on standard indices confirmed moderate-to-high resistance levels in the <em>Et</em>RPL27-overexpressing strain. These findings demonstrate that <em>Et</em>RPL27 is closely associated with both virulence and maduramicin resistance in <em>E. tenella,</em> indicating its potential utility as a molecular marker and therapeutic target.</div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"29 ","pages":"Article 100615"},"PeriodicalIF":3.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145154592","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2025-10-25DOI: 10.1016/j.ijpddr.2025.100623
Carlos Ospina , Tatiana Cáceres , Stivenn Gutiérrez , Luz Helena Patiño , Luis David Sáenz-Pérez , Karen Moreno Medina , Juan Carlos Villar , Juan David Ramírez
Chagas disease (CD), caused by the protozoan Trypanosoma cruzi, remains a major public health challenge due to limited treatment options, Benznidazole and Nifurtimox; which are associated with adverse effects and variable efficacy. The emergence of drug-resistant in T. cruzi strains, along with limited knowledge of the molecular mechanisms underlying resistance, hampers the development of more effective therapies. To explore these mechanisms, we performed a comparative transcriptomic analysis of two T. cruzi TcI strains: MG (naturally susceptible) and DA (naturally resistant) to Benznidazole. Parasites were cultured in LIT medium, and IC50 values were determined using the MTT assay. RNA was extracted and sequenced (RNA-seq), with reads aligned to a reference genome. Differential gene expressions were analyzed with DESeq2, functional enrichment through Gene Ontology (GO), and metabolic pathways were mapped via KAAS. The IC50 for Benznidazole in DA (28.92 μg/mL; 111.13 μM) was substantially higher than in MG (0.88 μg/mL; 3.39 μM), confirming differential susceptibility. DA showed 408 upregulated and 1515 downregulated genes, while MG had 153 upregulated and 866 downregulated (Log2FoldChange ≥ 2 or ≤ −2). GO analysis indicated divergent biological processes between strains: DA exhibited enrichment in electron transport and detoxification, while MG was enriched in DNA repair and energy metabolism. Metabolic mapping revealed significant differences in the pentose phosphate pathway, glycolysis/gluconeogenesis, and the tricarboxylic acid (TCA) cycle. Key genes potentially involved in resistance like prostaglandin F2α synthase, trypanothione synthase, thioredoxin, and prostaglandin F synthase were identified as candidate therapeutic targets. These findings suggest that Benznidazole resistance in T. cruzi involves multifactorial, strain-specific responses at the transcriptomic and metabolic levels. By analyzing naturally resistant and susceptible TcI strains of T. cruzi under identical experimental conditions, this study reveals strain-specific transcriptomic adaptations that have not been previously characterized in naturally resistant and susceptible populations. These findings expand our current understanding of intrinsic Benznidazole resistance in T. cruzi, moving beyond purely experimental models. Specifically, they highlight novel metabolic and redox pathways that could serve as therapeutic targets effective against diverse T. cruzi strains and Discrete Typing Units (DTUs).
{"title":"Comparative transcriptomics of naturally susceptible and resistant Trypanosoma cruzi strains in response to Benznidazole","authors":"Carlos Ospina , Tatiana Cáceres , Stivenn Gutiérrez , Luz Helena Patiño , Luis David Sáenz-Pérez , Karen Moreno Medina , Juan Carlos Villar , Juan David Ramírez","doi":"10.1016/j.ijpddr.2025.100623","DOIUrl":"10.1016/j.ijpddr.2025.100623","url":null,"abstract":"<div><div>Chagas disease (CD), caused by the protozoan <em>Trypanosoma cruzi</em>, remains a major public health challenge due to limited treatment options, Benznidazole and Nifurtimox; which are associated with adverse effects and variable efficacy. The emergence of drug-resistant in <em>T. cruzi</em> strains, along with limited knowledge of the molecular mechanisms underlying resistance, hampers the development of more effective therapies. To explore these mechanisms, we performed a comparative transcriptomic analysis of two <em>T. cruzi</em> TcI strains: MG (naturally susceptible) and DA (naturally resistant) to Benznidazole. Parasites were cultured in LIT medium, and IC50 values were determined using the MTT assay. RNA was extracted and sequenced (RNA-seq), with reads aligned to a reference genome. Differential gene expressions were analyzed with DESeq2, functional enrichment through Gene Ontology (GO), and metabolic pathways were mapped via KAAS. The IC50 for Benznidazole in DA (28.92 μg/mL; 111.13 μM) was substantially higher than in MG (0.88 μg/mL; 3.39 μM), confirming differential susceptibility. DA showed 408 upregulated and 1515 downregulated genes, while MG had 153 upregulated and 866 downregulated (Log<sub>2</sub>FoldChange ≥ 2 or ≤ −2). GO analysis indicated divergent biological processes between strains: DA exhibited enrichment in electron transport and detoxification, while MG was enriched in DNA repair and energy metabolism. Metabolic mapping revealed significant differences in the pentose phosphate pathway, glycolysis/gluconeogenesis, and the tricarboxylic acid (TCA) cycle. Key genes potentially involved in resistance like prostaglandin F2α synthase, trypanothione synthase, thioredoxin, and prostaglandin F synthase were identified as candidate therapeutic targets. These findings suggest that Benznidazole resistance in <em>T. cruzi</em> involves multifactorial, strain-specific responses at the transcriptomic and metabolic levels. By analyzing naturally resistant and susceptible TcI strains of <em>T. cruzi</em> under identical experimental conditions, this study reveals strain-specific transcriptomic adaptations that have not been previously characterized in naturally resistant and susceptible populations. These findings expand our current understanding of intrinsic Benznidazole resistance in <em>T. cruzi</em>, moving beyond purely experimental models. Specifically, they highlight novel metabolic and redox pathways that could serve as therapeutic targets effective against diverse <em>T. cruzi</em> strains and Discrete Typing Units (DTUs).</div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"29 ","pages":"Article 100623"},"PeriodicalIF":3.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145415542","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-12-01Epub Date: 2025-12-05DOI: 10.1016/j.ijpddr.2025.100627
Liana Theodoridis , Carlo Giannangelo , Farrah El-Saafin , MR Ranga Prabhath , Christopher A. Macraild , Pallavi Sharma , Delphine Merino , Darren J. Creek , Teresa G. Carvalho
Malaria is an infectious disease that imposes a significant global health burden. Increasing drug resistance creates an urgent demand for novel treatment options. We have previously synthesised a new class of heterospirocyclic compounds with novel chemical linkages, which have shown preliminary antimalarial activity. Compounds 25 and 26 display antimalarial activity within 24 h against a panel of drug-resistant strains of Plasmodium falciparum, the most virulent of human malaria parasites. Untargeted metabolomics analysis of P. falciparum-infected red blood cells revealed that the mechanism of action of compound 25 could involve disruption of the pyrimidine biosynthesis pathway and haemoglobin catabolism. Further, compounds 25 and 26 do not induce major toxicity in kidney- and hepatic-derived human cell lines, highlighting their specificity. These heterospirocyclic compounds represent a promising opportunity for antimalarial drug development and could prove relevant against drug-resistant malaria.
{"title":"Novel heterospirocyclic antimalarials with activity against artemisinin- and multidrug-resistant P. falciparum malaria","authors":"Liana Theodoridis , Carlo Giannangelo , Farrah El-Saafin , MR Ranga Prabhath , Christopher A. Macraild , Pallavi Sharma , Delphine Merino , Darren J. Creek , Teresa G. Carvalho","doi":"10.1016/j.ijpddr.2025.100627","DOIUrl":"10.1016/j.ijpddr.2025.100627","url":null,"abstract":"<div><div>Malaria is an infectious disease that imposes a significant global health burden. Increasing drug resistance creates an urgent demand for novel treatment options. We have previously synthesised a new class of heterospirocyclic compounds with novel chemical linkages, which have shown preliminary antimalarial activity. Compounds 25 and 26 display antimalarial activity within 24 h against a panel of drug-resistant strains of <em>Plasmodium falciparum</em>, the most virulent of human malaria parasites. Untargeted metabolomics analysis of <em>P. falciparum</em>-infected red blood cells revealed that the mechanism of action of compound 25 could involve disruption of the pyrimidine biosynthesis pathway and haemoglobin catabolism. Further, compounds 25 and 26 do not induce major toxicity in kidney- and hepatic-derived human cell lines, highlighting their specificity. These heterospirocyclic compounds represent a promising opportunity for antimalarial drug development and could prove relevant against drug-resistant malaria.</div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"29 ","pages":"Article 100627"},"PeriodicalIF":3.4,"publicationDate":"2025-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145722686","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-01Epub Date: 2025-05-28DOI: 10.1016/j.ijpddr.2025.100598
Phornyupa Sanguanwong , Ladawan Khowawisetsut , Lanaprai Kwathai , Peeraporn Varinthra , Chairat Turbpaiboon , Panapat Uawithya , Prasert Sobhon , Ingrid Y. Liu , Supin Chompoopong
Background
Astragaloside IV (ASIV), a natural compound from Astragalus membranaceus, exerts neuroprotective and anti-inflammatory effects in various pathologies. Its role in Plasmodium yoelii (Py) 17XL–induced inflammation leading to blood–brain barrier (BBB) damage remains undefined. Artesunate (ART), the frontline therapy for severe malaria, has encountered resistance and unresolved neurological sequelae. This study investigated the anti-inflammatory properties of ASIV combined with ART in Py-infected mice.
Methods
Sixty-five Institute of Cancer Research mice were randomized into 5 groups: sham, Py, Py-ART, Py-ASIV, and Py-ASIV + ART. Mice in Py groups were infected with Py 17XL. Either 25 mg/kg ASIV alone or 25 mg/kg ASIV plus 2.4 mg/kg ART was administered intraperitoneally for 5 days. Survival rate/time, parasitemia, neurological status, histopathology, and biochemical indices were evaluated.
Results
Although ASIV alone partially suppressed parasitemia, combination therapy significantly prolonged survival and mitigated neurological deficits. Both ASIV and ASIV + ART reduced IL-1β and TNF-α expression in serum and brain, attenuated BBB leakage (Evans blue assay), and preserved BBB integrity by decreasing astrocytic glial fibrillary acidic protein and aquaporin-4 while upregulating the tight junction proteins occludin and zonula occludens-1.
Conclusions
ASIV exhibited modest antiparasitic action and robust anti-inflammatory effects, alleviating BBB disruption when combined with ART in Py 17XL–infected mice. These findings provide an essential basis for further preclinical exploration of ASIV as an adjunct therapy in severe malaria.
{"title":"Combination astragaloside IV and artesunate preserves blood–brain barrier integrity by modulating astrocytes and tight junction proteins in Plasmodium yoelii infection","authors":"Phornyupa Sanguanwong , Ladawan Khowawisetsut , Lanaprai Kwathai , Peeraporn Varinthra , Chairat Turbpaiboon , Panapat Uawithya , Prasert Sobhon , Ingrid Y. Liu , Supin Chompoopong","doi":"10.1016/j.ijpddr.2025.100598","DOIUrl":"10.1016/j.ijpddr.2025.100598","url":null,"abstract":"<div><h3>Background</h3><div>Astragaloside IV (ASIV), a natural compound from <em>Astragalus membranaceus</em>, exerts neuroprotective and anti-inflammatory effects in various pathologies. Its role in <em>Plasmodium yoelii</em> (Py) 17XL–induced inflammation leading to blood–brain barrier (BBB) damage remains undefined. Artesunate (ART), the frontline therapy for severe malaria, has encountered resistance and unresolved neurological sequelae. This study investigated the anti-inflammatory properties of ASIV combined with ART in Py-infected mice.</div></div><div><h3>Methods</h3><div>Sixty-five Institute of Cancer Research mice were randomized into 5 groups: sham, Py, Py-ART, Py-ASIV, and Py-ASIV + ART. Mice in Py groups were infected with Py 17XL. Either 25 mg/kg ASIV alone or 25 mg/kg ASIV plus 2.4 mg/kg ART was administered intraperitoneally for 5 days. Survival rate/time, parasitemia, neurological status, histopathology, and biochemical indices were evaluated.</div></div><div><h3>Results</h3><div>Although ASIV alone partially suppressed parasitemia, combination therapy significantly prolonged survival and mitigated neurological deficits. Both ASIV and ASIV + ART reduced IL-1β and TNF-α expression in serum and brain, attenuated BBB leakage (Evans blue assay), and preserved BBB integrity by decreasing astrocytic glial fibrillary acidic protein and aquaporin-4 while upregulating the tight junction proteins occludin and zonula occludens-1.</div></div><div><h3>Conclusions</h3><div>ASIV exhibited modest antiparasitic action and robust anti-inflammatory effects, alleviating BBB disruption when combined with ART in Py 17XL–infected mice. These findings provide an essential basis for further preclinical exploration of ASIV as an adjunct therapy in severe malaria.</div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"28 ","pages":"Article 100598"},"PeriodicalIF":4.1,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144196339","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-01Epub Date: 2025-06-03DOI: 10.1016/j.ijpddr.2025.100600
Martin K. Nielsen , Alison Pyatt , Jodie Perrett , Eva Tydén , Deborah van Doorn , Tina H. Pihl , Jennifer S. Schmidt , Georg von Samson-Himmelstjerna , Anne Beasley , Ghazanfar Abbas , Abdul Jabbar
Equine parasite control has historically been characterized by confusing and conflicting information, posing significant challenges for veterinarians and horse owners to make evidence-based decisions. Since 2012, equine parasite control guidelines have been developed and published for different parts of the world to address this situation and provide trusted sources of current guidance. At the 2024 International Equine Infectious Disease Conference in Deauville, Normandy, France, lead authors of equine parasite control guideline documents published in the USA, UK, Sweden, Denmark, the Netherlands, Australia, and Europe convened and presented their guidelines. This led to a discussion of differences and similarities between the guidelines and an effort to identify current research needs in this area. In general, all guidelines recommend a surveillance-based approach for equine parasite control, emphasizing the importance of anthelmintic resistance testing. Some guidelines have a focus on controlling Strongylus vulgaris, while others primarily focus on cyathostomins, ascarids and tapeworms. Although the same four anthelmintic drug classes are marketed in most countries, there are some differences between product portfolios available, most notably between Australia and other countries. European countries have various degrees of prescription-only restrictions on anthelmintic products, whereas products are available over the counter in Australia and the USA. Commercially available diagnostic portfolios differed somewhat between countries and affected recommendations made as well. In conclusion, the guidelines are in general agreement and are based on the same general principles. One major challenge is communicating the recommendations effectively to end-users, which should be made a priority going forward.
{"title":"Global equine parasite control guidelines: Consensus or confusion?","authors":"Martin K. Nielsen , Alison Pyatt , Jodie Perrett , Eva Tydén , Deborah van Doorn , Tina H. Pihl , Jennifer S. Schmidt , Georg von Samson-Himmelstjerna , Anne Beasley , Ghazanfar Abbas , Abdul Jabbar","doi":"10.1016/j.ijpddr.2025.100600","DOIUrl":"10.1016/j.ijpddr.2025.100600","url":null,"abstract":"<div><div>Equine parasite control has historically been characterized by confusing and conflicting information, posing significant challenges for veterinarians and horse owners to make evidence-based decisions. Since 2012, equine parasite control guidelines have been developed and published for different parts of the world to address this situation and provide trusted sources of current guidance. At the 2024 International Equine Infectious Disease Conference in Deauville, Normandy, France, lead authors of equine parasite control guideline documents published in the USA, UK, Sweden, Denmark, the Netherlands, Australia, and Europe convened and presented their guidelines. This led to a discussion of differences and similarities between the guidelines and an effort to identify current research needs in this area. In general, all guidelines recommend a surveillance-based approach for equine parasite control, emphasizing the importance of anthelmintic resistance testing. Some guidelines have a focus on controlling <em>Strongylus vulgaris</em>, while others primarily focus on cyathostomins, ascarids and tapeworms. Although the same four anthelmintic drug classes are marketed in most countries, there are some differences between product portfolios available, most notably between Australia and other countries. European countries have various degrees of prescription-only restrictions on anthelmintic products, whereas products are available over the counter in Australia and the USA. Commercially available diagnostic portfolios differed somewhat between countries and affected recommendations made as well. In conclusion, the guidelines are in general agreement and are based on the same general principles. One major challenge is communicating the recommendations effectively to end-users, which should be made a priority going forward.</div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"28 ","pages":"Article 100600"},"PeriodicalIF":4.1,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144212083","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-01Epub Date: 2025-04-25DOI: 10.1016/j.ijpddr.2025.100590
Zheng Xiang , Mengxi Duan , Siqi Wang , Hui Zhao , Wei Zhao , Xiaosong Li , Xinxin Li , Weilin Zeng , Yanrui Wu , Fuxue Yang , Xinyu Liu , Cong Tang , Liwang Cui , Zhaoqing Yang
Drug resistance in Plasmodium falciparum represents a significant challenge in malaria treatment. Identifying the molecular markers associated with P. falciparum resistance will effectively detect resistance and enhance treatment efficiency. In this study, we utilized the advanced CRISPR/Cas9 technology to precisely insert one, two, or three asparagine residues into the Kelch 13(K13) gene of the 3D7 strain, positioned after the 142nd amino acid residue, resulting in 1N-3D7, 2N-3D7, and 3N-3D7. Using ring-stage survival assays (RSA), drug sensitivity evaluations, and in vitro developmental assessments, our findings revealed a trend: 1) the insertion of asparagine residues into the parasite genome increased RSA, with more asparagine insertions leading to higher RSA. 2) According to the IC50 values, 1N-3D7 and 2N-3D7 exhibited similar sensitivity profiles across all ten tested drugs, with both demonstrating resistance to Naphthoquine, indicating that the insertions of one or two asparagines played an equivalent role in conferring resistance. However, the insertion of three asparagine residues resulted in significantly higher IC50 values compared to the first two forms when tested with Artesunate, Artemether, Dihydroartemisinin, Pyronaridine Phosphate, and Naphthoquine, showing resistance to all five drugs. Furthermore, 3N-3D7 exhibited a prolonged ring phase and a shortened trophozoite phase within red blood cells; the schizont phase appeared synchronous with the others, yet its mature schizonts contained fewer merozoites. Additionally, 3N-3D7 exhibited a fitness defect, with the proportion decreasing gradually during co-culture with 3D7, its fitness cost calculated as 14.88 ± 2.87. All these results support the opinion that the insertion of three asparagines was a molecular marker of resistance to artemisinin derivatives, Pyronaridine Phosphate, and Naphthoquine in P. falciparum.
{"title":"Three Asparagine insertions in the K13-propeller led to Plasmodium falciparum becoming resistant to multiple antimalarial drugs","authors":"Zheng Xiang , Mengxi Duan , Siqi Wang , Hui Zhao , Wei Zhao , Xiaosong Li , Xinxin Li , Weilin Zeng , Yanrui Wu , Fuxue Yang , Xinyu Liu , Cong Tang , Liwang Cui , Zhaoqing Yang","doi":"10.1016/j.ijpddr.2025.100590","DOIUrl":"10.1016/j.ijpddr.2025.100590","url":null,"abstract":"<div><div>Drug resistance in <em>Plasmodium falciparum</em> represents a significant challenge in malaria treatment. Identifying the molecular markers associated with <em>P. falciparum</em> resistance will effectively detect resistance and enhance treatment efficiency. In this study, we utilized the advanced CRISPR/Cas9 technology to precisely insert one, two, or three asparagine residues into the Kelch 13(K13) gene of the 3D7 strain, positioned after the 142nd amino acid residue, resulting in 1N-3D7, 2N-3D7, and 3N-3D7. Using ring-stage survival assays (RSA), drug sensitivity evaluations, and in vitro developmental assessments, our findings revealed a trend: 1) the insertion of asparagine residues into the parasite genome increased RSA, with more asparagine insertions leading to higher RSA. 2) According to the IC50 values, 1N-3D7 and 2N-3D7 exhibited similar sensitivity profiles across all ten tested drugs, with both demonstrating resistance to Naphthoquine, indicating that the insertions of one or two asparagines played an equivalent role in conferring resistance. However, the insertion of three asparagine residues resulted in significantly higher IC50 values compared to the first two forms when tested with Artesunate, Artemether, Dihydroartemisinin, Pyronaridine Phosphate, and Naphthoquine, showing resistance to all five drugs. Furthermore, 3N-3D7 exhibited a prolonged ring phase and a shortened trophozoite phase within red blood cells; the schizont phase appeared synchronous with the others, yet its mature schizonts contained fewer merozoites. Additionally, 3N-3D7 exhibited a fitness defect, with the proportion decreasing gradually during co-culture with 3D7, its fitness cost calculated as 14.88 ± 2.87. All these results support the opinion that the insertion of three asparagines was a molecular marker of resistance to artemisinin derivatives, Pyronaridine Phosphate, and Naphthoquine in <em>P. falciparum.</em></div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"28 ","pages":"Article 100590"},"PeriodicalIF":4.1,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144204746","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-01Epub Date: 2025-04-15DOI: 10.1016/j.ijpddr.2025.100592
Anne-Charlotte Lenière , Amit Upadhyay , Jérôme Follet , Timothy P. O'Sullivan
The protozoan parasite Cryptosporidium is the etiological agent of cryptosporidiosis, a ubiquitous diarrheic disease affecting humans and animals. Treatment options are limited, highlighting an urgent need for novel therapeutics. Despite decades of research and a wide diversity of strategies to tackle parasite metabolic pathways, no completely effective drug has been identified to date. Within targeted parasite enzymatic and metabolic pathways, the synthesis of nucleotide mediated by the inosine 5′-monophosphate dehydrogenase (IMPDH) enzyme is the focus of significant research efforts. Based on our prior studies of bacterial IMPDH inhibitors, we report herein the development and characterisation of novel inhibitors targeting Cryptosporidium parvum IMPDH (CpIMPDH). Specifically, we synthesised heteroaryl-containing urea and squaramide analogues to evaluate their potential in vitro anti-Cryptosporidium activity. Initial screening identified nine active compounds with the most potent candidates achieving IC50 values as low as 2.2 μM. Subsequent time-course experiments revealed that the molecules effectively inhibit parasite invasion and early intracellular development but failed to tackle C. parvum growth when introduced at 30 h post infection. The present work introduces a new family of squaramide-derived IMPDH inhibitors and also interrogates the need to standardise commonly accepted protocols used for assessing anti-cryptosporidial drug activity.
{"title":"Effect of urea and squaramide IMPDH inhibitors on C. parvum: in vitro trial design impacts the assessment of drug efficacy","authors":"Anne-Charlotte Lenière , Amit Upadhyay , Jérôme Follet , Timothy P. O'Sullivan","doi":"10.1016/j.ijpddr.2025.100592","DOIUrl":"10.1016/j.ijpddr.2025.100592","url":null,"abstract":"<div><div>The protozoan parasite <em>Cryptosporidium</em> is the etiological agent of cryptosporidiosis, a ubiquitous diarrheic disease affecting humans and animals. Treatment options are limited, highlighting an urgent need for novel therapeutics. Despite decades of research and a wide diversity of strategies to tackle parasite metabolic pathways, no completely effective drug has been identified to date. Within targeted parasite enzymatic and metabolic pathways, the synthesis of nucleotide mediated by the inosine 5′-monophosphate dehydrogenase (IMPDH) enzyme is the focus of significant research efforts. Based on our prior studies of bacterial IMPDH inhibitors, we report herein the development and characterisation of novel inhibitors targeting <em>Cryptosporidium parvum</em> IMPDH (<em>Cp</em>IMPDH). Specifically, we synthesised heteroaryl-containing urea and squaramide analogues to evaluate their potential <em>in vitro</em> anti-<em>Cryptosporidium</em> activity. Initial screening identified nine active compounds with the most potent candidates achieving IC<sub>50</sub> values as low as 2.2 μM. Subsequent time-course experiments revealed that the molecules effectively inhibit parasite invasion and early intracellular development but failed to tackle <em>C. parvum</em> growth when introduced at 30 h post infection. The present work introduces a new family of squaramide-derived IMPDH inhibitors and also interrogates the need to standardise commonly accepted protocols used for assessing anti-cryptosporidial drug activity.</div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"28 ","pages":"Article 100592"},"PeriodicalIF":4.1,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143898678","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-01Epub Date: 2025-05-05DOI: 10.1016/j.ijpddr.2025.100596
Adrián Chávez-Cano , Scott C. Dawson , M. Guadalupe Ortega-Pierres
Albendazole resistance in Giardia duodenalis includes a complex and multifactorial challenge that potentially involves non-reported pathways such as the participation of metabolic regulators. In this context, sirtuins, known as metabolic sensors in various cellular processes, have emerged as promising candidates for novel anti-parasitic treatments. To investigate their role in albendazole (ABZ) resistance, initially we analyzed the expression of sirtuins in three Giardia strains resistant to 8 μM, 1.5 μM and 250 μM of ABZ that were obtained in our laboratory. Additionally, we used a CRISPRi-based knockdownstrategy to repress several sirtuins in Giardia and analyzed the effect of sirtuins on ABZ resistance. Our findings demonstrated a significant upregulation of sirtuins gdSir2.1, gdSir2.2 and gdSir2.3 in the three distinct albendazole-resistant lines. Knockdown of gdSir2.1 and gdSir2.3 resulted in heightened parasite susceptibility to both albendazole and hydrogen peroxide. Further, our study suggested that sirtuins contribute to the regulation of reactive oxygen species (ROS) levels, oxidative DNA damage, and the expression of oxidative stress response (OSR) genes within the parasite. Collectively, our results demonstrated that gdSir2.1 and gdSir2.3 play a significant role in mediating albendazole resistance, primarily through regulating the oxidative stress response.
{"title":"gdSir2.1 and gdSir2.3 are involved in albendazole resistance in Giardia duodenalis via regulation of the oxidative stress response","authors":"Adrián Chávez-Cano , Scott C. Dawson , M. Guadalupe Ortega-Pierres","doi":"10.1016/j.ijpddr.2025.100596","DOIUrl":"10.1016/j.ijpddr.2025.100596","url":null,"abstract":"<div><div>Albendazole resistance in <em>Giardia duodenalis</em> includes a complex and multifactorial challenge that potentially involves non-reported pathways such as the participation of metabolic regulators. In this context, sirtuins, known as metabolic sensors in various cellular processes, have emerged as promising candidates for novel anti-parasitic treatments. To investigate their role in albendazole (ABZ) resistance, initially we analyzed the expression of sirtuins in three <em>Giardia</em> strains resistant to 8 μM, 1.5 μM and 250 μM of ABZ that were obtained in our laboratory. Additionally, we used a CRISPRi-based knockdownstrategy to repress several sirtuins in <em>Giardia</em> and analyzed the effect of sirtuins on ABZ resistance. Our findings demonstrated a significant upregulation of sirtuins gdSir2.1, gdSir2.2 and gdSir2.3 in the three distinct albendazole-resistant lines. Knockdown of gdSir2.1 and gdSir2.3 resulted in heightened parasite susceptibility to both albendazole and hydrogen peroxide. Further, our study suggested that sirtuins contribute to the regulation of reactive oxygen species (ROS) levels, oxidative DNA damage, and the expression of oxidative stress response (OSR) genes within the parasite. Collectively, our results demonstrated that gdSir2.1 and gdSir2.3 play a significant role in mediating albendazole resistance, primarily through regulating the oxidative stress response.</div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"28 ","pages":"Article 100596"},"PeriodicalIF":4.1,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143947607","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-08-01Epub Date: 2025-05-20DOI: 10.1016/j.ijpddr.2025.100599
Josef Krátký , Markéta Zajíčková , Aya C. Taki , Oliver Michel , Petra Matoušková , Ivan Vokřál , Karolína Štěrbová , Ondřej Vosála , Beate Lungerich , Thomas Kurz , Robin B. Gasser , Karel Harant , Lenka Skálová
Parasitic nematodes cause a wide range of diseases in animals, including humans. However, the efficacy of existing anthelmintic drugs, commonly used to treat these infections, is waning due to the increasing prevalence of drug resistance in nematode populations. This growing challenge underscores the urgent need to discover and develop novel nematocidal drugs that target new molecular pathways. In the present study, 13 novel derivatives of benzhydroxamic acid (OMKs) were designed and synthesized. Their anthelmintic activity was tested in the parasitic nematode Haemonchus contortus (barber's pole worm) and the free-living nematode Caenorhabditis elegans and potential toxicity assessed in mammalian models. Compound OMK211 showed the most promising results. It decreased viability and motility of larval and adult stages of both nematode species and of both drug-sensitive and drug-resistant strains of H. contortus at micromolar concentrations with the highest efficacy in H. contortus adult males (IC50 ∼ 1 μM). Moreover, OMK211 was not toxic in mammalians cells in vitro and in mice in vivo. Consequently, thermal proteome profiling analysis was used to infer the putative molecular target of OMK211 in H. contortus. The results revealed C2-domain containing protein A0A6F7Q0A8, encoded by gene HCON_00184,900, as an interacting partner of OMK211. Using advanced structural prediction and docking tools, this protein is considered an interesting putative molecular target of new nematocidal drugs as its orthologs are present in several nematodes but not in mammals. In conclusion, novel derivatives of benzhydroxamic acid represent a promising new class of potential anthelmintics, which deserve further testing.
{"title":"New derivatives of benzhydroxamic acid with nematocidal activity against Haemonchus contortus and Caenorhabditis elegans","authors":"Josef Krátký , Markéta Zajíčková , Aya C. Taki , Oliver Michel , Petra Matoušková , Ivan Vokřál , Karolína Štěrbová , Ondřej Vosála , Beate Lungerich , Thomas Kurz , Robin B. Gasser , Karel Harant , Lenka Skálová","doi":"10.1016/j.ijpddr.2025.100599","DOIUrl":"10.1016/j.ijpddr.2025.100599","url":null,"abstract":"<div><div>Parasitic nematodes cause a wide range of diseases in animals, including humans. However, the efficacy of existing anthelmintic drugs, commonly used to treat these infections, is waning due to the increasing prevalence of drug resistance in nematode populations. This growing challenge underscores the urgent need to discover and develop novel nematocidal drugs that target new molecular pathways. In the present study, 13 novel derivatives of benzhydroxamic acid (OMKs) were designed and synthesized. Their anthelmintic activity was tested in the parasitic nematode <em>Haemonchus contortus</em> (barber's pole worm) and the free-living nematode <em>Caenorhabditis elegans</em> and potential toxicity assessed in mammalian models. Compound OMK211 showed the most promising results. It decreased viability and motility of larval and adult stages of both nematode species and of both drug-sensitive and drug-resistant strains of <em>H. contortus</em> at micromolar concentrations with the highest efficacy in <em>H. contortus</em> adult males (IC<sub>50</sub> ∼ 1 μM). Moreover, OMK211 was not toxic in mammalians cells <em>in vitro</em> and in mice <em>in vivo</em>. Consequently, thermal proteome profiling analysis was used to infer the putative molecular target of OMK211 in <em>H. contortus</em>. The results revealed C2-domain containing protein A0A6F7Q0A8, encoded by gene HCON_00184,900, as an interacting partner of OMK211. Using advanced structural prediction and docking tools, this protein is considered an interesting putative molecular target of new nematocidal drugs as its orthologs are present in several nematodes but not in mammals. In conclusion, novel derivatives of benzhydroxamic acid represent a promising new class of potential anthelmintics, which deserve further testing.</div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"28 ","pages":"Article 100599"},"PeriodicalIF":4.1,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144125291","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
This study explores the anti-proliferative and anti-parasitic properties of ursolic acid (UA) in Theileria annulata-infected bovine (TA) cells. Dose-response analyses determined an IC50 value of approximately 5 μg/mL for UA, demonstrating selective cytotoxicity toward infected cells with minimal impact on healthy cells. UA treatment induced pronounced morphological alterations and apoptosis in TA cells, as evidenced by light microscopy and a time-dependent increase in cell mortality. Notably, UA exhibited consistent efficacy against both buparvaquone (BPQ)-sensitive and BPQ-resistant TA cell lines, highlighting its broad-spectrum anti-parasitic potential. Mechanistic investigations revealed that UA triggers DNA damage, elevates reactive oxygen species (ROS) levels, disrupts mitochondrial function, and induces sub-G1 phase arrest, culminating in apoptosis primarily via the intrinsic pathway. Mass spectrometry-based proteomic profiling identified significant perturbations in host cell pathways, including DNA repair mechanisms, cell cycle regulation, and signaling networks, alongside direct interference with parasite metabolic processes. Western blot analysis further confirmed UA-mediated modulation of host cell signaling pathways and chromatin organization. Given the rising incidence of drug-resistant T. annulata strains, the development of novel therapeutic strategies is imperative. These findings highlight UA's multifaceted mechanism of action, targeting both parasitic and host cellular processes, and position it as a promising candidate for the treatment of bovine theileriosis.
{"title":"Ursolic acid induces apoptosis and disrupts host-parasite interactions in Theileria annulata-infected cells","authors":"Sakshi Singh , Madhusmita Subudhi , Vengatachala Moorthy A , Akash Suresh , Paresh Sharma","doi":"10.1016/j.ijpddr.2025.100593","DOIUrl":"10.1016/j.ijpddr.2025.100593","url":null,"abstract":"<div><div>This study explores the anti-proliferative and anti-parasitic properties of ursolic acid (UA) in <em>Theileria annulata</em>-infected bovine (TA) cells. Dose-response analyses determined an IC<sub>50</sub> value of approximately 5 μg/mL for UA, demonstrating selective cytotoxicity toward infected cells with minimal impact on healthy cells. UA treatment induced pronounced morphological alterations and apoptosis in TA cells, as evidenced by light microscopy and a time-dependent increase in cell mortality. Notably, UA exhibited consistent efficacy against both buparvaquone (BPQ)-sensitive and BPQ-resistant TA cell lines, highlighting its broad-spectrum anti-parasitic potential. Mechanistic investigations revealed that UA triggers DNA damage, elevates reactive oxygen species (ROS) levels, disrupts mitochondrial function, and induces sub-G1 phase arrest, culminating in apoptosis primarily via the intrinsic pathway. Mass spectrometry-based proteomic profiling identified significant perturbations in host cell pathways, including DNA repair mechanisms, cell cycle regulation, and signaling networks, alongside direct interference with parasite metabolic processes. Western blot analysis further confirmed UA-mediated modulation of host cell signaling pathways and chromatin organization. Given the rising incidence of drug-resistant <em>T. annulata</em> strains, the development of novel therapeutic strategies is imperative. These findings highlight UA's multifaceted mechanism of action, targeting both parasitic and host cellular processes, and position it as a promising candidate for the treatment of bovine theileriosis.</div></div>","PeriodicalId":13775,"journal":{"name":"International Journal for Parasitology: Drugs and Drug Resistance","volume":"28 ","pages":"Article 100593"},"PeriodicalIF":4.1,"publicationDate":"2025-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143860023","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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