Chemogenomic profiling of anti-leishmanial efficacy and resistance in the related kinetoplastid parasite Trypanosoma brucei

AbstractThe arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode-of-action and potential routes to resistance is limited. Forward genetic approaches have revolutionised our understanding of drug mode-of-action in the related kinetoplastid parasite, Trypanosoma brucei. Therefore, we screened our genome-scale T. brucei RNAi library in the current anti-leishmanial drugs, sodium stibogluconate (antimonial), paromomycin, miltefosine and amphotericin-B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin, TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin/aminoglycoside efficacy. The vesicle-associated membrane protein, TbVAMP7B, and a flippase contribute to amphotericin-B and miltefosine action, and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative β-subunit/CDC50 co-factor, and additional membrane-associated hits, affect amphotericin-B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of anti-leishmanial drug action and potential resistance mechanisms, and should facilitate the development of improved therapies, as well as surveillance for drug-resistant parasites.ImportanceLeishmaniasis is a devastating disease caused by the Leishmania parasites and is endemic to a wide swathe of the tropics and sub-tropics. While there are drugs available for the treatment of leishmaniasis, these suffer from various challenges, including the spread of drug resistance. Our understanding of anti-leishmanial drug action and the modes of drug resistance in Leishmania is limited. The development of genetic screening tools in the related parasite, Trypanosoma brucei, has revolutionised our understanding of these processes in this parasite. Therefore, we applied these tools to the anti-leishmanial drugs, identifying T. brucei orthologues of known Leishmania proteins that drive drug uptake, as well as a panel of novel proteins not previously associated with anti-leishmanial drug action. Our findings substantially advance our understanding of anti-leishmanial mode-of-action and provide a valuable starting point for further research.

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Chemogenomic Profiling of Antileishmanial Efficacy and Resistance in the Related Kinetoplastid Parasite Trypanosoma brucei

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00795-19 ◽

2019 ◽

Vol 63 (8) ◽

Cited By ~ 6

Author(s):

Clare F. Collett ◽

Carl Kitson ◽

Nicola Baker ◽

Heather B. Steele-Stallard ◽

Marie-Victoire Santrot ◽

...

Keyword(s):

Amphotericin B ◽

Trypanosoma Brucei ◽

Mode Of Action ◽

Drug Efficacy ◽

Resistance Mechanisms ◽

Major Facilitator Superfamily ◽

Drug Uptake ◽

Cross Resistance ◽

Content Type ◽

Kinetoplastid Parasite

ABSTRACT The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei. Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative β-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.

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Candida tropicalis Antifungal Cross-Resistance Is Related to Different Azole Target (Erg11p) Modifications

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00477-13 ◽

2013 ◽

Vol 57 (10) ◽

pp. 4769-4781 ◽

Cited By ~ 59

Author(s):

A. Forastiero ◽

A. C. Mesa-Arango ◽

A. Alastruey-Izquierdo ◽

L. Alcazar-Fuoli ◽

L. Bernal-Martinez ◽

...

Keyword(s):

Drug Resistance ◽

Amphotericin B ◽

Candida Tropicalis ◽

Antifungal Drug ◽

Cross Resistance ◽

Clinical Samples ◽

Content Type ◽

Antifungal Drug Resistance

ABSTRACTCandida tropicalisranks between third and fourth amongCandidaspecies most commonly isolated from clinical specimens. Invasive candidiasis and candidemia are treated with amphotericin B or echinocandins as first-line therapy, with extended-spectrum triazoles as acceptable alternatives.Candida tropicalisis usually susceptible to all antifungal agents, although several azole drug-resistant clinical isolates are being reported. However,C. tropicalisresistant to amphotericin B is uncommon, and only a few strains have reliably demonstrated a high level of resistance to this agent. The resistance mechanisms operating inC. tropicalisstrains isolated from clinical samples showing resistance to azole drugs alone or with amphotericin B cross-resistance were elucidated. Antifungal drug resistance was related to mutations of the azole target (Erg11p) with or without alterations of the ergosterol biosynthesis pathway. The antifungal drug resistance shownin vitrocorrelated very well with the results obtainedin vivousing the model hostGalleria mellonella. Using this panel of strains, theG. mellonellamodel system was validated as a simple, nonmammalian minihost model that can be used to studyin vitro-in vivocorrelation of antifungals inC. tropicalis. The development inC. tropicalisof antifungal drug resistance with different mechanisms during antifungal treatment has potential clinical impact and deserves specific prospective studies.

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Comparative Analysis of the Omics Technologies Used to Study Antimonial, Amphotericin B, and Pentamidine Resistance inLeishmania

Journal of Parasitology Research ◽

10.1155/2014/726328 ◽

2014 ◽

Vol 2014 ◽

pp. 1-11 ◽

Cited By ~ 18

Author(s):

Gagandeep Kaur ◽

Bhawana Rajput

Keyword(s):

Developing Countries ◽

Drug Resistance ◽

Comparative Analysis ◽

Amphotericin B ◽

Mode Of Action ◽

Omics Technologies ◽

Work Done ◽

Lack Of Knowledge

Leishmaniasis is a serious threat in developing countries due to its endemic nature and debilitating symptoms. Extensive research and investigations have been carried out to learn about the mechanism of drug resistance inLeishmaniabut results obtained in the laboratory are not in agreement with those obtained from the field. Also the lack of knowledge about the mode of action for a number of drugs makes the study of drug resistance more complex. A major concern in recent times has been regarding the role of parasitic virulence in drug resistance forLeishmania. Researchers have employed various techniques to unravel the facts about resistance and virulence inLeishmania. With advent of advanced and more specific means of detection, further hints about probable mechanisms of conferring resistance are expected. This review aims to provide a consolidated picture along with a comparative account of the work done so far to study the mechanism of antimony, amphotericin B, and pentamidine resistance using various techniques.

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The interactions between drugs and the parasite surface

Parasitology ◽

10.1017/s0031182000086029 ◽

1988 ◽

Vol 96 (S1) ◽

pp. S167-S193 ◽

Cited By ~ 12

Author(s):

L. H. Chappell

Keyword(s):

Drug Resistance ◽

Drug Target ◽

Drug Acquisition ◽

Drug Action ◽

Drug Uptake ◽

Specific Drug ◽

Permeability Changes ◽

And Function ◽

Uptake Mechanisms

SUMMARYThe interrelationships between drugs and parasite surfaces are considered under the headings of (a) effects on membrane transport, (b) drug uptake mechanisms and (c) effects on surface morphology and function: praziquantel is discussed under a separate heading. The range of chemotherapeutic compounds that cause permeability changes and concomitant morphological disruption is discussed in terms of mode of drug action. Interpretation of the available data renders it difficult to identify the primary mode of action in the drugs considered. Drug uptake mechanisms are known for relatively few compounds; drug resistance as a function of drug acquisition is discussed. The role of the parasite surface as a specific drug target is argued.

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Amphotericin B and Other Polyenes—Discovery, Clinical Use, Mode of Action and Drug Resistance

Journal of Fungi ◽

10.3390/jof6040321 ◽

2020 ◽

Vol 6 (4) ◽

pp. 321

Author(s):

Hans Carolus ◽

Siebe Pierson ◽

Katrien Lagrou ◽

Patrick Van Dijck

Keyword(s):

Drug Resistance ◽

Amphotericin B ◽

Mode Of Action ◽

Fungal Infections ◽

Resistance Mechanisms ◽

Antifungal Drugs ◽

Clinical Use ◽

Candida Auris ◽

Resistance Development ◽

Resistant Species

Although polyenes were the first broad spectrum antifungal drugs on the market, after 70 years they are still the gold standard to treat a variety of fungal infections. Polyenes such as amphotericin B have a controversial image. They are the antifungal drug class with the broadest spectrum, resistance development is still relatively rare and fungicidal properties are extensive. Yet, they come with a significant host toxicity that limits their use. Relatively recently, the mode of action of polyenes has been revised, new mechanisms of drug resistance were discovered and emergent polyene resistant species such as Candida auris entered the picture. This review provides a short description of the history and clinical use of polyenes, and focusses on the ongoing debate concerning their mode of action, the diversity of resistance mechanisms discovered to date and the most recent trends in polyene resistance development.

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Probing the methotrexate–protein interactions by proteomics and thermostability assay for drug resistance study

Analytical Methods ◽

10.1039/d0ay02099k ◽

2021 ◽

Author(s):

Wenbo Zhang ◽

Xiaoying Li ◽

Xiaolei Zhang ◽

Yan Dong ◽

Lianghai Hu

Keyword(s):

Drug Resistance ◽

Protein Interactions ◽

Quantitative Proteomics ◽

Drug Action ◽

Novel Strategy

Quantitative proteomics combined with thermostability assay provide a novel strategy for the study of mechanisms on drug action and resistance.

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Diversity and Drug Resistance of Filamentous Fungi Isolated from the Fresh Raspberries

Indian Journal of Microbiology ◽

10.1007/s12088-021-00966-y ◽

2021 ◽

Author(s):

Ewelina Farian ◽

Angelina Wójcik-Fatla

Keyword(s):

Drug Resistance ◽

Food Safety ◽

Human Health ◽

Amphotericin B ◽

Filamentous Fungi ◽

Rubus Idaeus ◽

Disc Diffusion ◽

Potential Threat ◽

Fungal Strains ◽

Strip Method

AbstractFungi are one of the most widely distributed microorganisms in the environment, including food such as fruits, vegetables and other crops, posing a potential threat to food safety and human health. The aim of this study was to determine the diversity, intensity and drug resistance of potentially pathogenic filamentous fungi isolated from the fresh raspberries (Rubus idaeus L.). A total of 50 strains belonging to genera Fusarium, Cladosporium, Alternaria, Penicillium, Mucor, Rhizopus, Aspergillus and Acremonium were tested for drug resistance against 11 antifungals by disc diffusion and gradient strips methods. The average mycological contamination in the examined samples of raspberries amounted to 4.34 log CFU/g. The Cladosporium was isolated from all tested samples, followed by Alternaria and Fusarium with a frequency of 61% and 34%, respectively. The highest level of drug resistance was observed for Acremonium genera and Fusarium strains recorded a wide variation in drug resistance as revealed by susceptibility with amphotericin B and voriconzole with MICs ranged from 0.5–4 µg/ml and posaconazole with MICs ranging from 3–8 µg/ml. All fungal strains showed 100% resistance to caspofungin, fluconazole and flucytosine with both the methods, and 100% resistance to micafungin and anidulafungin in the gradient strip method.

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Impact of genotypic diversity on selection of subtype-specific drug resistance profiles during raltegravir-based therapy in individuals infected with B and BF recombinant HIV-1 strains

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa042 ◽

2020 ◽

Vol 75 (6) ◽

pp. 1567-1574

Author(s):

Daniela Sánchez ◽

Solange Arazi Caillaud ◽

Ines Zapiola ◽

Silvina Fernandez Giuliano ◽

Rosa Bologna ◽

...

Keyword(s):

Drug Resistance ◽

Current Knowledge ◽

Phylogenetic Analyses ◽

Genotypic Diversity ◽

Resistance Testing ◽

Cross Resistance ◽

Resistance Mutations ◽

Middle Income ◽

Subtype B ◽

Hiv 1

Abstract Background Current knowledge on HIV-1 resistance to integrase inhibitors (INIs) is based mostly on subtype B strains. This contrasts with the increasing use of INIs in low- and middle-income countries, where non-B subtypes predominate. Materials and methods HIV-1 drug resistance genotyping was performed in 30 HIV-1-infected individuals undergoing virological failure to raltegravir. Drug resistance mutations (DRMs) and HIV-1 subtype were characterized using Stanford HIVdb and phylogenetic analyses. Results Of the 30 integrase (IN) sequences, 14 were characterized as subtype F (47%), 8 as subtype B (27%), 7 as BF recombinants (23%) and 1 as a putative CRF05_DF (3%). In 25 cases (83%), protease and reverse transcriptase (PR-RT) sequences from the same individuals confirmed the presence of different BF recombinants. Stanford HIVdb genotyping was concordant with phylogenetic inference in 70% of IN and 60% of PR-RT sequences. INI DRMs differed between B and F IN subtypes, with Q148K/R/H, G140S and E138K/A being more prevalent in subtype B (63% versus 0%, P = 0.0021; 50% versus 0%, P = 0.0096; and 50% versus 0%, P = 0.0096, respectively). These differences were independent of the time on raltegravir therapy or viral load at the time of genotyping. INI DRMs in subtype F IN genomes predicted a lower level of resistance to raltegravir and no cross-resistance to second-generation INIs. Conclusions Alternative resistance pathways to raltegravir develop in subtypes B and F IN genomes, with implications for clinical practice. Evaluating the role of HIV-1 subtype in development and persistence of mutations that confer resistance to INIs will be important to improve algorithms for resistance testing and optimize the use of INIs.

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Cross-Resistance to Nitro Drugs and Implications for Treatment of Human African Trypanosomiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00332-10 ◽

2010 ◽

Vol 54 (7) ◽

pp. 2893-2900 ◽

Cited By ~ 69

Author(s):

Antoaneta Y. Sokolova ◽

Susan Wyllie ◽

Stephen Patterson ◽

Sandra L. Oza ◽

Kevin D. Read ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Human African Trypanosomiasis ◽

Parent Drug ◽

Cross Resistance ◽

Pharmacokinetic Studies ◽

African Trypanosomiasis ◽

Trypanosoma Brucei Brucei ◽

Resistant Lines

ABSTRACT The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

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