Spontaneous selection of Cryptosporidium drug resistance in a calf model of infection

AbstractThe intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years with numerous lead compounds identified including several tRNA synthetase inhibitors. In this study, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (CpMetRS) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day five. Parasites shed by each recrudescent calf had different amino acid altering CpMetRS mutations, coding either an aspartate 243 to glutamate (D243E) or a threonine 246 to isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant expressing parasites respectively had 2093 EC50s of 613- or 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 by >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.ImportanceCryptosporidium is a leading protozoan cause of diarrhea in young children with no reliable treatment. We report results of a dairy calf drug efficacy trial and the spontaneous emergence of drug resistance. Cryptosporidium parvum infected calves initially improved with drug treatment, but infection relapsed in two animals. Parasites shed by each recrudescent calf had mutations in the gene encoding the drug target that altered its amino acid sequence. Recapitulation of the drug target mutations by CRISPR/Cas9 genome editing resulted in highly drug-resistant parasites, and recombinant mutant enzymes were resistant to inhibition. This is the first report of naturally emerging Cryptosporidium drug resistance. There is a currently a great opportunity to impact public health with new drugs to treat cryptosporidiosis, and this report highlights the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence in order to realize their full potential.One-sentence summaryDrug-target point mutations mediating anticryptosporidial resistance spontaneously arose in the dairy calf C. parvum infection model.

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Spontaneous selection of Cryptosporidium drug resistance in a calf model of infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00023-21 ◽

2021 ◽

Author(s):

Muhammad M. Hasan ◽

Erin E. Stebbins ◽

Robert K.M. Choy ◽

J. Robert Gillespie ◽

Eugenio L. de Hostos ◽

...

Keyword(s):

Drug Resistance ◽

Diarrheal Disease ◽

Trna Synthetase ◽

Dairy Calf ◽

Resistance Mutations ◽

Recombinant Enzymes ◽

Intestinal Protozoan ◽

Hydrophobic Residues ◽

Lead Compounds ◽

Transgenic Parasites

The intestinal protozoan Cryptosporidium is a leading cause of diarrheal disease and mortality in young children. There is currently no fully effective treatment for cryptosporidiosis, which has stimulated interest in anticryptosporidial development over the last ∼10 years with numerous lead compounds identified including several tRNA synthetase inhibitors. In this study, we report the results of a dairy calf efficacy trial of the methionyl-tRNA (CpMetRS) synthetase inhibitor 2093 and the spontaneous emergence of drug resistance. Dairy calves experimentally infected with Cryptosporidium parvum initially improved with 2093 treatment, but parasite shedding resumed in two of three calves on treatment day five. Parasites shed by each recrudescent calf had different amino acid altering CpMetRS mutations, coding either an aspartate 243 to glutamate (D243E) or a threonine 246 to isoleucine (T246I) mutation. Transgenic parasites engineered to have either the D243E or T246I CpMetRS mutation using CRISPR/Cas9 grew normally but were highly 2093 resistant; the D243E and T246I mutant expressing parasites respectively had 2093 EC50s of 613- or 128-fold that of transgenic parasites with wild-type CpMetRS. In studies using recombinant enzymes, the D243E and T246I mutations shifted the 2093 IC50 by >170-fold. Structural modeling of CpMetRS based on an inhibitor-bound Trypanosoma brucei MetRS crystal structure suggested that the resistance mutations reposition nearby hydrophobic residues, interfering with compound binding while minimally impacting substrate binding. This is the first report of naturally emerging Cryptosporidium drug resistance, highlighting the need to address the potential for anticryptosporidial resistance and establish strategies to limit its occurrence.

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Orphan designation: 505 amino acid protein, corresponding to amino acids 2-506 of the wild-type human histidyl-tRNA synthetase, Treatment of limb-girdle muscular dystrophy

Case Medical Research ◽

10.31525/cmr-2935ce2 ◽

2020 ◽

Author(s):

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Muscular Dystrophy ◽

Trna Synthetase ◽

Limb Girdle Muscular Dystrophy ◽

Wild Type ◽

Orphan Designation ◽

Acid Protein ◽

Amino Acid Protein

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Are Carbonic Anhydrases Suitable Targets to Fight Protozoan Parasitic Diseases?

Current Medicinal Chemistry ◽

10.2174/0929867325666180326160121 ◽

2019 ◽

Vol 25 (39) ◽

pp. 5266-5278 ◽

Cited By ~ 9

Author(s):

Katia D'Ambrosio ◽

Claudiu T. Supuran ◽

Giuseppina De Simone

Keyword(s):

Drug Resistance ◽

Animal Models ◽

Carbonic Anhydrase ◽

Drug Target ◽

Treatment Options ◽

Parasitic Diseases ◽

Carbonic Anhydrases ◽

Extensive Drug Resistance ◽

Protozoan Infections

Protozoans belonging to Plasmodium, Leishmania and Trypanosoma genera provoke widespread parasitic diseases with few treatment options and many of the clinically used drugs experiencing an extensive drug resistance phenomenon. In the last several years, the metalloenzyme Carbonic Anhydrase (CA, EC 4.2.1.1) was cloned and characterized in the genome of these protozoa, with the aim to search for a new drug target for fighting malaria, leishmaniasis and Chagas disease. P. falciparum encodes for a CA (PfCA) belonging to a novel genetic family, the η-CA class, L. donovani chagasi for a β-CA (LdcCA), whereas T. cruzi genome contains an α-CA (TcCA). These three enzymes were characterized in detail and a number of in vitro potent and selective inhibitors belonging to the sulfonamide, thiol, dithiocarbamate and hydroxamate classes were discovered. Some of these inhibitors were also effective in cell cultures and animal models of protozoan infections, making them of considerable interest for the development of new antiprotozoan drugs with a novel mechanism of action.

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Nucleotide and deduced amino acid sequence of human threonyl-tRNA synthetase reveals extensive homology to the Escherichia coli and yeast enzymes

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)92906-6 ◽

1991 ◽

Vol 266 (15) ◽

pp. 9919-9923

Author(s):

M.E. Cruzen ◽

S.M. Arfin

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Trna Synthetase

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Transferability of N-terminal mutations of pyrrolysyl-tRNA synthetase in one species to that in another species on unnatural amino acid incorporation efficiency

Amino Acids ◽

10.1007/s00726-020-02927-z ◽

2020 ◽

Author(s):

Thomas L. Williams ◽

Debra J. Iskandar ◽

Alexander R. Nödling ◽

Yurong Tan ◽

Louis Y. P. Luk ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Genetic Code ◽

Unnatural Amino Acids ◽

Trna Synthetase ◽

Unnatural Amino Acid ◽

Amino Acid Incorporation ◽

Acid Incorporation ◽

Genetic Code Expansion ◽

Incorporation Efficiency

AbstractGenetic code expansion is a powerful technique for site-specific incorporation of an unnatural amino acid into a protein of interest. This technique relies on an orthogonal aminoacyl-tRNA synthetase/tRNA pair and has enabled incorporation of over 100 different unnatural amino acids into ribosomally synthesized proteins in cells. Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNA from Methanosarcina species are arguably the most widely used orthogonal pair. Here, we investigated whether beneficial effect in unnatural amino acid incorporation caused by N-terminal mutations in PylRS of one species is transferable to PylRS of another species. It was shown that conserved mutations on the N-terminal domain of MmPylRS improved the unnatural amino acid incorporation efficiency up to five folds. As MbPylRS shares high sequence identity to MmPylRS, and the two homologs are often used interchangeably, we examined incorporation of five unnatural amino acids by four MbPylRS variants at two temperatures. Our results indicate that the beneficial N-terminal mutations in MmPylRS did not improve unnatural amino acid incorporation efficiency by MbPylRS. Knowledge from this work contributes to our understanding of PylRS homologs which are needed to improve the technique of genetic code expansion in the future.

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A randomized, single-blind and crossover study of an amino acid-based milk formula in treating young children with atopic dermatitis

Pediatric Allergy and Immunology ◽

10.1111/j.1399-3038.2004.00197.x ◽

2004 ◽

Vol 15 (6) ◽

pp. 558-561 ◽

Cited By ~ 19

Author(s):

Ting Fan Leung ◽

Kwok Chiu Ma ◽

Lorena T. F. Cheung ◽

Christopher W. K. Lam ◽

Eric Wong ◽

...

Keyword(s):

Atopic Dermatitis ◽

Amino Acid ◽

Young Children ◽

Crossover Study ◽

Milk Formula ◽

Single Blind

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Nucleotide and deduced amino acid sequence of human cysteinyl-tRNA synthetase

DNA Sequence ◽

10.3109/10425179409020847 ◽

1994 ◽

Vol 4 (4) ◽

pp. 243-248 ◽

Cited By ~ 4

Author(s):

Matt E. Cruzen ◽

Stuart M. Arfin

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Trna Synthetase

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Structural Basis of Furan-Amino Acid Recognition by a Polyspecific Aminoacyl-tRNA-Synthetase and its Genetic Encoding in Human Cells

ChemBioChem ◽

10.1002/cbic.201402006 ◽

2014 ◽

Vol 15 (12) ◽

pp. 1755-1760 ◽

Cited By ~ 16

Author(s):

Moritz J. Schmidt ◽

Annemarie Weber ◽

Moritz Pott ◽

Wolfram Welte ◽

Daniel Summerer

Keyword(s):

Amino Acid ◽

Trna Synthetase ◽

Human Cells ◽

Structural Basis ◽

Aminoacyl Trna Synthetase ◽

Genetic Encoding ◽

Amino Acid Recognition

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A Combined Genetic-Proteomic Approach Identifies Residues within Dengue Virus NS4B Critical for Interaction with NS3 and Viral Replication

Journal of Virology ◽

10.1128/jvi.00867-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7170-7186 ◽

Cited By ~ 26

Author(s):

Laurent Chatel-Chaix ◽

Wolfgang Fischl ◽

Pietro Scaturro ◽

Mirko Cortese ◽

Stephanie Kallis ◽

...

Keyword(s):

Amino Acid ◽

Dengue Virus ◽

Virus Replication ◽

Strong Evidence ◽

Drug Target ◽

Nonstructural Protein ◽

Rna Replication ◽

Replication Cycle ◽

Infected Cells ◽

Ns3 Protease

ABSTRACTDengue virus (DENV) infection causes the most prevalent arthropod-borne viral disease worldwide. Approved vaccines are not available, and targets suitable for the development of antiviral drugs are lacking. One possible drug target is nonstructural protein 4B (NS4B), because it is absolutely required for virus replication; however, its exact role in the DENV replication cycle is largely unknown. With the aim of mapping NS4B determinants critical for DENV replication, we performed a reverse genetic screening of 33 NS4B mutants in the context of an infectious DENV genome. While the majority of these mutations were lethal, for several of them, we were able to select for second-site pseudoreversions, most often residing in NS4B and restoring replication competence. To identify all viral NS4B interaction partners, we engineered a fully viable DENV genome encoding an affinity-tagged NS4B. Mass spectrometry-based analysis of the NS4B complex isolated from infected cells identified the NS3 protease/helicase as a major interaction partner of NS4B. By combining the genetic complementation map of NS4B with a replication-independent expression system, we identified the NS4B cytosolic loop—more precisely, amino acid residue Q134—as a critical determinant for NS4B-NS3 interaction. An alanine substitution at this site completely abrogated the interaction and DENV RNA replication, and both were restored by pseudoreversions A69S and A137V. This strict correlation between the degree of NS4B-NS3 interaction and DENV replication provides strong evidence that this viral protein complex plays a pivotal role during the DENV replication cycle, hence representing a promising target for novel antiviral strategies.IMPORTANCEWith no approved therapy or vaccine against dengue virus infection, the viral nonstructural protein 4B (NS4B) represents a possible drug target, because it is indispensable for virus replication. However, little is known about its precise structure and function. Here, we established the first comprehensive genetic interaction map of NS4B, identifying amino acid residues that are essential for virus replication, as well as second-site mutations compensating for their defects. Additionally, we determined the NS4B viral interactome in infected cells and identified the NS3 protease/helicase as a major interaction partner of NS4B. We mapped residues in the cytosolic loop of NS4B as critical determinants for interaction with NS3, as well as RNA replication. The strong correlation between NS3-NS4B interaction and RNA replication provides strong evidence that this complex plays a pivotal role in the viral replication cycle, hence representing a promising antiviral drug target.

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