title: Cytochrome b mutation Y268S conferring atovaquone resistance phenotype in malaria parasite results in reduced parasite bc1 catalytic turnover and protein expression.
abstract: Atovaquone is an anti-malarial drug used in combination with proguanil (e.g. Malarone(TM)) for the curative and prophylactic treatment of malaria. Atovaquone, a 2-hydroxynaphthoquinone, is a competitive inhibitor of the quinol oxidation (Q(o)) site of the mitochondrial cytochrome bc(1) complex. Inhibition of this enzyme results in the collapse of the mitochondrial membrane potential, disruption of pyrimidine biosynthesis, and subsequent parasite death. Resistance to atovaquone in the field is associated with point mutations in the Q(o) pocket of cytochrome b, most notably near the conserved Pro(260)-Glu(261)-Trp(262)-Tyr(263) (PEWY) region in the ef loop). The effect of this mutation has been extensively studied in model organisms but hitherto not in the parasite itself. Here, we have performed a molecular and biochemical characterization of an atovaquone-resistant field isolate, TM902CB. Molecular analysis of this strain reveals the presence of the Y268S mutation in cytochrome b. The Y268S mutation is shown to confer a 270-fold shift of the inhibitory constant (K(i)) for atovaquone with a concomitant reduction in the V(max) of the bc(1) complex of ∼40% and a 3-fold increase in the observed K(m) for decylubiquinol. Western blotting analyses reveal a reduced iron-sulfur protein content in Y268S bc(1) suggestive of a weakened interaction between this subunit and cytochrome b. Gene expression analysis of the TM902CB strain reveals higher levels of expression, compared with the 3D7 (atovaquone-sensitive) control strain in bc(1) and cytochrome c oxidase genes. It is hypothesized that the observed differential expression of these and other key genes offsets the fitness cost resulting from reduced bc(1) activity.
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom and.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Fisher
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom and.
authors.forename: Roslaini
authors.initials: R
authors.lastname: Abd Majid
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom and.
authors.forename: Thomas
authors.initials: T
authors.lastname: Antoine
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom and.
authors.forename: Mohammed
authors.initials: M
authors.lastname: Al-Helal
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom and.
authors.forename: Ashley J
authors.initials: AJ
authors.lastname: Warman
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom and.
authors.forename: David J
authors.initials: DJ
authors.lastname: Johnson
authors.affiliation: Department of Chemistry, University of Liverpool, Liverpool L69 7ZD, United Kingdom.
authors.forename: Alexandre S
authors.initials: AS
authors.lastname: Lawrenson
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom and.
authors.forename: Hilary
authors.initials: H
authors.lastname: Ranson
authors.affiliation: Department of Chemistry, University of Liverpool, Liverpool L69 7ZD, United Kingdom.
authors.forename: Paul M
authors.initials: PM
authors.lastname: O'Neill
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom and. Electronic address: saward@liv.ac.uk.
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Ward
authors.affiliation: Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, United Kingdom and. Electronic address: Biagini@liverpool.ac.uk.
authors.forename: Giancarlo A
authors.initials: GA
authors.lastname: Biagini
mesh_terms: D019943:Amino Acid Substitution; D000962:Antimalarials / Q000494:pharmacology*; D053626:Atovaquone / Q000494:pharmacology*; D045303:Cytochromes b / Q000096:biosynthesis* / Q000235:genetics; D004351:Drug Resistance; D014450:Electron Transport Complex III / Q000235:genetics / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D020125:Mutation, Missense; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics; D002727:Proguanil / Q000494:pharmacology; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000962:Antimalarials; D015800:Protozoan Proteins; D045303:Cytochromes b; D014450:Electron Transport Complex III; D002727:Proguanil; D053626:Atovaquone