Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole

Giardia duodenalis causes giardiasis, a major diarrheal disease in humans worldwide whose treatment relies mainly on metronidazole (MTZ) and albendazole (ABZ). The emergence of ABZ resistance in this parasite has prompted studies to elucidate the molecular mechanisms underlying this phenomenon. G. duodenalis trophozoites convert ABZ into its sulfoxide (ABZSO) and sulfone (ABZSOO) forms, despite lacking canonical enzymes involved in these processes, such as cytochrome P450s (CYP450s) and flavin-containing monooxygenases (FMOs). This study aims to identify the enzyme responsible for ABZ metabolism and its role in ABZ resistance in G. duodenalis. We first determined that the iron-containing cofactor heme induces higher mRNA expression levels of flavohemoglobin (gFlHb) in Giardia trophozoites. Molecular docking analyses predict favorable interactions of gFlHb with ABZ, ABZSO and ABZSOO. Spectral analyses of recombinant gFlHb in the presence of ABZ, ABZSO and ABZSOO showed high affinities for each of these compounds with Kd values of 22.7, 19.1 and 23.8 nM respectively. ABZ and ABZSO enhanced gFlHb NADH oxidase activity (turnover number 14.5 min-1), whereas LC-MS/MS analyses of the reaction products showed that gFlHb slowly oxygenates ABZ into ABZSO at a much lower rate (turnover number 0.01 min-1). Further spectroscopic analyses showed that ABZ is indirectly oxidized to ABZSO by superoxide generated from the NADH oxidase activity of gFlHb. In a similar manner, the superoxide-generating enzyme xanthine oxidase was able to produce ABZSO in the presence of xanthine and ABZ. Interestingly, we find that gFlHb mRNA expression is lower in albendazole-resistant clones compared to those that are sensitive to this drug. Furthermore, all albendazole-resistant clones transfected to overexpress gFlHb displayed higher susceptibility to the drug than the parent clones. Collectively these findings indicate a role for gFlHb in ABZ conversion to its sulfoxide and that gFlHb down-regulation acts as a passive pharmacokinetic mechanism of resistance in this parasite.

[1]  A. Jex,et al.  In vitro selection of Giardia duodenalis for Albendazole resistance identifies a β-tubulin mutation at amino acid E198K , 2021, International journal for parasitology. Drugs and drug resistance.

[2]  P. R. Gardner,et al.  Allostery in the nitric oxide dioxygenase mechanism of flavohemoglobin , 2020, The Journal of biological chemistry.

[3]  G. Panayotou,et al.  Formation of novel N-acetylcysteine-hemin adducts abrogates hemin-induced cytotoxicity and suppresses the NRF2-driven stress response in human pro-erythroid K562 cells. , 2020, European journal of pharmacology.

[4]  M. Bermejo,et al.  Giardiasis: Characteristics, Pathogenesis and New Insights About Treatment. , 2018, Current topics in medicinal chemistry.

[5]  Fabian Sievers,et al.  Clustal Omega for making accurate alignments of many protein sequences , 2018, Protein science : a publication of the Protein Society.

[6]  M. Couture,et al.  Resonance Raman studies on the flavohemoglobin of the protist Giardia intestinalis: evidence of a type I/II-peroxidase-like heme environment and roles of the active site distal residues , 2017, JBIC Journal of Biological Inorganic Chemistry.

[7]  M. McConville,et al.  Transcriptomics Indicates Active and Passive Metronidazole Resistance Mechanisms in Three Seminal Giardia Lines , 2017, Front. Microbiol..

[8]  G. Dayer,et al.  Heme proteins of Giardia intestinalis. , 2015, Experimental parasitology.

[9]  John J. Irwin,et al.  ZINC 15 – Ligand Discovery for Everyone , 2015, J. Chem. Inf. Model..

[10]  E. Redman,et al.  Genetic evidence for the spread of a benzimidazole resistance mutation across southern India from a single origin in the parasitic nematode Haemonchus contortus. , 2015, International journal for parasitology.

[11]  E. Saavedra,et al.  Albendazole induces oxidative stress and DNA damage in the parasitic protozoan Giardia duodenalis , 2015, Front. Microbiol..

[12]  Yang Zhang,et al.  I-TASSER server: new development for protein structure and function predictions , 2015, Nucleic Acids Res..

[13]  R. Argüello-García,et al.  An antioxidant response is involved in resistance of Giardia duodenalis to albendazole , 2015, Front. Microbiol..

[14]  A. Hehl,et al.  Comparative characterisation of two nitroreductases from Giardia lamblia as potential activators of nitro compounds , 2015, International journal for parasitology. Drugs and drug resistance.

[15]  Sanjay Gupta,et al.  Type I flavohemoglobin of mycobacterium smegmatis is a functional nitric oxide dioxygenase , 2014, IUBMB life.

[16]  Yang Zhang,et al.  The I-TASSER Suite: protein structure and function prediction , 2014, Nature Methods.

[17]  J. Tachezy,et al.  Giardia intestinalis Incorporates Heme into Cytosolic Cytochrome b5 , 2013, Eukaryotic Cell.

[18]  P. Nejsum,et al.  Genetic variations in the beta-tubulin gene and the internal transcribed spacer 2 region of Trichuris species from man and baboons , 2013, Parasites & Vectors.

[19]  M. Basáñez,et al.  Association between Response to Albendazole Treatment and β-Tubulin Genotype Frequencies in Soil-transmitted Helminths , 2013, PLoS neglected tropical diseases.

[20]  R. Argüello-García,et al.  Proteomic and transcriptional analyses of genes differentially expressed in Giardia duodenalis clones resistant to albendazole. , 2013, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[21]  E. Warkentin,et al.  Active site analysis of yeast flavohemoglobin based on its structure with a small ligand or econazole , 2012, The FEBS journal.

[22]  A. Mauk,et al.  Cytochrome b5 from Giardia lamblia. , 2012, Metallomics : integrated biometal science.

[23]  L. Quihui-Cota,et al.  Persistence of intestinal parasitic infections during the national de-worming campaign in schoolchildren of northwestern Mexico: a cross-sectional study , 2012, Annals of gastroenterology.

[24]  A. Landar,et al.  Hemin causes mitochondrial dysfunction in endothelial cells through promoting lipid peroxidation: the protective role of autophagy. , 2012, American journal of physiology. Heart and circulatory physiology.

[25]  Sanjay Gupta,et al.  An Unconventional Hexacoordinated Flavohemoglobin from Mycobacterium tuberculosis* , 2012, The Journal of Biological Chemistry.

[26]  H. Jung-Cook,et al.  A simple LC-MS/MS method to determine plasma and cerebrospinal fluid levels of albendazole metabolites (albendazole sulfoxide and albendazole sulfone) in patients with neurocysticercosis. , 2012, Biomedical chromatography : BMC.

[27]  S. Robinson,et al.  The metabolism and toxicity of hemin in astrocytes , 2011, Glia.

[28]  F. Gillin,et al.  Impaired Parasite Attachment as Fitness Cost of Metronidazole Resistance in Giardia lamblia , 2011, Antimicrobial Agents and Chemotherapy.

[29]  S. Robinson,et al.  Uptake, metabolism and toxicity of hemin in cultured neurons , 2011, Neurochemistry International.

[30]  Aurélien Grosdidier,et al.  SwissDock, a protein-small molecule docking web service based on EADock DSS , 2011, Nucleic Acids Res..

[31]  T. Lithgow,et al.  The Minimal Proteome in the Reduced Mitochondrion of the Parasitic Protist Giardia intestinalis , 2011, PloS one.

[32]  E. Warkentin,et al.  Structure of Ralstonia eutropha flavohemoglobin in complex with three antibiotic azole compounds. , 2011, Biochemistry.

[33]  L. Pucillo,et al.  Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis. , 2010, Biochemical and biophysical research communications.

[34]  R. March,et al.  Giardia lamblia encodes a functional flavohemoglobin. , 2010, Biochemical and biophysical research communications.

[35]  J. Wen,et al.  Giardiasis, drug resistance, and new target discovery. , 2010, Infectious disorders drug targets.

[36]  S. Svärd,et al.  Behind the smile: cell biology and disease mechanisms of Giardia species , 2010, Nature Reviews Microbiology.

[37]  L. Robertson,et al.  Giardiasis--why do the symptoms sometimes never stop? , 2010, Trends in parasitology.

[38]  E. Oldfield,et al.  Binding of Azole Antibiotics to Staphylococcus aureus Flavohemoglobin Increases Intracellular Oxidative Stress , 2010, Journal of bacteriology.

[39]  Arthur J. Olson,et al.  AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading , 2009, J. Comput. Chem..

[40]  R. Argüello-García,et al.  In vitro resistance to 5-nitroimidazoles and benzimidazoles in Giardia duodenalis: variability and variation in gene expression. , 2009, Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases.

[41]  Pascal Benkert,et al.  QMEAN server for protein model quality estimation , 2009, Nucleic Acids Res..

[42]  Eileen Kraemer,et al.  GiardiaDB and TrichDB: integrated genomic resources for the eukaryotic protist pathogens Giardia lamblia and Trichomonas vaginalis , 2008, Nucleic Acids Res..

[43]  A. Boffi,et al.  Flavohemoglobin: Structure and reactivity , 2007, IUBMB life.

[44]  A. Boffi,et al.  Structural studies on flavohemoglobins. , 2008, Methods in enzymology.

[45]  Feng Chen,et al.  Genomic Minimalism in the Early Diverging Intestinal Parasite Giardia lamblia , 2007, Science.

[46]  A. Escobedo,et al.  Giardiasis: a pharmacotherapy review , 2007, Expert opinion on pharmacotherapy.

[47]  Joachim Müller,et al.  Characterization of Giardia lamblia WB C6 clones resistant to nitazoxanide and to metronidazole. , 2007, The Journal of antimicrobial chemotherapy.

[48]  C. Hess,et al.  Therapy-Resistant Diarrhea due to Giardia lamblia in a Patient with Common Variable Immunodeficiency Disease , 2007, Digestion.

[49]  Manfred J. Sippl,et al.  Thirty years of environmental health research--and growing. , 1996, Nucleic Acids Res..

[50]  A. Thompson,et al.  Giardia and Cryptosporidium join the 'Neglected Diseases Initiative'. , 2006, Trends in parasitology.

[51]  P. Upcroft,et al.  5-Nitroimidazole Drugs Effective against Metronidazole-Resistant Trichomonas vaginalis and Giardia duodenalis , 2006, Antimicrobial Agents and Chemotherapy.

[52]  J. Gough,et al.  A phylogenomic profile of globins , 2006, BMC Evolutionary Biology.

[53]  P. R. Gardner,et al.  Imidazole Antibiotics Inhibit the Nitric Oxide Dioxygenase Function of Microbial Flavohemoglobin , 2005, Antimicrobial Agents and Chemotherapy.

[54]  J. Skolnick,et al.  TM-align: a protein structure alignment algorithm based on the TM-score , 2005, Nucleic acids research.

[55]  J. Farrés,et al.  Ligand binding properties of bacterial hemoglobins and flavohemoglobins. , 2005, Biochemistry.

[56]  P. R. Gardner.  Nitric oxide dioxygenase function and mechanism of flavohemoglobin, hemoglobin, myoglobin and their associated reductases. , 2005, Journal of inorganic biochemistry.

[57]  I. Fairweather,et al.  A possible model of benzimidazole binding to beta-tubulin disclosed by invoking an inter-domain movement. , 2004, Journal of molecular graphics & modelling.

[58]  A. Armson,et al.  Characterisation of benzimidazole binding with recombinant tubulin from Giardia duodenalis, Encephalitozoon intestinalis, and Cryptosporidium parvum. , 2004, Molecular and biochemical parasitology.

[59]  R. Poole,et al.  Escherichia coli Hmp, an “oxygen-binding flavohaemoprotein”, produces superoxide anion and self-destructs , 2004, Archives of Microbiology.

[60]  P. R. Gardner,et al.  Nitric oxide metabolism in mammalian cells: substrate and inhibitor profiles of a NADPH-cytochrome P450 oxidoreductase-coupled microsomal nitric oxide dioxygenase. , 2004, Free radical biology & medicine.

[61]  P. R. Gardner,et al.  Inducible Defense Mechanism against Nitric Oxide in Candida albicans , 2004, Eukaryotic Cell.

[62]  A. Pesce,et al.  Mapping protein matrix cavities in human cytoglobin through Xe atom binding. , 2004, Biochemical and biophysical research communications.

[63]  Anders Björkman,et al.  Identification of human cytochrome P450s that metabolise anti-parasitic drugs and predictions of in vivo drug hepatic clearance from in vitro data , 2003, European Journal of Clinical Pharmacology.

[64]  R. Regan,et al.  Hemin induces an iron‐dependent, oxidative injury to human neuron‐like cells , 2003, Journal of neuroscience research.

[65]  A. Boffi,et al.  Escherichia coli Flavohemoglobin Is an Efficient Alkylhydroperoxide Reductase* , 2003, Journal of Biological Chemistry.

[66]  M. Avery,et al.  Giardiasis: recent progress in chemotherapy and drug development. , 2003, Current pharmaceutical design.

[67]  O. Dym,et al.  Sequence‐structure analysis of FAD‐containing proteins , 2001, Protein science : a publication of the Protein Society.

[68]  T. Moore,et al.  Treatment of patients with refractory giardiasis. , 2001, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[69]  R. Poole,et al.  Flavohemoglobin, a Globin with a Peroxidase-like Catalytic Site* , 2001, The Journal of Biological Chemistry.

[70]  P. Upcroft,et al.  Drug Targets and Mechanisms of Resistance in the Anaerobic Protozoa , 2001, Clinical Microbiology Reviews.

[71]  C. Mascie-Taylor,et al.  Anthelmintic treatment of rural Bangladeshi children: effect on host physiology, growth, and biochemical status. , 2001, The American journal of clinical nutrition.

[72]  J. Reynoldson,et al.  The binding and distribution of albendazole and its principal metabolites in Giardia duodenalis. , 2000, Journal of veterinary pharmacology and therapeutics.

[73]  G. Kokwaro,et al.  Relative contribution of cytochromes P-450 and flavin-containing monoxygenases to the metabolism of albendazole by human liver microsomes. , 2000, British journal of clinical pharmacology.

[74]  P. R. Miles,et al.  Hemoglobin potentiates the production of reactive oxygen species by alveolar macrophages. , 2000, Experimental lung research.

[75]  D T Jones,et al.  Protein secondary structure prediction based on position-specific scoring matrices. , 1999, Journal of molecular biology.

[76]  M. N. Hughes,et al.  Anoxic function for the Escherichia coli flavohaemoglobin (Hmp): reversible binding of nitric oxide and reduction to nitrous oxide , 1999, FEBS letters.

[77]  P. R. Gardner,et al.  Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[78]  N. Buisson,et al.  Flavohemoglobin Expression and Function in Saccharomyces cerevisiae , 1998, The Journal of Biological Chemistry.

[79]  T. Nash,et al.  Episomal and integrated maintenance of foreign DNA in Giardia lamblia. , 1998, Molecular and biochemical parasitology.

[80]  D. Eisenberg,et al.  VERIFY3D: assessment of protein models with three-dimensional profiles. , 1997, Methods in enzymology.

[81]  D. Raitt,et al.  Function and Expression of Flavohemoglobin in Saccharomyces cerevisiae , 1996, The Journal of Biological Chemistry.

[82]  R. Poole,et al.  The flavohaemoglobin (HMP) of Escherichia coli generates superoxide in vitro and causes oxidative stress in vivo , 1996, FEBS letters.

[83]  P. Upcroft,et al.  Quinacrine-resistant Giardia duodenalis , 1996, Parasitology.

[84]  W. Pryor.  Cytochrome P450: Structure, mechanism, and biochemistry , 1996 .

[85]  P. Upcroft,et al.  Albendazole resistance in Giardia is correlated with cytoskeletal changes but not with a mutation at amino acid 200 in beta-tubulin. , 1996, Microbial drug resistance.

[86]  D. Goldberg,et al.  Regulation of Saccharomyces cerevisiae Flavohemoglobin Gene Expression (*) , 1995, The Journal of Biological Chemistry.

[87]  G. Wiedermann,et al.  Albendazole is highly effective against cutaneous larva migrans but not against Giardia infection: results of an open pilot trial in travellers returning from the tropics. , 1993, Transactions of the Royal Society of Tropical Medicine and Hygiene.

[88]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[89]  J. Reynoldson,et al.  Activities of several benzimidazoles and tubulin inhibitors against Giardia spp. in vitro , 1993, Antimicrobial Agents and Chemotherapy.

[90]  A. Martínez-Palomo,et al.  Giardia lamblia: ultrastructural study of the in vitro effect of benzimidazoles. , 1992, The Journal of protozoology.

[91]  D. Eisenberg,et al.  Assessment of protein models with three-dimensional profiles , 1992, Nature.

[92]  J. Eaton,et al.  Hemin: a possible physiological mediator of low density lipoprotein oxidation and endothelial injury. , 1991, Arteriosclerosis and thrombosis : a journal of vascular biology.

[93]  D. Eisenberg,et al.  A method to identify protein sequences that fold into a known three-dimensional structure. , 1991, Science.

[94]  R. Prichard,et al.  Specific interaction of benzimidazole anthelmintics with tubulin: high-affinity binding and benzimidazole resistance in Haemonchus contortus. , 1990, Molecular and biochemical parasitology.

[95]  J. Murrell.  Structure and Reactivity , 1989 .

[96]  I. Fridovich,et al.  Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. , 1971, Analytical biochemistry.