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UntitledJ. Eukaryot. Microbiol., 54(1), 2007 pp. 38–41r 2006 The Author(s)Journal compilation r 2006 by the International Society of ProtistologistsDOI: 10.1111/j.1550-7408.2006.00140.x Analysis of the b-Tubulin Genes from Enterocytozoon bieneusi Isolates from a DONNA E. AKIYOSHI,a LOUIS M. WEISS,b XIAOCHUAN FENG,a BRYONY A. P. WILLIAMS,c PATRICK J. KEELING,c aDivision of Infectious Diseases, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts 01536, and bDepartment of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA, and cCanadian Institute for Advanced Research, Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada Enterocytozoon bieneusi is the most common and clinically significant microsporidium associated with chronic diarrhea and wasting in immunocompromised humans. Albendazole, which is effective against several helminths, protozoa, and microsporidia, isrelatively ineffective against infections due to E. bieneusi. A likely explanation for the observed clinical resistance to albendazole wasdiscovered from sequence analysis of the E. bieneusi b-tubulin from isolates from an infected human and a naturally infected rhesusmacaque. The b-tubulin of E. bieneusi has a substitution at Glu198, which is one of six amino acids reported to be associated with benz-imidazole sensitivity.
Key Words. Albendazole resistance, beta-tubulin gene, microsporidia.
THE microsporidia represent a large and diverse group of positive for Enterocytozoon bieneusi. Spores were purified from obligate intracellular eukaryotic parasites and to date, 1,200 the human and rhesus macaque stool samples (Sheoran et al. 2005; species have been identified. Of these, Enterocytozoon bieneusi is Zhang et al. 2005), and were confirmed to be E. bieneusi by elec- the most clinically significant species associated with AIDS-re- tron microscopy and sequencing of the internal transcribed spacer lated human microsporidiosis (reviewed in Cali 1991; Curry and (ITS) of the small subunit rRNA gene. The spores isolated from Canning 1993; Didier et al. 2004; Keeling and Fast 2002; Mathis, the human patient and rhesus macaque will be referred to as the Weber, and Deplazes 2005; Wittner 1999), with symptoms includ- H206 isolate and M231 isolate, respectively.
ing chronic diarrhea, wasting, and malabsorption. Enterocytozoon Cloning and sequencing of the b-tubulin genes. The b-tubulin bieneusi has also been identified in immunocompetent patients gene from the M231 isolate was first isolated from a whole genome (Albrecht and Sobottka 1997; Gainzarain et al. 1998; Sandfort et amplified (Molecular Staging Inc., New Haven, CT) library cloned al. 1994), in individuals receiving immunosuppressive therapy into the pHCSmart-Kan vector (Lucigen Corp., Middleton, WI).
(Guerard et al. 1999; Rabodonirina et al. 1996), and in macaques, The E. bieneusi b-tubulin genes from the H206 and M231 isolates both immunocompetent and those infected with simian immuno- were then amplified from purified genomic DNA (1 ng; DNeasy deficiency virus (SIV) (Green et al. 2004; Mansfield et al. 1997).
Tissue Kit; Qiagen Inc., Valencia, CA) using primers, MEb btub-I Microtubules, which are formed by polymerization of the (50-AACGGGCAGCTGAGTAGTTTAAGTGATT-30) and MEb a- and b-tubulin subunits, are a major component of the mito- tic spindle. The benzimidazoles have been found to prevent using the Expand High Fidelity PCR System (Roche Diagnostics both the polymerization of the tubulin subunits by binding to the Corp., Indianapolis, IN). The PCR products were cloned into b-tubulin subunit, thus preventing elongation of the microtubules, pCR4TOPO (Invitrogen Corp., Carlsbad, CA) and used to trans- and depolymerization of the two subunits. The benzimidazoles form E. coli TOP10 cells. Inserts from four independent clones are toxic to fungi and helminths (Davidse 1986; Lacey 1988), and were double-strand sequenced for each isolate.
have been used to effectively treat both microsporidiosis due toEncephalitozoon spp. (De Groote et al. 1995; Katiyar and Edlind 1997; Molina et al. 1998; Ridoux and Drancourt 1998), and someprotozoan infections including Giardia intestinalis (Katiyar et al.
Analysis of the b-tubulin of Enterocytozoon bieneusi. The 1994; Lemee et al. 2000). However, these benzimidazole deriv- E. bieneusi b-tubulin gene was first identified in the M231 amp- atives, including albendazole, are relatively ineffective against E.
lified genomic library. One clone, with an open reading frame bieneusi infections (Blanshard et al. 1992; Conteas et al. 2000; of 1,314 bp, had significant similarity (E-value 5 e À 177) to other b-tubulin sequences in the NCBI database using the BLASTp In this communication, we report the analysis of the b-tubulin program (Altschul et al. 1990; McGinnis and Madden 2004). PCR gene from E. bieneusi isolated from an HIV-infected human and primers, Meb btub-I and MEb-btub-J, were designed to amplify an SIV-infected rhesus macaque (Macaca mulatta). The sequence the b-tubulin gene from purified spores isolated from a human data provide an explanation for the reported resistance of E.
patient (H206) and a rhesus macaque (M231). These genes were compared using the ClustalW algorithm (Thompson, Higgins, andGibson 1994). Both genes were 1,314 bp and were identical ex-cept for five synonymous transitions (99.62% sequence identity).
The A-T content was 55.4% and 55.6% for the M231 and H206 b- Parasite strains. An HIV-positive adult patient admitted to the tubulin genes, respectively. Analysis of the 50—and 30-flanking Mulago Hospital in Kampala, Uganda, and an SIV-infected rhesus regions revealed no obvious canonical elements, such as promoter macaque (Macaca mulatta), housed at the New England Regional elements or polyadenylation sequences.
Primate Research Center (Southborough, MA), were found to be The E. bieneusi b-tubulin gene encoded a 438-amino acid polypeptide with a molecular weight of 49,116 daltons. A highdegree of sequence identity (68%–73%) was observed between Corresponding Author: D. Akiyoshi, Division of Infectious Diseases, the E. bieneusi b-tubulin and b-tubulins from the family Enceph- Tufts Cummings School of Veterinary Medicine, North Grafton, Mas-sachusetts 01536—Telephone number: 508-839-7935; FAX number: alitozoonidae, Antonospora locustae, Trachipleistophora homin- 508-839-7911; e-mail: firstname.lastname@example.org is, Saccharomyces cerevisiae, and Homo sapiens. However, the AKIYOSHI ET AL.—b-TUBULIN GENES FROM E. BIENEUSI + E. bieneusi MREIIHVQAGQCGNQIGTKFWEEISREHGIDENGKKCGCDGDDGWCDETNRISVYYNQSSSNKYVPRAVLVDLEPGTMEAIRNHPMGNIFRPDNFIFGQS 100E. cuniculi MREIIHLQTGQCGNQVGCKFWETISGEHGIDQTGRYVGTSDN-----QLERINVYYNEASSKKYVPRAVLIDLEPGTMDAVRQGPFGELFRPDNFVFGQS 95E. hellem MREIIHLQTGQCGNQVGCKFWETISGEHGIDQTGKYVGTSDN-----QLERVNVYYNEASSKKYVPRAVLIDLEPGTMDAVRQGPFGDLFRPDNFVFGQS 95E. intestinalis REIIHIQTGQCGNQIGAKFWETISGEHGVDPSGRYVGTSDL-----QIERINVYYNEASGKKYVPRAVLIDLEPGTMDSVRAGPFGELFRPDNFVFGQS 94N. locustae VGSKFWEVISEEHGINNEGHFVGHSSN-----QLERINVYYNEASSSKYVPRAVLIDLEPGTMDSVRAGPLGRLFRPDNFIFGQS 80T. hominis IGTKFWEVISEEHGINNLGQYTGTKDN-----QLDRISVYYNESSTKQYVPRAVLVDLEPGTMDTLRSGPLGSLFRPDNYVFGQS 80S. cerevisiae MREIIHISAGQYGNQIGAAFWETICGEHGLDFNGTYHGHDDI-----QKERLNVYFNEASSGKWVPRSINVDLEPWTIDAVRNSAIGNLFRPDNYIFGQS 95H. sapiens MREIVHIQAGQCGNQIGAKFWEVISDEHGIDPTGSYHGDSDL-----QLERINVYYNEAAGNKYVPRAILVDLEPGTMDSVRSGPFGQIFRPDNFVFGQS 95 .*. *** *. ***. * * . *. **.*. . ***. .**** *.* * .*****.**** + + E. bieneusi GAGNNWAKGHYTEGAELCEQVLECIRKEAEKTDCLQGFQLTHSLGGGTGSGMGTLLVSKIKEEFPDRMLATFSVVPSPKVSDTVVEPYNATLSFHQLVEN 200E. cuniculi GAGNNWAKGHYTEGAELIDSVMDVVRKEAESSDCLQGFQITHSLGGGTGAGMGTLLLSKIREDFPDRMICTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 195E. hellem GAGNNWAKGHYTEGAELIDSVMDVVRKEAESSDCLQGFQITHSLGGGTGAGMGTLLLSKIREDFPDRMICTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 195E. intestinalis GAGNNWAKGHYTEGAELIDAVMDVVRKEAESCDCLQGFQITHSLGGGTGAGMGTLLIAKIREDFPDRMICTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 194N. locustae GAGNNWAKGHYTEGAELIDSVLDVVRKEAESSDCLQGFQFTHSLGGGTGAGMGTLLISKIREEYPDRMMCTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 180T. hominis GAGNNWAKGHYTEGAELIENVMDCVRREAEKSNCLQGFQITHSLGGGTGAGMGTLLISKIREEFPDRMMCTFSVVPSPKVSDTVVEPYNATLSIHQLVEN 180S. cerevisiae SAGNVWAKGHYTEGAELVDSVMDVIRREAEGCDSLQGFQITHSLGGGTGSGMGTLLFSKIKEELPDRMMATFSVLPSPKTSDTVVEPYNATLSVHQLVEH 195H. sapiens GAGNNWAKGHYTEGAELVDSVLDVVRKESESCDCLQGFQLTHSLGGGTGSGMGTLLISKIREEYPDRIMNTFSVMPSPKVSDTVVEPYNATLSVHQLVEN 195 *** ************ . *. .*.*.* . .***** *********.****** .**.*. ***. ****.**** ************* *****. + + + E. bieneusi ANQTFCIDNDALYEICTKTLKLKNPSYNDLNSLVSKVMSGITTCLRFPGQLNSDLRKLAVNMIPFPRLHFFCVGYAPLCSEASTQYRNITVSDLTAQLFD 300E. cuniculi ADETFCIDNEALYDICFRTLKLNNPGYGDLNHLVSLVMSGVTTCLRFPGQLNADLRKLAVNMIPFPRLHFFVAGFAPLIAIGTQKFKTYSVSELTQQMFD 295E. hellem ADETFCIDNEALYDICFRTLKMSNPGYGDLNHLVSLVMSGVTTCLRFPGQLNADLRKLAVNMIPFPRLHFFVVGSAPLIAIGTQKFKTYSVSELTQQMFD 295E. intestinalis ADEVFCIDNEALYDICFRTLKLSNPGYGDLNHLVSLVMSGVTSCLRFPGQLNADLRKLAVNMIPFPRLHFFLVGFAPLTAVGSQKFKTYSVSELTQQMFD 294N. locustae ADETFCIDNEALYDICFRTLKLSTPGYGELNRLVSLVMSGVTTCLRFPGQLNADLRKLAVNMVPFPRLHFFIVGFAPLIAQGTSQYRTYSVSELTSQMFD 280T. hominis ADETFCIDNEALYNICFNILKLKNPGYADLNRLVSLVMSGVTTCLRFPGQLNADLRKLAVNMIPFPRLHFFMIGFAPLIAEGMASYRSYSVSELTQQMFD 280S. cerevisiae SDETFCIDNEALYDICQRTLKLNQPSYGDLNNLVSSVMSGVTTSLRYPGQLNSDLRKLAVNLVPFPRLHFFMVGYAPLTAIGSQSFRSLTVPELTQQMFD 295H. sapiens TDETYCIDNEALYDICFRTLKLTTPTYGDLNHLVSATMSGVTTCLRFPGQLNADLRKLAVNMVPFPRLHFFMPGFAPLTSRGSQQYRALTVPELTQQMFD 295 . . .****.*** ** **. * * .** *** ***.*.**.*****.********.******** * *** . . .* .** *.** E. bieneusi SKNMMTACNPRDGRYLTAAVYFRGKMSMKEVDEQMNLMQTRTMDSFVEWIPNNVQTAVCSVPPKDVEMSATFIGNTTSIQEIFKRVGEQFSSMFKRKAFL 400E. cuniculi SKNMMTACDPRKGRYLTVAAMFRGKISMKDVDEQMSMVQSKNSSLFVEWIPSNVKTAVCDIAPTGLEMSATFVGNTTSIQELFKRISDQFTVMFRRKAFL 395E. hellem SKNMMTACDPRKGRYLTVAAMFRGKISMKDVDEQMSMVQSKNSTLFVEWIPSNVKTAVCDIAPTGLEMSATFVGNTTSIQELFKRISDQFTVMFRRKAFL 395E. intestinalis SKNMMTASDPRKGRYLTAAAMFRGRISTKDVDEQMSMVQSKNSSYFVEWIPSNIKVAVCDIAPTGLEMSATFVGNSTSIQELFKRVSDQFTVMFRRKAFL 394N. locustae SKNMMAASDPRHGRYLTVAAVFRGKISMKDVDEQMLQVQTRNSAHFVEWIPNNVKTAVCDIPPSGLDMSATFIGNSTSIQELFKRISDQFSVMFRRKAFL 380T. hominis SKNMMAASDPKHGRYLTVATIFRGNISMKDVDEQLHNIQSRNASNFVEWIPNNVKTAVCDIPPSTLDMSATFIGNTTAIQELFKRIAEQFQLMFRRKAFL 380S. cerevisiae AKNMMAAADPRNGRYLTVAAFFRGKVSVKEVEDEMHKVQSKNSDYFVEWIPNNVQTAVCSVAPQGLDMAATFIANSTSIQELFKRVGDQFSAMFKRKAFL 395H. sapiens SKNMMAACDPRHGRYLTVAAIFRGRMSMKEVDEQMLNVQNKNSSYFVEWIPNNVKTAVCDIPPRGLKMSATFIGNSTAIQELFKRISEQFTTMFRRKAFL 395 .****.*. *. ***** * *** .* *.*. .* . ****** *. *** . * . *.***. *.*.***.***. .** **.***** E. bieneusi HWYTGEGMDEAEFTEAEANLQDLLSEYQQYRDSGVGGYN 439E. cuniculi HWYTGEGMDEMEFSEAESNMNDLLSEYQQYQDATIEDAEEFLVN 439E. hellem HWYTGEGMDEMEFSEAESNMNDLLSEYQQYQDATVEDAEEFLVN 439E. intestinalis HWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATVEDAEEFLVN 438N. locustae HWYTGEG 387T. hominis HWYTQEG 387S. cerevisiae HWYTSEGMDELEFSEAESNMNDLVSEYQQYQEATVEDDEEVDENGDFGAPQNQDEPITENFE 457H. sapiens HWYTGEGMDEMEFTEAESNMNDLVSEYQQYQDATADEQGEFEEEEGEDEA 445 ClustalW alignment of the b-tubulin amino acid sequences from Enterocytozoon bieneusi, Encephalitozoon cuniculi (GenBank Accession no. NP_597591), Encephalitozoon hellem (GenBank Accession no. AAB12034), Encephalitozoon intestinalis (GenBank Accession no. AAN78302),Nosema locustae (GenBank accession no. AAN35161), Trachipleistophora hominis (GenBank Accession no. AAF31660), Saccharomyces cerevisiae(GenBank Accession no. CAA24603), and Homo sapiens (GenBank Accession no. T08726). Positions with identical residues in all eight sequences areindicated by asterisks (below). Conserved changes are indicated by a dot (below). The b-tubulin sequence from E. bieneusi had an apparent five aminoacid insertion (boxed), which was not found in other fungal, microsporidial or mammalian sequences. Changes in the six amino acids (His6, Phe167,Glu198, Phe200, Arg241; numbering based on S. cerevisiae), which are associated with benzimidazole resistance, are indicated by a cross (1) above.
Glu198, which has a high association with benzimidazole sensitivity, is changed to a glutamine in E. bieneusi. Nucleotide sequences of the b-tubulin genesare available in the GenBank database under Accession numbers, DQ242639 (human) and DQ242640 (rhesus macaque).
E. bieneusi b-tubulin has near its amino terminus an additional Phe200, which confer resistance to benzimidazole (Katiyar et al.
five residues (DGWCD) that are unlikely to represent an intron 1994; Kwa, Veenstra, and Roos 1994). With respect to Ala165, the because of the absence of a canonical GT-AG splice boundary, as b-tubulins of the Encephalitozoonidae have five of the six resi- well as, an AT-AC boundary (Fig. 1).
dues, but with Ala165 changed to a Cys165. Yet, they are highly The most significant finding was a molecular explanation for sensitive to albendazole (Cruz, Bartlett, and Edlind 1994; Katiyar the observed clinical resistance of E. bieneusi to benzimidazoles.
et al. 1994). This residue is also a cysteine in Giardia lamblia The E. bieneusi b-tubulin has only five of the six amino acids that (Kirk-Mason, Turner, and Chakrabory 1988) and Cryptococcus have been reported to be associated with benzimidazole activity neoformans (Li et al. 1996), which are also sensitive to albenda- (His6, Phe167, Glu198, Phe200 or Arg241; numbering based on the zole. Contrary to this, Ala165 is replaced by a glycine in thiaben- S. cerevisiae sequence, Fig. 1) (Jung, Wilder, and Oakley 1992; dazole-resistant strains of Aspergillus nidulans. Of these six Ohrbach, Porro, and Yanofsky 1986; Thomas, Neff, and Botstein amino acids, His6 and Arg241, are not highly predictive of benz- 1985). The one residue difference occurred at position 198 in imidazole sensitivity because these two residues are conserved in which glutamine was substituted for glutamic acid.
both benzimidazole-resistant and benzimidazole-sensitive organ- Organisms resistant to benzimidazole lack one or both of isms. Phe167 is also not highly predictive of benzimidazole sensi- Glu198 or Phe200. Both E. bieneusi and Entamoeba histolytica tivity because the Trichomonas vaginalis b-tubulins have tyrosine have changes at Glu198, and both are relatively resistant to alben- instead of phenylalanine at this position (Katiyar and Edlind dazole. Cryptosporidium parvum and Acanthamoeba polyphaga 1997), yet the organism is sensitive to the benzimidazoles. Over- have changes at both Glu198 and Phe200, and both are resistant to all, the data for these six amino acids show that substitutions of benzimidazole. The helminths, fungi, and humans have changes at either Glu198 or Phe200 are highly associated with benzimidazole J. EUKARYOT. MICROBIOL., VOL. 54, NO. 1, JANUARY– FEBRUARY 2007 sensitivity, and changes of one or both of these amino acids results of disseminated Encephalitozoon cuniculi in a patient with AIDS: in resistance to benzimidazole. The E. bieneusi b-tubulin data are successful therapy. J. Infect. Dis., 171:1375–1378.
Didier, E. S., Stovall, M. E., Green, L. C., Brindley, P. J., Sestak, K. & The microsporidia have been placed within the fungal clade Didier, P. J. 2004. Epidemiology of microsporidiosis: sources and based on phylogenetic analyses of the a- and b-tubulin genes modes of transmission. Vet. Parasitol., 126:145–166.
Dieterich, D., Lew, E., Kotler, D., Poles, M. & Orenstein, J. 1994.
(Edlind et al. 1996; Keeling and Doolittle 1996), sequencing of Treatment with albendazole for intestinal disease due to Enterocytozo- the E. cuniculi genome (Katinka et al. 2001; Vivares et al. 2002), on bieneusi in patients with AIDS. J. Infect. Dis., 169:178–183.
the existence of relic mitochondrial genes in the nuclear genome, Edlind, T. D., Li, J., Visvesvara, G. S., Vodkin, M. H., McLaughlin, G. L.
and the presence of mitosomes and Golgi-like membranes. In ad- & Katiyar, S. K. 1996. Phylogenetic analysis of b-tubulin sequences dition, morphological and life cycle data are consistent with their from amitochondrial protozoa. Mol. Phylogenet. Evol., 5:359–367.
placement within the fungal clade (reviewed in Germot, Philippe, Gainzarain, J. C., Canut, A., Lozano, M., Labora, A., Carreras, F., Fenoy, and Le Guyander 1997; Keeling and Doolittle 1996). We attempt- S., Navajas, R., Pieniazek, N. J., da Silva, A. J. & del Aguila, C. 1998.
ed to align the E. bieneusi b-tubulin to 67 other b-tubulin se- Detection of Enterocytozoon bieneusi in two human immunodeficiency quences representative of the major eukaryotic groups, and then virus-negative patients with chronic diarrhea by polymerase chainreaction in duodenal biopsy specimens and review. Clin. Infect. Dis., use maximum likelihood methods (TREE-PUZZLE 5.2 and PHYML; Guindon and Gascuel 2003; Schmidt et al. 2002) to de- Germot, A., Philippe, H. & Le Guyader, H. 1997. Evidence for loss of termine the phylogenetic position of E. bieneusi. Unfortunately, mitochondria in Microsporidia from a mitochondrial-type HSP70 in the E. bieneusi b-tubulin sequence was found to be highly diver- Nosema locustae. Mol. Biochem. Parasitol., 87:159–168.
gent and its position within the tree was not well resolved (data Green, L. C., Didier, P. J., Bowers, L. C. & Didier, E. S. 2004. Natural and experimental infection of immunocompromised rhesus macaques While E. bieneusi is clinically the most significant human (Macaca mulatta) with the microsporidian Enterocytozoon bieneusi microsporidium, it is also the least understood with respect to genotype D. Microbes Infect., 6:996–1002.
its biology and epidemiology. Greater than 50 genotypes have Guerard, A., Rabodonirina, M., Cotte, M. L., Liguory, O., Piens, M. A., Daoud, S., Picot, S. & Touraine, J. L. 1999. Intestinal microsporidiosis been identified using the ITS sequence (Mathias et al. 2005; Sulai- occurring in two renal transplant recipients treated with mycophenolate man et al. 2004). A recent study characterized E. bieneusi isolates mofetil. Transplantation, 68:699–707.
from cattle and found some genotypes were host-adapted while Guindon, S. & Gascuel, O. 2003. A simple, fast, and accurate algorithm others were identical to the human genotype K, suggesting that to estimate large phylogenies by maximum likelihood. Syst. Biol., 52: these isolates might have the potential to infect humans (Sulaiman et al. 2004). Additional independent genetic markers are needed to Jung, M. K., Wilder, I. B. & Oakley, B. R. 1992. Amino acid alterations in provide more genotyping tools, which would also aid in clarifying the benA (b-tubulin) of Aspergillus nidulans that confer benomyl the genetic structure of E. bieneusi. The determination of the se- resistance. Cell Motil. Cytoskeleton, 22:170–174.
quence of the b-tubulin gene provides the first such independent Katinka, M. D., Duprat, S., Cornillot, E., Me´te´nier, G., Thomarat, F., marker for studies on the heterogeneity of E. bieneusi populations.
Prensier, G., Bare, V., Peyretaillade, E., Brottier, P., Wincker, P.,Delbac, F., El Alaoui, H., Peyret, P., Saurin, W., Gouy, M., Weissen- But more importantly, the significant finding of this study was the bach, J. & Vivares, C. P. 2001. Genome sequence and gene compaction correlation between the observed clinical resistance of E. bieneusi of the eukaryote parasite Encephalitozoon cuniculi. Nature, 414: to benzimidazoles and its b-tubulin sequence.
Katiyar, S. K. & Edlind, T. D. 1997. In vitro susceptibilities of the AIDS- associated microsporidian Encephalitozoon intestinalis to albendazole, its sulfoxide metabolite, and 12 additional benzimidazole derivatives.
This work was supported by National Institutes of Health grants Antimicrob. Agents Chemother., 41:2729–2732.
R21 AI52792 and R21 AI064118. LMW was supported by RO1 Katiyar, S. K., Gordon, V. R., McLaughlin, G. L. & Edlind, T. D. 1994.
Antiprotozoal activities of benzimidazoles and correlations with AI31788. The authors thank Joel Hanawalt for technical assistance.
b-tubulin sequence. Antimicrob. Agents Chemother., 38:2086–2090.
Keeling, P. J. & Doolittle, W. F. 1996. Alpha-tubulin from early diverging eukaryotic lineages and the evolution of the tubulin family. Mol.
Albrecht, H. & Sobottka, I. 1997. Enterocytozoon bieneusi infection in Keeling, P. J. & Fast, N. M. 2002. Microsporidia: biology and evolution patients who are not infected with human immunodeficiency virus. Clin.
of highly reduced intracellular parasites. Ann. Rev. Microbiol., 56: Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. 1990.
Kirk-Mason, K. E., Turner, M. J. & Chakrabory, P. R. 1988. Cloning and Basic local alignment search tool. J. Mol. Biol., 215:403–410.
sequence of b-tubulin cDNA from Giardia lamblia. Nucleic Acids Res., Blanshard, C., Ellis, D. S., Tovey, D. G., Dowell, S. & Gazzard, B. G.
1992. Treatment of intestinal microsporidiosis with albendazole in Kwa, M. S., Veenstra, J. G. & Roos, M. H. 1994. Benzimidazole resistance patients with AIDS. AIDS, 6:311–313.
in Haemonchus contortus is correlated with a conserved mutation at Cali, A. 1991. General microsporidian features and recent findings on amino acid 200 in beta-tubulin isotype 1. Mol. Biochem. Parasitol., AIDS isolates. J. Protozool., 38:625–630.
Conteas, C. N., Berlin, O. G., Ash, L. R. & Pruthi, J. S. 2000. Therapy for Lacey, E. 1988. The role of the cytoskeletal protein, tubulin, in the mode human gastrointestinal microsporidiosis. Am. J. Trop. Med. Hyg., of action and mechanism of drug resistance to benzimidazoles. Int.
Cruz, M. C., Bartlett, M. S. & Edlind, T. D. 1994. In vitro susceptibility of Lemee, V., Zaharia, I., Nevez, G., Rabodonirina, M., Brasseur, P., Ballet, the opportunistic fungus Cryptococcus neoformans to anthelmintic J. J. & Favennec, L. 2000. Metronidazole and albendazole susceptibility benzimidazoles. Antimicrob. Agents Chemother., 38:378–380.
of 11 clinical isolates of Giardia duodenalis from France. J. Antimicrob.
Curry, A. & Canning, E. U. 1993. Human microsporidiosis. J. Infect., Li, J., Katiyar, S. K., Hamelin, A., Visvesvara, G. S. & Edlind, T. D. 1996.
Davidse, L. C. 1986. Benzimidazole fungicides: mechanism of action and Tubulin genes from AIDS-associated microsporidia and implications biological impact. Ann. Rev. Phytopathol., 24:43–65.
for phylogeny and benzimidazole sensitivity. Mol. Biochem. Parasitol., De Groote, M. A., Visvesvara, G., Wilson, M. L., Pieniazek, N. J., Siemenda, S. B., deSilva, A. J., Leitch, G. J., Bryan, R. T. & Mansfield, K. G., Carville, A., Shvetz, D., MacKey, J., Tzipori, S. & Reves, R. 1995. Polymerase chain reaction and culture confirmation Lackner, A. A. 1997. Identification of an Enterocytozoon bieneusi-like AKIYOSHI ET AL.—b-TUBULIN GENES FROM E. BIENEUSI microsporidian parasite in simian-immunodeficiency-virus-inoculated Schmidt, H. A., Strimmer, K., Vingron, M. & von Haeseler, A. 2002.
macaques with hepatobiliary disease. Am. J. Pathol., 150:1395–1405.
TREE-PUZZLE: maximum-likelihood phylogenetic analysis using Mathias, A., Weber, R. & Deplazes, P. 2005. Zoonotic potential of the quartets and parallel computing. Bioinformatics, 18:502–504.
microsporidia. Clin. Microbiol. Rev., 18:423–445.
Sheoran, A. S., Feng, X., Kitaka, S., Green, L., Pearson, C., Didier, E. S., McGinnis, S. & Madden, T. L. 2004. BLAST: at the core of a powerful Chapman, S., Tumwine, J. K. & Tzipori, S. 2005. Purification of En- and diverse set of sequence analysis tools. Nucleic Acids Res., 32: terocytozoon bieneusi from stools and production of specific antibodies.
Molina, J. M., Chastang, C., Goguel, J., Michiels, J. F., Sarfati, C., Sulaiman, I. M., Fayer, R., Yang, C., Santin, M., Matos, O. & Xiao, L.
Desportes-Livage, I., Horton, J., Derouin, F. & Modai, J. 1998.
2004. Molecular characterization of Enterocytozoon bieneusi in cattle Albendazole for treatment and prophylaxis of microsporidiosis due to indicates that only some isolates have zoonotic potential. Parasitol.
Encephalitozoon intestinalis in patients with AIDS. J. Infect. Dis., Thomas, J. H., Neff, N. F. & Botstein, D. 1985. Isolation and character- Orbach, M. J., Porro, E. B. & Yanofsky, C. 1986. Cloning and character- ization of mutations in the b-tubulin gene of Saccharomyces cerevisiae.
ization of the gene for b-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as dominant selectable marker. Mol. Cell.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment Rabodonirina, M., Bertocchi, M., Desportes-Livage, I., Cotte, L., Levrey, through sequence weighting, position-specific gap penalties and weight H., Piens, M. A., Monneret, G., Celard, M., Mornex, J. F. & Mojon, M.
matrix choice. Nucleic Acids Res., 22:4673–4680.
1996. Enterocytozoon bieneusi as a cause of chronic diarrhea in a heart- Vivare`s, C. P., Gouy, M., Thomarat, F. & Me´te´nier, G. 2002. Functional lung transplant recipient who was seronegative for human immunodefi- and evolutionary analysis of a eukaryotic parasitic genome. Curr. Opin.
ciency virus. Clin. Infect. Dis., 23:114–117.
Ridoux, O. & Drancourt, M. 1998. In vitro susceptibilities of the micro- Wittner, M. (ed.). 1999. The Microsporidia and Microsporidiosis. ASM sporidia Encephalitozoon cuniculi, Encephalitozoon hellem, and En- cephalitozoon intestinalis to albendazole and its sulfoxide and sulfone Zhang, Q., Singh, I., Sheoran, A., Feng, X., Nunnari, J., Carville, A. & metabolites. Antimicrob. Agents Chemother., 42:3301–3303.
Tzipori, S. 2005. Production and characterization of monoclonal anti- Sandfort, J., Hannemann, A., Gelderblom, H., Stark, K., Owen, R. L. & bodies against Enterocytozoon bieneusi purified from rhesus macaques.
Ruf, B. 1994. Enterocytozoon bieneusi infection in an immunocompe- tent patient who had acute diarrhea and who was not infected with thehuman immunodeficiency virus. Clin. Infect. Dis., 19:514–516.
Received: 01/09/05, 06/13/06; accepted: 06/25/06
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