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J. 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: donna.akiyoshi@tufts.edu 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.
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Received: 01/09/05, 06/13/06; accepted: 06/25/06

Source: http://www3.botany.ubc.ca/keeling/PDF/07EbtubulinJEM.pdf

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