Aspergillus species

A. fumigatus Fres. (Sartorya fumigata)

Biochemical and molecular characterizations used in species determination.

The need for a better taxonomic definition of the species A. fumigatus and the possible misidentification of a teleomorph stage of A. fumigatus among the Neosartorya species have led to the study of selected biochemical and molecular criteria, in addition to morphological data. as adjuncts to species determination. Biochemical characterizations which have been studied include the detection and identification of secondary metabolites (9), the identification the ubiquinone system (10), and the examination of isoenzyme patterns (10, 11, 12, 13). Molecular data have been obtained on total DNA (14, 15, 16), mitochondrial DNA (mtDNA) (17, 12) or ribosomal DNA (rDNA) (14, 17, 18, 12, 19) by using various methodological approaches, mainly restriction fragment length polymorphisms (RFLP) visualized with or without hybridization to specific probes and sequencing of characteristic DNA regions. Criteria which have been suggested as useful in the identification of A. fumigatus are summarized in Table 1.

^a Isoenzvme patterns (11, 10, 12, 13), ethidium-bromide visualized RFLP (25, 26), and Igs probes (27, 19) can be used to rank strains at a subspecies level.

^b Used directly for comparative analysis of sequence data obtained with unique nuclear (20) or Its 1 and Its 2 ribosomal (14, 18, 21) genes or indirectly in Southern hybridization experiments (15, 1).

The profiles of secondary metabolites, including mycotoxins and antibiotics, produced by A. fumigatusand its morphological variants listed above are similar. Fumagillin, fumitoxin, fumigaclavines, fumigatin, fumitremorgins, gliotoxin, monotrypacidin, tryptoquivaline, helvolic acid, and metabolites of two chromophore families uncharacterized chemically (FUA and FUB) are the secondary metabolites most commonly found in A. fumigatus (9). In contrast, anamorphs of Neosartorya species, as well as other members of Aspergillus sect. fumigati (A. brevipes, A. duricaulis, A. unilateralis. and A. viridinutans), produce few of these secondary metabolites. The number of isoprene side chains of the ubiquinone molecules, which is 10 in the Aspergillus sect. fumigati, cannot be used for identification at the species level, since all species of this section, including all Neosartorya anamorphs, have the same number of isoprene units (10)

Several investigators have attempted to use the analysis of isoenzyme patterns as a taxonomic tool (11, 10, 12, 13). The only enzyme pattern common to all strains of A. fumigatus is glutamate dehydrogenase. Some enzymes (lactate dehydrogenase, superoxide dismutase, isocitrate dehydrogenase, aspartate aminotransferase. gIucose-6-phosphate dehydrogenase, and phosphogluconate dehydrogenase) have been reported to be monomorphic, although data vary from study, to study, and other enzymes (malate dehydrogenase, glucose phosphate isomerase, phosphoglucomutase, hexokinase, esterase, malate dehydrogenase, peptidases. fructose kinase, purine nucleoside phosphorylase, and phosphatases) display polymorphic patterns. However, since multilocus enzyme electrophoresis patterns of closely related species have not been investigated, their usefulness as a general taxonomic tool is unknown.

Analytical approaches which involve the analysis of DNA have shown more promise in the characterization of A. fumigatus. One useful criteria is DNA-DNA reassociation values, wherein values higher than 92% have been found for strains of A. fumigatus, while values lower than 70% have been calculated for A. fumigatus and Neosartorya species, indicating that the two genera are genetically distinct (16). Another helpful approach to the study of nuclear DNA has been the analysis of introns and the sequencing of entire or significant portions of unique genes such as the ß -tubulin and hydrophobin genes (14, 20). A third successful method is the hybridization of endonuclease-digested DNA with various A. fumigatus-specific unique or repeated (see below) DNA sequences (15, 1). Amplification by PCR of specific sequences, originally identified by random amplification, seems promising (28). While sequencing of the internally transcribed spacers Its 1 and Its 2 of rDNA has not been completed, there appear to be sufficient differences to distinguish Neosartorya species and A. fumigatus (14, 18, 21).

Other DNA-based approaches have not been useful in the speciation of A. fumigatus. For example, pulse field gel electrophoresis has shown the presence of five chromosomal-sized DNA bands ranging in size from 1.7 to 4.8 Mb (29). It is not known, however, if each of these bands corresponds to one or two chromosomes, as has been shown with Aspergillus spp. other than A. fumigatus (30). Moreover, comparisons of chromosomal banding patterns of taxonomically related species have not been done. The analyses of mtDNA and rDNA have produced limited results. RFLP's in mtDNA were not observed among 60 strains of A. fumigatus when DNA was digested with HaeIII alone or in combination with other enzymes. Moreover, the same pattern was observed with the closely related species, Neosartorya fischeri fischeri. An approach which has not been attempted, but which could be helpful, is the use of AT-rich recognition enzymes for digesting mtDNA. The use of such enzymes has proved beneficial in characterizing the flavi section of the aspergilli (17, 12). As with other fungi, investigations of the sequences of the 18S and 28S subunits of RDNA have shown that there is insufficient variability for this method to be useful taxonomically. Southern hybridization with intergenic spacer (Igs) probes from non-fumigatus species showed that all isolates of A. fumigatus tested had common major fragments with a variable number of 200-bp repeat units, suggesting that the Igs region was too heterogeneous to be used at a species level (19).

In summary, secondary metabolites and sequencing data, as well as DNA-DNA reassociation values and Southern hybridizations patterns with singe and repeated sequences or PCR amplicons have been useful criteria for the taxonomic characterization of A. fumigatus (Table 1). They prove that A. fumigatus and N. fischeri, whose anamorphic stage is very closely related to A. fumigatus. are two separate species Genetically and biochemically. Therefore. the search for a teleomorph stage of A. fumigatus must continue.

References

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14 - Chrzavzez, E. (1995). D.Sc. Thesis. Universite Paris XI, Paris. France.

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21 - Peterson, S.W. Unpublished data.

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27 - Radford, S.A., Johnson, E.M., Leeming, J.P., Millar, M.R., Cornish, J.M., Foot, A.B.M. & Warnock, D.W. (1998). Molecular epidemiological study of Aspergillus fumigatus in a bone marrow transplantation unit by PCR amplification of ribosomal intergenic spacer sequences. J. Clin. Microbiol. 36: 1294 -1299.

28 - Brandt, M.E., Padhye, A.A., Mayer, L.W. & Holloway, B.P. (1998). Utility of random amplified polymorphic DNA PCR and Taqman automated detection in molecular identification of Aspergillus fumigatus. J. Clin. Microbiol. 36: 2057-2062.

29 - Tobin, M.B., Peery, R.B. & Skatrud, P.L. (1997). An electrophoretic molecular karyotype of a clinical isolate of Aspergillus fumigatus and localisation of the MDR-like genes AfuMDR1 and AfuMDR2. Microbiol. Infect. Dis. 29: 67-71.

30 - Swart, K., Debets, A.J.M., Holub, E.F., Bos, C.J. & Hoekstra, R.F. (1994). Physical karyotyping: genetic and taxonomic applications in aspergilli, p. 233-240. In K.A. Powell, A. renwick and J.F. Peberdy (ed.), The genus Aspergillus: from taxonomy to genetics to industrial application. Plenum Press, London, United Kingdom.

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