Aspergillus species
A. fumigatus Fres. (Sartorya fumigata)
Molecular Analyses in Strain Typing
Aside from its fundamental interest, intraspecific characterization of this species has potent epidemiological and clinical implications. Since strain typing requires methods that are highly discriminative, reproducible, and independent of growth conditions, phenotypic analysis based on protein patterns detected by antibodies or enzymatic substrates should be discouraged (32, 33). At best, protein patterns can be used to rank strains at a subspecies level. In contrast, genotypic methods are independent of the external milieu. Some of the earlier molecular methods, however, are helpful only for analysis at the subspecies level. RFLP following digestion of total genomic DNA by XbaI, Sal l, or Xhol shows a limited degree of discrimination among strains. The complex banding patterns with large numbers of faint bands displayed in ethidium bromide stained gels are difficult to interpret, and only major bands can be used to designate subspecific clusters (25, 26). Similarly, heterogeneity in the Igs region can be used to group strains of A. fumigatus only at a subspecific level (27, 19).
Only three methods can be used to genotypically type A. fumigatus strains. Two of these methods use PCR, each with different primers and amplification protocols ( microsatellite and random amplified polymorphic DNAs [RAPD]), whereas the third uses RFLP visualized after hybridization with a repeated DNA sequence.
RAPD is the method most commonly used to type strains of A. fumigatus (34, 35, 36, 37, 11, 38, 39, 40). To date, the decamer primer RIOS (GTATTGCCCT) generated the best strain differentiation (35). However, RAPD patterns are difficult to repeat or interpret due to the low annealing temperature (41, 42, 43). Moreover, the distance of migration scanned is only a few centimeters, and the variability in banding pattern is too limited to make the comparison of a large number of strains feasible. The second PCR-based method involves microsatellites. This method, which has been used to construct the physical map of the human genome, has been successfully applied recently to A. fumigatus (22). The method is rapid and highly reproducible and, in contrast to RAPD, uses unique primers and specific sequences flanking the microsatellite. Four CA repeats have been identified to date: (CA)9(GA)25, (CA)2C(CA)23, (CA)8, and (CA)21.
Hybridization of restriction enzyme fragments with repeated DNA sequences, a method successfully used to type other funeral pathogens, has also been used to type A. fumigants strains. Screening of a phage library resulted in the isolation of a phage (l 3.9) which contains a species-specific repeat sequence. Use of this phage as a probe provides unique and highly discriminative Southern blot hybridization patterns for each strain tested (23, 24). The repeat sequence Afu t 1, inserted into phage l 3.9 and used for strain fingerprinting, is a defective retrotransposon element of 6.9 kb bounded by two long terminal repeats (LTR) of 282 bp (44) (Fig. 2).
FIG. 2. Schematic representation of the l 3.9 probe of A. fumigatus used for molecular studies and typical hybridization patterns obtained with EcoR1-digested total DNA probed with the entire Sal1-Sal1 fragment (A) and EcoR1 fragments of the repeated sequence (B to E). The repeated element Afu t 1 (squares) is an inactive retroelement of 6.9 kb bounded by two LTRs (>) and with sequences homologous to reverse transcriptase (RT). RNase H, and endonuclease (endo) encoded bv the pol genes of retrotransposons.
The 5' and 3' LTRs are not totally homologous, since they have only 90% identity. Moreover, the, 5' LTR of another copy of Afu t 1, isolated from a different phage (l 4.11). which crosshybridizes with l 3.9. is 86.5% identical to the 5'LTR of the retrotransposon isolated from the l 3.9 phage. A 5-bp duplication site was found at the border of Afu t 1. Afu t 1 encodes amino acid sequences homologous to the reverse transcriptase, RNase H, and endonuclease encoded by the pol gene of retroviruses.
Comparison of Afu t 1 with other fungal and nonfungal LTR retrotransposons showed that Afu t 1 has a sequence and organization characteristic of the gypsy family of Drosophila (44). At least 10 copies of the retrotransposon element are found in the genome of A. fumigatus However, Afu t 1 is a defective element; the putative coding domains contain multiple stop codons due exclusively to transitions from C . G to T . A. Such a pattern of nucleotide variation is reminiscent of the repeated-induced point mutation (Rip) in Neosartorya repeated sequences. However, no sexual reproduction is known in A. fumigatus. and no methylation of cytosine, an event typically associated with mutations in sequences affected by Rip, was detected (44). This result would suggest that Afu t 1 was subjected to Rip at a time when A. fumigatus possessed a functional sexual cycle and an active DNA methylation process. The copies of this repeated sequence found today could be relics of Rip consecutive to and fixed at a time where A. fumigatus had lost its sexual stage.
Although most researchers have used the PCR- and RFLP based typing methods separately, a study is under way to compare their discriminatory potential and to evaluate if combination of data obtained by more than one typing method will lead to better strain discrimination (11). To date, strain typing has been most successful by using microsatellite polymorphism or analysis of Southern hybridization patterns obtained with repeated DNA sequences.
References
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