of DNA), and the second filter was hybridized with 106 cpm ..... Goodwin, S. B., A. Drenth, and W. E. Fry. 1992. ... Perfect, J. R., B. B. Magee, and P. T. Magee.
JOURNAL OF CLINICAL MICROBIOLOGY, June 1993, p. 1547-1554
Vol. 31, No. 6
0095-1137/93/061547-08$02.00/0 Copyright © 1993, American Society for Microbiology
Development of DNA Probes for Fingerprinting Aspergillus fumigatus HELENE GIRARDIN,1t JEAN-PAUL LATGE,2 THYAGARAJAN SRIKANTHA,3 BRIAN MORROW,3 AND DAVID R. SOLL3*
Laboratoire du Genie de l'Hygiene et des Procedes Alimentaires, Institut National de Recherche Agronomique, 91300 Massy, and Unite de Mycologie, Institut Pasteur, 75724 Paris,2 France, and Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242-13243 Received 19 January 1993/Accepted 10 March 1993
Several different DNA fragments containing nonribosomal repetitive sequences have been isolated from the of Aspergiflus fumigatus and tested as potential DNA fingerprinting probes. Eight of these clones generate 19 or more bands when hybridized to EcoRI-digested DNA of a reference strain in Southern blots, and they fall into four families. Individual clones from two families were tested and were found to generate complex Southern blot hybridization patterns which are stable within a single strain over many generations, which vary among unrelated strains, and which are amenable to computer-assisted analyses involving large numbers of strains in epidemiological studies. Clones from three of the families clustered a majority of test strains in a similar fashion in individual dendrograms based on similarity coefficients computed from band positions in Southern blot hybridization patterns. These clones therefore fulfill the major requisites for effective DNA fingerprinting probes. genome
Aspergillus fumigatus represents the most common airborne fungal pathogen. It commonly grows in damp environments such as soil, decaying vegetation, and organic debris, from which it releases high numbers of conidia into the atmosphere (2), where they are inhaled by potential hosts. There are three severe clinical manifestations of A. fumigatus: (i) allergic bronchopulmonary aspergillosis resulting from repeated inhalation by and limited growth in the airways of atopic patients, (ii) colonization of a preexisting pulmonary cavity, forming a fungal ball, or aspergilloma, and (iii) invasive pulmonary aspergillosis, in whichAspergillus organisms invade lung parenchyma and eventually disseminate to other organs (15). The last, systemic form of the disease occurs in immunocompromised hosts and is usually life threatening. Aspergillosis has recently become a major concern in transplant units. In a recent study, it was found that 94% of bone marrow transplant recipients infected with Aspergillus spp. died from their infections (7). Therefore, in each pathological situation, detailed epidemiological studies are needed in order to assess whether particular strains in a geographical locale predominate in particular types of infections and to identify the source of infection. A requisite for such studies is a DNA fingerprinting system which accurately provides a measure of strain relatedness or unrelatedness (27, 29). A number of fingerprinting systems based on genomic differences have been applied to the fungi, including restriction fragment length polymorphism (RFLP) patterns (5, 6, 20, 25), electrophoretically separated chromosome patterns (19, 21), randomly amplified polymorphic DNA patterns (1, 4, 9), Southern blots probed with ribosomal (18, 32) or mitochondrial (34) DNA, and Southern blots probed with moderately repetitive, nonribosomoal genomic sequences (10, 11, 13, 14, 26, 31). The last of these methods has proven to be effective
in large-scale fingerprinting studies (e.g., see reference 30), and the relatively complex patterns have proven accessible to analysis by computer-assisted automated DNA fingerprinting systems (27). We have, therefore, used a strategy first employed in the cloning of moderately repetitive DNA probes of Candida albicans (23, 26) to clone a number of potential fingerprinting probes for A. fumigatus. The majority of these probes are species specific and appear to fulfill all of the requisites for an effective fingerprinting system. MATERIALS AND METHODS
Strains. The strains of Aspergillus used in this study are listed in Table 1, along with their origins of isolation and abbreviations. All strains were maintained on 2% malt extract agar at 25°C. The reference strain AF1 was originally isolated and freeze-dried in 1971 (AF1-1971). It was recultured in 1986 (AF1-1986). The strain was deposited at the Centraalbureau voor schimmelcultures in 1989 as CBS 14389. Since 1989, the strain has been continuously transferred approximately 60 times. In 1992, nine serial subcultures were isolated at 3-day intervals, and these are referred to as AF1-1992-1 to AF1-1992-9. Unless noted otherwise, AF1 will refer to AF1-1992. Cloning of moderately repetitive sequences. Conidia from A. fumigatus strain AF1 were inoculated into Sabouraud's medium (2% glucose, 1% peptone [wt/vol]) at a concentration of 106/ml and were cultured at 30°C for 15 h. Mycelia were collected by filtration, washed with sterile distilled water, and resuspended at 100 mg (wet weight) per ml in 10 mM citrate-phosphate buffer (pH 5.2) containing 0.5 M MgSO4 and 3 mg of Novozym 234 (Sigma, St. Louis, Mo.) per ml. After 9 h of incubation, floating, buoyant protoplasts were purified as described by Srikantha and Rao (33). Protoplasts were lysed in 50 mM Tris-HCl (pH 8.0)-20 mM EDTA-1% sodium dodecyl sulfate (SDS) at 65°C for 20 min. Potassium acetate was added to a final concentration of 1.3 M, and following centrifugation, DNA was precipitated from the supernatant with isopropanol. DNA was resuspended in
* Corresponding author. t Present address: Unite de Mycologie, Institut Pasteur, 75724 Paris, France.
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TABLE 1. Strains of Aspergillus used in this study Nomenclaturea Abbreviatione Origin Species
A. fumigatus
CBS 143.89
Human
Plant Human Plant Soil Soil Human Plant Human Human
A. nidulans A. flavus A. ochraceus A. restrictus
D300 CHUV 192-88 CBS 148.89 CBS 132.54 CBS 192.65 CBS 147.89 CBS 150.89 CBS 331.90 CBS 144.89 CBS 589.65 CBS 569.65 CBS 547.65 CBS 177.33
AF1-1971, AF1-1986, AF1-1989, AFl1992 (AF1), and AFl-1992-1 to -9 AF2 AF3 AF4 AF5 AF6 AF7 AF8 AF9 AF10 AN1 AF11 AOcl AR1
a CBS, Centraalbureau voor schimmelcultures, Baarn, The Netherlands; D300, Institut Pasteur, Unit6 de Mycologie, Paris, France; CHUV, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland. b AFl-1971, a subculture of the original AF1 strain stored in 1971; AFl1986, a subculture of the stored culture of strain AF1-1971 stored in 1986; AF1-1989, a subclone of AF1-1971 carried as a laboratory culture and subcloned in 1989; AF1-1992, the present laboratory culture of AF1, which is referred to as AF1 in the text and which was subcultured from AF1-1989; AF1-1992-1 to -9, sequential subcultures of the laboratory culture of AF1-1992 removed at 3-day intervals.
10 mM Tris-HCl (pH 8.0)-i mM EDTA containing 2 mg of RNase A (Boehringer, Mannheim, Germany) per ml and incubated at 37°C for 1 h. Proteinase K (Merck, Darmstadt, Germany) was then added to a final concentration of 100 mglml, and the DNA preparation was incubated at 65°C for 2 h. DNA was further purified by phenol-chloroform extraction and isopropanol precipitation. Purified DNA was partially digested with Sau3A (GIBCO BRL, Gaithersburg, Md.), and fragments averaging 10 kb were purified and ligated into the bacteriophage vector lambda EMBL 3A according to established protocols (24). The genomic library was amplified and then plated onto 150-mm-diameter plates. Duplicate nitrocellulose filters were prepared from each plate and prehybridized at 65°C for 5 h in a solution of 5 x SSC (1 x SSC contains 0.15 M sodium chloride and 0.015 M sodium citrate [pH 7.0]), 5x Denhardt's solution (lx Denhardt's solution contains 0.02% Ficoll, 0.02% polyvinylpyrrolidone, and 0.02% bovine serum albumin), 0.5% SDS, and 100 ,g of sheared, denatured calf thymus DNA per ml. One filter of each set was then hybridized with a total of 106 cpm of nick-translated total cellular DNA of A. fumigatus AF1 per ml (2.5 x 106 cpm/,g of DNA), and the second filter was hybridized with 106 cpm of nick-translated ribosomal DNA of A. nidulans (5 x 106 cpm/,Lg of DNA), a gift from W. Timberlake, University of Georgia. Clones which exhibited the more intense levels of hybridization expected of repeat sequences and which did not hybridize to the ribosomal probe in the duplicate filter were selected. Selected clones were rescreened under the same conditions and were plaque purified (24). Southern blot hybridization for fingerprinting. DNA was isolated from Aspergillus strains by a method adapted from that of Raeder and Broda (22). Briefly, strains were cultured in 50 ml of Sabouraud medium for 20 to 24 h at 37°C. Mycelia were collected, washed, frozen in liquid nitrogen, and ground with a mortar and pestle. The mycelial powder was
suspended in 5 ml of lysis buffer (20 mM Tris-HCl [pH 8.01, 250 mM NaCl, 25 mM EDTA [pH 8], 1% SDS, 50 ±Lg of proteinase K per ml) and incubated for 10 min at 65°C. The slurry was then extracted with 1 volume of phenol-chloroform (7:3, vol/vol) and centrifuged for 1 h in an SS34-Sorvall rotor at 9,681 x g. The aqueous phase was treated with RNase A (6 ug/ml) for 2 to 3 h at 37°C and extracted with 1 volume of chloroform. DNA was precipitated by adding 1 volume of isopropanol. The pellet was washed with 70% ethanol and dried under a vacuum. The DNA was resuspended in 300 ,ul of 10 mM Tris-HCl (pH 8.0) containing 1 mM EDTA. DNA from Saccharomyces cerevisiae was kindly provided by A. Thierry and C. d'Enfert (Institut Pasteur, Paris, France). DNA from each strain was digested with 4 U of EcoRI per ,ug of DNA at 37°C for 16 h. DNA fragments were separated on a 0.7% agarose gel (1 ,ug of DNA per lane; 16 h at 25 V) and transferred to a nylon filter (Hybond N+; Amersham) with a vacuum transfer unit. Each gel contained digested DNA of the reference strain AF1 in either the leftmost lane or both the left- and rightmost lanes for assisting in computer-assisted analysis. DNA from selected clones was labelled using the random priming method (Megaprime; Amersham). Prehybridization and hybridization were performed at 65°C in a solution of 5 x SSPE (lx SSPE contains 10 mM sodium phosphate [pH 7.5], 10 mM EDTA, and 0.18 M NaCl), 5% dextran sulfate, 150 ,ug of sheared denatured salmon sperm DNA per ml, and 0.3% SDS. Filters were washed with a solution of 2x SSPE-0.3% SDS at 65°C and then exposed to Kodak X-Omat AR film with an intensifying screen for 1 to 3 h at 80°C. Analysis of fingerprints. Southern blot hybridization patterns of a set of test strains generated with each probe were compared with the Dendron software package (Solltech, Iowa City, Iowa). Autoradiograms were scanned into the data file of Dendron, normalized, and unwarped. Band positions were identified automatically and then verified and modified when necessary by human intervention. The similarity coefficient (SAB) was then automatically computed for every pair of strains A and B on the basis of the positions of bands in the noted molecular weight range according to the following formula: SAB = 2E/2E + a + b, where E is the number of bands shared by strains A and B, a is the number of bands unique to strain A, and b is the number of bands unique to strain B. In this case, an SAB of 1.0 represents identical banding patterns, an SAB of 0.0 represents patterns with no matching bands, and SABs from 0.01 to 0.99 represent banding patterns of increasing similarity. Dendrograms based upon SAB values were generated with Dendron software according to the unweighted group method (28). Neighboring Southern blot hybridization patterns. To align lanes from different gels or from different regions of the same gel for side-by-side comparison, autoradiogram images were digitized into a data file in a Macintosh II computer with a Sharp scanner, normalized, and straightened with the imageprocessing capabilities of the Dendron software package. Lanes of interest were then windowed, neighbored (placed side by side), and in turn normalized by the common standard in the independent gels. Neighbored lanes were then rephotographed from the monitor screen. RESULTS RFLP patterns and ribosomal probes do not effectively discriminate among strains of A. fumigatus. In Fig. 1A, the ethidium bromide-stained patterns of EcoRI-digested DNA
FINGERPRINTING A. FUMIGATUS
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FIG. 1. RFLP (A) and Southern blot hybridization with a ribosomal probe (B) for 10 strains of A. fumigatus, four additional species of Aspergillus, and S. cerevisiae. The ethidium bromidestained master gel (A) and the Southern blot hybridized with a genomic clone of ribosomal DNA from A. nidulans (B) are shown. Lanes: AF1 to AF10, individual strains of A. fumigatus; AN1, A. nidulans; AFl1, A. flavus; AOcl, A. ochraceus; AR1, A. restnctus; SC, S. cerevisiae. See Table 1 for descriptions of the Aspergllus strains used. The molecular sizes (in kilobases) of markers are noted to the left of panel A, and those of the major hybridizing bands of A. fumigatus are noted to the right of panel B.
from 10 strains of A. fumigatus (AF1 to AF10) and one strain each of A. nidulans, A. flavus, A. ochraceus, A. restrictus, and S. cerevisiae are compared. The ethidium bromide staining patterns of the five different species of Aspergillus differed dramatically. However, the patterns of AF1 to AF8 were identical, as were the patterns for AF9 and AF10. The patterns of the two groups of A. fumigatus differed by two band positions. Therefore, the RFLP patterns of EcoRIdigested DNA did not provide the level of discrimination necessary for distinguishing among unrelated strains of the same species, a requisite in an effective fingerprinting system (27). Hybridization of the same gel with a 4.5-kb fragment of ribosomal DNA from A. nidulans (17) also failed to discriminate adequately among the 10 test strains of A. fumigatus (Fig. 1B). The two major hybridization bands were invariant in the 10 test strains, but variation was observed in a few minor bands, which separated the 10 strains into three groups. Just as with RFLPs (Fig. 1A), the hybridization pattern generated with the ribosomal probe discriminated among A. fumigatus, A. nidulans, A. flavus, and S. cerevisiae but did not readily discriminate between A. restnctus and A. fumigatus. Therefore, as with RFLPs, the patterns generated by Southern blot hybridization with a ribosomal probe did not provide the level of discrimination requisite for an effective fingerprinting system. Isolation of moderately repetitive sequences as potential fingerprinting probes. A genomic library of five genomic equivalents was constructed in EMBL 3A from a Sau3A partial digest of A. fumigatus genomic DNA containing fragments of approximately 10 kb. Duplicate blots of the library were hybridized in parallel with radiolabelled A. fumigatus AF1 total cellular DNA or ribosomal DNA of A. nidulans. Clones which hybridized intensely with the genomic DNA probe but not with the ribosomal DNA probe were selected. An example of a differential screen identifying putative repetitive sequences of interest (examples 1, 2, and 3) and ribosomal sequences (R) is presented in Fig. 2. Among the 20,000 clones screened, 365 contained ribosomal sequences (1.90%) and 30 contained nonribosomal repetitive sequences of interest (0.15%). Fifteen of the latter clones were randomly selected and were used to probe
2
FIG. 2. Hybridization screens used to select clones of nonribosomal, moderately repetitive genomic sequences. A genomic library of A. fumigatus strain AF1 was blotted in duplicate and hybridized with either radiolabelled genomic DNA of A. fumigatus (A) or a radiolabelled ribosomal DNA probe of A. nidulans (B). 1, 2, 3, potential nonribosomal repetitive sequences; R, ribosomal sequences.
Southern blots of EcoRI-digested DNA of four or more test strains of A. fiumigatus. The characteristics of the Southern blot hybridization patterns of the 15 probes are presented in Table 2. The number of hybridization bands with EcoRIdigested DNA of reference strain AF1 ranged from 2 for probe 4.12 to at least 25 for probes 3.9, 4.10, and 4.11. Band variability for a particular probe was estimated by counting the bands in the patterns of AF1 which were absent or not in the same position in the hybridization patterns of one or more of the other probed strains. No or low variability was observed for the five probes generating patterns of five or fewer bands (probes 4.12, 4.15, TABLE 2. Characteristics of the Southern blot hybridization patterns generated by 15 randomly selected genomic clones of A. fumigatus isolated with the initial screen' Probe
No. of bandsb
No. of bands in AF1 pattern which vary among strainsc
4.12 4.16 4.15 4.18 4.20 4.19 3.16 3. 17d
0 0 0
3.19d 3.8d 3.11
2 3 5 5 5 8 9 19 19 21 21 22
1 5 5 12 12 17 17 12
3.9e
25
13
4.11
25 25
13 21
3.2d
4.10w a
0
The initial comparison of pattern variability was performed with EcoRI-
di,ested DNA of four test strains ofA. fumigatus (AF1, AF4, AF5, and AF7).
Minimum estimates of band numbers for the more complex probes (19 bands or more). c Obtained by comparing each band in the pattern of reference strain AF1 with the patterns of the three additional test strains. If the band in the AF1 pattern was missing from at least one other strain pattern, it was considered varying. d Pattern was highly similar to those of other probes marked with a
superscript d. e Pattern was highly similar to those of other probes marked with a superscript e.
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FIG. 3. Southern blot patterns of EcoRI-digested DNA of four test strains of A. fiumigatus hybridized with four low-complexity probes as indicated. Lanes: AF1, AF7, AF5, and AF4,A. fimigatus test strains (see Table 1 for description of strains). Molecular sizes (in kilobases) of markers are presented to the left of panel A and indicated by arrows in other panels.
4.16, 4.18, and 4.20). Hybridization patterns with four strains of A. fumigatus are presented in Fig. 3 for probe 4.12 (Fig. 3A), which contains two invariant bands; probe 4.15 (Fig. 3B), which contains five major bands and one faint band, all invariant; and probe 4.20 (Fig. 3C), which contains five bands in reference strain AF1, four of which were invariant and one variant, and an additional band in strains AF7 and AF4. Variability increased in the banding patterns of probes generating eight and nine bands (probes 4.19 and 3.16, respectively [Table 2]). In Fig. 3D, the banding patterns are presented for probe 4.19, in which five of the eight bands in reference strain AF1 vary in the three other test strains. In addition, the intensities of the bands obtained with probe 4.19 varied among strains, most notably when the hybridization pattern of strain AF1 was compared with that of strain AF4 (Fig. 3D). Eight probes generated Southern blot hybridization patterns which included 19 bands or more for strain AF1 and the additional test strains of A. fumigatus. For each of the probes, at least half of the bands in reference strain AF1 varied in one or more of the other test strains (Table 2). In Fig. 4, examples of the Southern blot hybridization patterns of probes 3.11, 3.19, and 3.9 are presented. In each pattern, a limited number of invariant bands can be distinguished. For example, an invariant band is positioned at 2.0 kb in the A. 3.11 C4 co -;
90
C.3.9
B. 3.19 LO to r- CO
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cn
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patterns generated by probe 3.11 (Fig. 4A) and at 1.6 kb in the patterns generated by probe 3.9 (Fig. 4C). It is clear from casual examination of the patterns generated by probes 3.11, 3.19, and 3.9 that each probe discriminates among all test strains of A. fumigatus except for AF9 and AF10, which represent laboratory strains of a common origin. Since the cloned probes contain sequences which are members of repetitive gene families, one would expect to isolate related but nonidentical probes by the screening technique used in this study. The patterns generated by clones 3.2, 3.8, 3.17, and 3.19 shared many bands, as did the patterns of clones 3.9 and 4.10. To assess the level of similarity among patterns in each of the two groups, we took advantage of the neighboring program in the Dendron software package. The digitized images of the hybridization patterns of strain AF1 probed with probes 3.2, 3.8, 3.17, and 3.19 in one group and probes 3.9 and 4.10 in the second group were moved from separate gel images next to one another, normalized by aligning common bands, and scanned for pixel density with Dendron scanning software. In Fig. 5, the neighbored lanes and scans are presented for AF1 probed with probes 3.19, 3.8, 3.2, and 3.17. The patterns generated by probes 3.19 and 3.8 were identical, suggesting that they represent clones of the same genomic sequence. However, the patterns of probes 3.19 and 3.8 differed from those of probes 3.2 and 3.17 by the presence or intensity of five bands, bands a, b, c, d, and e. Band a is absent from the 3.2 pattern, band b is present in the 3.19 and 3.8 but absent in the 3.2 and 3.17 patterns, band c is present only in the 3.2 pattern, and bands d and e exhibit differences in intensity. In Fig. 6, the neighbored lanes and scans are presented for AF1 probed with probes 4.10 and 3.9. The patterns are identical except for that of one major band, band a. Species specificity of the complex probes. All complex probes, generating 19 bands or more, were hybridized to four additional species of Aspergillus and S. cerevisiae. No significant hybridization was observed for any of the complex probes with EcoRI-digested DNA of A. nidulans, A. flavus, A. ochraceus, A. restictus, or S. cerevisiae (Fig. 4). Stability of the Southern blot hybridization pattern over time for an individual strain of A. fimigatus. To assess the stability of the Southern blot hybridization patterns generated with probes 3.19 (Fig. 7A) and 3.9 (Fig. 7B), DNA was extracted from the original 1971 stored culture, AF1-1971 (lanes a), from the 1986 stored subculture, AF1-1986 (lanes b), from a subculture of the working strain stored in 1989 as
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