Development and characterization of complex DNA fingerprinting probes for the infectious yeast Candida dubliniensis

Abstract

Using a strategy to clone large genomic sequences containing repetitive elements from the infectious yeast Candida dubliniensis, the three unrelated sequences Cd1, Cd24, and Cd25, with respective molecular sizes of 15,500, 10,000, and 16,000 bp, were cloned and analyzed for their efficacy as DNA fingerprinting probes. Each generated a complex Southern blot hybridization pattern with endonuclease-digested genomic DNA. Cd1 generated an extremely variable pattern that contained all of the bands of the pattern generated by the repeat element RPS of Candida albicans. We demonstrated that Cd1 does not contain RPS but does contain a repeat element associated with RPS throughout the C. dubliniensis genome. The Cd1 pattern was the least stable over time both in vitro and in vivo and for that reason proved most effective in assessing microevolution. Cd24, which did not exhibit microevolution in vitro, was highly variable in vivo, suggesting in vivo-dependent microevolution. Cd25 was deemed the best probe for broad epidemiological studies, since it was the most stable over time, was the only truly C. dubliniensis-specific probe of the three, generated the most complex pattern, was distributed throughout all C. dubliniensis chromosomes, and separated a worldwide collection of 57 C. dubliniensis isolates into two distinct groups. The presence of a species-specific repetitive element in Cd25 adds weight to the already substantial evidence that C. dubliniensis represents a bona fide species.

FIG. 1

Southern blot hybridization patterns and resulting dendrograms of EcoRI-digested DNA of nine C. dubliniensis isolates and C. albicans 3153A probed with Cd1(A), Cd24 (B), and Cd25 (C). The DNA was electrophoresed in a 0.65% agarose gel, and the Southern blot was sequentially hybridized with the three probes. The arrowheads to the right of each gel represent prominent invariant (monomorphic) bands. Key molecular sizes are presented in kilobases to the left of each gel. The origins of these test isolates are presented in Table 1. A dendrogram (shown below each gel) for each set of hybridization patterns was generated from the similarity coefficient (S_AB) computed for all possible pairs of the nine unrelated C. dubliniensis isolates. Since the M3 lane was underloaded, analyses of that pattern were performed on autoradiograms exposed for longer periods. The average S_AB is indicated at the top of each dendrogram.

FIG. 2

Hybridization patterns of the nine test isolates of C. dubliniensis and C. albicans 3153A probed with the Cd1 probe (A), the C1 fragment of the C. albicans probe Ca3 (B) (1), the RPS39 element of C. albicans (C) (27a), and the C. albicans probe 27A (30). The origins of the test isolates are presented in Table 1. Molecular sizes are presented in kilobases to the left of each set of hybridization patterns.

FIG. 3

Hybridization of CHEF-separated chromosomes of C. dubliniensis (C.d.) and C. albicans (C.a.) with the C. dubliniensis probes Cd1, Cd24, and Cd25 and the C. albicans repeat element RPS39. Chromosomes of C. albicans 3153A and C. dubliniensis M6 and d126423 were separated by CHEF, and the gel was stained with ethidium bromide (EtBr) (A). The gels were then Southern blotted and probed with Cd1(B), Cd24 (C), Cd25 (D), and RPS39 (E). C. albicans bands are numbered to the left of the EtBr-stained image, and C. dubliniensis bands are numbered to the right of the EtBr-stained image. m, minichromosomal band.

FIG. 4

Species specificity of the three C. dubliniensis probes. EcoRI-digested DNA of 14 different yeast species was sequentially probed with Cd1 (A), Cd24 (B), Cd25 (C), and the C. albicans repeat element RPS39 (D). Lanes: C.g., Candida guillermondii; C.r., Candida rugosa; C.kr., C. krusei; C.l., Candida lusitaniae; C.gl., C. glabrata; P., Pichia sp.; C.t., C. tropicalis; Y.l., Yarrowia lipolytica; S.c., Saccharomyces cerevisiae; C.a., C. albicans; T.b., Trichosporon beigelii; C.k., Candida kefyr; C.d., C. dubliniensis; C.p., C. parapsilosis.

FIG. 5

In vitro analysis of the stability of the patterns generated by C. dubliniensis probes Cd1, Cd24, and Cd25. Southern blots of EcoRI-digested DNA from clones of C. dubliniensis d1419-2 (A), B71507 (B), and Co5 (C), at zero hours (0) and after 200 generations, were probed with Cd1, Cd24, and Cd25. Nine individual clones of each strain were selected randomly for analysis at 200 generations. Variant patterns are numbered at the bottom of each blot. Key molecular sizes in kilobases are presented to the left of each blot.

FIG. 6

The variability of probe-generated patterns in a population colonizing an HIV-positive individual over time. EcoRI-digested DNA from multiple isolates from patient 50 (Table 1) were probed with Cd1 (A), Cd24 (B), and Cd25 (C). The isolates were obtained at 0, 5, 7, and 12 months. The patient presented with oral thrush at 12 months. Variations in patterns are indicated at the bottom of each blot. This patient was infected by two different strains displaying the general genotypes a and b. Changes in the patterns are numbered in chronological order of occurrence. Molecular sizes are presented in kilobases to the left of the gels. On the right side of each hybridized Southern blot the representative dendrogram is generated. The average S_AB is presented at the top of each dendrogram.

FIG. 7

Dendrograms generated from the similarity coefficients (S_ABs) computed for all pairs of 57 unrelated isolates collected worldwide and fingerprinted with Cd25. The origins of the isolates are presented in Table 1. The letters (A through H) to the right of the dendrogram indicate the hospitals of origin in cases where several isolates, each from a different individual, were collected from the same hospital: A, Brussels, Belgium; B, Leicester, United Kingdom; C, London, United Kingdom; D, Antwerp, Belgium; E, Frankfurt, Germany; F, Victoria, Australia; G, Lausanne, Switzerland; H, Leeds, United Kingdom. The two main clusters are delineated to the right of the dendrogram (groups I and II). The average S_ABs for each group, and for the total collection, are presented in the middle of the dendrogram.

FIG. 8

Examples of Southern blot hybridization patterns of group I and group II isolates probed with Cd25. Hybridization patterns are presented in A, and a model generated by the DENDRON program is presented in B. Molecular sizes are presented in kilobases to the left of the hybridization pattern. The arrowheads to the right of the model indicate the prominent invariant bands shared by group I isolates but not by group II isolates.