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Differentiation of Candida albicans and Candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae.

ABSTRACT: To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones (scFv5 and scFv12) were characterized in detail. Filament-specific antigen expression was detected by an indirect immunofluorescence assay. ScFv5 reacted with C. dubliniensis filaments, while scFv12 did not. Neither scFv reacted with C. glabrata, C. parapsilosis, C. rugosa, C. tropicalis, or Saccharomyces cerevisiae grown under conditions that stimulated filament formation in C. albicans and C. dubliniensis. Epitope detection by the two scFv was sensitive to proteinase K treatment but not to periodate treatment, indicating that the cognate epitopes were composed of protein. The antigens reactive with scFv5 and scFv12 were extractable from the cell surface with Zymolyase, but not with sodium dodecyl sulfate (SDS) and 2-mercaptoethanol, and migrated as polydisperse, high-molecular-weight bands on SDS-polyacrylamide gel electrophoresis gels. The epitopes were detected on clinical specimens obtained from infants with thrush and urinary candidiasis without passage of the organisms on laboratory media, confirming epitope expression in human infection. The availability of a monoclonal immunologic reagent that recognizes filaments from both C. albicans and C. dubliniensis and another specific only to C. albicans adds to the repertoire of potential diagnostic reagents for differentiation between these closely related species.

SUBMITTER: Bliss JM

PROVIDER: S-EPMC150300 | biostudies-literature | 2003 Mar

REPOSITORIES: biostudies-literature

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Differentiation of Candida albicans and Candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae.

Bliss Joseph M JM Sullivan Mark A MA Malone Jane J Haidaris Constantine G CG

Journal of clinical microbiology 20030301 3

To identify antigens specific for the filamentous form of Candida albicans, a combinatorial phage display library expressing human immunoglobulin heavy and light chain variable regions was used to select phage clones capable of binding to the surfaces of viable C. albicans filaments. Eight distinct phage clones that bound specifically to filament surface antigens not expressed on blastoconidia were identified. Single-chain antibody variable fragments (scFv) derived from two of these phage clones ...[more]

PMID: 12624045

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Project description:Because Candida dubliniensis is closely related to Candida albicans, we tested whether it underwent white-opaque switching and mating and whether white-opaque switching depended on MTL homozygosity and mating depended on switching, as they do in C. albicans. We also tested whether C. dubliniensis could mate with C. albicans. Sequencing revealed that the MTLalpha locus of C. dubliniensis was highly similar to that of C. albicans. Hybridization with the MTLa1, MTLa2, MTLalpha1, and MTLalpha2 open reading frames of C. albicans further revealed that, as in C. albicans, natural strains of C. dubliniensis exist as a/alpha, a/a, and alpha/alpha, but the proportion of MTL homozygotes is 33%, 10 times the frequency of natural C. albicans strains. C. dubliniensis underwent white-opaque switching, and, as in C. albicans, the switching was dependent on MTL homozygosis. C. dubliniensis a/a and alpha/alpha cells also mated, and, as in C. albicans, mating was dependent on a switch from white to opaque. However, white-opaque switching occurred at unusually high frequencies, opaque cell growth was frequently aberrant, and white-opaque switching in many strains was camouflaged by an additional switching system. Mating of C. dubliniensis was far less frequent in suspension cultures, due to the absence of mating-dependent clumping. Mating did occur, however, at higher frequencies on agar or on the skin of newborn mice. The increases in MTL homozygosity, the increase in switching frequencies, the decrease in the quality of switching, and the decrease in mating efficiency all reflected a general deterioration in the regulation of developmental processes, very probably due to the very high frequency of recombination and genomic reorganization characteristic of C. dubliniensis. Finally, interspecies mating readily occurred between opaque C. dubliniensis and C. albicans strains of opposite mating type in suspension, on agar, and on mouse skin. Remarkably, the efficiency of interspecies mating was higher than intraspecies C. dubliniensis mating, and interspecies karyogamy occurred readily with apparently the same sequence of nuclear migration, fusion, and division steps observed during intraspecies C. albicans and C. dubliniensis mating and Saccharomyces cerevisiae mating.

Project description:There have been increased reports of the isolation of unusual genotypic groups of Candida albicans (groups C and D) based on a well-defined genotypic method; this method uses cellular DNA digested with the EcoRI enzyme and the restriction fragment length polymorphisms (RFLPs) generated by agarose gel electrophoresis. The aim of the present study was to use additional molecular tools to characterize these unusual strains and to compare them with authentic strains of C. dubliniensis, a recently delineated species, and type I C. stellatoidea. The RFLPs of PCR products generated from the intergenic transcribed spacer (ITS) region did not differentiate among C. albicans genotypes A, B, and C and type I C. stellatoidea. However, this method did differentiate the C. albicans genotype D strains, which were identical to C. dubliniensis. The RFLPs generated by HaeIII digestion of the PCR products of the V3 region of the 25S rRNA gene (rDNA) could differentiate the same groups as RFLP analysis of the PCR amplicon of the ITS region. C. albicans genotype B isolates have been shown to have a transposable intron in the 25S rDNA, whereas genotype A isolates do not; C. dubliniensis strains also have an intron that is larger than that in genotype B C. albicans strains but that is in the same location. PCR designed to span this region resulted in a single product for C. albicans genotype A (450 bp), B (840 bp), type 1 C. stellatoidea (840 bp), and C. dubliniensis (1,080 bp), whereas the C. albicans genotype C isolates had two major products (450 and 840 bp). All C. albicans genotype D isolates gave a PCR product identical to that given by C. dubliniensis. These results indicate that those strains previously designated C. albicans genotype D are in fact C. dubliniensis, that no differences were found between type 1 C. stellatoidea and C. albicans genotype B strains, and that the C. albicans genotype C strains appear to have the transposable intron incompletely inserted throughout the ribosomal repeats in their genomes. The results of the antifungal susceptibility testing of 105 of these strains showed that, for fluconazole, strains of C. dubliniensis were significantly more susceptible than strains of each of the C. albicans genotypes (genotypes A, B, and C). The flucytosine susceptibility results indicated that strains of C. albicans genotype A were significantly less susceptible than either C. albicans genotype B or C. albicans genotype C strains. These results indicate that there is a correlation between the Candida groups and antifungal susceptibility.

Dataset Information

Differentiation of Candida albicans and Candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae.

Publications

Differentiation of Candida albicans and Candida dubliniensis by using recombinant human antibody single-chain variable fragments specific for hyphae.

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