Cloning and characterization of PRA1, a gene encoding a novel pH-regulated antigen of Candida albicans

Abstract

Candida albicans is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of C. albicans was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designated PRA1, for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The PRA1 protein was homologous to surface antigens of Aspergillus spp. which react with serum from aspergillosis patients, suggesting that the PRA1 protein may have a role in the host-parasite interaction during candidal infection.

FIG. 1

Separation of chromosomes of C. albicans ATCC 26555 (lanes 1), SGY243 (lanes 2), 996 (lanes 3), and FC18 (lanes 4). The gel was stained with ethidium bromide (A) and blotted onto nylon and hybridized with PRA1 (B). C. albicans chromosomal designations (on the right) are those proposed by Wickes et al. (48).

FIG. 2

Nucleotide sequence and deduced amino acid sequence of PRA1. A putative TATA sequence (boxed), the translation start site (boxed by a broken line), four potential N-glycosylation sites (circled with thick lines), noncanonical CTG codons (circled with thin lines), the predicted signal cleavage site (arrow), and the serine-rich regions (underlined) are indicated.

FIG. 3

Northern blot analysis of the effects of temperature and pH on PRA1 expression. Strain SC5314 was inoculated in the medium of Lee et al. (29) or medium 199 adjusted to the indicated pH and incubated for 3 h at 37 or 25°C. The results of hybridization with the 8M cDNA insert (top panel) and of hybridization with actin DNA (bottom panel) are shown. The electrophoretic position of 18s rRNA is indicated on the right.

FIG. 4

Alignment of the amino acid sequences of Pra1p and related proteins. Identical residues present in at least five of the eight sequences (boxes), residues conserved specifically between Pra1p and the Aspergillus sp. antigens (lines above the sequences), and the zinc-binding motif conserved in metalloproteinases (thick bar) are shown. AspndI, A. nidulans antigen (6); AspfII, A. fumigatus antigen (3); ScAOB249, S. cerevisiae open reading frame; AspNPII, neutral protease of A. oryzae (46, 47); Pnclysin, penicillinolysin of Penicillium citricum (30); Afumep20, metalloproteinase from A. fumigatus (35); Aflmep20 metalloproteinase from Aspergillus flavus (35).

FIG. 5

Disruption of the PRA1 locus. (A) Restriction map of the 4.3-kb SacI genomic fragment containing the PRA1 gene (pMBW3) and the deletion-disruption construct (pMBW8). (B) Southern blot analysis of SacI-digested DNA from the parental strain CAI4, the Δpra1 heterozygote CAMB and its Urd⁻ segregant, CAMB11, and the Δpra1/Δpra1 null mutant CAMB43 and its Urd⁻ segregant, CAMB435. The blot was probed with the SacI fragment containing PRA1. The lengths and structures of the hybridizing fragments are shown on the left. S, SacI.

FIG. 6

Chitin distribution in CAI4 and CAMB435 after 6-h induction in SD medium with N-acetylglucosamine. The cells were labelled with WGA-FITC as described in Materials and Methods. Phase-contrast (a, c, and e) and fluorescence (b, d, and f) micrographs of CAMB435 at 37°C (a and b), CAI4 at 42°C (c and d), and CAMB435 at 42°C (e and f) are shown.

FIG. 7

Localization of Pra1p. A Western blot of mycelial fractions of strain ATCC 26555 was reacted with monospecific polyclonal antibodies against Pra1p. Lanes: 1, Zymolyase-solubilized cell wall fraction; 2, spent medium from protoplasts after 3 h of regeneration; 3, wall material solubilized with SDS; and 4, a mixed membrane preparation. Molecular masses (in kilodaltons) are indicated on the right.

FIG. 8

Structure and localization of Pra1p. (A) Western blot of SDS extracts of cell walls (lanes 1 and 2) and culture supernatants (lanes 3 and 4) prepared from S. cerevisiae W303-1B (lanes 1 and 3) and W303-1B transformed with pADH-PRA1 (lanes 2 and 4). The blot was reacted with polyclonal antiserum against Pra1p (PAb-M) or AspNDI (PabAspnD1). CF, culture filtrates. (B) Effect of endoglycosidase H treatment. Culture filtrates from strain W303-1B transformed with pADH-PRA1 were separated by PAGE before (lanes 1 and 3) or after (lanes 2 and 4) endoglycosidase H treatment. The gel was blotted and either reacted with PAb-M or stained with ConA-peroxidase.