Cloning and characterization of CSP37, a novel gene encoding a putative membrane protein of Candida albicans

Abstract

In the course of an analysis of the functions and assembly of the cell wall of Candida albicans, we have cloned and characterized a gene, which we designated CSP37 (cell surface protein), encoding a 37-kDa polypeptide which is a membrane-associated protein. Candida albicans is an imperfect fungus capable of causing life-threatening infections in immunocompromised patients as well as a variety of mucosal infections in generally healthy individuals (50). Due to its importance as a human pathogen and the limited number of safe drugs to control deep-rooted infections, many laboratories have undertaken cellular, molecular, and genetic studies to understand the mechanisms governing C. albicans biology. However, the diploid nature of this fungus and the failure to identify a sexual cycle have hampered many classical genetic approaches (59).A number of factors are thought to contribute to the virulence of C. albicans, but their relative importance during pathogenesis remains unclear (14). Potential virulence determinants include the ability to switch from a yeast to a mycelial form (31,62) and also between different colonial morphologies (64), the production of extracellular hydrolytic enzymes (28, 36), synthesis of receptor-ligand molecules required for recognition, and adhesion to the host tissues (7, 8), etc. Since the cell wall is involved in these processes, some groups have attempted to elucidate the mechanism of synthesis of its different constituents, their assembly, and the modifications that occur during the morphological transition. Thus, a number of cell surface antigens have been reported to be preferentially expressed in hyphal or blastoconidial cell morphologies (10,52,66,67), and several genes related to cell wall architecture (assembly and functions) have been cloned. These genes have been identified by various methods, including differential hybridization screening (27), complementation in Saccharomyces cerevisiae (3), cross-hybridization with genes cloned from other organisms (heterologous genes) (20, 65), hybridization with sequence-specific oligonucleotides (9), and PCR amplification (11,12). We have used another approach to clone morphologyspecific C. albicans genes related to the cell wall. We have isolated cDNA clones by screening with polyclonal antibodies raised against isolated cell walls of blastoconidia and mycelial cells (17,61).In this paper, a cDNA clone that reacted with polyclonal antibodies specific for mycelial cell walls was studied. It encodes a novel protein with no significant homology to known sequences and absent from the S. cerevisiae genome. ⌬csp37 null mutants were constructed and subsequently phenotypic analysis and virulence testing were conducted. Location of the protein codified and potential functions are discussed. MATERIALS AND METHODSMicroorganisms and growth conditions. The C. albicans strains used in this study are listed in Table 1. Cells were routinely grown in YPD (2% glucose, 1% yeast extract, 2% Bacto Peptone [Difco, Detroit, Mich.]) with shaking at the selecte...

The Candida albicans pH-regulated KER1 gene encodes a lysine/glutamic-acid-rich plasma-membrane protein that is involved in cell aggregation

“…PHR1 and its functional homologue PHR2 encode glycosidases that are localized at the plasma membrane, and are involved in cross-linking cell-wall glucans, a process required for the maintenance of cell shape and morphology (Fonzi, 1999). PRA1 encodes a protein that was found to be homologous to surface antigens of Aspergillus spp., but whose importance in C. albicans remains to be elucidated (Sentandreu et al, 1998). Overall, these observations indicate that gene expression patterns, cell morphology and virulence are coordinated by pH-responsive signalling pathways in C. albicans.…”

“…The pH response in C. albicans involves the differential expression of at least three genes, PHR1, PHR2 and PRA1 (Mühlschlegel & Fonzi, 1997;Saporito-Irwin et al, 1995;Sentandreu et al, 1998). PHR1 is expressed at a pH above 5?5 and is required for normal morphology at these pH levels.…”

“…The transcription factor of A. nidulans homologous to PacC in Saccharomyces cerevisiae and C. albicans is Rim101p, the product of the RIM101 gene (also designated PRR2 in C. albicans) (Davis et al, 2000;Porta et al, 1999;Ramó n et al, 1999). Genes known to be under RIM101 regulation in C. albicans include PHR1, PHR2 and PRA1 (Ramó n et al, 1999; Sentandreu et al, 1998).…”

How Doratomyces stemonitis copes with Benzoxazolin-2(3H)-one (BOA), its derivatives and detoxification products

“…Glucanases are therefore essential for continuous rearrangements of 1,3 glucans accompanying cell wall degradation and reconstruction during the development and perhaps during the construction of chlamidospores. Surface located PRA1, a glycoprotein with motifs characteristic of secreted proteins, seems to have a role in morphogenesis as deletion of the gene resulted in temperature dependent defects of hyphae formation (Sentandreu et al 1998). The observed changes in hyphae and colony morphology as well as the formation of chlamidospores when D. stemonitis is exposed to BOA derivatives, such as MBOA and BOA-6-OH or phenoxazinone may point to a glucanase function.…”

Secreted pH-Regulated Antigen 1 of Candida albicans Blocks Activation and Conversion of Complement C3

“…PRA1, PHR1, and PHR2 are three pH-regulated genes of C. albicans that are involved in morphogenesis (45). Pra1 is located on the fungal surface and is also secreted into culture supernatant by both yeast and hyphal forms of C. albicans (46)(47)(48). Pra1, which was originally identified as a fibrinogen-binding protein (46), binds human complement regulator factor H, FHL-1, and plasminogen (23), and is also a ligand for the integrin CR3 (alternative names: a M b 2 or CD11b/CD18), which is expressed on the surface of human leukocytes (47).…”