Structure and regulation of a Candida albicans RP10 gene which encodes an immunogenic protein homologous to Saccharomyces cerevisiae ribosomal protein 10

Abstract

, Infect. Immun. 61:4263-4271, 1993). cDNA10 was used to isolate its cognate gene, and both the cDNA and gene were sequenced, revealing a major open reading frame with the potential to encode a basic protein of 256 amino acids with a predicted molecular weight of 29 kDa. Over its entire length, the open reading frame showed strong homology at both the nucleic acid (75 to 78%) and amino acid (79 to 81%) levels to two Saccharomyces cerevisiae genes encoding the 40S ribosomal protein, Rp10. Therefore, our C. albicans gene was renamed RP10. Northern (RNA) analyses in C. albicans 3153 revealed that RP10 expression is regulated in a manner very similar to that of S. cerevisiae ribosomal genes. The level of the RP10 mRNA decreased upon heat shock (from 25 to 45؇C) and was tightly regulated during growth. Maximal levels of the mRNA were reached during mid-exponential phase before they decreased to negligible levels in stationary phase. The level of the RP10 mRNA was induced only transiently during the yeast-to-hyphal morphological transition but did not appear to respond to hyphal development per se.Candida albicans is a commensal fungus of humans which can cause irritating infections of the epithelial tissues, particularly in the mouth and urogenital tract (39). In the immunocompromised host, it can cause deep-seated and systemic infections, which may be life threatening. Little is known about the relative importance of the various potential virulence factors of C. albicans during pathogenesis. These factors include the production of extracellular hydrolytic enzymes, especially proteases and phospholipases (5,10,22,26,32,41), the ability to adhere to host tissues (8), the immunomodulatory effects of various cell wall components (34,58), and the ability to undergo a dimorphic transition from a budding yeast to a hyphal form (39,52). The relationship between the dimorphic transition and pathogenicity remains unclear (10,33,39,42,51). Both forms are found in infected tissues (40, 42), but C. albicans hyphae appear to be better adapted to penetrate epithelia (48) and therefore might play a role in establishing deepseated infections.Although numerous factors can stimulate C. albicans dimorphism in vitro (39), little is known about the mechanisms which control the transition. Different approaches are being taken to identify genes involved in the control of dimorphism. For example, the identification of S. cerevisiae genes that influence pseudohyphal growth in this yeast (17, 30) may lead ultimately to the isolation of homologs that are important for dimorphism in C. albicans. Also, differential hybridization strategies are proving successful in the identification of genes which are regulated during dimorphism despite the fact that the expression of many genes fluctuates during the yeast-to-hyphal form transition (54, 55). Such strategies have led to the isolation of the C. albicans ECE1 and PHR1 genes, which are expressed specifically in the hyphal growth form (4, 14), but these genes are not required for the transition unde...

The Candida albicans HYR1 gene, which is activated in response to hyphal development, belongs to a gene family encoding yeast cell wall proteins

“…The expression of many other C. albicans genes fluctuates in response to the inducing conditions during the yeast-to-hypha transition rather than to morphogenesis itself. These genes encode proteins involved in wideranging functions such as glycolysis (ADH1, PYK1, GPM1, and PGK1 [67]), protein synthesis and folding (TEF3, RP10, and HSP90 [65,66,69]), cell wall biosynthesis (CHS1, CHS2, CHS3, and GFA1 [10,24,59]), and formation of the cytoskeleton (ACT1 [13,66]). We know of only one C. albicans mRNA (HST7) that remains at relatively constant levels during the yeast-to-hypha transition.…”

“…Hence, we attempted to identify C. albicans genes which are regulated specifically in response to morphogenesis. Using various approaches, we identified numerous genes that are regulated during morphogenesis, including HSP90, TEF3, ADH1, PYK1, RP10, and GFA1 (59,[65][66][67]69). However, in all cases we showed that these genes were responding to changing growth conditions rather than to morphogenesis.…”

“…Following electrophoresis on formaldehyde-containing gels, the RNA was transferred to nylon membranes by vacuum blotting, and hybridizations were performed under conditions of probe excess (42). To date, there has been no report of a reasonably abundant C. albicans mRNA that remains at sufficiently constant levels during the dimorphic transition to use as an internal loading control on Northern blots (3,11,59,(65)(66)(67)69). Therefore, mRNA levels were measured relative to those of the rRNAs by loading equal amounts of total RNA in each lane of the Northern gels (3,11,59,(65)(66)(67)69).…”

Genetically engineered human salivary histatin genes are functional in Candida albicans: development of a new system for studying histatin candidacidal activity

“…After amplification (2 min at 94 mC followed by three-step cycling of 30 s denaturation at 94 mC, 15 s annealing at 57 mC, 30 s extension at 72 mC, and 2 min final extension at 72 mC) the resulting PCR fragment was cut with HindIII and SalI and ligated into the abovedescribed pMAL\EFB3hUTR\URA3 to yield pCaOSS. The sequence of the 1674 bp fragment bearing CaRP10 was amplified from C. albicans SGY-243 genomic DNA with RP10-5h\RP10-3h primers (Table 2), which were designed based on the fragment 3156 of Contig5 of the C. albicans sequencing project at Stanford University (sequencewww.stanford.edu\group\candida), and was obtained by using the GenBank X82017 entry (Swoboda et al, 1995) as a query against the C. albicans database. PCR conditions were : initial denaturation for 3 min at 94 mC, followed by three-step cycling of 30 s denaturation at 94 mC, 30 s annealing at 57 mC, 1 min 30 s extension at 72 mC, for 30 cycles, and 7 min final extension at 72 mC.…”

“…The final plasmid construction step was amplification and cloning of a target region into the vectors carrying the above-described genes to ensure stable integration into the genome of the recipient C. albicans SGY-243 cells following transformation. The CaRP10 locus was chosen to serve this purpose (Swoboda et al, 1995 ;Care et al, 1999 ;Backen et al, 2000). We chose SGY-243 as the recipient strain since it has a very similar profile of histatin sensitivity as the clinical isolate (DS1) we have previously studied (Edgerton et al, 1998).…”