The Function Of Rps4p And Ste18p In Candida Albicans

Candida albicans, a pleiomorphic fungus, is one of the most common opportunistic fungalpathogens in humans, causing various diseases from superficial mucosal infections tolife-threatening systemic disorders. In the recent years, Candida infections have increaseddramatically with increase in immunosuppressive treatments, long-term catheterization,using of broad-spectrum antibiotics and longer survival of immunologically compromisedindividuals. C. albicans, as a dimorphic species, is capable of a morphological transitionfrom an unicellular budding yeast to a filamentous form upon various environmental cues.The process of morphological transition is known to be important for the biofilm formationand virulence in C. albicans. Another switching system in C. albicans consisted of areversible transition between two phases: a white hemispherical colony morphology,referred to as the “white phase,” and a grey flat colony morphology, referred to as the“opaque phase”. C. albicans has recently been demonstrated to be able to mating betweenMTLa and MTLα strains in vivo and in vitro, and have a pheromone response pathwaywhich is similar with Saccharomyces cerevisiae.The ribosome, which is a complex molecular machine, is the site of mRNA translation andprotein synthesis. The eukaryotic ribosome is composed of ribosomal RNAs and ribosomalproteins. Ribosomal proteins are important components of the ribosome, and play key rolesin protein synthesis. In eukaryotes, loss of cytoplasmic ribosomal proteins results in areduced growth rate and other phenotypic defects. Many ribosomal proteins haveextra-ribosomal functions in addition to their roles in protein synthesis on ribosomalparticles. The eukaryotic ribosomal protein S4(Rps4p) is thought to carry out potentialextra-ribosomal functions. In S. cerevisiae, ScRps4p is encoded by duplicated genesRPS4A and RPS4B. These two paralogous ribosomal protein genes, RPS4A and RPS4B,exhibit completely unique phenotypes. Loss of RPS4A can give phenotypes showingdifferences in cell size and CG4-theopalaumide sensitivity, while deletion of RPS4B canlead to abnormal telomere length, changed neomycin sulfate sensitivity and hydrogen peroxide sensitivity. The ribosomal protein S4of wheat is an cysteine proteases. In humans,Rps4p might be involved in Turner syndrome.In the present study we have focused on the function of Rps4p of the fungal pathogen C.albicans. In S. cerevisiae, ScRps4p is encoded by duplicated genes RPS4A and RPS4B.These two paralogous ribosomal protein genes, RPS4A and RPS4B, exhibit completelyunique phenotypes. In C.albicans, Rps4p is encoded by two isogenes, RPS41and RPS42.The CaRPS41gene locus is on chromosome II, while CaRPS42is on chromosome I inC.albicans. Sequence analysis of the CaRPS41shows that its ORF only contains an exon,while CaRPS42reveals that its ORF is interrupted by an intron of569bp, at position15after ATG. Analysis of the deduced primary structure of the protein showed99.6%sequence identity and code for two almost identical proteins of262amino acids. Thededuced Gly2residue of Rps41p is substituted for Ala2in Rps42p.To assess the function of Rps4p in C. albicans, we used the “Ura-blaster” technology todisruption RPS41gene and RPS42gene in C. albicans strain CAI4, as described above.Applications of “Ura-blaster” technology result in different genomic positions for theURA3gene in the mutant strains, wild type strains and the strains complemented for thegenes of interest. Studies using animal models of systemic candidiasis pointed to possibledifferences in URA3gene expression, depending on its genomic location, whichconfounded interpretation of the role of the gene of interest in lethality. Fortunately,position effects on URA3expression can be avoided by placement at a common locus in allstrains for comparison. For this reason, we constructed mutant strains, in which the URA3was at the same RP10locus, to investigate the function of the Rps4p in C. albicans.To assess whether there are differences between the two RPS4isogenes, the rps41Δ andrps42Δ mutant strains of C. albicans were studied in YPD/Uridine-liquid medium. Theaverage of the growth rates of rps41Δ strain tested was μ=0.48h-1corresponding to a28%reduction in growth in comparison to μ=0.68h-1for the rps42Δ, and μ=0.66h-1forthe wild-type strain CAI4.To test the ability of biofilm formation of the rps41Δ mutant cells, we used the SEM to monitor the characteristics of48-h biofillms formed on silicon discs by wild-type andmutant C. albicans strains. SEM showed the intricate network of spatially dispersedfilamentous hyphal elements that intertwine to form a coherent three-dimensional structure.We observed rps41Δ mutant cells biofilm formation dramaticaly decreased compared withwild-type strain by the SEM. SEM showed that wild-type strain biofilm exhibited a typicalthree-dimensional nature, composed mainly of true hyphae, while rps41Δ biofilmdevelopment was inhibited and growth was predominantly composed of yeast cells andpseudohyphae. True hyphae were rarely observed, a factor that contributed the poorbiofilm archiectures. Dimorphism has been shown to play a relevant role in the bio-filmformation in C.albicans. In order to characterize the phenotype of rps41Δ and rps42Δmutants, we examined the ability to undergo the dimorphic transition. These resultsindicate that RPS41，but not RPS42, was required for the dimorphic transition in C.albicans. We inculated the wild-type strain and rps41Δ mutant strain in the biofilmformation conditions, and harvested the bio-film and extracted the whole proteins. Andthen we used the Two-dimensional electrophoresis to analysis the different expressionproteins in biofilm between wild-type strain and rps41Δ mutant strain. We included theabsent of RPS41caused the filamentous associated proteins and biofilm formationassociated proteins synthesis decreased. To investigate the virulence of the rps41nullmutant, a murine model of systemic candidiasis was constructed. We found that miceinjected with CAI4cells started to die the seventh day and all were dead within nine days.In contrast, the rps41Δ showed dramatically decreased virulence compared to CAI4. RPS4mRNAs of the wild-type and rps41Δ or rps42Δ mutant strains were compared to testwhether the RPS41and RPS42genes are differently transcribed. Our results demonstratednearly similar amounts of RPS41mRNA in the wild-type and the rps42Δ null mutants. Forthe RPS42transcript, a dramatically strong induction that the RPS42gene mRNA level inrps41Δ null mutants was20more times than in wild-type strains, was found. These resultsstrongly suggested that Rps4p a component of the40S ribosomal subunit, plays a pivotalrole in morphological transition, biofilm formation and virulence in C. albicans by controlling expression of morphological transition and biofilm formation genes via an asyet undefined process involving the ribosome.The fungal pathogen C. albicans has been demonstrated to show mating between MTLaand MTLα strains in vivo and in vitro, and to have a pheromone response pathway similarto that of S. cerevisiae. In C. albicans, the STE18gene (ORF19.6551.1) encodes a potentialγ subunit of a heterotrimeric G protein; this protein contains the C-terminal CAAX boxcharacteristic of γ subunits, and has sequence similarity to γ subunits implicated in themating pathways of a variety of fungi. Disruption of this gene was shown to cause sterilityof MTLa mating cells, and deletion of the CAAX box residues is sufficient to inactivate itsfunction in the mating process. Intriguingly, overproduction of either the Gα or the Gβsubunit of the heterotrimeric G protein is able to partially suppress the mating defectcaused by deletion of the Gγ subunit.