白念珠菌Rps4p和Ste18p的功能研究
摘要
白念珠菌是重要的机会性致病真菌。它可以引起包括粘膜感染和危及生命的系统性疾病等多种疾病。最近几年，随着免疫抑制剂治疗的增加，长期的导尿技术的运用，以及广谱抗生素的使用，导致了念珠菌感染病例的逐年上升，其中白念珠菌占了很高的比例。白念珠菌作为具有多种形态的真菌，一种重要的形态转换就是在各种菌丝诱导条件下从单一细胞的酵母态转变为多细胞的菌丝态。白念珠菌的这种“酵母—菌丝”形态转变在白念珠菌被膜的形成过程中起着至关重要的作用。白念珠菌的另一种形态转换是在纯合子的状态下，从白色形态到不透明态的转换，这种转换在白念珠菌的交配过程中起着重要的作用。
核糖体是细胞合成蛋白质的重要场所。成熟的核糖体由核糖体RNA(rRNA)和核糖体蛋白（RP）组成。核糖体蛋白是核糖体的重要组成部分，其主要功能是保证蛋白质合成的顺利进行。在真核生物中，缺失胞浆核糖体蛋白将会导致生长率的降低和其他生理表型的缺陷。最近的研究发现，核糖体蛋白不仅具有组成核糖体蛋白的功能，还可能参加了其他的生命活动，即具有核糖体外功能。最近的研究表明，真核生物的核糖体蛋白S4（Rps4p）具有潜在的核糖体外功能。在酿酒酵母中，Rps4p与细胞大小，端粒长度，过氧化氢敏感度和硫酸新霉素的敏感度有关。小麦的Rps4p具有半胱氨酸酶活性。而研究也表明，人类的唐氏综合征也可能与Rps4p相关。这些研究结果提示了真核生物的Rps4p具有潜在的核糖体外功能。
本课题主要关注了白念珠菌核糖体蛋白S4的功能。在酿酒酵母中，Rps4p由同源基因RPS4A和RPS4B编码。虽然两个基因编码的蛋白质的序列的一致程度相当高，但两个基因却表现出了不同的表型。这些现象为我们研究白念珠菌的Rps4p的功能提供了很好的线索。在白念珠菌中，Rps4p由同源基因RPS41和RPS42编码，长度为262个氨基酸。基因RPS41位于白念珠菌染色体的2号染色体上，全长为789bp,不含有内含子；基因RPS42位于白念珠菌染色体的1号染色体上，全长为1358bp,含有一个长度为569bp的内含子。基因RPS41和RPS42所编码的蛋白仅有一个氨基酸的差异。
为了深入研究Rps4p的功能，我们用“Ura-Blaster”基因敲除策略在CAI4菌中分别构建了rps41Δ和rps42Δ基因缺失菌和相应的回复菌。“Ura-blaster”基因敲除策略虽然需要构建重组质粒，具有一定的工作量；营养选择标记URA3对表型也有一定的影响等缺点。但本方法也具有其无可取代的优点。首先，该方法成熟稳定，应用范围广，在技术操作上易于实现。其次，可以在基因敲除片段两端构建较长的目的基因同源序列，增加同源重组过程的特异性，较易获得基因敲除菌株，特别是对较难敲除的基因尤为有效。再次，URA3营养选择标记可以重复利用，可以实现对多拷贝基因的敲除或者同一菌株的多基因敲除，其他基因敲除方法很难实现。本课题中的RPS41和RPS42均具有三拷贝，所以“Ura-blaster”基因敲除策略比较适合本课题。虽然有文献报道，URA3基因的表达量和在染色体的位置对白念珠菌的毒力等表型有所影响，但这些缺点是可以克服的。有文献报道，将URA3插在RP10，ENO1， ARG4等基因位置或者URA3在染色体中的原始位置，这样可在一定程度上消除URA3的影响。本课题统一将URA3营养选择标记回复在RP10的位置，来消除URA3的影响。
我们的实验结果表明，RPS41基因的缺失将导致白念珠菌很多方面的功能缺陷。通过生长率测定实验，发现rps41Δ基因缺失菌表现为生长速率显著降低，而rps42Δ基因缺失菌的生长速率与野生菌没有明显的差别。通过测定，rps41Δ基因缺失菌的平均生长率为μ=0.48h-1，rps42Δ基因缺失菌的生长率为μ=0.68h-1，而野生菌CAI4的生长率为μ=0.66h-1。rps41Δ基因缺失菌的生长速率相对于rps42Δ基因缺失菌和野生菌降低了28%左右。
为了检测rps41Δ基因缺失菌的被膜形成能力，我们利用扫描电镜来检测成熟生物被膜的结构。结果表明，rps41Δ基因缺失菌的被膜形成较野生菌大量降低。野生菌的生物被膜呈经典的三维结构，主要由菌丝构成。而rps41Δ基因缺失菌的生物被膜的形成受到了抑制，以酵母态和假菌丝态为主，很少看见真菌丝。为了寻求RPS41基因影响生物被膜形成的机制，我们从影响生物被膜形成的各个方面进行了研究。我们首先考察了影响被膜形成的关键因素，菌丝形成能力。利用菌丝形成实验，我们发现rps41Δ基因缺失菌的菌丝形成能力下降，而rps42Δ基因缺失菌的菌丝形成没有受到明显的影响。其次，我们考察了影响生物被膜形成的另一重要因素，抗氧化能力。我们发现rps41Δ基因缺失菌的对过氧化氢的敏感度是升高的，说明了其抗氧化能力的下降。而rps42Δ基因缺失菌与野生菌相比没有显著的变化。再次，我们利用二维电泳技术分析了野生型菌和rps41Δ基因缺失菌生物被膜状态下的蛋白质组。蛋白质组分析结果表明，rps41Δ基因缺失菌的能量代谢主要以无氧呼吸为主，抗氧化的相关蛋白表达水平上升，表明了其体内的ROS水平较野生菌低，从而表明了其菌丝形成能力是降低的。同时我们也发现菌丝形成相关蛋白也是低表达的。实验结果表明，白念珠菌生物被膜的形成时依赖于Rps4p的。我们还进行了体内毒力实验来检测两个基因缺失菌的毒力。实验结果表明，rps41Δ基因缺失菌的毒力是显著降低的。野生菌和rps42Δ基因缺失菌感染组的小鼠平均生长时间只有4-7天，而rps41Δ基因缺失菌感染组的小鼠平均生长时间却达到16天左右。我们还考察了RPS41和RPS42两个基因的表达水平。结果表明，基因RPS42的缺失对基因RPS41的表达水平影响并不是很明显，而基因RPS41的缺失会强烈的诱导基因RPS42的表达，达到正常水平的20多倍。虽然基因RPS42被强烈的诱导，其所表达的Rps4p的mRNA也只有正常水平的20%.这些结果说明，在正常情况下，主要由基因RPS41表达Rps4p，基因RPS42基本上处于沉默状态。基因RPS41和RPS42的表达水平是不对称的，基因RPS41在白念珠菌的生命活动中比基因RPS42发挥更加关键的作用。RPS41基因的缺失并不是对所有的表型都有影响，rps41Δ基因缺失菌对粘附生长能力的影响不是很显著，对唑类药物敏感度也没有明显的变化。Rps4p的量的降低会影响核糖体的形成，可能会使某些蛋白质的合成受到影响，但是这种影响是特异的，这表明了Rps4p功能的特异性，也揭示了Rps4p潜在的核糖体外功能。
最近的研究表明白念珠菌具有交配的能力。参与交配的信号通路和酿酒酵母的是相似的。在白念珠菌中，缺失G蛋白的α亚基和β亚基将会导致白念珠菌完全丧失交配能力。但是G蛋白γ亚基在白念珠菌交配过程中的作用还不是很清楚。为了明确G蛋白γ亚基在白念珠菌交配过程中的作用，本课题对STE18基因的功能进行了研究。在白念珠菌中，基因STE18编码经典的G蛋白的γ亚基，氨基酸长度为90个氨基酸。G蛋白γ亚基的氨基酸序列在裂殖酵母中有很高的保守性，并且C末端含有CAAX盒。我们利用同源重组的原理，构建了ste18Δ基因缺失菌和STE18基因C末端敲除菌。结果表明，G蛋白的γ亚基和γ亚基的C末端在白念珠菌的交配中都是必需的。我们还考察了G蛋白三个亚基之间的关系。结果表明，在ste18Δ基因缺失菌过量表达G蛋白的α亚基或者β亚基可以使白念珠菌在缺少γ亚基的情况下进行交配。这说明Gγ的作用可以被过量的Gα或者Gβ取代。我们的结果揭示了白念珠菌的G蛋白3个亚基之间的相互作用关系与其它菌株相比具有特异性。
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.
核糖体是细胞合成蛋白质的重要场所。成熟的核糖体由核糖体RNA(rRNA)和核糖体蛋白（RP）组成。核糖体蛋白是核糖体的重要组成部分，其主要功能是保证蛋白质合成的顺利进行。在真核生物中，缺失胞浆核糖体蛋白将会导致生长率的降低和其他生理表型的缺陷。最近的研究发现，核糖体蛋白不仅具有组成核糖体蛋白的功能，还可能参加了其他的生命活动，即具有核糖体外功能。最近的研究表明，真核生物的核糖体蛋白S4（Rps4p）具有潜在的核糖体外功能。在酿酒酵母中，Rps4p与细胞大小，端粒长度，过氧化氢敏感度和硫酸新霉素的敏感度有关。小麦的Rps4p具有半胱氨酸酶活性。而研究也表明，人类的唐氏综合征也可能与Rps4p相关。这些研究结果提示了真核生物的Rps4p具有潜在的核糖体外功能。
本课题主要关注了白念珠菌核糖体蛋白S4的功能。在酿酒酵母中，Rps4p由同源基因RPS4A和RPS4B编码。虽然两个基因编码的蛋白质的序列的一致程度相当高，但两个基因却表现出了不同的表型。这些现象为我们研究白念珠菌的Rps4p的功能提供了很好的线索。在白念珠菌中，Rps4p由同源基因RPS41和RPS42编码，长度为262个氨基酸。基因RPS41位于白念珠菌染色体的2号染色体上，全长为789bp,不含有内含子；基因RPS42位于白念珠菌染色体的1号染色体上，全长为1358bp,含有一个长度为569bp的内含子。基因RPS41和RPS42所编码的蛋白仅有一个氨基酸的差异。
为了深入研究Rps4p的功能，我们用“Ura-Blaster”基因敲除策略在CAI4菌中分别构建了rps41Δ和rps42Δ基因缺失菌和相应的回复菌。“Ura-blaster”基因敲除策略虽然需要构建重组质粒，具有一定的工作量；营养选择标记URA3对表型也有一定的影响等缺点。但本方法也具有其无可取代的优点。首先，该方法成熟稳定，应用范围广，在技术操作上易于实现。其次，可以在基因敲除片段两端构建较长的目的基因同源序列，增加同源重组过程的特异性，较易获得基因敲除菌株，特别是对较难敲除的基因尤为有效。再次，URA3营养选择标记可以重复利用，可以实现对多拷贝基因的敲除或者同一菌株的多基因敲除，其他基因敲除方法很难实现。本课题中的RPS41和RPS42均具有三拷贝，所以“Ura-blaster”基因敲除策略比较适合本课题。虽然有文献报道，URA3基因的表达量和在染色体的位置对白念珠菌的毒力等表型有所影响，但这些缺点是可以克服的。有文献报道，将URA3插在RP10，ENO1， ARG4等基因位置或者URA3在染色体中的原始位置，这样可在一定程度上消除URA3的影响。本课题统一将URA3营养选择标记回复在RP10的位置，来消除URA3的影响。
我们的实验结果表明，RPS41基因的缺失将导致白念珠菌很多方面的功能缺陷。通过生长率测定实验，发现rps41Δ基因缺失菌表现为生长速率显著降低，而rps42Δ基因缺失菌的生长速率与野生菌没有明显的差别。通过测定，rps41Δ基因缺失菌的平均生长率为μ=0.48h-1，rps42Δ基因缺失菌的生长率为μ=0.68h-1，而野生菌CAI4的生长率为μ=0.66h-1。rps41Δ基因缺失菌的生长速率相对于rps42Δ基因缺失菌和野生菌降低了28%左右。
为了检测rps41Δ基因缺失菌的被膜形成能力，我们利用扫描电镜来检测成熟生物被膜的结构。结果表明，rps41Δ基因缺失菌的被膜形成较野生菌大量降低。野生菌的生物被膜呈经典的三维结构，主要由菌丝构成。而rps41Δ基因缺失菌的生物被膜的形成受到了抑制，以酵母态和假菌丝态为主，很少看见真菌丝。为了寻求RPS41基因影响生物被膜形成的机制，我们从影响生物被膜形成的各个方面进行了研究。我们首先考察了影响被膜形成的关键因素，菌丝形成能力。利用菌丝形成实验，我们发现rps41Δ基因缺失菌的菌丝形成能力下降，而rps42Δ基因缺失菌的菌丝形成没有受到明显的影响。其次，我们考察了影响生物被膜形成的另一重要因素，抗氧化能力。我们发现rps41Δ基因缺失菌的对过氧化氢的敏感度是升高的，说明了其抗氧化能力的下降。而rps42Δ基因缺失菌与野生菌相比没有显著的变化。再次，我们利用二维电泳技术分析了野生型菌和rps41Δ基因缺失菌生物被膜状态下的蛋白质组。蛋白质组分析结果表明，rps41Δ基因缺失菌的能量代谢主要以无氧呼吸为主，抗氧化的相关蛋白表达水平上升，表明了其体内的ROS水平较野生菌低，从而表明了其菌丝形成能力是降低的。同时我们也发现菌丝形成相关蛋白也是低表达的。实验结果表明，白念珠菌生物被膜的形成时依赖于Rps4p的。我们还进行了体内毒力实验来检测两个基因缺失菌的毒力。实验结果表明，rps41Δ基因缺失菌的毒力是显著降低的。野生菌和rps42Δ基因缺失菌感染组的小鼠平均生长时间只有4-7天，而rps41Δ基因缺失菌感染组的小鼠平均生长时间却达到16天左右。我们还考察了RPS41和RPS42两个基因的表达水平。结果表明，基因RPS42的缺失对基因RPS41的表达水平影响并不是很明显，而基因RPS41的缺失会强烈的诱导基因RPS42的表达，达到正常水平的20多倍。虽然基因RPS42被强烈的诱导，其所表达的Rps4p的mRNA也只有正常水平的20%.这些结果说明，在正常情况下，主要由基因RPS41表达Rps4p，基因RPS42基本上处于沉默状态。基因RPS41和RPS42的表达水平是不对称的，基因RPS41在白念珠菌的生命活动中比基因RPS42发挥更加关键的作用。RPS41基因的缺失并不是对所有的表型都有影响，rps41Δ基因缺失菌对粘附生长能力的影响不是很显著，对唑类药物敏感度也没有明显的变化。Rps4p的量的降低会影响核糖体的形成，可能会使某些蛋白质的合成受到影响，但是这种影响是特异的，这表明了Rps4p功能的特异性，也揭示了Rps4p潜在的核糖体外功能。
最近的研究表明白念珠菌具有交配的能力。参与交配的信号通路和酿酒酵母的是相似的。在白念珠菌中，缺失G蛋白的α亚基和β亚基将会导致白念珠菌完全丧失交配能力。但是G蛋白γ亚基在白念珠菌交配过程中的作用还不是很清楚。为了明确G蛋白γ亚基在白念珠菌交配过程中的作用，本课题对STE18基因的功能进行了研究。在白念珠菌中，基因STE18编码经典的G蛋白的γ亚基，氨基酸长度为90个氨基酸。G蛋白γ亚基的氨基酸序列在裂殖酵母中有很高的保守性，并且C末端含有CAAX盒。我们利用同源重组的原理，构建了ste18Δ基因缺失菌和STE18基因C末端敲除菌。结果表明，G蛋白的γ亚基和γ亚基的C末端在白念珠菌的交配中都是必需的。我们还考察了G蛋白三个亚基之间的关系。结果表明，在ste18Δ基因缺失菌过量表达G蛋白的α亚基或者β亚基可以使白念珠菌在缺少γ亚基的情况下进行交配。这说明Gγ的作用可以被过量的Gα或者Gβ取代。我们的结果揭示了白念珠菌的G蛋白3个亚基之间的相互作用关系与其它菌株相比具有特异性。
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.
引文
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