Priming with FLO8-deficient Candida albicans induces Th1-biased protective immunity against lethal polymicrobial sepsis.

The morphological switch between yeast and hyphae of Candida albicans is essential for its interaction with the host defense system. However, the lack of understanding of host-pathogen interactions during C. albicans infection greatly hampers the development of effective immunotherapies. Here, we found that priming with the C. albicans FLO8-deficient (flo8) mutant, locked in yeast form, protected mice from subsequent lethal C. albicans infection. Deficiency of Dectin-2, a fungus-derived α-mannan recognition receptor, completely blocked flo8 mutant-induced protection. Mechanistically, the flo8 mutant-induced Dectin-2/CARD9-mediated IL-10 production in DCs and macrophages to block thymus atrophy by inhibiting the C. albicans-induced apoptosis of thymic T cells, which facilitated the continuous output of naive T cells from the thymus to the spleen. Continuous recruitment of naive T cells to the spleen enhanced Th1-biased antifungal immune responses. Consequently, depletion of CD4+ T cells or blockade of IL-10 receptor function using specific antibodies in mice completely blocked the protective effects of flo8 mutant priming against C. albicans infection. Moreover, mannans exposed on the surface of the flo8 mutant were responsible for eliciting protective immunity by inhibiting the C. albicans-induced apoptosis of thymic T cells to sustain the number of naive T cells in the spleen. Importantly, priming with the flo8 mutant extensively protected mice from polymicrobial infection caused by cecal ligation and puncture (CLP) by enhancing Th1-biased immune responses. Together, our findings imply that targeting FLO8 in C. albicans elicits protective immune responses against polymicrobial infections and that mannans extracted from the flo8 mutant are potential immunotherapeutic candidate(s) for controlling infectious diseases.

Cloning and Functional Analysis of ALS Family Genes from Candida albicans.

With a 0.5 kb probe of CX2, distribution of CX2 tandem repeats was studied in different C.albicans strains. Results suggest that all the C.albicans strains tested contained the tandem repeat. In order to verify if the expression of CX2 was hyphal specific, its expression was analyzed under various inductive and non-inductive conditions. With CX2 0.5 kb probe, Northern hybridization analysis confirmed that it was specifically in hyphae. The result of chromosomal localizationtion and genomic Southern blot analysis suggested that there were other genes containing the tandem repeat besides of ALS1. A C.albicans s genomic DNA library was screened with the CX2 0.5 kb probe and several positive recombinant lambda phages were obtained. After endonuclease identification, subcloning, and sequence analysis, several ALS family genes were cloned. No.1 lambda phage DNA contained ALS4, No.4 lambda phage DNA contained ALS1, No.6 lambda phage DNA contained ALS3. To study the role of ALS family genes in yeast-hyphal transition, als1/ALS1 mutant was constructed by homologous recombination. The ability to form hyphae was tested in different inductive culturing conditions. Defective hyphal growth were observed in some solid media.

Using Kl LAC4 as a Reporter Gene in Candida albicans.

The lacZ gene which codes for beta-galactosidase from E.coli does not work in Candida albicans. In this work a reporter system with Kl LAC4 gene was contructed, coding for beta-galactosidase from Kluyveromyces lactis. Kl LAC4 gene was fused to the promoter of ADH1 of Candida albicans with the terminator sequence of ADH1 at the tail. The constructed plasmid, named pYPB1-LAC4, was used to transform Candida albicans. beta-galactosidase activity was measured by plate assay, when cultured on solid medium, and by photometric assay, when cultured in liquid medium. The results suggest that LAC4 of Kluyveromyces lactis can serve as a reporter gene in Candida albicans.

Cloning and Identification of Genes Related with Morphogenesis of Candida albicans.

A Candida albicans cDNA library was constructed and screened by differential hybridization. In hybridization using probes derived from population of yeast cells or hyphae, 67 recombinant phages exhibited more intense signal with the probe derived from hyphae than with the probe from yeast cells. One phage behaved vice versa. Physical map analysis and nucleotide sequence analysis suggested that cDNA of the CX1, a clone specific for yeast form, coded for cytochrome P450 L1A1 (Lanosterol 14alpha-demethylase). Its specific expression pattern was confirmed by Northern analysis. Inhibition of serum on the expression of CX1 cDNA was observed. CX2 cDNA was one of those giving intensive signal with hyphae probes. The cDNA sequence contained a tandem repeat sequence, which was also found in ALS1, another Candida albicans gene identified, whose expression was related with morphogenesis. Northern analysis proved that it was expressed intensively with hyphae probes, however the expression could not be detected in those strongly hybridized to yeast cell probes. The locations of both cDNA on chromosome were analyzed.

CEK2, a Novel MAPK from Candida albicans Complement the Mating Defect of fus3/kss1 Mutant.

A novel MAPK(mitogen-activated protein kinase) gene, CEK2(Candida albicans extracellular signal-regulated kinase 2), was isolated by screening the Candida albicans library based on oligonucleotide probe hybridization and degenerated PCR. The CEK2 gene is 1 119 bp in length, and coding for a 373 aa protein. The CEK2 shares 56% homology with CEK1 from Candida albicans, 55 % homology with FUS3 from S. cerevisiae. From the deduced amino acid sequence, the Cek2 protein contains a conserved ATP binding site and a Ser/Thr kinase activity signal and a conserved TEY sequence was located at L(12) region. In vitro kinase activity assay showed Cek2 could phosphorylate MBP(myelin bovine protein) but not histone H1. CEK2 gene could complement the fus3/kss1 mutant and underwent mating signal induction by a tester strain, but CEK1 gene could not complement with the fus3/kss1 mutant. CEK2 is therefore a FUS3 homolog in Candida albicans.

Molecular Cloning of MAPK Gene Family Using Degenerate PCR.

MAPK(Mitogen-activated protein kinase) pathways play an important role in morphorgenesis of Candida albicans. According to the conserved amino acid sequence of the known MAPKs, two degenerate primers attaching to subdomain VIB and IX were designed to screen novel MAPKs in C. albicans. The PCR was performed under non-strigent conditons. 100 PCR fragments were sequenced and among 25 novel gene fragments, two novel MAPK gene fragments were obtained. Using these two PCR fragments as probes to screen a Candida albicans genomic DNA library, two novel MAPK genes designated CSK1(Candida albicans sporulation-related MAP kinase1) and CEK2(Candida albicans extracellular signal-regulated kinase 2) were cloned. These two genes share high similarity with three cloned MAPK genes CEK1 and MKC1 and CaHOG1. The CSK1 gene is 1 193 bp in length, containing a 92 bp intron, coding for a 367 aa protein. The CSK1 shares highest similarity with SMK1, with homology 55.3% in nucleotide sequence and 50% in amino acid sequence. SMK1 encodes a MAPK involved in the sporulation pathway in S. cerevisiae. In vitro kinase activity assay showed that the Csk1 kinase exhibited a phosphorylation ability when using MBP as a substrate but not histone H1.

Functions of CRK1 Gene of Candida albicans as Studied by Gene Knock-out.

In the course of screening MAPK related protein kinase genes from Candida albicans DNA library, a CRK1 (CDC2-related protein kinase) gene was identified. Deletion of the CRK1 gene significantly decreased the growth. rate of cells. CRK1 knock out strains precipitated more rapidly in YPD liquid medium. The CRK1 null mutant stimulated hyphae formation in Lee's liquid medium at pH 4.5 and 25 degrees, which is the medium that maintains the wild type Candida albicans strain in yeast form. These phenomena suggest that CRK1 gene may be involved in cell cycle, flocculation and morphogenesis of Candida albicans. Overexpression of the half length CRK1 gene could induce the invasive growth into agar faintly, the cells that was capable of the invasive growth also changed their cell shapes to long, thin pseudohyphal-like cells, but the full-length CRK1 had only weak effect. The Crk1p might promote the invasive growth or cell elongation through a route different from the MAPK signal transduction pathway.

[Cloning of Candida albicans CaBEM1 and its role in filamentous growth of Saccharomyces cerevisiae].

The pathogenic fungus Candida albicans has a dimorphic transition in various environmental conditions. Many regulatory factors and several transduction pathways have been identified in controlling filamentous growth. G(1) cyclins Cln1 and Cln2 have been reported as involved in the control of morphogenesis in Saccharomyces cerevisiae. Diploid cln1/cln1 and cln2/cln2 strains completely lost the ability to form pseudohyphae. A C. albicans genomic DNA library was introduced into cln1/cln1 and cln2/cln2 mutants to screen genes which could complement its filamentous growth defect. In this screening a BEM1 homolog, CaBEM1, was identified. The CaBEM1 gene has an ORF of 1 899 bp, encoding a putative protein of 632 amino acids. The CaBem1 protein shares highest homology in amino acids with Bem1 (38%) of S. cerevisiae and Scd2 (32%) of Schizosaccharomyces pombe. Sequence analysis showed that the CaBem1 contains two N-terminal SH3 domains, a PX domain and a C-terminal PB1 domain. It is believed these domains are required for binding to proteins involved in polarized growth in S. cerevisiae and S. pombe. Ectopic expression of the CaBEM1 gene in diploid S. cerevisiae suppressed defects in filamentous growth of some mutants under nitrogen starvation conditions. This suppression bypassed MAPK pathway and cAMP/PKA pathway in filamentous growth. These results suggest that the CaBem1 protein may be a downstream component of these two signal transduction pathways of filament formation.

KIAA0202, a human septin family member, interacting with hPFTAIRE1.

hPFTAIRE1 is a member of Cdc2-related kinase family localized in cytoplasm. To search its substrates and regulatory proteins, the hPFTAIRE1 was fused to LexA and used as bait to screen a human brain LexA two-hybrid library. In this screening, 7 hPFTAIRE1 interacting proteins, including KIAA0202, were obtained. The interaction between the KIAA0202 and the hPFTAIRE1 was confirmed by using immunoprecipitation. The KIAA0202 is a 508 aa protein and is a member of septin family. The central portion of the KIAA0202 is a GTP-CDC domain, which is conserved among almost all septins. Its C-terminus is a coiled-coil structure and its N-terminus is a variable region. Our results suggest that the KIAA0202 may play important roles in functional regulation of hPFTAIRE1.

Molecular Cloning of MAPK Gene Family Using Synthetic Oligonucleotide Probe.

MAPK(mitogen activated protein kinase) is a kind of Ser/Thr protein kinase. The MAPKs play an important role in several different signal transduction pathways. The MAPKs may also have a role in morphorgenesis of Candida albicans. An oligonucleotide probe was used to screen novel MAPKs in C. albicans. All MAPKs shared high homogeneity in their eleven kinase subdomains, especially subdoman VII and VIII. In subdomain VII, nearly all MAPKs have the same KIDFGLAR sequence, and the two known MAPKs in C. albicans CEK1 and MKC1 have only one different nucleotide in that DNA sequence. This probe was hybridized with C. albicans genomic DNA. Under stringent conditions, the probe could only hybridize with CEK1 and MKC1 gene fragment. But when hybridized at 40 degrees in non-SDS solution, two novel bands appeared. This condition was used to screen SC5314 DNA library, and many positive clones with different hybridization density were obtained. The strongest hybridization clones were identified to contain CEK1 and MKC1 gene. From the stronger positive hybridization clones, two novel genes were identified. The first gene, named CRK1(CDC2-related protein kinase 1), shared high homogeneity to MAPKs, but was not of them. It is closest to SGV1 from S. cerevisiae (with homology 47%) and PITALRE from human (with homology 41%), both of which are CDC2-related protein kinases. The second gene called CEK2(Candida albicans extracelluar signal-regulated kinase 2) is a novel MAPK of Candida albicans, which shares the highest identity with CEK1 and its S. cerevisiae homologs, FUS3 and KSS1, two redundant MAPKs in yeast pheromone response and morphogenesis.

Construction of Candida albicans Two-hybrid Library and Screening for Proteins Interacting with Crk1.

In order to study interactions among proteins involved in Candida albicans morphorgenesis, C.albicans genomic DNA was digested with Sau3AI and fused to activating domain(AD) of LEXA to construct an AD-fused C.albicans genomic DNA expression library. The library contained 7.7x10(4) independent clones and included 1.2x10(5) kb DNA which was 10 fold of the C.albicans whole genomic DNA. The CRK1 gene encodes a CDC2-related protein, that was involved in morphorgenesis of C.albicans. The Crk1 fused to DNA binding domain of LexA was used as the bait to screen the LexA library. Among 9x10(5) transformants, eight Leu and LacZ double positive clones were selected. With restriction enzyme analysis, three kinds of fragments were identified. The clones NJ1, NJ2, NJ3 were sequenced and analyzed with BLAST program. It was found that NJ1, NJ2, NJ3 were homologs of S.cerevisiae gene STI1, RET2 and NFI1, respectively. The kinase domain of Crk1(Crk1N)interacted weakly with protein encoded by NJ1, while the noncatalytic domain of Crk1(Crk1C) interacted strongly with proteins encoded by NJ2 and NJ3. The interaction of NJ3 and Crk1C was verified by the coimmunoprecipition of NJ3-HA with Crk1C-LexA. These proteins may be involved in maturation, transport, localization, and activity regulation of the Crk1.

[Expression of a human ribonuclease inhibitor variant in Escherichia coli and silkworm insect cell (Bombyx mori)].

Ribonuclease inhibitor (RI) is an acidic 50 kD protein with a high content of leucine and cysteine residues. RI inhibits RNases of the pancreatic type. A variant of RI was cloned from human fetal liver cDNA library by polymerase chain reaction (PCR). Compared with the reported RI, only two variations Arg(359)Ala and Leu(365)Pro were found in RIv amino acid sequence. Recombinant RIv has been expressed both in Escherichia coli and silkworm insect cells (Bombyx mori). The recombinant RIv exhibited inhibition activity on ribonucleolytic activity of RNase A in vitro system.

Analysis of the interaction between hPFTAIRE1 and PLZF in a yeast two-hybrid system.

hPFTAIRE1 is a Cdc2-related kinase family member. To search its substrates and regulatory proteins, hPFTAIRE1 was fused to LexA and used as a bait to screen a human brain LexA two-hybrid library. In this screening, seven hPFTAIRE1 interacting proteins, including promyelocytic leukemia zinc finger (PLZF), were obtained. The interaction between PLZF and hPFTAIRE1 was confirmed by beta-galactosidase assay and Leu growth activity. PLZF encodes a transcription factor belonging to the POZ/BTB domain and Krüpel zinc finger (POK) family. The highly conserved POZ/BTB domain plays a critical role in protein-protein interaction. We deleted the POZ/BTB and Krüpel zinc finger domains, respectively, and observed the interaction between hPFTAIRE1 and truncated PLZFs by liquid beta-galactosidase activity assay. A weak interaction was detected between hPFTAIRE1 and PLZF. We also observed the interaction between PLZF and another Cdc2-related kinase, PCTAIRE1. A similar result was observed. The interaction between PLZF and hPFTAIRE1 or PCTAIRE1 was confirmed by co-immunoprecipitation assay in a yeast system. PLZF is a phosphoprotein and plays multiple roles during cell growth. Our results suggest that hPFTAIRE1 and PCTAIRE1 may play important roles in the functional regulation of PLZF.

CaMac1, a Candida albicans copper ion-sensing transcription factor, promotes filamentous and invasive growth in Saccharomyces cerevisiae.

Molecular mechanisms of morphogenesis share many common components between Candida albicans and Saccharomyces cerevisiae. The Kss1-associated MAPK cascade and the cAMP/PKA pathway are two important signal transduction pathways that control morphogenesis in S. cerevisiae. A C. albicans copper ion-sensing transcription factor gene, CaMAC1, was cloned from C. albicans SC5314. Ectopic expression of CaMAC1 in S. cerevisiae promoted filamentous and invasive growth. In diploid cells, CaMac1 could suppress the filamentous growth defect of mutants in the Kss1-associated MAPK pathway and the cAMP/PKA pathway. In haploid strains, ectopic expression of CaMAC1 suppressed the invasive growth defect of mutants in the MAPK pathway (ste7, ste12 and tec1), but failed to suppress the invasive growth defect of the flo8 mutant. Our results suggest that the activation of CaMac1 is independent of the MAPK and cAMP/PKA pathways in filament formation, but requires Flo8 factor for invasive growth. In the media containing a high concentration of CuSO4, the yeast filamentous and invasive growth was blocked. The activating effect of CaMac1 is inhibited by copper ions.

Transiently expressed short hairpin RNA targeting 126 kDa protein of tobacco mosaic virus interferes with virus infection.

RNA interference (RNAi) silences gene expression by guiding mRNA degradation in a sequence-specific fashion. Small interfering RNA (siRNA), an intermediate of the RNAi pathway, has been shown to be very effective in inhibiting virus infection in mammalian cells and cultured plant cells. Here, we report that Agrobacterium tumefaciens-mediated transient expression of short hairpin RNA (shRNA) could inhibit tobacco mosaic virus (TMV) RNA accumulation by targeting the gene encoding the replication-associated 126 kDa protein in intact plant tissue. Our results indicate that transiently expressed shRNA efficiently interfered with TMV infection. The interference observed is sequence-specific, and time- and site-dependent. Transiently expressed shRNA corresponding to the TMV 126 kDa protein gene did not inhibit cucumber mosaic virus (CMV), an unrelated tobamovirus. In order to interfere with TMV accumulation in tobacco leaves, it is essential for the shRNA constructs to be infiltrated into the same leaves as TMV inoculation. Our results support the view that RNAi opens the door for novel therapeutic procedures against virus diseases. We propose that a combination of the RNAi technique and Agrobacterium-mediated transient expression could be employed as a potent antiviral treatment in plants.nt antiviral treatment in plants.

[Cloning and functional study of CaPPe1 in Candida albicans by using Saccharomyses cerevisiae model system].

The dimorphic transition of yeast and hyphal forms is one of most determinants in Candida albicans for its pathogenicity. This transition is regulated by several signal transduction pathways. Transcriptional factor Flo8 plays an important role in morphogenesis of Saccharomyces cerevisiae. In this work, a C. albicans genomic DNA library was introduced into a S. cerevisiae flo8/flo8 mutant and genes which could suppress invasive growth defect were isolated. A novel gene was isolated and designated CaPPE1 (Candida albicans PPE1 gene). CaPPE1 encoded for a 361 amino acid protein CaPpe1, shared highest similarity in amino acids (35% identity) with the protein phosphatase methylesterase Ppel of S. cerevisiae. In haploid of S. cerevisiae, ectopic expressed CaPPE1 could partially suppress the invasive growth defect of the flo8 mutant but failed to suppressed the invasive growth defects of the mutants in MAPK pathway(ste12/ste12 and tec1/ tecl). Ectopic expression of the CaPPE1 in diploid of S. cerevisiae suppress the filamentous growth defect of some mutants in MAPK pathway, but not in flo8/flo8 mutant under nitrogen starvation condition. It is suggested that CaPpe1 may be involved in different regulating pathways in diploid filamentous growth and in haploid invasive growth.

A viral protein suppresses siRNA-directed interference in tobacco mosaic virus infection.

Plant viruses encode suppressors of post-transcriptional gene silencing (PTGS), an adaptive defense response that limits virus replication and its spread in plants. The helper component proteinase (HC-Pro) of the potato virus A (PVA, genus Potyvirus) suppresses PTGS of silenced transgenes. Here, the effect of HC-Pro on siRNA-directed interference in the tobacco mosaic virus (TMV) was examined by using a transient Agrobacterium tumefaciens-based delivery system in intact tissues. It was shown that the interference effect was completely blocked by co-infiltration with HC-Pro plus siRNA constructs in both systemic and hypersensitive hosts. In the system host, all plants agro-infiltrated with HC-Pro plus siRNA constructs displayed the same symptoms as the negative control. Meanwhile, TMV RNA accumulation was found to be abundant in the upper leaves using reverse transcriptase-PCR (RT-PCR) and Northern blot assays. On the contrary, plants agro-infiltrated with the siRNA construct alone were free of symptoms. Therefore, our study suggests that the transient expression of HC-Pro inhibited the siRNA-directed host defenses against TMV infection.

[Long terminal repeats of retroposons in Candida albicans].

25 Candida albicans strains have been analysed on Southern hybridization patterns with alpha, beta, gamma, kappa probes. The alpha, beta, gamma, kappa, elements can be used to distinguish the differences and relations between C. albicans strains. Chromosomal localization analysis indicated that the alpha, beta, gamma, kappa, elements were located on several different chromosomes in Candida albicans strains. Northern hybridization showed that alpha, beta, gamma were transcribed actively and regulated by different stimuli. The kappa was inactive. Our results indicated that in Candida albicans there were many kinds of retroposons whose numbers and distribution were different. These retroposons could be used to identify Candida albicans strains as moleculer markers.

[The role of Cln3 in filamentous growth and invasive growth of Saccharomyces cerevisiae].

Cln3 is one of G1 cyclins in Saccharomyces cerevisiae. In order to study the function of Cln3 in cell cycle and morphogenesis, we constructed a cln3 null mutant and analyzed its phenotype. Our results indicated that the cln3 null mutant was more sensitive to alpha pheromone, and arrested at G1 phase. The hypersensitivity to alpha-pheromone was not suppressed by overexpression of SGV1. The null mutant showed a different phenotype with that of the other two G1 cyclin mutants. The filamentous growth in diploid cells of cln3 mutant was stronger than that in wild type cells, while invasive growth of the haploid cells was partially inhibited. The results suggested that the Cln3 plays a unique function in morphogenesis under a different mechanism with that used by Cln1 and Cln2.