Mating Factor Research Articles

Cyclic AMP-dependent protein kinase catalytic subunits have divergent roles in virulence factor production in two varieties of the fungal pathogen Cryptococcus neoformans.

Our earlier findings established that cyclic AMP-dependent protein kinase functions in a signaling cascade that regulates mating and virulence of Cryptococcus neoformans var. grubii (serotype A). Mutants lacking the serotype A protein kinase A (PKA) catalytic subunit Pka1 are unable to mate, fail to produce melanin or capsule, and are avirulent in animal models, whereas mutants lacking the PKA regulatory subunit Pkr1 overproduce capsule and are hypervirulent. Because other mutations have been observed to confer different phenotypes in two diverged varieties of C. neoformans (grubii variety [serotype A] and neoformans variety [serotype D]), we analyzed the functions of the PKA genes in the serotype D neoformans variety. Surprisingly, the Pka1 catalytic subunit was not required for mating, haploid fruiting, or melanin or capsule production of serotype D strains. Here we identify a second PKA catalytic subunit gene, PKA2, that is present in both serotype A and D strains of C. neoformans. The divergent Pka2 catalytic subunit was found to regulate mating, haploid fruiting, and virulence factor production in serotype D strains. In contrast, Pka2 has no role in mating, melanin production, or capsule formation in serotype A strains. Our studies illustrate how different components of signaling pathways can be co-opted and functionally specialized during the evolution of related but distinct varieties or subspecies of a human fungal pathogen.

Regional and local population structure of the pioneer wood-decay fungus Trichaptum abietinum

The population structure of 11 Fennoscandian geographic populations of the pioneer wood-decay basidiomycete Trichaptum abietinum was assessed with PCR-RFLPs, intersequence simple repeats (ISSRs) and mating studies. The three codominant PCR-RFLP markers (1) internal transcribed spacer 2 (nrDNA), (2) glyceraldehyde-3-phosphate dehydrogenase and (3) translation elongation factor 1α showed that genotype distributions in most cases (94%) agreed with Hardy-Weinberg expectations and that random association of alleles occurred across loci. The molecular data suggest that T. abietinum is a highly outcrossing fungus that regularly proliferates and spreads by sexual spores. Interstock mating reactions suggest a high number of mating factors among individuals and that biological barriers to gene flow are nonexistent in the region. The three PCR-RFLP loci gave an overall FST = 0.03, indicating a low level of genetic differentiation and presumably high gene flow among the geographic populations. The ISSR markers revealed no systematic substructuring and the among-population variance component was low (6.1%) in AMOVA. However, all PCR-RFLP and most ISSR markers (7/12) showed significant deviation from the null hypothesis of an even distribution of allele frequencies across the 11 geographic populations. Allele frequencies varied in an apparently random manner, suggesting that genetic drift might be an important structuring factor in T. abietinum. The spatial small-scale distribution of heterokaryons on three selected substrate units (logs) showed that most isolates represented discrete individuals and that a number of genets (19) may occupy a single log. The small-scale genotype distributions (within logs) were in agreement with panmictic Hardy-Weinberg expectations.

Production of biologically active tethered ovine FSHbetaalpha by the methylotrophic yeast Pichia pastoris.

The pituitary-derived glycoprotein hormone FSH plays a central role in controlling vertebrate gonadal function. In female mammals the maturation of ovarian follicles is critically dependent upon stimulation by FSH. Moreover, injection of exogenous FSH is used extensively to stimulate increased numbers of follicles to ovulate. Structurally FSH is a heterodimeric glycoprotein composed of two non-covalently associated polypeptide subunits. The tertiary structures of both the alpha- and beta-subunits are constrained by intramolecular disulphide bonds and are post-translationally modified with two N-linked carbohydrate moieties, the structure of which appears to modulate in vivo biological activity. Here we report the expression of ovine FSH (oFSH) as a biologically active single-chain polypeptide using the methylotrophic yeast Pichia pastoris. Sequences encoding the mature oFSH alpha- and beta-proteins were fused to form a gene encoding a fusion protein with the C-terminus of the beta-chain joined to the N-terminus of the alpha-chain, with the chains separated by a two amino acid linker sequence. This fusion gene was itself fused to two alternative Pichia leader sequences (mating factor alpha and acid phosphatase) and transformed into the Pichia strains GS115 and SMD1168. The recombinant fusion protein (oFSHbetaalpha) was expressed at approximately 0.1 microg/ml in 'shake-flask' cultures. The Pichia-expressed tethered protein was biologically active in an in vitro bioassay, had a molecular mass of 28 kDa, as determined by SDS-PAGE, and bound the bovine FSH receptor with a binding profile similar to that of native oFSH.

The Glc7p-interacting protein Bud14p attenuates polarized growth, pheromone response, and filamentous growth in Saccharomyces cerevisiae.

A genetic selection in Saccharomyces cerevisiae for mutants that stimulate the mating pathway uncovered a mutant that had a hyperactive pheromone response pathway and also had hyperpolarized growth. Cloning and segregation analysis demonstrated that BUD14 was the affected gene. Disruption of BUD14 in wild-type cells caused mild stimulation of pheromone response pathway reporters, an increase in sensitivity to mating factor, and a hyperelongated shmoo morphology. The bud14 mutant also had hyperfilamentous growth. Consistent with a role in the control of cell polarity, a Bud14p-green fluorescent protein fusion was localized to sites of polarized growth in the cell. Bud14p shared morphogenetic functions with the Ste20p and Bni1p proteins as well as with the type 1 phosphatase Glc7p. The genetic interactions between BUD14 and GLC7 suggested a role for Glc7p in filamentous growth, and Glc7p was found to have a positive function in filamentous growth in yeast.

항균활성을 보유한 재조합 Pichia pastoris 균주의 개발

곤충에서 유래한 항균 펩티드, defensin을 항균활성이 있는 활성적인 형태로 분비하는 Pichia 균주를 개발하기 위한 일환으로서 MF<TEX>$\alpha$</TEX>1 prerpo sequence와 defensin 합성유전자를 pichia 발현 벡터에 재조합하여 형질전환하고 아미노산 histidine을 첨가하지 않은 최소 배지에서 형질전환체를 일차적으로 선별하였다. 선별한 형질전환체를 대상으로 항생제 G-418에 대한 내성과 M. luteus를 시험균으로 사용하여 생육환 저해를 통한 defensin의 세포 외 분비를 조사하여 4 균주를 선택하고 분석하였다. Southern hybridizaion을 통해 숙주의 염색체 DNA에 삽입한 defensin 유전자가 유지됨을 확인하였으며 전체 RNA를 분리하고 RT-PCR을 수행하여 defensin mRNA를 증폭하고 Southern hybridization을 실시한 결과 증폭된 밴드는 probe로 사용한 defensin 유전자에 의해 양성 신호가 나타났다. 배양시간에 따른 4 균주의 세포성장과 항균활성을 비교하기 위해 BMMY 배지에서 96시간 배양하였다. 세포성장은 모두 유사한 양상을 보였다. 세포성장은 48시간 동안 급격하게 증가한 후 그 이후로는 정지기에 도달되었다. 항균활성도 48시간까지 급격히 증가하였으며 항균활성이 가장 높은 형질전환체 균주 3는 72시간 배양 시 550 AU/<TEX>$m\ell$</TEX>을 나타내었다. To develop a yeast strain of Pichia pastoris producing an antibacterial peptide, we have attempted the expression and secretion of an insect defensin. The nucleotide sequences corresponding to mature defensin were chemically synthesized by 6 oligomers, assembled in vitro and the synthesized gene was identified by nucleotide sequencing. The prepro sequence of yeast mating factor <TEX>$\alpha$</TEX>1 and the defensin gene were recombined into a Pichia expression vector, pPIC9K. The resulting plasmid, pPIDE, was transformed into P. pastoris GSl15 and transformants selected on histidine-deficient minimal plates were tested for antibacterial activity against Micrococcus luteus. Four strains with different antibacterial activity were selected for further analysis. Southern hybridization and RT-PCR verified the defensin gene was maintained and transcribed in a host. Four strains were cultivated in YPD broth for 96 hours to compare cell growth and antibacterial activity, They showed no difference in cell growth, however, each strain showed different antibacterial activity pattern with culture time. The maximal activity was about 550 AU/ <TEX>$m\ell$</TEX>.

Characterization of a fungal protein kinase from Cryphonectria parasitica and its transcriptional upregulation by hypovirus.

The chestnut blight fungus Cryphonectria parasitica and its hypovirus comprise useful model system to study the mechanisms of hypoviral infection. We used degenerate primers based on fungal protein kinases to isolate a gene, cppk1, which encodes a novel Ser/Thr protein kinase of C. parasitica. The gene showed highest homology to ptk1, a Ser/Thr protein kinase from Trichoderma reesei. The encoded protein had a predicted mass of 70.5 kDa and a pI of 7.45. Northern blot analyses revealed that the cppk1 transcript was expressed from the beginning of culture, with a slight increase by 5 days of culture. However, its expression was specifically affected by the presence of virus, and it was transcriptionally upregulated in the fungal strain infected with the hypovirus. A kinase assay using Escherichia coli-derived CpPK1 revealed CpPK1-specific phosphorylated proteins with estimated masses of 50 kDa and 44 kDa. In addition, the phosphorylation of both proteins was higher in a cell-free extract from the hypovirulent strain. The increased expression of cppk1 by the introduction of an additional copy results in a subset of viral symptoms of reduced pigmentation and conidiation in a virus-free isolate. cppk1 overexpression also causes the downregulation of mating factor genes Mf2/1 and Mf2/2, resulting in female sterility. The present study suggests that the hypovirus disturbs fungal signalling by transcriptional upregulation of cppk1, which results in reduced pigmentation and conidiation and female sterility.

High-level expression of recombinant house dust mite allergen Der p 1 in Pichia pastoris.

The major house dust mite Der p 1 allergen is associated with allergic disease. Heterologous over-expression of biologically active Der p 1 was previously attempted but with limited success. The aim of this study was to establish an efficient system for the production of recombinant Der p 1. The proform of Der p 1 was expressed in Pichia pastoris as a fusion with the alpha mating factor signal sequence. The recombinant product was purified from culture medium and compared to Der p 1 isolated from mite culture, in terms of enzymatic activity as well as IgE binding capacity. ProDer p 1 was efficiently secreted into culture medium as a hyperglycosylated protein of 40-60 kDa. Postpurification dialysis in acidic buffer was required for the autocatalytic processing of Der p 1. During this treatment, the prosequence was efficiently removed to give highly glycosylated recombinant mature Der p 1. Competition ELISA experiments as well as cysteine proteinase activity assays indicated that recombinant processed Der p 1 was similar to natural Der p 1 isolated from mite cultures in terms of IgE binding and enzymatic activities. However, the histamine releasing activity of recombinant Der p 1 was slightly weaker than that of natural Der p 1. This efficient system for recombinant Der p 1 expression leads the way for the design of new diagnostics for house dust mite allergy, epitope mapping, allergen engineering, structural and immunological studies and new immunotherapeutic treatments.

Mating Effect on Sex Pheromone Production of the Oriental tobacco budworm, Helicoverpa assulta

This study was undertaken to clarify the suppression phenomenon of sex pheromone production after mating and its relationship to the physiological mechanism in adult females of Helicoverpa assulta, and determine the mating factor from males causing depletion of sex pheromonc production. Sex pheromone production of H. assulta females was mostly terminated in 3 hours after mating. Mated females maintained with a low titer of sex pheromone until 3 days when it started to increase again, which showed a characteristic of species mating more than once. The mated female again produced pheromone upon injection of pheromone biosynthesis activating neuropeptide (PBAN) or extracts of brain-suboesophageal ganglion complexes (Br-Sg) of mated female, which were shown similar pheromonotropic activities as compared with virgin females. These results indicated that the mating did not inhibit the receptivity of pheromone gland itself and PBAN biosynthesis in suboesophageal ganglion of the mated females. And it seems to support that the depletion of sex pheromone production is responsible for blocking of PBAN release from head. To investigate the mating factor from adult males, when extracts of reproductive organs of male were injected into hemocoel of virgin females evoking depletion of sex pheromone production as shown in mated female. The results suggest that a chemical substance(s) from the male reproductive organs could be responsible for the loss of sex pheromone biosynthesis in H. assulta.

T-SNARE phosphorylation regulates endocytosis in yeast.

Earlier we demonstrated that activation of a ceramide-activated protein phosphatase (CAPP) conferred normal growth and secretion to yeast lacking their complement of exocytic v-SNAREs (Snc1,2) or bearing a temperature-sensitive mutation in an exocytic t-SNARE (Sso2). CAPP activation led to Sso dephosphorylation and enhanced the assembly of t-SNAREs into functional complexes. Thus, exocytosis in yeast is modulated by t-SNARE phosphorylation. Here, we show that endocytic defects in cells lacking the v- and t-SNAREs involved in endocytosis are also rescued by CAPP activation. Yeast lacking the Tlg1 or Tlg2 t-SNAREs, the Snc v-SNAREs, or both, undergo endocytosis after phosphatase activation. CAPP activation correlated with restored uptake of FM4-64 to the vacuole, the uptake and degradation of the Ste2 receptor after mating factor treatment, and the dephosphorylation and assembly of Tlg1,2 into SNARE complexes. Activation of the phosphatase by treatment with C(2)-ceramide, VBM/ELO gene inactivation, or by the overexpression of SIT4 was sufficient to confer rescue. Finally, we found that mutation of single PKA sites in Tlg1 (Ser31 to Ala31) or Tlg2 (Ser90 to Ala90) was sufficient to restore endocytosis, but not exocytosis, to snc cells. These results suggest that endocytosis is also modulated by t-SNARE phosphorylation in vivo.

Adenylyl cyclase functions downstream of the Galpha protein Gpa1 and controls mating and pathogenicity of Cryptococcus neoformans.

The signaling molecule cyclic AMP (cAMP) is a ubiquitous second messenger that enables cells to detect and respond to extracellular signals. cAMP is generated by the enzyme adenylyl cyclase, which is activated or inhibited by the Galpha subunits of heterotrimeric G proteins in response to ligand-activated G-protein-coupled receptors. Here we identified the unique gene (CAC1) encoding adenylyl cyclase in the opportunistic fungal pathogen Cryptococcus neoformans. The CAC1 gene was disrupted by transformation and homologous recombination. In stark contrast to the situation for Saccharomyces cerevisiae, in which adenylyl cyclase is essential, C. neoformans cac1 mutant strains were viable and had no vegetative growth defect. Furthermore, cac1 mutants maintained the yeast-like morphology of wild-type cells, in contrast to the constitutively filamentous phenotype found upon the loss of adenylyl cyclase in another basidiomycete pathogen, Ustilago maydis. Like C. neoformans mutants lacking the Galpha protein Gpal, cac1 mutants were mating defective and failed to produce two inducible virulence factors: capsule and melanin. As a consequence, cac1 mutant strains were avirulent in animal models of cryptococcal meningitis. Reintroduction of the wild-type CAC1 gene or the addition of exogenous cAMP suppressed cac1 mutant phenotypes. Moreover, the overexpression of adenylyl cyclase restored mating and virulence factor production in gpal mutant strains. Physiological studies revealed that the Galpha protein Gpa1 and adenylyl cyclase controlled cAMP production in response to glucose, and no cAMP was detectable in extracts from cac1 or gpa1 mutant strains. These findings provide direct evidence that Gpal and adenylyl cyclase function in a conserved signal transduction pathway controlling cAMP production, hyphal differentiation, and virulence of this human fungal pathogen.

The yeast cell-wall salvage pathway

The integrity of the cell wall depends on the synthesis and correct assembly of its individual components. Several environmental factors, such as temperature up-shift, treatments with mating factors or with specific cell wall-perturbing drugs, or genetic factors, such as inactivation of cell wall-related genes (for example FKS1 or GAS1) can impair construction of the cell wall. As the cell wall is essential for preserving the osmotic integrity of the cell, several responses are triggered in response to cell-wall damage. This review focuses on the activation of salvage pathways that guarantee cell survival through remodeling of the extracellular matrix. These researches have useful implication for the study of similar pathways in human fungal pathogens, and for the evaluation of the efficacy of new antifungal drugs.

Secretory expression of human growth hormone inSaccharomyces cerevisiae using three different leader sequences

A recombinant human growth hormone (hGH) was expressed as a secretory product in the yeastSaccharomyces cerevisiae. Three different leader sequences derived from the mating factor α1 (MFα1), inulinase and invertase were used to direct the secretion of hGH into the extracellular medium. Among three leader sequences tested, the inulinase leader sequence was found to be the most efficient in the secretory expression of hGH. In contrast, no hGH was detected in the extracellular medium with the invertase leader sequence. After 48 h shake-flask culture, the yields of hGH secreted into the medium by the invertase, MFα1, inulinase and invertase leader sequences were approximately 0, 0.3 and 0.9 mg/L, respectively. The secretion efficiencies were also found to be 0, 3.8 and 13% for the invertase, MFα1 and inulinase leader sequences, respectively.

Isolation and characterization of WHI3, a size-control gene of Saccharomyces cerevisiae.

WHI3 is a gene affecting size control and cell cycle in the yeast Saccharomyces cerevisiae. The whi3 mutant has small cells, while extra doses of WHI3 produce large cells, and a large excess of WHI3 produces a lethal arrest in G1 phase. WHI3 seems to be a dose-dependent inhibitor of Start. Whi3 and its partially redundant homolog Whi4 have an RNA-binding domain, and mutagenesis experiments indicate that this RNA-binding domain is essential for Whi3 function. CLN3-1 whi3 cells are extremely small, nearly sterile, and largely nonresponsive to mating factor. Fertility is restored by deletion of CLN2, suggesting that whi3 cells may have abnormally high levels of CLN2 function.

The ste3 pheromone receptor gene of Pneumocystis carinii is surrounded by a cluster of signal transduction genes.

Although the clinical aspects of Pneumocystis carinii pneumonia are well characterized, the basic biology of the causative organism is poorly understood. Most proposed life cycles of P. carinii include both asexual and sexual replicative cycles. The two most prominent morphological forms are a trophic form, thought to undergo asexual replication by binary fission, and a cystic form or ascus containing intracystic bodies or ascospores, the products of sexual replication. To facilitate the Pneumocystis genome project, a P. carinii f. sp. carinii genomic cosmid library and an additional lambda cDNA library were generated. A partial expressed sequence tag database, created as part of the genome project, revealed the transcription of meiosis-specific genes and other genes related to sexual reproduction. The ortholog of Ste3, an a-factor pheromone receptor, was cloned and genes surrounding the ste3 locus were examined. Clustered around the ste3 gene are genes encoding elements functional in the pheromone response signal transduction cascade of model fungal organisms. These include the Ste20 protein kinase, the Ste12 homoeodomain transcriptional regulator, a potential pheromone mating factor, and other DNA-binding proteins. The genomic organization of the ste3 locus bears significant similarity to that of the mating locus recently described in Cryptococcus neoformans. The P. carinii genome contains much of the genetic machinery necessary for pheromone responsiveness, and these data support the existence of a sexual replication cycle.

The yeast cell-wall salvage pathway

The integrity of the cell wall depends on the synthesis and correct assembly of its individual components. Several environmental factors, such as temperature up-shift, treatments with mating factors or with specific cell wall-perturbing drugs, or genetic factors, such as inactivation of cell wall-related genes (for example FKS1 or GAS1) can impair construction of the cell wall. As the cell wall is essential for preserving the osmotic integrity of the cell, several responses are triggered in response to cell-wall damage. This review focuses on the activation of salvage pathways that guarantee cell survival through remodeling of the extracellular matrix. These researches have useful implication for the study of similar pathways in human fungal pathogens, and for the evaluation of the efficacy of new antifungal drugs.

Comparison of Three Signals for Secretory Expression of Recombinant Human Midkine in Pichia pastoris

The secretion signals of Saccharomyces cerevisiae alpha mating factor, human midkine itself, and Pichia pastoris acid phosphatase, were tried for the expression of human midkine under the control of the AOX1 gene promoter in P. pastoris. Approximately 28 mg/l, 1.5 mg/l, and 0.2 mg/l of midkine were secreted by using the a mating factor pre-pro-sequence, the midkine signal sequence, and the phosphatase signal sequence in flask cultures, respectively.

Post-translational modifications of heterologously expressed cholecystokinin in Saccharomyces cerevisiae

The vertebrate neuroendocrine peptide cholecystokinin (CCK) is subjected to numerous post-translational modifications upon maturation to bioactive CCK. However, the current knowledge of the proteolytic processing of proCCK, as reviewed here, is not complete. We have chosen Saccharomyces cerevisiae as a model to study these endoproteolytic processing events. Expression of proCCK as a fusion protein to the prepro leader peptide of f -mating factor directed the protein through the secretory pathway and resulted in nanomolar concentrations of secreted glycine-extended CCK. The CCK peptides showed many correlations to the known endoproteolytic processing products of proCCK in endocrine cells. Especially the processing to the abundant form, CCK-22, was investigated and it is suggested that a novel enzyme is responsible for its production. Thus, yeast may be used to study the proteolysis of proCCK with the aim to identify mammalian homologues involved in maturation of CCK.

Improved Secretion of Native Human Insulin-Like Growth Factor 1 from gas1 Mutant Saccharomyces cerevisiae Cells

We studied the secretion of recombinant human insulin-like growth factor 1 (rhIGF-1) from transformed yeast cells. The hIGF-1 gene was fused to the mating factor alpha prepro- leader sequence under the control of the constitutive ACT1 promoter. We found that the inactivation of the GAS1 gene in the host strain led to a supersecretory phenotype yielding a considerable increase, from 8 to 55 mg/liter, in rhIGF-1 production.

Coexpression of the Bacillus pumilus beta-xylosidase (xynB) gene with the Trichoderma reesei beta xylanase 2 (xyn2) gene in the yeast Saccharomyces cerevisiae.

The xynB gene encoding the Bacillus pumilus beta-xylosidase was expressed separately and jointly with the Trichoderma reesei beta-xylanase (xyn2) gene in the yeast Saccharomyces cerevisiae. Both genes were placed under the transcriptional control of the glucose-derepressible alcohol dehydrogenase 2 promoter (ADH2p) and terminator (ADH2T) sequences. The xynB gene was fused in frame to the yeast mating factor alpha1 secretion sequence (MFalpha1s) to effect secretion in S. cerevisiae. The fusion protein was designated Xlo1. Xlo1 produced in S. cerevisiae exhibited low affinity for xylobiose, but eventually hydrolyzed xylobiose and xylotriose to the monomeric constituent, D-xylose. Coproduction of Xyn2 and Xlo1 by S. cerevisiae led to a 25% increase in the amount of reducing sugars released from birchwood xylan compared to S. cerevisiae producing only the Xyn2 beta-xylanase. However, no D-xylose was produced from birchwood xylan, presumably due to very low Xlo1 beta-xylosidase activity and its low affinity for xylobiose.

Effects of mating factors on reproduction and sex-ratio of the predacious miteamblyseius zaheri yous. &amp; el-bor. (acari, phytoseiidae)

Three factors were examined to determine their effect on the reproduction and sex-ratio in the predacious miteAmblyseius zaheri Yousef & El-Borolossy in the laboratory. The factors studied included multiple matings, disturbed mating and age of mating females. Fecundity of females mated more than once was 3 times higher than in females mated only once. Artificial curtailment of copulation to 120, 60 and SO min resulted in reduction of fecundity and oviposition period. The Age of mated females has an influence also on fecundity, old females produce fewer eggs compared with young females under constant condition of abundant prey. All 3 factors studied have no influence on the sex-ratio of the progeny.