Identification and characterization of rad51 of Pneumocystis

Rad51, a eukaryotic homolog of RecA, is an important protein involved in DNA recombination and repair. We have characterized rad51 of Pneumocystis carinii and Pneumocystis murina. rad51 is a single copy gene that encodes a 1.2 kb mRNA, which contains an open reading frame encoding 343 amino acids. Rad51 from Pneumocystis showed high homology to those from yeast. ATP binding motifs GEFRTGKS and LLIVD, similar to those of Saccharomyces cerevisiae and Schizosaccharomyces pombe, are conserved in Pneumocystis Rad51. The recombinant protein when expressed in E. coli showed DNA-dependent ATPase activity. Since Rad51 is a key enzyme in DNA repair and recombination, it potentially plays an important role in the recombination process leading to antigenic variation and thereby resistance to host immune responses in Pneumocystis.     

硫腺苷甲硫氨酸合成酶基因的克隆及其在盐胁迫条件下的不同表达

硫腺苷甲硫氨酸作为甲基供体在转甲基反应中起到重要作用.为了解硫腺苷甲硫氨酸在盐地碱蓬(Suaedasalsa (L.)Pall)耐盐中的作用,我们对可能编码硫腺苷甲硫氨酸合成酶的基因(SsSAMS2)进行了分析.该基因在经400 mmol/L NaCl处理的盐地碱蓬地上部分的λ-Zap cDNA文库中克隆到,其插入片段全长1 531 bp,包含一个395个氨基酸的开放阅读框架,该基因推断的分子量约为43 kD.SsSAMS2与长春花(Catharanthus roseus)的SAMS2在氨基酸水平上的一致性为93%.Southern杂交显示,SsSAMS2在盐地碱蓬基因组中可能是两个拷贝.Northern分析显示硫腺苷甲硫氨酸合成酶基因受NaCl等胁迫的正调控.酶活性检测表明,NaCl胁迫条件下该酶活性增强.     

嗜热古菌S-腺苷甲硫氨酸合成酶的序列分析、基因克隆与异源表达

将Methanopyrus sp.SNP6进行功能基因组测序,获得S-腺苷甲硫氨酸合成酶基因(sam)序列,并进行序列分析。将sam基因扩增后连接至表达质粒p ET-28b(+),转化E.coli BL21(DE3),获得重组菌后进行诱导表达。生物信息学分析表明,sam基因编码蛋白的理论分子量为44 086.4Da,三级结构为同源四聚体,与其他相关古菌来源的S-腺苷甲硫氨酸合成酶的蛋白序列较保守。实验结果显示,构建的重组表达质粒p ET28b(+)-sam可在E.coli BL21(DE3)宿主菌中高水平表达。重组蛋白的分子量与预期值基本一致,部分为胞内可溶性表达,另一部分以包涵体形式存在。本研究首次实现了Methanopyrus sp.SNP6菌株S-腺苷甲硫氨酸合成酶的异源表达,为后期的蛋白纯化、酶学性质研究和酶促转化法生产S-腺苷甲硫氨酸奠定了理论基础。     

Pneumocystis murina MSG gene family and the structure of the locus associated with its transcription

Analysis of the Pneumocystis murina MSG gene family and expression-site locus showed that, as in Pneumocystis carinii, P. murina MSG genes are arranged in head-to-tail tandem arrays located on multiple chromosomes, and that a variety of MSG genes can reside at the unique P. murina expression site. Located between the P. murina expression site and attached MSG gene is a block of 132 basepairs that is also present at the beginning of MSG genes that are not at the expression site. The center of this sequence block resembles the 28 basepair CRJE of P. carinii, but the block of conserved sequence in P. murina is nearly five times longer than in P. carinii, and much shorter than in P. wakefieldiae. These data indicate that the P. murina expression-site locus has a distinct structure.     

Molecular phylogeny of Pneumocystis based on 5.8S rRNA gene and the internal transcribed spacers of rRNA gene sequences

To clarify the phylogenetic relationships and species status of Pneumocystis, the 5.8S rRNA gene and the internal transcribed spacers (ITS, 1 and 2) of Pneumocystis rRNA derived from rat, gerbil and human were amplified, cloned and sequenced. The genetic distance matrix of six Pneumocystis species compared with other fungi like Taphrina and Saccharomyces indicated that the Pneumocystis genus contained multiple species including Pneumocystis from gerbil. The phylogenetic tree also showed that Pneumocystis from human and monkey formed one group and four rodent Pneumocystis formed another group. Among the four members, Pneumocystis wakefieldiae was most closely related to Pneumocystis murina and Pneumocystis carinii, and was least related to gerbil Pneumocystis.     

The multifunctional peptide synthetase performing the first step of penicillin biosynthesis in Penicillium chrysogenum is a 421,073 dalton protein similar to Bacillus brevis peptide antibiotic synthetases.

The nucleotide sequence of the Penicillium chrysogenum Oli13 acvA gene encoding delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine synthetase, which performs the first step in penicillin biosynthesis, has been determined. The acvA gene contains an open reading frame of 11,238 bp encoding a protein of 3746 amino acids with a predicted mol. wt of 421,073 dalton. Three domains within the protein of approximately 570 amino acids have between 38% and 43% identity with each other and share similarity with two antibiotic peptide synthetases from Bacillus brevis as well as two other enzymes capable of performing ATP-pyrophosphate exchange reactions. The acvA gene is located close to the pcbC gene encoding isopenicillin N synthetase, the enzyme for the second step of beta-lactam biosynthesis, and is transcribed in the opposite orientation to it. The intergenic region of 1107 bp from which the acvA and pcbC genes are divergently transcribed has also been sequenced.     

Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments yielded two additional isozymic peaks, I and III, thereby suggesting its heterodimeric nature. We envisage that the three isozymes in GA3-treated wheat aleurone layers are formed by the random dimerization of two classes of enzyme subunits. The two enzyme subunits which differ in their net charge could be the product of two genes of AdoMet synthetase (SAM1 and SAM2). Based on this assumption, we propose that a single isozyme I in water imbibed control wheat aleurones is the product of SAM1 gene of AdoMet synthetase. The occurrence of three isozymes in GA3-treated aleurones could be ascribed to the expression of an alternate gene of AdoMet synthetase (SAM2 gene).(ABSTRACT TRUNCATED AT 400 WORDS)     

S-adenosylmethionine and Pneumocystis carinii

We previously reported that S-adenosylmethionine (AdoMet), a key molecule in methylation reactions and polyamine biosynthesis, enhances axenic culture of the AIDS-associated opportunistic fungal pathogen Pneumocystis carinii. Here we report that AdoMet is absolutely required for continuous growth. Two transporters are present, one high affinity, K(m) = 4.5 microm, and one low affinity, K(m) = 333 microm. The physiologically relevant high affinity transporter has a pH optimum of 7.5 and no related natural compounds compete for uptake. Transport is 98% inhibited at 4 degrees C, 24% inhibited by 20 mm sodium azide, and 95% inhibited by the combination of 20 mm sodium azide and 1 mm salicylhydroxamic acid; thus transport is active and dependent on both a cytochrome chain and an alternative oxidase. In vitro, AdoMet is used at a rate of 1. 40 x 10(7) molecules cell(-1) min(-1). AdoMet synthetase activity was not detected by a sensitive radiolabel incorporation assay capable of detecting 0.1% of the activity in rat liver. In addition, the AdoMet plasma concentration of rats is inversely correlated with the number of P. carinii in the lungs. These findings demonstrate that P. carinii is an AdoMet auxotroph. The uptake and metabolism of this compound are rational chemotherapeutic targets.     

Antigenic conservation of primary structural regions of S-adenosylmethionine synthetase.

Although the physical and kinetic properties of S-adenosylmethionine (AdoMet) synthetases from different sources are quite different, it appears that these enzymes have structurally or antigenically conserved regions as demonstrated by studies with AdoMet synthetase specific antibodies. Polyclonal anti-human lymphocyte AdoMet synthetase crossreacted with enzyme from rat liver (beta isozyme), Escherichia coli and yeast. In addition, polyclonal anti-E. coli enzyme and antibodies to synthetic peptides copying several regions of the yeast enzyme reacted with the human gamma and rat beta isozymes. Antibodies to yeast SAM1 encoded protein residues 6-21, 87-113 and 87-124 inhibited the activity of human lymphocyte AdoMet synthetase, while antibodies to residues 272-287 had no effect on the enzyme activity. Our results suggest that these conserved regions may be important in enzyme activity.     

Functional cloning of a Dictyostelium discoideum cDNA encoding GMP synthetase

A functional cloning procedure has been used to recover a cDNA coding for the GMP synthetase of Dictyostelium discoideum. The enzyme is encoded by a single gene, which is actively transcribed during growth, but not during development. The open reading frame encodes a protein of 718 amino acids with a predicted molecular mass of 79.6 kDa. The Dictyostelium enzyme has extensive homology with the GMP synthetase of Escherichia coli and regional homology to other glutamine amidotransferases.     

The core element of the EcoRII methylase as defined by protease digestion and deletion analysis.

Binding of the EcoRII DNA methyltransferase to azacytosine-containing DNA protects the enzyme from digestion by proteases. The limit digest yields a product having a Mr on SDS-PAGE 20% less than the intact protein. The N terminus of the tryptic digestion product was sequenced and found to be missing the N terminal 82 amino acids. Under the conditions used unbound enzyme was digested to small peptides. Protection of the enzyme from protease digestion implies that the enzyme undergoes major conformational changes when bound to DNA. The trypsin sensitive region of the EcoRII methyltransferase occurs prior to the first constant region shared with other procaryotic DNA(cytosine-5)methyltransferases. To determine if this region played a role in substrate binding or specificity, N-terminal deletion mutants were studied. Deletion of 97 amino acids resulted in a decrease of enzyme activity. Further deletions caused a complete loss of activity. Enzyme deleted through amino acid 85 was purified and found to have the same specificity as wild type however there was an increase in Km for both S-adenosylmethionine (AdoMet) and DNA of 27 and 18 fold respectively. The N-terminus of the EcoRII methylase, although a variable region present in many procaryotic DNA(cytosine-5)methylases, plays no role in determining enzyme specificity, although it does contribute to the interaction with both AdoMet and DNA.     

Effects of Surfactant Protein-A on the Interaction of Pneumocystis murina with its Host at Different Stages of the Infection in Mice

ABSTRACT. We examined the effects of surfactant protein A (SP-A), a collectin, on the interaction of Pneumocystis murina with its host at the beginning, early to middle, and late stages of infection. Pneumocystis murina from SP-A wild-type (WT) mice inoculated intractracheally into WT mice (WT_S-WT_R) adhered well to alveolar macrophages, whereas organisms from SP-A knockout (KO) mice inoculated into KO mice (KO_S-KO_R) did not. Substitution of WT mice as the source of organisms (WT_S-KO_R) or recipient host macrophages (KO_S-WT_R) restored adherence to that found with WT_S-WT_R mice. In contrast, when immunosuppressed KO and WT mice were inoculated with P. murina from a homologous source (KO_S-KO_R, WT_S-WT_R) or heterologous source (WT_S-KO_R, KO_S-WT_R) and followed sequentially, WT_S-KO_R mice had the highest levels of infection at weeks 3 and 4; these mice also had the highest levels of the chemokine macrophage inflammatory protein-2 and neutrophils in lavage fluid at week 3. Surfactant protein-A administered to immunosuppressed KO_S-KO_R mice with Pneumocystis pneumonia for 8 wk as a therapeutic agent failed to lower the organism burden. We conclude that SP-A can correct the host immune defect in the beginning of P. murina infection, but not in the middle or late stages of the infection.     

Pulsatile growth hormone secretion decreases S-adenosylmethionine synthetase in rat liver

S-adenosylmethionine synthetase (AdoMet synthetase) is responsible for the synthesis of the major methyl donor S-adenosylmethionine. The AdoMet synthetase gene was identified by subtractive suppressive hybridization as being expressed at higher levels in the liver of rats continuously exposed to growth hormone (GH) than in rats intermittently exposed to the hormone. Further studies on the regulation of AdoMet synthetase showed that the activity and mRNA levels were higher in female than in male rats. Hypophysectomy increased AdoMet synthetase mRNA in both male and female rats. Combined thyroxine and cortisol treatment of hypophysectomized rats had no effect on AdoMet synthetase mRNA levels. Two daily injections of GH for 7 days, mimicking the male secretory pattern of GH, decreased AdoMet synthetase activity and mRNA levels. A continuous infusion of GH, mimicking the female secretory pattern of GH, had small or no effects on AdoMet synthetase activity and decreased the mRNA levels to a lesser degree than two daily injections. It is concluded that the lower AdoMet synthetase activity in male rats is due to an inhibitory effect of the male characteristic pulsatile secretory pattern of GH on AdoMet synthetase mRNA expression.     

IL-33 and M2a alveolar macrophages promote lung defense against the atypical fungal pathogen Pneumocystis murina

We have recently reported that mice deficient in the myeloid Src-family tyrosine kinases Hck, Fgr, and Lyn (Src triple knockout [TKO]) had augmented innate lung clearance of Pneumocystis murina that correlated with a higher ability of alveolar macrophages (AMs) from these mice to kill P. murina. In this article, we show that despite possessing enhanced killing, AMs from naive Src TKO mice did not demonstrate enhanced inflammatory responses to P. murina. We subsequently discovered that both AMs and lungs from P. murina-infected Src TKO mice expressed significantly greater levels of the M2a markers RELM-α and Arg1, and the M2a-associated chemokines CCL17 and CCL22 than did wild-type mice. IL-4 and IL-13, the primary cytokines that promote M2a polarization, were not differentially produced in the lungs between wild-type and Src TKO mice. P. murina infection in Src TKO mice resulted in enhanced lung production of the novel IL-1 family cytokine IL-33. Immunohistochemical analysis of IL-33 in lung tissue revealed localization predominantly in the nucleus of alveolar epithelial cells. We further demonstrate that experimental polarization of naive AMs to M2a resulted in more efficient killing of P. murina compared with untreated AMs, which was further enhanced by the addition of IL-33. Administration of IL-33 to C57BL/6 mice increased lung RELM-α and CCL17 levels, and enhanced clearance of P. murina, despite having no effect on the cellular composition of the lungs. Collectively, these results indicate that M2a AMs are potent effector cells against P. murina. Furthermore, enhancing M2a polarization may be an adjunctive therapy for the treatment of Pneumocystis.     

Differential splicing of Pneumocystis carinii f. sp. carinii inosine 5'-monophosphate dehydrogenase pre-mRNA

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme in guanine nucleotide metabolism that has drawn attention as a drug target in several organisms. Pneumocystis carinii f. sp. carinii IMPDH mRNA (GeneBank Accession No: U42442) previously identified from cultured organisms yielded a predicted amino acid sequence about 70 amino acids shorter at the amino terminus than IMPDH from other species. Recent research has shown that the amino terminal region is important for enzyme activity, suggesting that the previous putative P. carinii IMPDH might not represent full length, functional enzyme. To test this hypothesis, RT-PCR was performed with total RNA isolated from P. carinii f. sp. carinii. Three IMPDH splicing variants were found and splicing preference was observed: P. carinii isolated from infected rat lung contained primarily splicing variant one (introns two and four deleted), but organisms from spinner flask culture contained primarily splicing variant three (all four introns deleted). Importantly, splicing variant one (GeneBank Accession No: AF196975) contained an open reading frame for 529 amino acids, a size comparable to that of other eukaryotic IMPDH forms. The other variants contained the same open reading frame (454 amino acids) previously reported. Sequence analysis and complementation studies suggest variant one represents the full length, catalytically active form of P. carinii IMPDH. The differential splicing of the enzyme may reflect a mechanism by which the organism regulates the expression of IMPDH in response to environmental stresses.     

Pneumocystis carinii BCK1 functions in a mitogen-activated protein kinase cascade regulating fungal cell-wall assembly

Pneumocystis pneumonia remains the most common AIDS-defining opportunistic infection in people with HIV. The process by which Pneumocystis carinii constructs its cell wall is not well known, although recent studies reveal that molecules such as beta-1-3-glucan synthetase (GSC1) and environmental pH-responsive genes such as PHR1 are important for cell-wall integrity. In closely related fungi, a specific mitogen-activated protein kinase (MAPK) cascade regulates cell-wall assembly in response to elevated temperature. The upstream mitogen-activated protein kinase kinase kinase (MAPKKK, or MEKK), BCK1, is an essential component in this pathway for maintaining cell-wall integrity and preventing fungal cell lysis. We have identified a P. carinii MEKK gene and have expressed it in Saccharomyces cerevisiae to gain insights into its function. The P. carinii MEKK, PCBCK1, corrects the temperature-sensitive cell lysis defect of bck1Delta yeast. Further, at elevated temperature PCBCK1 restored the signaling defect in bck1Delta yeast to maintain expression of the temperature-inducible beta-1-3-glucan synthetase gene, FKS2. PCBCK1, as a functional kinase, is capable of autophosphorylation and substrate phosphorylation. Since glucan machinery is not present in mammals, a better understanding of this pathway in P. carinii might aid in the development of novel medications which interfere with the integrity of the Pneumocystis cell wall.