Cloning and sequence of a β-tubulin cDNA from Pneumocystis carinii: possible implications for drug therapy

This work describes the isolation and characterization of a full-length cDNA clone encoding beta-tubulin from the pathogen Pneumocystis carinii. P. carinii contains a single gene encoding beta-tubulin. The complete sequence of this cDNA has been determined and its inferred amino acid sequence compared with the beta-tubulins from other organisms. This analysis augments the data indicating that P. carinii should be classified as a fungal organism. Further comparisons between the P. carinii beta-tubulin and those of fungal beta-tubulins resistant to benomyl, a beta-tubulin-binding drug, indicate a difference which may be exploited in the development of a new drug therapy for P. carinii pneumonitis. These results suggest that, theoretically, a drug presently administered for treatment of nematode worm infections may be an effective agent against P. carinii, without being toxic to the mammalian host. This possibility is currently being investigated.     

Characterization of the beta-tubulin gene of Pneumocystis carinii.

To examine the potential of antimicrotubule drugs for treating Pneumocystis carinii infections, and to learn more about this unusual organism on a molecular level, we are studying its tubulin genes. A 0.3 kbp fragment of the P. carinii beta-tubulin gene was amplified by the polymerase chain reaction. Sequence analysis of this DNA revealed that P. carinii beta-tubulin is most closely related to those of the fungal molds. Consistent with these results, P. carinii growth in vitro was sensitive to the antifungal benzimidazoles benomyl and carbendazim.     

A comparison of the antigenic characteristics of rat and human Pneumocystis carinii by immunoblotting

The antigenic characteristics of rat Pneumocystis carinii obtained from infected lungs and grown in tissue culture were compared with the properties of human P. carinii obtained from the lungs of AIDS and non-AIDS patients by the immunoblotting technique, using different sources of antibody. Major immunoreactive bands of 45, 50, and 116 kd were found in both lung and tissue culture-derived rat P. carinii, suggesting the organism retains its antigenic characteristics in short-term culture. The principal immunoreactive bands in human P. carinii included a band of 40 kd, and to a lesser extent, a band of 66 kd; these antigens were found in the lungs of six and seven AIDS patients but in only one of eight non-AIDS patients with pneumocystosis. The rat and human P. carinii antigens reacted with sera from immunized rabbits, from rats with pneumocystosis and prolonged environmental exposure to the organism, from AIDS and non-AIDS P. carinii patients, and from healthy blood donors. Reactivity of these antigens could be removed by adsorption of antisera with P. carinii-infected lungs but not with normal lungs or lungs infected with bacteria and fungi. We conclude that rat and human P. carinii have shared, as well as species-specific, antigenic determinants, which should be useful for a variety of studies with this organism.     

Isolation and sequence analysis of a beta-tubulin gene from Aspergillus flavus and its use as a selectable marker

An altered beta-tubulin gene that confers resistance to benomyl [whose active ingredient is 2-(methoxycarbonylamino)benzimidazole (MBC)] was isolated from a DNA library of Aspergillus flavus and used as a selectable marker for transformation. The beta-tubulin gene was cloned into a plasmid vector containing the pyr-4 gene of Neurospora crassa, and transformants were selected either for uracil prototrophy or MBC resistance. Transformants selected for uracil prototrophy were of three phenotypic classes: sensitive, intermediate, and resistant to MBC. Transforming DNA appeared to integrate at several sites in the genome, with the more resistant phenotypes having more copies of the altered beta-tubulin gene than the sensitive and intermediate phenotypes. Transformants were also selected on medium containing MBC. The average frequency of transformation (1 to 3 transformants per micrograms of transforming DNA) was lower than that obtained by selection for uracil prototrophy, presumably because of failure to select transformants that contained few copies of the altered beta-tubulin gene. The sequence of the beta-tubulin gene was determined and compared with the published sequence of the benA gene of A. nidulans; the beta-tubulin gene was found to be highly conserved between the two Aspergillus species. Notable differences were that the beta-tubulin gene of A. flavus lacks intron 6 present in benA and has an additional leucine at position 148. This is the first gene sequence reported from an aflatoxin-producing fungus and adds to the growing body of knowledge of the beta-tubulin genes and their use as selectable markers for transformation of filamentous fungi.     

Mutations affecting assembly and stability of tubulin: evidence for a nonessential beta-tubulin in CHO cells.

Eight strains of Chinese hamster ovary (CHO) cells having an assembly-defective beta-tubulin were found among revertants of strain Cmd 4, a mutant with a conditional lethal mutation in a beta-tubulin gene (F. Cabral, M. E. Sobel, and M. M. Gottesman, Cell 20:29-36, 1980). The altered beta-tubulins in these strains have electrophoretically silent alterations or, in some cases, an increase or a decrease in apparent molecular weight based on their migration in two-dimensional gels. The identity of these variant proteins as beta-tubulin was confirmed by peptide mapping, which also revealed the loss of distinct methionine-containing peptides in the assembly-defective beta-tubulins of lower apparent molecular weight. The altered mobility of these beta-tubulin polypeptides was not the result of a posttranslational modification, since the altered species could be labeled in very short incubations with [35S]methionine and were found among in vitro-translated polypeptides by using purified mRNA. In at least one strain, an altered DNA restriction fragment could be demonstrated, suggesting that an alteration occurred in one of the structural genes for beta-tubulin. Assembly-defective beta-tubulin was unstable and turned over with a half-life of only 1 to 2 h in exponentially growing cells. This rapid degradation of a tubulin gene product resulted in approximately 30% lower steady-state levels of both alpha- and beta-tubulin yet did not affect the growth rate of the cells or the distribution of the microtubules as judged by immunofluorescence microscopy. These results argue that CHO cells possess a beta-tubulin gene product that is not essential for survival.     

Identification of antigens and antibodies specific for Pneumocystis carinii

To increase understanding of Pneumocystis carinii and its interaction with its hosts, Ag specific for rodent and human P. carinii were identified by the immunoblot method after PAGE of P. carinii organism extracts. The m.w. of the major Ag of rat P. carinii were 45,000, 110,000, and a broad band of 49,000 to 64,000, and of human P. carinii were 22,000, 24,000, and a broad band of 35,000 to 45,000 daltons. Human and rat pneumocystis were not antigenically identical. Specific antibodies against rat P. carinii Ag were found in 18 of 79 rats by the immunoblot method. Specific antibodies against human P. carinii Ag were found in 32 of 33 adult human sera, but in only 1 of 8 sera from infants and children. Specific antibodies were found in sera of 13 of the 14 adults with no history of P. carinii pneumonia, and all 19 patients with recently diagnosed P. carinii pneumonia, including 9 patients with P. carinii pneumonia associated with AIDS. The results of this study support previous suggestions that a large proportion of adults have been exposed to P. carinii and provide a basis for the further investigations of host-P. carinii interactions.     

Homologous expression of a mutated beta-tubulin gene does not confer benomyl resistance on Trichoderma virens

AIMS: To clone the beta-tubulins and to induce resistance to benzimidazoles in the biocontrol fungus Trichoderma virens through site-directed mutagenesis. METHODS AND RESULTS: Two beta-tubulin genes have been cloned using PCR amplification followed by the screening of a T. virens cDNA library. The full-length cDNA clones, coding for 445 and 446 amino acids, have been designated as T. virens tub1 and T. virens tub2. A sequence alignment of these two tubulins with tubulins from other filamentous fungi has shown the presence of some unique amino acid sequences not found in those positions in other beta-tubulins. Constitutive expression of the tub2 gene with a histidine to tyrosine substitution at position 6 (known to impart benomyl/methyl benzimadazol-2-yl carbamate resistance in other fungi), under the Pgpd promoter of Aspergillus nidulans, did not impart resistance to benomyl. CONCLUSIONS: The homologous expression of tub2 gene with a histidine to tyrosine mutation at position +6, which is known to impart benomyl tolerance in other fungi, does not impart resistance in T. virens. SIGNIFICANCE AND IMPACT OF THE STUDY: Unlike other Trichoderma spp., T. virens, has been difficult to mutate for benomyl tolerance. The present study, through site-directed mutagenesis, shows that a mutation known to impart benomyl tolerance in T. viride and other fungi does not impart resistance in this fungus. Understanding the mechanisms of this phenomenon will have a profound impact in plant-disease management, as many plant pathogenic fungi develop resistance to this group of fungicides forcing its withdrawal after a short period of use.     

Detection of Pneumocystis carinii in serum of AIDS patients with Pneumocystis pneumonia by the polymerase chain reaction.

Amplification of DNA by the polymerase chain reaction (PCR) offers a highly sensitive and specific method for detecting DNA sequences in biological samples. We applied this technology to develop an assay for the P. carinii dihydrofolate reductase (DHFR) gene. This assay was found to be sensitive enough to detect as little as 1 organism-'equivalent' of DHFR DNA. In rats with experimentally-induced P. carinii pneumonia, DHFR DNA amplification demonstrated the presence of pulmonary P. carinii 2 wk prior to the onset of histopathological changes. When rat serum was analyzed by PCR, serum P. carinii DNA was found in 5 of 14 experimental rats. Finally, P. carinii DNA was detected in the serum of 7 of 18 patients (39%) with AIDS and active P. carinii pneumonia. These results suggest that circulating serum P. carinii DNA can be detected frequently in the course of pulmonary infection and may represent a blood-borne phase of infection. The PCR detection of P. carinii DNA provides a useful tool to study the natural history of P. carinii infection and may offer a non-invasive diagnostic procedure in some patients with P. carinii pneumonia.     

禾谷镰孢菌β-微管蛋白基因克隆及其与多菌灵抗药性关系的分析

应用3对引物,从禾谷镰孢菌(Gibberella zeae)对多菌灵(MBC)的敏感菌株(MBC^R)和田间及室内诱导抗药性菌株(MBC^R)中扩增β-微管蛋白基因。该基因全长1631bp,包含3个内含子,编码447aa,与其他常见植物病原丝状真菌β-微管蛋白基因的氨基酸同源性达95．12％～99．30％。MBC^R和MBC^R菌株核苷酸序列分析表明,MBCR菌株未发生任何位点的突变,说明G．zeae对MBC的抗药性机制并非像其他丝状真菌一样由β-微管蛋白198位氨基酸突变所致。     

Pneumocystis carinii shows DNA homology with the ustomycetous red yeast fungi

Pneumocystis carinii causes life-threatening pneumonia in T-lymphocyte-immunodeficient subjects in transplant and oncology units or with acquired immune deficiency syndrome (AIDS). Recent DNA homology studies show P. carinii to be a fungus. To investigate the biology and epidemiology of this parasite further, we elected to determine for it a more precise taxonomic assignment within the fungal kingdom. We screened a wide range of organisms representing the major orders of fungi using DNA amplification and subsequently sequenced a portion of the mitochondrial gene encoding the large subunit ribosomal RNA. Our data show that the opportunistic pulmonary pathogen P. carinii is closely related to the ustomycetous red yeast fungi, a group which includes organisms that are extensively distributed throughout the environment and which release many widely dispersed airborne spores.     

Evidence from beta-tubulin phylogeny that microsporidia evolved from within the fungi

Microsporidia are obligate intracellular parasites that were thought to be an ancient eukaryotic lineage based on molecular phylogenies using ribosomal RNA and translation elongation factors. However, this ancient origin of microsporidia has been contested recently, as several other molecular phylogenies suggest that microsporidia are closely related to fungi. Most of the protein trees that place microsporidia with fungi are not well sampled, however, and it is impossible to resolve whether microsporidia evolved from a fungus or from a protistan relative of fungi. We have sequenced beta-tubulins from 3 microsporidia, 4 chytrid fungi, and 12 zygomycete fungi, expanding the representation of beta-tubulin to include all four fungal divisions and a wide diversity of microsporidia. In phylogenetic trees including these new sequences, the overall topology of the fungal beta-tubulins generally matched the expected relationships among the four fungal divisions, although the zygomycetes were polyphyletic in some analyses. The microsporidia consistently fell within this fungal diversification, and not as a sister group to fungi. Overall, beta-tubulin phylogeny suggests that microsporidia evolved from a fungus sometime after the divergence of chytrids. We also found that chytrid alpha- and beta-tubulins are much less divergent than are tubulins from other fungi or microsporidia. In trees in which the only fungal representatives were the chytrids, microsporidia still branched with fungi (i.e., with chytrids), suggesting that the affiliation between microsporidian and fungal tubulins is not an artifact of long-branch attraction.     

Proliferation patterns of latent Pneumocystis carinii in rat organs during progressive stages of immunosuppression

Pneumocystis carinii is known to proliferate mainly in the lung of an immunocompromised host. In AIDS and other immune disorders sporadic extrapulmonary presence of this organism has been documented. Occasionally, P. carinii does not appear to infect the lung. These observations have been based on the detection of P. carinii by conventional staining techniques. We have sought to determine the extent of these infections by the polymerase chain reaction (PCR) in a rat model. Harlan Sprague-Dawley rats weighing between 110 and 130 g were immunosuppressed with dexamethasone (1.2 mg/l) in drinking water. During progressive stages of immunosuppression 2 rats were sacrificed at 2, 3, 4 and 5 wk, and the lung, liver, kidney, spleen and bone marrow were taken. Sonicated crude extracts of the tissues were used as template DNA for the amplification of the dihydrofolate reductase (DHFR) gene of P. carinii. All the PCR products were analyzed by Southern hybridization with radiolabelled DHFR DNA. These analyses revealed a general trend of P. carinii proliferation first in bone marrow at 2 wk, followed by liver at 3 wk, and lung at 5 wk on immunosuppression. Kidney and spleen infections were infrequent. Although P. carinii appears to proliferate in the lung at later stages of immunosuppression, the degree of proliferation is several-fold greater than in extrapulmonary organs. The extrapulmonary proliferation of P. carinii, however small, may possibly suppress hematopoietic stem cell differentiation in bone marrow, and may also contribute to the pathology present in various organs.     

76 The Pneumocystis sam:smt, an atypical fungal enzyme protein

Pneumocystis , an opportunistic fungal protist, causes a type of pneumonia in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii and human-derived P. jiroveci contain a large number of sterols with C-24 alkyl groups. S-Adenosyl-L-methionine:sterol C-24 methyl transferase (SAM:SMT) is the enzyme that transfers methyl groups from SAM to the C-24 position of the sterol side chain. An alkyl group at the C-24 sterol side chain position appears to be essential for the organism to proliferate. Thus SAM:SMT, which is absent in mammals, is an attractive target for chemotherapeutic attack against the pathogen. The P. carinii erg6 gene that codes for SAM:SMT has been sequenced, cloned, and the protein expressed in E. coli . Since bacteria do not synthesize sterols, and do not have SAM:SMT, the P. carinii erg6 gene product expressed in E. coli would only transmethylate exogenously provided sterol substrates. The P. carinii recombinant SAM:SMT is unique because lanosterol, a central intermediate in sterol biosynthesis, is its preferred substrate for enzyme activity. Most SAM:SMT from other organisms do not bind lanosterol and prefer other sterol substrates produced from lanosterol. Furthermore, it appears that this unusual P. carinii SAM:SMT can also methylate cholesterol, which is readily scavenged from the lungs of its rat host. The recombinant enzyme protein is being purified by affinity chromatography techniques, which will be used to obtain definitive structural analyses of the sterol compounds formed by the enzyme reaction using different sterols substrates and allow detailed structural analysis of this unusual SAM:SMT enzyme protein.     

Characterisation of a beta-tubulin gene from the liver fluke, Fasciola hepatica

This study represents the first beta-tubulin sequence from a trematode parasite, namely, the liver fluke, Fasciola hepatica. PCR of genomic DNA showed that at least one beta-tubulin gene from F. hepatica contains no introns. A number of amino acids in the primary sequence of fluke tubulin are different from those described previously in various nematode species and the cestode, Echinococcus multilocularis. beta-Tubulin is an important target for benzimidazole anthelmintics, although (with the exception of triclabendazole) they show limited activity against F. hepatica. The amino acid differences in fluke beta-tubulin are discussed in relation to the selective toxicity of benzimidazoles against helminths and the mechanism of drug resistance.     

Amino-acid sequence data of beta-tubulin from Physarum polycephalum myxamoebae

Starting with 7.7 mg of a beta-tubulin isolated from myxamoebae of the slime mould Physarum polycephalum, 90% of the sequence has been determined by the Edman degradation of peptides generated by cyanogen bromide, trypsin and Staphylococcus aureus protease. Differences to other beta-tubulins are mainly conservative and spread evenly throughout the chain except for a high concentration at the C-terminus. The Physarum beta-tubulin shows most homology to Chlamydomonas beta-tubulin (90.5%) and least homology to yeast beta-tubulin (S. cerevisiae, 73.4%). Two tryptic peptides were isolated in approximately equal quantities which were identical except in one position (S/ALTVPELTQRMFDA) showing that at least two beta-tubulins are present in myxamoebae. However, since this was the only heterogeneity found, these beta-tubulins are probably very similar.