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1.
Med Mycol ; 39(1): 81-6, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11270411

ABSTRACT

A putative Candida albicans homologue of Saccharomyces cerevisiae MOT2 (modulator of transcription) has been cloned and analyzed. A cDNA fragment corresponding to a portion of S. cerevisiae MOT2 was used to isolate a similar C. albicans gene (CaMOT2). CaMOT2 is comprised of two exons of 50 bp and 1,714 bp, respectively, with a single 82 bp intron located near the 5' end of the gene. The gene encodes a protein (CaMot2p) with an estimated mass of 67 kDa. The 5' region of the gene shows sequence homology with S. cerevisiae MOT2, whereas no significant similarity was observed in the 3' region. Similarly, the N-terminal portion of C. albicans Mot2p exhibits approximately 80% homology with S. cerevisiae Mot2p, while no significant homology to any known protein was observed in the carboxy-terminal half of the C. albicans protein. The N-terminal portion of CaMot2p contains a cysteine-rich domain (amino acids 18-62). The distribution of the cysteine residues identifies CaMot2p as a zinc-finger protein. The data suggest two potential Zn-binding sites, similar to the arrangement found in S. cerevisiae. Reverse-transcriptase polymerase chain reaction was used to compare the level of CaMOT2 expression between C. albicans grown in vitro and growth during in vivo infection in the rat model of oral candidiasis. The results showed CaMOT2 is down-regulated during growth in the rat oral cavity compared to in vitro culture. Although the function of C. albicans MOT2 has not been determined, comparison to S. cerevisiae MOT2 suggests the gene product may act as a general negative regulator.


Subject(s)
Candida albicans/genetics , Fungal Proteins/genetics , Repressor Proteins , Saccharomyces cerevisiae Proteins , Amino Acid Sequence , Animals , Base Sequence , Candidiasis, Oral/microbiology , Cloning, Molecular , Culture Media , DNA, Fungal , Gene Expression Regulation, Fungal , Genes, Fungal , Humans , Molecular Sequence Data , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Saccharomyces cerevisiae/genetics , Sequence Homology, Amino Acid , Ubiquitin-Protein Ligases
2.
Microb Pathog ; 25(3): 121-9, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9790871

ABSTRACT

Candida albicans is an opportunistic fungal pathogen responsible for the largest percentage of fungal-mediated oral and oesophageal disease. In this regard, knowledge concerning patterns of gene expression during the establishment and/or maintenance of infection may be the key to the design of new strategies for treatment, as well as providing insight into pathogenesis. To address this issue, experiments were performed that utilized differential display to compare the spectrum of C. albicans genes expressed during oral infection versus growth in in vitroculture. Experimentally, the rat model of oral candidiasis served as the in vivo source. After initiation of infection and subsequent harvesting of C. albicans from the rat oral cavity, RNA was isolated, and used with a small number of primers in reverse-transcriptase polymerase chain reaction (RT-PCR) and differential display experiments. Fragments unique to in vivo samples were subcloned and sequenced. Southern blot analysis verified the origin of seven fragments as fromC. albicans. Additionally, specific RT-PCR confirmed that two of these fragments represented genes that were up-regulated during C. albicans in vivo growth in the rat model. Database searches indicated the fragments share homology with a member of the C. albicans agglutinin gene family and to a bacterial gene (gidB) possibly involved in cell division.


Subject(s)
Candida albicans/genetics , Candida albicans/pathogenicity , Candidiasis, Oral/microbiology , Genes, Fungal , Up-Regulation , Amino Acid Sequence , Animals , Blotting, Southern , Candida albicans/growth & development , Candida albicans/isolation & purification , DNA, Complementary/analysis , Disease Models, Animal , False Positive Reactions , Molecular Sequence Data , Oropharynx/microbiology , RNA, Fungal/analysis , RNA, Fungal/isolation & purification , RNA, Messenger/analysis , Rats , Reverse Transcriptase Polymerase Chain Reaction
3.
Antimicrob Agents Chemother ; 42(9): 2342-6, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9736560

ABSTRACT

The candidacidal activity of nitric oxide (NO) as delivered by a class of compounds termed diazeniumdiolates has been investigated. Diazeniumdiolates are stable agents capable of releasing NO in a biologically usable form at a predicted rate, and three such compounds were examined for activity. One compound, (Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (DETA-NO), proved to be most suitable for examining NO activity due to its relatively long half-life (20 h) and because of limited candidacidal activity of the uncomplexed DETA nucleophile. DETA-NO was active against six species of Candida for which the MICs necessary to inhibit 50% growth (MIC50s) ranged from 0.25 to 1.0 mg/ml. C. parapsilosis and C. krusei were the most susceptible to the compound. In addition to a determination of NO effects alone, the complex was utilized to investigate the synergistic potential of released NO in combination with ketoconazole, fluconazole, and miconazole. Activity was investigated in vitro against representative strains of Candida albicans, C. krusei, C. parapsilosis, C. tropicalis, C. glabrata, and C. dubliniensis. Determination of MIC50, MIC80 and MICs indicated that DETA-NO inhibits all strains tested, with strains of C. parapsilosis and C. krusei being consistently the most sensitive. The combination of DETA-NO with each azole was synergistic against all strains tested as measured by fractional inhibitory concentration indices that ranged from 0.1222 to 0.4583. The data suggest that DETA-NO or compounds with similar properties may be useful in the development of new therapeutic strategies for treatment of Candida infections.


Subject(s)
Antifungal Agents/pharmacology , Candida/drug effects , Triazenes/pharmacology , Drug Synergism , Fluconazole/pharmacology , Ketoconazole/pharmacology , Miconazole/pharmacology , Microbial Sensitivity Tests
4.
Infect Immun ; 65(2): 829-32, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9009352

ABSTRACT

Disruption of both alleles of the Candida albicans FAS2 gene abolishes the ability of the organism to establish infection in a murine model of systemic candidiasis. Within 72 h all mice inoculated with 10(6) CFU of the parental C. albicans strain had died. In contrast, all animals inoculated with the mutant strain CFD2 survived for the course of the experiment (21 days). Animals infected with either mutant strain CFD1 or CFD3, in which only one FAS2 allele was disrupted, also succumbed to infection, but mortality was not observed until 4 days postinfection and survivors remained for up to 20 days postinfection. The results demonstrate that FAS2 is required for successful C. albicans infection.


Subject(s)
Candida albicans/genetics , Candida albicans/pathogenicity , Candidiasis/etiology , Candidiasis/microbiology , Fatty Acid Synthases/genetics , Mutagenesis , Animals , Candida albicans/enzymology , Candidiasis/enzymology , Mice , Mice, Inbred BALB C , Virulence
5.
Microbiology (Reading) ; 142 ( Pt 9): 2509-14, 1996 Sep.
Article in English | MEDLINE | ID: mdl-8828218

ABSTRACT

The virulence of Candida albicans strains deficient in fatty acid synthase activity by virtue of disruption/deletion of the FAS2 gene was examined in a rat model of oropharyngeal candidiasis. The FAS2 alleles of C. albicans CAI4 (delta ura3::imm434/delta ura3::imm434) were sequentially disrupted with a cassette that included a portion of FAS2 from which a 984 bp fragment containing the FAS condensing reaction domain was deleted and replaced with hisG-URA3-hisG sequences. Verification of fatty acid synthase inactivation was obtained from assays of enzyme activity. Strains in which a single allele was disrupted (CFD1 and CFD3) exhibited an approximately 20% reduction in activity, when compared to wild-type. In addition, fatty acid synthase activity was abolished in a FAS2 null mutant strain (CFD2), and growth of CFD2 occurred only when the growth medium was supplemented with Tween 40 and certain fatty acids. Strain CFD2 was avirulent in the rat model, indicating that fatty acid synthase activity is required for C. albicans oropharyngeal infection. Strains with a single FAS2 allele disruption colonized the oral cavity, but the number of cells recovered from infected animals was approximately fivefold less than for the parental strain. The results suggest that FAS may be exploited as a possible target for the development of new antifungal agents.


Subject(s)
Candida albicans/genetics , Candida albicans/pathogenicity , Candidiasis, Oral/genetics , Fatty Acid Synthases/genetics , Alleles , Animals , Blotting, Southern , Candida albicans/growth & development , Cloning, Molecular , Culture Media , Fatty Acid Synthases/metabolism , Fatty Acids/metabolism , Mutagenesis, Insertional , Plasmids , Polysorbates/metabolism , Rats , Rats, Sprague-Dawley , Sequence Deletion
6.
Microbiology (Reading) ; 141 ( Pt 7): 1553-8, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7551023

ABSTRACT

The basis of cerulenin resistance of Candida albicans strains 4918-2 and 4918-10 has been investigated. Parasexual genetic analyses established that cerulenin resistance to concentrations of at least 5 micrograms ml-1 is dominant in both strains. The results also showed that strain 4918-2 is heterozygous for resistance, while the change from resistance to sensitivity of strain 4918-10 is reversible. Experiments to define the mechanism(s) responsible for resistance focused on cerulenin uptake and fatty-acid synthase activity. Cerulenin uptake by strains 4918-2 and 4918-10 was 24% of that of the wild-type (strain 4918). Uptake was restored in UV-induced cerulenin-sensitive segregants of strains 4918-2 and 4918-10, and varied from 63% to 200% of parental values. Fatty-acid synthase from strains 4918-2 and 4918-10 was resistant to cerulenin as judged by differences in the inactivation of the enzyme by the agent. However, inactivation kinetics of fatty-acid synthase of cerulenin-sensitive segregants did not revert to the parental inactivation profile. Further investigation showed that nine out of ten segregants were resistant to cerulenin at concentrations between 1 and 4 micrograms ml-1 while strain 4918 was sensitive to cerulenin at all concentrations tested. Thus, the results suggest that alteration of fatty-acid synthase and changes in permeability contribute to total cerulenin resistance of each strain.


Subject(s)
Candida albicans/genetics , Cerulenin/metabolism , Cerulenin/pharmacology , Fatty Acid Synthases/metabolism , Candida albicans/drug effects , Candida albicans/metabolism , Candida albicans/radiation effects , Cell Membrane Permeability , Drug Resistance, Microbial , Fatty Acid Synthases/antagonists & inhibitors , Fatty Acid Synthases/genetics , Heterozygote , Kinetics , Microbial Sensitivity Tests , Recombination, Genetic , Time Factors , Ultraviolet Rays
7.
Gene ; 156(1): 133-8, 1995 Apr 14.
Article in English | MEDLINE | ID: mdl-7737507

ABSTRACT

Candida albicans (Ca) FAS2, the gene encoding the alpha-subunit of fatty acid synthase (FAS), has been isolated and characterized. Saccharomyces cerevisiae (Sc) FAS2 was used as a probe to screen genomic libraries of Ca, strain 4918. Clones were obtained that contained all but the first 1 kb of the gene. The 5' end, as well as upstream sequences, were subsequently isolated by PCR. The Ca FAS2 gene is comprised of an open reading frame (ORF) of 5655 bp that is free of introns and encodes a 207, 587-Da protein of 1885 amino acids. The Ca FAS2 gene and the encoded polypeptide exhibit 70 and 67% homology with the Sc FAS2 gene and protein, respectively. The gene specifies a single transcript of approx. 6 kb, and transcript levels are regulated independently of morphogenesis. Chromosome analysis localized the gene to chromosome 3. In addition, no differences were noted when comparing the FAS2 sequence, that encompasses the cerulenin-binding domain of FAS, between strain 4918 and two derived cerulenin-resistant (CerR) mutants. Thus, alteration within this region is not responsible for the increased CerR of the mutant strains.


Subject(s)
Candida albicans/genetics , Fatty Acid Synthases/genetics , Genes, Fungal/genetics , Amino Acid Sequence , Base Sequence , Candida albicans/enzymology , Cerulenin/metabolism , Chromosome Mapping , Chromosomes, Fungal , Cloning, Molecular , Drug Resistance, Microbial/genetics , Fatty Acid Synthases/chemistry , Gene Expression Regulation, Fungal , Molecular Sequence Data , Morphogenesis , RNA, Fungal/analysis , RNA, Messenger/analysis , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
8.
FEMS Microbiol Lett ; 126(2): 177-80, 1995 Feb 15.
Article in English | MEDLINE | ID: mdl-7705609

ABSTRACT

The virulence of Candida albicans strain SC5413 and two isogenic derivatives have been investigated in a rat model of oropharyngeal candidiasis. The results demonstrate that both mutant strains are avirulent in this animal model while the parental strain readily initiates infection. Avirulence is not related to altered growth characteristics or the inability of the strains to undergo yeast-to-hyphal morphogenesis. The potential importance of nutritional sufficiency as a virulence factor as well as the possibility of utilizing such strains in the development of an in vitro expression technology system for Candida albicans is discussed.


Subject(s)
Candida albicans/genetics , Candidiasis, Oral/microbiology , Animals , Candida albicans/pathogenicity , Disease Models, Animal , Mutation , Rats , Rats, Sprague-Dawley , Virulence
9.
Gene ; 147(1): 119-24, 1994 Sep 15.
Article in English | MEDLINE | ID: mdl-8088535

ABSTRACT

The gene (FAS1) encoding the beta-subunit of fatty-acid synthase (FAS) of Candida albicans has been isolated and analyzed. The gene was isolated on the basis of homology to the Saccharomyces cerevisiae FAS1 gene. Sequence analysis showed that the gene contained an intron-free open reading frame of 6111 bp encoding a protein of 2037 amino acids (aa) (227 916 Da). C. albicans FAS1 and its product exhibit a high degree of overall sequence relatedness to their counterparts in S. cerevisiae, with identities of 68 and 63% at the nucleotide (nt) and aa level, respectively. In addition, the beta-subunits of both organisms contain the catalytic domains in an identical sequential order. Northern blots demonstrated that the gene encodes a single mRNA of approx. 6.1 kb, and that changes in transcript levels are not a prerequisite of the yeast-to-hyphal transition. Southern blot analysis of C. albicans chromosomes separated by pulsed-field gel electrophoresis showed that FAS1 resides on chromosome 5.


Subject(s)
Candida albicans/genetics , Fatty Acid Synthases/genetics , Genes, Fungal , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Cloning, Molecular , Gene Expression , Molecular Sequence Data , RNA, Fungal/genetics , RNA, Messenger/genetics , Sequence Alignment , Sequence Homology, Amino Acid
10.
J Med Vet Mycol ; 32(1): 13-20, 1994.
Article in English | MEDLINE | ID: mdl-8207619

ABSTRACT

Fatty acid synthase from three strains of Candida albicans (parental strain 4918, and two spontaneous cerulenin-resistant mutants, 4918-2 and 4918-10) has been purified and characterized. In all three cases the purification protocol included ammonium sulfate precipitation, fractionation with butyl-Toyopearl, differential centrifugation and sedimentation velocity centrifugation. Inclusion of protease inhibitors, aprotinin, leupeptin and pepstatin was a prerequisite to maximize recoveries. Polyacrylamide gel electrophoresis analysis demonstrated protocol efficacy and showed the apparent molecular mass of the two enzyme sub-units from each strain to be 195 kDa and 210 kDa. The Km (malonyl-CoA) and Vmax of each fatty acid synthase were similar. In contrast, inactivation kinetics of the respective enzymes in the presence of cerulenin showed enzyme activity from both mutants to differ significantly from the parent and from each other. Other experiments suggested in vivo cerulenin resistance of mutant strains is not solely attributable to enzyme alteration.


Subject(s)
Candida albicans/enzymology , Cerulenin/pharmacology , Fatty Acid Synthases/isolation & purification , Candida albicans/drug effects , Candida albicans/growth & development , Drug Resistance, Microbial , Fatty Acid Synthases/drug effects , Fatty Acid Synthases/metabolism , Mutation , Species Specificity
11.
J Med Vet Mycol ; 30(6): 421-32, 1992.
Article in English | MEDLINE | ID: mdl-1287161

ABSTRACT

Prolonged exposure of several Candida albicans strains to inhibitory concentrations of Cd, Cu, or Zn resulted in the appearance of resistant colonies at frequencies and with kinetics significantly different than expected based solely upon the predicted spontaneous mutation rate. Characteristics of the response included: (i) a delay usually of 4-10 days in the emergence of the first resistant colonies; (ii) continued accumulation of resistant colonies for a minimum of 21 days after initial exposure to selection; and (iii) final mutation frequencies ranging from 7.0 x 10(-6) to 9.8 x 10(-4). Further examination of the response of one of the strains to Cd, demonstrated that pretreatment with either ultraviolet irradiation or hydroxyurea resulted in approximately a 10-fold increase in the number of resistant colonies detected. While the distribution and identity of colony phenotypes was altered for all strains after exposure to the heavy metals, no specific morphologies could be correlated to development of resistance.


Subject(s)
Cadmium Compounds , Candida albicans/drug effects , Metals/pharmacology , Cadmium/pharmacology , Candida albicans/growth & development , Candida albicans/radiation effects , Copper/pharmacology , Copper Sulfate , Culture Media , Hydroxyurea/pharmacology , Kinetics , Phenotype , Sulfates/pharmacology , Ultraviolet Rays , Zinc/pharmacology , Zinc Sulfate
12.
J Med Vet Mycol ; 29(3): 145-55, 1991.
Article in English | MEDLINE | ID: mdl-1890561

ABSTRACT

The effect of media and temperature of incubation on colony phenotype of Candida albicans strain 4918, and two relatively avirulent mutant strains, designated 4918-2 and 4918-10, has been investigated. In addition, the strains were characterized on the basis of morphotyping pattern. Colony phenotypes were determined for cultures grown on either Lee's medium supplemented with arginine and zinc, or M63 medium supplemented with casamino acids. Incubation was at either 24 or 37 degrees C for 7 days. The results demonstrated that the predominant colony phenotype observed at 24 degrees C was different from that at 37 degrees C for all three strains, irrespective of the medium. While the growth medium influenced the specific colony phenotypes observed, as well as their categorical distribution, no significant medium effect on switching frequency was apparent. The switching repertoire of strain 4918-10 was consistently more varied than either the parental strain or 4918-2 under the conditions examined. However, categorization of the colony phenotypes shown by the three strains suggested that the pattern exhibited by strain 4918-2 was distinct from that of the other two strains. In addition, individual primary colonies of each phenotype observed were clonally plated in order to examine further the switching frequencies. The results established that all three strains were capable of high frequency switching. Other experiments demonstrated that morphotypes of all three strains were different from one another as expected from the differences in their virulence reported previously.


Subject(s)
Candida albicans/pathogenicity , Genes, Fungal , Amino Acids , Arginine , Candida albicans/cytology , Candida albicans/genetics , Candida albicans/growth & development , Colony Count, Microbial , Culture Media , DNA, Fungal/genetics , Phenotype , Temperature , Virulence , Zinc
13.
Infect Immun ; 58(7): 2061-6, 1990 Jul.
Article in English | MEDLINE | ID: mdl-2194957

ABSTRACT

We have previously reported the characteristics of mannans isolated from the yeast forms of two relatively avirulent Candida albicans strains, designated 4918-2 and 4918-10. Investigations have been expanded to include an analysis of mannans from the hyphal form of these strains as well as from the hyphal form of the parental strain, 4918. After extraction, mannans were further purified by high-pressure liquid chromatography on a Bio-gel TSK DEAE-5-PW column. Subsequent to either mild acid hydrolysis, alkali hydrolysis, or acetylation followed by acetolysis, the resulting products were fractionated by high-pressure liquid chromatography on an Aminex HPX-42A column. The results of acid hydrolysis showed only minor quantitative differences in the products released from each strain, with mannose constituting the vast majority of product liberated. The profiles of mannooligosaccharides obtained from either alkali hydrolysis or acetolysis for strain 4918-2 showed distinct quantitative differences compared with profiles of the other two strains. Finally, a general characteristic noted is a decrease in the average chain length of mannooligosaccharides in hyphal mannans compared with the yeast counterpart.


Subject(s)
Candida albicans/analysis , Mannans/analysis , Acetylation , Candida albicans/genetics , Candida albicans/pathogenicity , Chromatography, High Pressure Liquid , Hydrochloric Acid , Hydrolysis , Mannans/isolation & purification , Mutation , Sodium Hydroxide
14.
Infect Immun ; 57(2): 413-9, 1989 Feb.
Article in English | MEDLINE | ID: mdl-2643567

ABSTRACT

We previously reported the isolation of two cerulenin-resistant mutant strains of Candida albicans 4918 that differ in adherence properties and are less virulent than the parental strain. In addition, biochemical characterization demonstrated significant differences in both protein and polysaccharide composition of cell wall material between the mutant and wild-type strains. These observations prompted studies concerning the chemical structure of mannans in these strains. After extraction and subsequent purification by ion-exchange chromatography, mannan fractions were subjected to either mild acid hydrolysis, alkali hydrolysis, or acetylation followed by acetolysis. Acid- and alkali-modified mannans were studied by proton magnetic resonance spectroscopy, and released products were analyzed by high-performance liquid chromatography on an Aminex HPX-42A column. The results demonstrated quantitative and qualitative differences between mannooligosaccharides of the wild-type and mutant strains in the identity of released oligosaccharides as well as in linkage of the oligosaccharides to the protein backbone.


Subject(s)
Candida albicans/pathogenicity , Mannans/analysis , Mutation , Acetylation , Candida albicans/analysis , Candida albicans/genetics , Chromatography, High Pressure Liquid , Hydrogen-Ion Concentration , Hydrolysis , Magnetic Resonance Spectroscopy , Mannans/genetics , Mannose/analysis , Virulence
15.
Can J Microbiol ; 33(6): 546-50, 1987 Jun.
Article in English | MEDLINE | ID: mdl-2957042

ABSTRACT

Experiments were conducted to gain insight concerning the mechanism(s) whereby cerulenin and sodium butyrate affect chitin synthesis in Candida albicans. In vitro studies with isolated membrane-bound chitin synthase from C. albicans, strain 4918, showed that neither agent affected the level of either unactivated or trypsin-activated enzyme activity. Subsequent studies utilizing protoplasts revealed that early in the cell wall regeneration process, cells treated with cerulenin or butyrate synthesized chitin at a rate equal to untreated controls, as measured by the incorporation of [3H]-N-acetylglucosamine (GlcNAc) into acid-alkali insoluble material. However, after 40 min of incubation, the incorporation of [3H]GlcNAc into chitin is reduced in cells treated with either agent. On the other hand, samples taken during the same time intervals and analyzed by flow cytometry suggested that the amount of chitin synthesis in treated and untreated cells was identical. A marked decrease in fluorescence was observed in similar experiments using polyoxin D, a direct inhibitor of chitin synthase activity. Experiments that measured uptake of [3H]GlcNAc into both whole cells and protoplasts demonstrated that cerulenin and butyrate had no effect on the transport of the chitin precursor.


Subject(s)
Antifungal Agents/pharmacology , Butyrates/pharmacology , Candida albicans/drug effects , Cerulenin/pharmacology , Chitin Synthase/metabolism , Chitin/biosynthesis , Glucosyltransferases/metabolism , Butyric Acid , Candida albicans/enzymology , Candida albicans/metabolism , Flow Cytometry , Protoplasts/drug effects , Protoplasts/enzymology , Protoplasts/metabolism , Pyrimidine Nucleosides/pharmacology
16.
J Virol ; 60(2): 645-52, 1986 Nov.
Article in English | MEDLINE | ID: mdl-3021997

ABSTRACT

The transforming (focus forming) activity of defined cloned DNA fragments from human cytomegalovirus Towne and AD169 was carried out in immortalized rodent cells. The frequency of focus formation in NIH 3T3 cells by Towne XbaI fragment E was 80- to 100-fold higher than that observed with Towne XbaI fragments AO, O, C, or carrier DNA alone but was similar to that observed with pCM4127, a transforming fragment from HCMV AD169 (J. A. Nelson, B. Fleckenstein, D. A. Galloway, and J. K. McDougall, J. Virol. 43:83-91, 1982; J. A. Nelson, B. Fleckenstein, G. Jahn, D. A. Galloway, and J. K. McDougall, J. Virol. 49:109-115, 1984). Foci were first detected in Towne XbaI fragment E-transfected NIH 3T3 cells at 5 to 6 weeks posttransfection, whereas foci were detected at 2 to 3 weeks after transfection with AD169 pCM4127. Digestion of Towne XbaI fragment E with BamHI did not significantly reduce its focus-forming activity. When BamHI subclones of Towne XbaI fragment E were assayed individually for focus formation in NIH 3T3 and Rat-2 cells, transforming activity was localized within each terminal fragment (EJ and EM). Foci induced by EJ or EM DNA alone were smaller compared with those induced by Towne XbaI fragment E. Isolated focal lines exhibited growth in soft agar and were tumorigenic in immunocompetent syngeneic animals. High-molecular-weight DNAs from transformed and tumor-derived lines were analyzed by Southern blot hybridization with intact EM and a 1.5-kilobase subfragment lacking cell-related sequences. Virus-specific EM sequences were detected at less than one copy per cell in Towne XbaI fragment E-transformed NIH 3T3 cells and at multiple copies in rat tumor-derived cell lines. In contrast, virus-specific EJ sequences were barely detected in EJ-transformed and tumor-derived lines with intact EJ as probe.


Subject(s)
Cell Transformation, Neoplastic , Cell Transformation, Viral , Cytomegalovirus/genetics , DNA, Viral/genetics , Deoxyribonucleases, Type II Site-Specific , Animals , Base Sequence , Cell Line , DNA Restriction Enzymes , Humans , Mice , Rats , Transfection
17.
J Med Vet Mycol ; 24(2): 127-31, 1986 Apr.
Article in English | MEDLINE | ID: mdl-3522840

ABSTRACT

A spontaneous, cerulenin-resistant mutant of Candida albicans (strain 4918-10) was found to adhere less readily to human vaginal mucosal cells in vitro than a wild type C. albicans (strain 4918). In a murine model of vaginal infection, strain 4918-10 was found to be less virulent than wild type C. albicans, i.e., the infection rate caused by 4918-10 was only 31% of that observed with wild type, 4918. A chitin-soluble extract (CSE) prepared from 4918 blocked attachment of yeast cells to human vaginal epithelial cells, while CSE from 4918-10 did not significantly reduce the attachment of yeasts to vaginal cells. Both 4918 and 4918-10 produced hyphae in vitro and in vivo, were negative for proteinase production and grew equally well at 28 degrees C and 37 degrees C. The data suggest that adherence to vaginal mucosa may be an important determinant in the pathogenesis of vaginal infection caused by C. albicans.


Subject(s)
Candida albicans/pathogenicity , Candidiasis, Vulvovaginal/microbiology , Vagina/microbiology , Adhesiveness , Animals , Candida albicans/genetics , Candida albicans/physiology , Cells, Cultured , Cerulenin/pharmacology , Chitin/pharmacology , Epithelium/microbiology , Female , Humans , Mice , Mucous Membrane/microbiology , Mutation , Virulence
18.
Infect Immun ; 51(1): 102-9, 1986 Jan.
Article in English | MEDLINE | ID: mdl-3510162

ABSTRACT

Cerulenin, an inhibitor of fatty acid biosynthesis, has been used to study the role of the plasma membrane in germination of Candida albicans. To further elucidate this association, spontaneous, cerulenin-resistant mutants of C. albicans were isolated. Two of the mutants, 4918-2 and 4918-10, were compared biochemically with wild-type cells (4918). All strains grew equally well at 37 degrees C and synthesized fatty acids at comparable rates in the absence of the drug. In the presence of cerulenin, wild-type cells did not proceed through a logarithmic growth stage and exhibited a significantly impaired ability to incorporate [3H]acetate into newly synthesized lipid material. All strains were examined ultrastructurally. Alterations were observed in the membranous structures of cerulenin-treated wild-type cells. Such changes were not observed in cerulenin-treated mutant strains. Further examination of mutant strains revealed differences in cell wall protein and polysaccharide compositions when compared with those of wild-type cells. These apparent alterations in cell surface components may be correlated with the reduced abilities of mutant strains to adhere, in vitro, to mammalian cells.


Subject(s)
Antifungal Agents/pharmacology , Candida albicans/genetics , Cerulenin/pharmacology , Adhesiveness , Candida albicans/drug effects , Candida albicans/pathogenicity , Candida albicans/ultrastructure , Cell Membrane/ultrastructure , Cell Wall/ultrastructure , Ceruletide , Drug Resistance, Microbial , Fungal Proteins/metabolism , Lipids/biosynthesis , Mitochondria/ultrastructure , Polysaccharides/metabolism
19.
J Infect Dis ; 152(4): 710-5, 1985 Oct.
Article in English | MEDLINE | ID: mdl-3900233

ABSTRACT

Two spontaneous cerulenin-resistant mutants of Candida albicans, 4918-2 and 4918-10, were unable to adhere in vitro in fibrin-platelet clots. Because in vitro adherence correlates well with colonization of nonbacterial thrombotic endocarditis on traumatized valvular endocardium, 50% infectious dose studies were performed with a rabbit model of endocarditis. Wild-type C. albicans required 10(3.6) +/- 0.12 cfu in comparison with 10(5.73) +/- 0.31 and 10(7.3) +/- 0.21 cfu for mutants 4918-2 and 4918-10, respectively. The relative avirulence of mutant strains in producing endocarditis was not attributed to accelerated clearance of these strains from the bloodstream. In fact, clearance of wild-type and mutant strains was almost identical. In the same animals renal candidiasis was observed with all strains of C. albicans, although the number of cfu per gram of kidney was higher after infection with wild-type C. albicans. Thus, strains of C. albicans with reduced ability to adhere in vitro to a fibrin-platelet matrix are relatively avirulent in the rabbit endocarditis model.


Subject(s)
Candida albicans/pathogenicity , Endocarditis/etiology , Adhesiveness , Animals , Candida albicans/immunology , Candidiasis/etiology , Cerulenin/pharmacology , Female , Immunization , Kidney Diseases/etiology , Macrophages/immunology , Mutation , Phagocytosis , Rabbits , Virulence
20.
J Bacteriol ; 162(1): 438-40, 1985 Apr.
Article in English | MEDLINE | ID: mdl-3980442

ABSTRACT

A dispersed repetitive DNA sequence has been identified within the genome of the fungus Mucor racemosus. Recombinant phage clones, as well as a plasmid harboring the sequence, have been isolated. Examination of cloned fragments comprising part of the repetitive sequence has led to a partial characterization of the element. The sequence has been detected in other Mucor species, and although the apparent number and chromosomal position of the repetitive sequence vary from strain to strain, it is clear that at least portions of the element have been conserved.


Subject(s)
DNA, Fungal/analysis , Mucor/genetics , Repetitive Sequences, Nucleic Acid
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