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Biomedical subjects

R D Cannon

Publications and source records attributed to R D Cannon.

At least 37 records · Page 2Linked to original sources
PubMed1996

Multiple efflux mechanisms are involved in Candida albicans fluconazole resistance.

Fluconazole-susceptible Candida albicans strains accumulated [3H]fluconazole at a rate of approximately 2 pmol/min per 10(9) cells. Fluconazole accumulation was not affected by the pretreatment of cells with sodium azide or with 2-deoxyglucose. The rate of fluconazole accumulation became saturated at high fluconazole concentrations and was not affected by the addition of ketoconazole, and there was no fluconazole accumulation in cells incubated at 4 degrees C. A fluconazole-resistant mutant of C. albicans SGY-243 was isolated following growth enrichment in fluconazole-containing medium. Cells of the mutant strain, designated FR2, showed a reduced rate of fluconazole accumulation compared with SGY-243 and were not resistant to other azole antifungal agents. The rates of fluconazole accumulation by C. albicans FR2 and the other azole-resistant strains, B59630, AD, and KB, were increased in the presence of sodium azide, suggesting that fluconazole resistance in these strains may be associated with an energy-dependent drug efflux. Fluconazole-resistant C. albicans strains all contained elevated amounts (2- to 17-fold) of mRNA encoding Cdr1, and an ATP-binding cassette-type transporter. In addition, C. albicans FR2 also contained increased amounts of mRNA encoding Benr, a major facilitator superfamily transporter. These results suggest that fluconazole enters C. albicans cells by facilitated diffusion and that fluconazole resistance may involve energy-dependent drug efflux associated with increased expression of Benr and/or Cdr1.

Antifungal Agents↗
PubMed1995

Interactions of Candida albicans with bacteria and salivary molecules in oral biofilms.

The yeast Candida albicans coaggregates with a variety of streptococcal species, an interaction that may promote oral colonization by yeast cells. C. albicans and Candida tropicalis are the yeasts most frequently isolated from the human oral cavity and our data demonstrate that both these species bind to Streptococcus gordonii NCTC 7869 while two other Candida species (Candida krusei and Candida kefyr) do not. Adherence of C. albicans was greatest when the yeast had been grown at 30 degrees C to mid-exponential growth phase. For 21 strains of C. albicans there was a positive correlation between the ability to adhere to S. gordonii and adherence to experimental salivary pellicle. Whole saliva either stimulated or slightly inhibited adherence of C. albicans to S. gordonii depending on the streptococcal growth conditions. The results suggest that the major salivary adhesins and coaggregation adhesins of C. albicans are co-expressed.

Bacterial Adhesion↗
PubMed1995

Adherence of Candida albicans to human salivary components adsorbed to hydroxylapatite.

Colonization of the oral cavity by Candida albicans involves adherence of yeast cells to oral surfaces. An assay was developed to measure the attachment of C. albicans cells, metabolically labelled with [35S]methionine, to saliva-coated hydroxylapatite (SHA) beads--a model for the tooth surface. Using this assay approximately 1.26 x 10(6) C. albicans cells (50.5% of input cells) attached to the SHA beads (12 mg). Different strains of C. albicans adhered to varying degrees to SHA beads, but in general adherence was promoted by growth of cells at 28 degrees C and by starvation of cells for glucose. Proteins in human whole, or parotid, saliva samples were fractionated by gel filtration (Sephacryl S-200 column) and fractions were adsorbed to hydroxylapatite beads. Fractions that contained proline-rich proteins or statherin promoted attachment of C. albicans ATCC 10261 cells. Neuraminidase treatment of SHA beads, but not of cells, significantly increased yeast cell binding to the SHA beads. Neuraminidase activity in the oral cavity may unmask glycoprotein receptors involved in the adhesion of C. albicans to saliva-coated surfaces.

Adsorption↗
PubMed1995

Physiological traits associated with success of Candida albicans strains as commensal colonizers and pathogens.

DNA fingerprinting with the moderately repetitive sequence Ca3 has repeatedly identified groups of genetically similar strains of Candida albicans that are more frequently isolated than other groups of strains from human hosts in a geographical locale. Members of these groups are found in approximately 30% of healthy individuals and in up to 70% of patients suffering from candidiasis. The high prevalence of these strains implies that they are more successful in colonizing human hosts and in causing disease than other strains (J. Schmid, Clin. Adv. Treatment Fungal Infect. 4(6):12-16, 1993). In the present study, we have compared one such group of highly prevalent strains with other strains from the same locale to identify physiological traits a larger number of chemicals than other strains in a resistogram assay. When resistance to individual chemicals used in the resistogram assay was analyzed, strains from the group of highly prevalent strains were significantly more often resistant to boric acid, cetrimide, chlorhexidine, 5-fluorocytosine, and high sodium chloride concentrations than other strains. Strains from the group of highly prevalent strains also adhered significantly (1.5 times) better to saliva-coated surfaces than did other strains. Because members of highly prevalent groups of strains are the most common infectious agents in candidiasis, these physiological traits may be involved in determining not only the success of C. albicans in colonizing human hosts in general but also its ability to cause disease. Sodium chloride resistance and increased adherence were also associated with infectious isolates outside the group of highly prevalent strains, indicating that they may be of particular importance in pathogenesis.

Anti-Infective Agents↗
PubMed1995

Oral Candida: clearance, colonization, or candidiasis?

Candida albicans is frequently isolated from the human mouth, yet few carriers develop clinical signs of candidiasis. Oral candidiasis presents clinically in many forms. This reflects the ability of the yeast to colonize different oral surfaces and the variety of factors which predispose the host to Candida colonization and subsequent infection. Colonization of the oral cavity appears to be facilitated by several specific adherence interactions between C. albicans and oral surfaces which enable the yeast to resist host clearance mechanisms. Thus, Candida has been shown to adhere to complement receptors, various extracellular matrix proteins, and specific sugar residues displayed on host or bacterial surfaces in the oral cavity. Oral candidiasis results from yeast overgrowth and penetration of the oral tissues when the host's physical and immunological defenses have been undermined. Tissue invasion may be assisted by secreted hydrolytic enzymes, hyphal formation, and contact sensing. While these and other phenotypic characteristics may endow certain Candida species or strains with a competitive advantage in the oral cavity, it is the host's immune competence that ultimately determines whether clearance, colonization, or candidiasis occurs.

Antifungal Agents↗
PubMed1994

Purification and characterization of two forms of N-acetylglucosaminidase from Candida albicans showing widely different outer chain glycosylation.

Two forms of N-acetylglucosaminidase were purified to homogeneity by ion exchange (TSK DEAE-3SW, Aquapore CX-300) and gel filtration (TSK G4000 SW) HPLC of Candida albicans ATCC 10261 culture filtrates. Synthesis and secretion of N-acetylglucosaminidase were induced by incubating starved yeast cells at 37 degrees C in medium containing N-acetylglucosamine (GlcNAc). The form of the enzyme depended on the cell growth and starvation conditions before GlcNAc induction. N-Acetylglucosaminidase A (32% total carbohydrate, M(r) 85,000 subunit) was isolated from cells grown in glucose/salts/biotin medium, and N-acetylglucosaminidase B (56% carbohydrate, M(r) 132,000 subunit) was isolated from cells grown in yeast extract/peptone/dextrose. The estimated relative molecular masses of the native enzymes, based on Sephacryl S-300 gel filtration were: A form, 350,000; B form, 600,000; A and B forms after endoglycosidase H (endo H) treatment, 180,000. The purified enzymes migrated on SDS polyacrylamide gels as heterogeneous glycoproteins of M(r) centred at approximately 100,000 (A) and approximately 150,000 (B) but were reduced to a single 58,000 band after denaturation with SDS and cleavage of asparagine-linked sidechains by endo H. When the native glycoproteins were treated with endo H, both enzyme forms had three oligosaccharide sidechains of M(r) approximately 3000 that were endo H resistant. Therefore the difference in the size of N-acetylglucosaminidase A and B was due to variations in outer chain glycosylation of endo H-sensitive inner core structures. N-Acetylglucosaminidase was active and stable over a broad pH range with maximum activity against both p-nitrophenylGlcNAc (pNPGlcNAc) and pNPGalNAc at pH 4.0. The kinetic parameters kcat (s-1) and Km (mM) of N-acetylglucosaminidase A using the following substrates were, respectively: pNPGlcNAc, 740, 0.77; pNPGalNAc, 910, 1.26; N,N'-diacetylchitobiose 620, 0.20; and N,N',N"-triacetylchitotriose, 170, 0.044. The enzyme showed substrate inhibition with all substrates above 0.5 mM except for pNPGalNAc.

Acetylglucosaminidase↗
PubMed1994

Molecular cloning and expression of the Candida albicans beta-N-acetylglucosaminidase (HEX1) gene.

beta-N-Acetylglucosaminidase was purified from the spent culture medium of Candida albicans A72 grown in the presence of N-acetylglucosamine (GlcNAc). The N-terminal amino acid sequence of the protein was determined, two degenerate oligonucleotide probes were constructed, and a 3.9-kb BamHI fragment of DNA that hybridized to both probes was subcloned from a lambda EMBL4 library of C. albicans A72 genomic DNA. This fragment of DNA contained the entire beta-N-acetylglucosaminidase (HEX1) gene, which consisted of an open reading frame coding for a polypeptide precursor of 562 amino acids with a putative 22-amino-acid leader sequence. The deduced HEX1 amino acid sequence showed similarity to hexosaminidases from a variety of organisms. Growth of C. albicans on GlcNAc induced transcription of HEX1, resulting in increased specific beta-N-acetylglucosaminidase activity. HEX1 mRNA (2.35 kb) from GlcNAc-grown cells was approximately 200 bp larger than HEX1 mRNA from cells grown on glucose. This size difference was suggested to result from the use of alternative transcription termination sites. The cloned HEX1 gene introduced into C. albicans SGY-243 on a plasmid also responded to GlcNAc induction.

Acetylglucosaminidase↗
PubMed1994

Detection of Candida albicans and other yeasts in blood by PCR.

Primers complementary to the region of genes coding for rRNA in Candida albicans were used in PCRs to detect yeast DNA extracted from blood samples containing various Candida species. One fragment (105 bp) was amplified from all yeasts tested, whereas a second (684 bp) was only amplified when C. albicans DNA was present. The level of sensitivity was 15 +/- 5 (mean +/- standard error) CFU of C. albicans per ml of blood.

Base Sequence↗
PubMed1993

Interactions of Actinomyces naeslundii strains T14V and ATCC 12104 with saliva, collagen and fibrinogen.

Approximately similar numbers of actinomyces cells adhered to hydroxylapatite beads coated with saliva, collagen or fibrinogen. Adherence generally was unaffected by the presence of free saliva. Binding of cells to collagen- or fibrinogen-coated beads was reduced in the presence of either free collagen or fibrinogen. Glucan inhibited bacterial adherence only to collagen-coated hydroxylapatite beads. It is suggested that actinomyces bind to saliva-, collagen- or fibrinogen-coated surfaces by different mechanisms, but that these mechanisms involve some common bacterial cell-surface components.

Actinomyces↗
PubMed1993

An exo-beta-(1,3)-glucanase of Candida albicans: purification of the enzyme and molecular cloning of the gene.

A nucleotide sequence encoding an exo-beta-(1,3)-glucanase was cloned from a library of genomic DNA of Candida albicans ATCC 10261. The sequenced gene encodes a protein of 438 amino acid residues. The amino terminal and an internal peptide sequence of the enzyme matched with deduced sequences within the cloned gene. Analysis of the sequence indicated that the nascent protein is processed during secretion by the signal peptidase and a Kex2-like proteinase, yielding a predicted mature enzyme of 400 residues. There is 58% identity and 85% similarity between the amino acid sequences of this exoglucanase and the homologous enzyme of Saccharomyces cerevisiae. An antiserum to the purified exoglucanase cross-reacted with the S. cerevisiae exoglucanase and a similar protein secreted by other C. albicans strains and Candida species. There are no sites for N-linked glycosylation in the sequence and this is consistent with the carbohydrate content of the secreted enzyme. Putative upstream promoter elements are associated with the gene. Southern analysis of the gene indicated that it was present at one copy per genome and that the diploid genome of C. albicans ATCC 10261 is heterozygous at this locus for a BglII RFLP. A 2.5 kb mRNA transcript was detected by Northern analysis and gene expression, as monitored by Northern and Western blots, reflected the growth rates of the cultures.

Amino Acid Sequence↗
PubMed1992

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu-) was transformed by pRC2312 to Leu+ at a frequency of 1.41 x 10(5) colonies per microgram DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura+ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 x 10(3) per microgram DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per microgram DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15 +/- 3 per haploid genome in S. cerevisiae and 2-3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7-12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade- strains of both C. albicans and S. cerevisiae to Ade+. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.

Candida albicans↗
PubMed1992

Yeast-specific DNA probes and their application for the detection of Candida albicans.

Two DNA fragments cloned from the genome of Candida albicans ATCC 10261 may be useful in the rapid diagnosis of disseminated candidosis. One sequence (probe EOB1) was specific for C. albicans (positive hybridisation with 45 strains tested). The second sequence (probe EOB2) detected C. albicans, as well as five other pathogenic Candida spp. and Saccharomyces cerevisiae, but did not react with human or bacterial DNA. Both probes were repetitive sequences in the genome of C. albicans. Probe EOB1 was used to detect, without DNA amplification, 500 C. albicans yeast cells in 1 ml of human blood.

Candida albicans↗
PubMed1992

Mechanisms of aggregation accompanying morphogenesis in Candida albicans.

Cultures of Candida albicans yeast cells do not normally aggregate, but extensive aggregation accompanies the induction of mycelial growth, indicating the occurrence of cell surface changes during the yeast to mycelial transition. Aggregation correlated with the formation of germ tubes as did changes in surface charge determined by attachment to ion exchange sepharose beads. Yeast cells of all strains examined were negatively charged and attachment to positively charged (DEAE) sepharose beads increased following germ tube formation. If Mg2+ was present during germ tube formation, a high degree of clumping occurred that could only be dispersed by treatment with protein-disrupting agents. Trypsin, chymotrypsin, SDS, urea, guanidine HCl and dithiothreitol but not EDTA or EGTA caused irreversible dispersal of aggregates, although germ tube aggregates dispersed by treatment with buffers at high pH reaggregated if neutralized or if calcium ions were added. Germ tube cultures produced in divalent cation-deprived medium formed aggregates that were readily dispersed by washing. However, the addition of Mg2+ or other divalent cations (Ca2+, Zn2+, Cu2+, Fe2+) caused immediate aggregation of these cultures. These results suggest that divalent cation crossbridging between opposing anionic sites and protein interactions act synergistically to promote aggregation of C. albicans germ tube cells.

Candida albicans↗
PubMed1991

Effect of calcium ion uptake on Candida albicans morphology.

In liquid culture using a synthetic medium, added magnesium but not calcium was required for exponential growth of Candida albicans yeast cells. However, medium without added divalent cations supported 2-3 generations of yeast growth or germ tube induction. The addition of calcium ions (1.0 mM) at any stage during the induction of germ tube formation caused reversion to a yeast mode of growth, in contrast to the effect of zinc and cobalt ions which were toxic to all growth. Inhibition of germ tube formation by calcium was not observed in the presence of either magnesium (10 microM) or manganese (100 microM). The presence of either of these ions caused inhibition of 45Ca uptake in yeast cultures. We conclude that unrestricted calcium uptake resulted in the specific inhibition of C. albicans mycelial growth, indicating a critical role for calcium in the regulation of C. albicans morphogenesis.

Biological Transport↗
PubMed1990

Isolation and nucleotide sequence of an autonomously replicating sequence (ARS) element functional in Candida albicans and Saccharomyces cerevisiae.

An 8.6-kb fragment was isolated from an EcoRI digest of Candida albicans ATCC 10261 genomic DNA which conferred the property of autonomous replication in Saccharomyces cervisiae on the otherwise non-replicative plasmid pMK155 (5.6 kb). The DNA responsible for the replicative function was subcloned as a 1.2-kb fragment onto a non-replicative plasmid (pRC3915) containing the C. albicans URA3 and LEU2 genes to form plasmid pRC3920. This plasmid was capable of autonomous replication in both S. cerevisiae and C. albicans and transformed S. cerevisiae AH22 (leu2-) to Leu+ at a frequency of 2.15 x 10(3) transformants per microgram DNA, and transformed C. albicans SGY-243 (delta ura3) to Ura+ at a frequency of 1.91 x 10(3) transformants per microgram DNA. Sequence analysis of the cloned DNA revealed the presence of two identical regions of eleven base pairs (5'TTTTATGTTTT3') which agreed with the consensus of autonomously replicating sequence (ARS) cores functional in S. cerevisiae. In addition there were two 10/11 and numerous 9/11 matches to the core consensus. The two 11/11 matches to the consensus, CaARS1 and CaARS2, were located on opposite strands in a non-coding AT-rich region and were separated by 107 bp. Also present on the C. albicans DNA, 538 bp from the ARS cores, was a gene for 5S rRNA which showed sequence homology with several other yeast 5S rRNA genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1988

Correlation between the sterol composition of membranes and morphology in Candida albicans.

The opportunistic pathogenic fungus Candida albicans was examined in three distinct morphological forms: yeast, mycelia, and stunted mycelia obtained by growing cells under mycelial-inducing conditions in the presence of ketoconazole at concentrations below the MIC. Comparison of the sterols of the parental strain with those from a mutant unable to produce mycelia, by TLC and GLC, showed no significant differences in sterol composition between the yeast and mycelial morphological forms. However, ketoconazole-treated cells possessed a lower ergosterol content and an increased amount of 14-methylated sterol precursors. The effect of ketoconazole on mycelial cells was quantitatively different from the effect on yeast cells in that the ratio of 4, 14-dimethylsterols to 4-methylsterols was significantly lower in the stunted mycelial morphological forms. A comparison of the effect of terbinafine on sterol biosynthesis and morphology suggests that the effect on ergosterol content is of greater importance than the increase in sterol precursors in determining cell shape.

Candida albicans↗
PubMed1986

Isolation of a mycelial mutant of Candida albicans.

A mutant of Candida albicans strain MEN, which was unable to produce mycelia in SSV medium and in horse serum at 37 degrees C, was isolated by a physical separation procedure. The mutant was shown to be derived from the parental strain by growth and morphology studies, sugar uptake and fermentation patterns, and the presence of genetic markers.

Candida albicans↗