Biomedical subjects

T C White

Publications and source records attributed to T C White.

At least 19 recordsLinked to original sources

PubMed2005

Regulation of sterol metabolism in Candida albicans by the UPC2 gene.

Candida albicans is an important pathogenic fungus of humans, causing a range of infections. These infections are usually treated with antifungal drugs that target sterol metabolism. Resistance to these antifungals can result from overexpression of sterol biosynthetic genes. Therefore it is of interest to understand transcriptional regulation of sterol biosynthesis in C. albicans. Recently two reports [Silver, Oliver and White (2004) Eukaryot. Cell 3, 1391-1397; MacPherson, Akache, Weber, De Deken, Raymond and Turcotte (2005) Antimicrob. Agents Chemother. 49, 1745-1752] have identified and characterized a single C. albicans transcription factor gene UPC2 that regulates sterol metabolism. The details of both characterizations are compared and contrasted. These reports extend our understanding of sterol regulation in this important human pathogen.

Antifungal Agents↗

PubMed2002

Persistence of oral Candida albicans carriage in healthy Portuguese schoolchildren followed for 3 years.

Little is known about carriage of Candida albicans, the predominant pathogenic yeast in oral infection, in children. We cultured buccal mucosal and gingival swabs from 150 Portuguese children to investigate the prevalence of C. albicans at baseline (before dental treatment), post-treatment, and 12, 24, and 36 months post-baseline. The children, aged 8 to 11 years at baseline, had no systemic disease or clinical symptoms of oral candidiasis. At each successive visit, respectively, 47, 32, 21, 27, and 28% of children were C. albicans positive, resulting in an almost 50% reduction in prevalence from baseline to post-treatment (P < 0.0005). Children who carried C. albicans at one visit had 3 to 20 times greater odds of carrying C. albicans at another visit. C. albicans was cultured from 12 children at all time-points and from 10 children at four time-points. Children with oral C. albicans frequently maintained carriage over time, even with regular dental care.

Age Factors↗

PubMed2002

Transcription initiation of genes associated with azole resistance in Candida albicans.

Oral infections with the opportunistic pathogenic yeast Candida albicans are one of the earliest and most frequent infections in immunosuppressed individuals. In these populations, drug-resistant isolates have emerged with the widespread use of antifungal azole drugs. Many molecular mechanisms of resistance have been identified, including overexpression of two types of efflux pumps, the major facilitator MDR1 and the ABC-transporters (CDR1 and CDR2), and the overexpression or mutation of the target enzyme, ERG11. With overexpression of these four genes implicated in multidrug resistance, identification of regulatory regions of the promoters is important. 5' rapid amplification of cDNA ends (RACE) was used to identify transcription initiation sites for genes associated with multidrug resistance (CDR1, CDR2, MDR1 and ERG11). These results were confirmed by cloning and sequencing of 5' RACE products and by primer extension. This research will allow further analysis of the regulation of transcription for these genes.

ATP-Binding Cassette Transporters↗

PubMed2001

Therapeutic alteration of insulin-dependent diabetes mellitus progression by T cell tolerance to glutamic acid decarboxylase 65 peptides in vitro and in vivo.

We have reported previously that nonobese diabetic (NOD) fetal pancreas organ cultures lose the ability to produce insulin when maintained in contact with NOD fetal thymus organ cultures (FTOC). Initial studies indicated that exposure to glutamic acid decarboxylase (GAD65) peptides in utero resulted in delay or transient protection from insulin-dependent diabetes mellitus (IDDM) in NOD mice. We also found that exposure of young adult NOD mice to the same peptides could result in acceleration of the disease. To more closely examine the effects of early and late exposure to diabetogenic Ags on T cells, we applied peptides derived from GAD65 (GAD AA 246-266, 509-528, and 524-543), to our "in vitro IDDM" (ivIDDM) model. T cells derived from NOD FTOC primed during the latter stages of organ culture, when mature T cell phenotypes are present, had the ability to proliferate to GAD peptides. ivIDDM was exacerbated under these conditions, suggesting that GAD responsiveness correlates with the ivIDDM phenotype, and parallels the acceleration of IDDM we had seen in young adult NOD mice. When GAD peptides were present during the initiation of FTOC, GAD proliferative responses were inhibited, and ivIDDM was reduced. This result suggests that tolerance to GAD peptides may reduce the production of diabetogenic T cells or their capacity to respond, as suggested by the in utero therapies studied in NOD mice.

Adjuvants, Immunologic↗

PubMed2001

Characterization of cellobiohydrolase I (Cel7A) glycoforms from extracts of Trichoderma reesei using capillary isoelectric focusing and electrospray mass spectrometry.

Capillary isoelectric focusing (CIEF) was used to profile the cellulase composition in complex fermentation samples of secreted proteins from Trichoderma reesei. The enzyme cellobiohydrolase I (CBH I, also referred to as Cel7A), a major component in these extracts, was purified from different strains and characterized using analytical methods such as CIEF, high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and capillary liquid chromatography-electrospray mass spectrometry (cLC-ESMS). ESMS was also used to monitor the extent of glycosylation in CBH I isolated from T. reesei strain RUT-C30 and two derivative mutant strains. Selective identification of tryptic N-linked glycopeptides was achieved using LC-ESMS on a quadrupole/time-of-flight instrument with a mixed scan function. The suspected glycopeptides were further analyzed by on-line tandem mass spectrometry to determine the nature of N-linked glycans and their attachment sites. This strategy enabled the identification of a high mannose glycan attached to Asn270 (predominantly Man8GlcNAc2) and single GlcNAc occupancy at Asn45 and Asn384 with some site heterogeneity depending on strains and fermentation conditions. The linker region of CBH I was shown to be extensively glycosylated with di-, and tri-saccharides at Thr and Ser residues as indicated by MALDI-TOF and HPAEC-PAD experiments. Additional heterogeneity was noted in the CBH I linker peptide of RUT-C30 strain with the presence of a phosphorylated di-saccharide.

Amino Acid Sequence↗

PubMed2001

Inducible azole resistance associated with a heterogeneous phenotype in Candida albicans.

The development of azole resistance in Candida albicans is most problematic in patients with AIDS who receive long courses of drug for therapy or prevention of oral candidiasis. Recently, the rapid development of resistance was noted in other immunosuppressed patients who developed disseminated candidiasis despite fluconazole prophylaxis. One of these series of C. albicans isolates became resistant, with an associated increase in mRNA specific for a CDR ATP-binding cassette transporter efflux pump (K. A. Marr, C. N. Lyons, T. R. Rustad, R. A. Bowden, and T. C. White, Antimicrob. Agents Chemother. 42:2584-2589, 1998). Here we study this series of C. albicans isolates further and examine the mechanism of azole resistance in a second series of C. albicans isolates that caused disseminated infection in a recipient of bone marrow transplantation. The susceptible isolates in both series become resistant to fluconazole after serial growth in the presence of drug, while the resistant isolates in both series become susceptible after serial transfer in the absence of drug. Population analysis of the inducible, transiently resistant isolates reveals a heterogeneous population of fluconazole-susceptible and -resistant cells. We conclude that the rapid development of azole resistance occurs by a mechanism that involves selection of a resistant clone from a heterogeneous population of cells.

Antifungal Agents↗

PubMed2000

Candidemia in allogeneic blood and marrow transplant recipients: evolution of risk factors after the adoption of prophylactic fluconazole.

The prophylactic use of fluconazole is common in blood and marrow transplant (BMT) recipients. To evaluate how fluconazole has influenced the development of azole resistance and candidemia, weekly mouthwashings were done, and fluconazole susceptibility was determined for 1475 colonizing and invasive isolates obtained from patients undergoing BMT. Of 585 patients, 256 (44%) were colonized with Candida species during the course of BMT. Of these, 136 patients (53%) had at least 1 mouthwashing sample that yielded Candida species other than C. albicans on culture. Only 4.6% of patients developed candidemia. Overall, C. albicans was the most common colonizing isolate, but it caused only 7% of cases of candidemia. About 5% of colonizing C. albicans strains and 100% (2 of 2) invasive C. albicans strains were fluconazole-resistant. Colonization, cytomegalovirus disease, and bacteremia are risk factors for the development of candidemia. The use of prophylactic fluconazole is associated with a low incidence of candidemia and attributable mortality, despite colonization with azole-resistant Candida species in BMT recipients.

Adolescent↗

PubMed2000

The R467K amino acid substitution in Candida albicans sterol 14alpha-demethylase causes drug resistance through reduced affinity.

The cytochrome P450 sterol 14alpha-demethylase (CYP51) of Candida albicans is involved in an essential step of ergosterol biosynthesis and is the target for azole antifungal compounds. We have undertaken site-directed mutation of C. albicans CYP51 to produce a recombinant mutant protein with the amino acid substitution R467K corresponding to a mutation observed clinically. This alteration perturbed the heme environment causing an altered reduced-carbon monoxide difference spectrum with a maximum at 452 nm and reduced the affinity of the enzyme for fluconazole, as shown by ligand binding studies. The specific activity of CYP51(R467K) for the release of formic acid from 3beta-[32-(3)H]hydroxylanost-7-en-32-ol was 70 pmol/nmol of P450/min for microsomal protein compared to 240 pmol/nmol of P450/min for microsomal fractions expressing wild-type CYP51. Furthermore, inhibition of activity by fluconazole revealed a 7.5-fold-greater azole resistance of the recombinant protein than that of the wild type. This study demonstrates that resistance observed clinically can result from the altered azole affinity of the fungal CYP51 enzyme.

Antifungal Agents↗

PubMed2000

Transcriptional analyses of antifungal drug resistance in Candida albicans.

Oral infections with the pathogenic yeast Candida albicans are one of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug-resistant isolates. Several molecular mechanisms that contribute to drug resistance have been identified, including increased mRNA levels for two types of efflux pump genes: the ATP binding cassette transporter CDRs (CDR1 and CDR2) and the major facilitator MDR1. Using Northern blot analyses, the expression patterns of these genes have been determined during logarithmic and stationary phases of cell growth and during growth in different carbon sources in a set of matched susceptible and fluconazole-resistant isolates that have been characterized previously. MDR1, CDR1, and CDR2 are expressed early during logarithmic growth, CDR4 is expressed late during logarithmic growth, and CDR1 is preferentially expressed in stationary-phase cells. There is a small decrease in expression of these genes when the cells are grown in carbon sources other than glucose. While increased mRNA levels of efflux pump genes are commonly associated with azole resistance, the causes of increased mRNA levels have not yet been resolved. Southern blot analysis demonstrates that the increased mRNA levels in these isolates are not the result of gene amplification. Nuclear run-on assays show that MDR1 and CDR mRNAs are transcriptionally overexpressed in the resistant isolate, suggesting that the antifungal drug resistance in this series is associated with the promoter and trans-acting factors of the CDR1, CDR2, and MDR1 genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1999

In vitro antifungal activity of BMS-207147 and itraconazole against yeast strains that are non-susceptible to fluconazole.

The activities of itraconazole and the new triazole BMS-207147 were determined against Candida strains that were susceptible-dose dependent (fluconazole MICs 16 to 32 micrograms/mL) or resistant (MICs > or = 64 micrograms/mL) to fluconazole. These strains included clinical isolates of Candida krusei, Candida glabrata, and Candida albicans. In addition, 16 isogenic, genetically characterized isolates of C. albicans, with progressively decreased susceptibility to fluconazole, were tested. BMS-207147 MICs to C. krusei, a species considered intrinsically resistant to fluconazole, were at 0.13 to 0.5 microgram/mL. The population distribution of the fluconazole-nonsusceptible C. glabrata was bimodal with BMS-207147/itraconazole MICs at 0.5 to 2 micrograms/mL and > or = 16 micrograms/mL. The BMS-207147 MICs to the majority of fluconazole-nonsusceptible C. albicans strains tested were < or = 1 microgram/mL. The activity of BMS-207147 was minimally affected by overexpression of the gene encoding the efflux pump MDR1, but MIC increases were observed with changes in ERG11 and with overexpression of the CDR transporter gene. Nonetheless, BMS-207147 can be active against C. albicans mutants containing cumulative resistance mechanisms to azoles. In other words, fluconazole-resistant candidal strains may be susceptible to BMS-207147.

Antifungal Agents↗

PubMed1999

Effects of azole antifungal drugs on the transition from yeast cells to hyphae in susceptible and resistant isolates of the pathogenic yeast Candida albicans.

Oral infections caused by the yeast Candida albicans are some of the most frequent and earliest opportunistic infections in human immunodeficiency virus-infected patients. The widespread use of azole antifungal drugs has led to the development of drug resistance, creating a major problem in the treatment of yeast infections in AIDS patients and other immunocompromised individuals. Several molecular mechanisms that contribute to drug resistance have been identified. In C. albicans, the ability to morphologically switch from yeast cells (blastospores) to filamentous forms (hyphae) is an important virulence factor which contributes to the dissemination of Candida in host tissues and which promotes infection and invasion. A positive correlation between the level of antifungal drug resistance and the ability to form hyphae in the presence of azole drugs has been identified. Under hypha-inducing conditions in the presence of an azole drug, resistant clinical isolates form hyphae, while susceptible yeast isolates do not. This correlation is observed in a random sample from a population of susceptible and resistant isolates and is independent of the mechanisms of resistance. 35S-methionine incorporation suggests that growth inhibition is not sufficient to explain the inhibition of hyphal formation, but it may contribute to this inhibition.

Antifungal Agents↗

PubMed1999

The trailing end point phenotype in antifungal susceptibility testing is pH dependent.

The interpretation of end points in azole antifungal drug susceptibility testing is problematic, in part due to incomplete growth inhibition of Candida species. Such trailing growth can cause the MICs of fluconazole for some isolates to be low (<1 microg/ml) after 24 h of growth but much higher (>64 microg/ml) after 48 h. Isolates having this type of growth have been described as having a low-high phenotype. Although these isolates would be considered resistant by current National Committee of Clinical Laboratory Standards definitions, growing evidence suggests that they are susceptible in vivo. To further characterize these isolates in vitro, microdilution susceptibility testing comparing the complex defined medium RPMI 1640 to a defined minimal medium (yeast nitrogen broth) was performed. Isolates having trailing growth in MOPS (morpholinepropanesulfonic acid)-buffered RPMI 1640 (pH 7.0) were found to have clear end points in the minimal medium at its native pH of 4.5. The pH of the medium influenced the low-high phenotype, as these same isolates trailed in minimal medium adjusted to a pH of >/=6.0 but did not trail in RPMI 1640 adjusted to a pH of </=5.0. This pH effect was independent of the medium buffering capacity, as trailing was decreased in both minimal medium and RPMI 1640 (pH 4.5) buffered in citrate. Adjustment in the pH of MOPS-buffered RPMI 1640 reduced trailing in multiple strains of Candida albicans without affecting the MICs for isolates having known susceptible (low-low) and resistant (high-high) phenotypes. Adjustment of the medium pH could be considered to eliminate trailing in azole drug susceptibility testing.

Antifungal Agents↗

PubMed1999

In vivo analysis of secreted aspartyl proteinase expression in human oral candidiasis.

Secreted aspartyl proteinases are putative virulence factors in Candida infections. Candida albicans possesses at least nine members of a SAP gene family, all of which have been sequenced. Although the expression of the SAP genes has been extensively characterized under laboratory growth conditions, no studies have analyzed in detail the in vivo expression of these proteinases in human oral colonization and infection. We have developed a reliable and sensitive procedure to detect C. albicans mRNA from whole saliva of patients with oral C. albicans infection and those with asymptomatic Candida carriage. The reverse transcription-PCR protocol was used to determine which of the SAP1 to SAP7 genes are expressed by C. albicans during colonization and infection of the oral cavity. SAP2 and the SAP4 to SAP6 subfamily were the predominant proteinase genes expressed in the oral cavities of both Candida carriers and patients with oral candidiasis; SAP4, SAP5, or SAP6 mRNA was detected in all subjects. SAP1 and SAP3 transcripts were observed only in patients with oral candidiasis. SAP7 mRNA expression, which has never been demonstrated under laboratory conditions, was detected in several of the patient samples. All seven SAP genes were simultaneously expressed in some patients with oral candidiasis. This is the first detailed study showing that the SAP gene family is expressed by C. albicans during colonization and infection in humans and that C. albicans infection is associated with the differential expression of individual SAP genes which may be involved in the pathogenesis of oral candidiasis.

AIDS-Related Opportunistic Infections↗

PubMed1998

Rapid, transient fluconazole resistance in Candida albicans is associated with increased mRNA levels of CDR.

Fluconazole-resistant Candida albicans, a cause of recurrent oropharyngeal candidiasis in patients with human immunodeficiency virus infection, has recently emerged as a cause of candidiasis in patients receiving cancer chemotherapy and marrow transplantation (MT). In this study, we performed detailed molecular analyses of a series of C. albicans isolates from an MT patient who developed disseminated candidiasis caused by an azole-resistant strain 2 weeks after initiation of fluconazole prophylaxis (K. A. Marr, T. C. White, J. A. H. vanBurik, and R. A. Bowden, Clin. Infect. Dis. 25:908-910, 1997). DNA sequence analysis of the gene (ERG11) for the azole target enzyme, lanosterol demethylase, revealed no difference between sensitive and resistant isolates. A sterol biosynthesis assay revealed no difference in sterol intermediates between the sensitive and resistant isolates. Northern blotting, performed to quantify mRNA levels of genes encoding enzymes in the ergosterol biosynthesis pathway (ERG7, ERG9, and ERG11) and genes encoding efflux pumps (MDR1, ABC1, YCF, and CDR), revealed that azole resistance in this series is associated with increased mRNA levels for members of the ATP binding cassette (ABC) transporter superfamily, CDR genes. Serial growth of resistant isolates in azole-free media resulted in an increased susceptibility to azole drugs and corresponding decreased mRNA levels for the CDR genes. These results suggest that C. albicans can become transiently resistant to azole drugs rapidly after exposure to fluconazole, in association with increased expression of ABC transporter efflux pumps.

ATP-Binding Cassette Transporters↗

PubMed1998

Distinct patterns of gene expression associated with development of fluconazole resistance in serial candida albicans isolates from human immunodeficiency virus-infected patients with oropharyngeal candidiasis.

Resistance to fluconazole is becoming an increasing problem in the management of oropharyngeal candidiasis in human immunodeficiency virus-infected patients. Strains obtained from five patients developed decreased fluconazole susceptibility over time. DNA strain typing confirmed the high degree of relatedness among isolates from one patient and the variability among isolates from different patients. Expression of genes involved in development of fluconazole resistance was monitored in each isolate using probes specific for ERG11 (lanosterol 14alpha-demethylase), MDR1 (a major facilitator), and CDR (ATP-binding cassette or ABC transporter) genes. Increased expression of CDR genes was detected in the series of isolates from two patients. Isolates from one of the two patients also demonstrated increased ERG11 expression, whereas isolates from the other patient did not. Increased levels of MDR1 mRNA correlated with increased resistance in sequential isolates from another patient. Initial overexpression of MDR1 with subsequent overexpression of CDR genes and a final isolate again overexpressing MDR1 were detected in serial isolates from another patient. In another patient, overexpression of these genes was not detected despite an eightfold increase in fluconazole MIC. In this patient, sequence data of the ERG11 gene revealed no point mutations associated with decreased susceptibility. Five different patterns of gene expression were observed in isolates recovered from five patients who developed resistance. Therefore, these experiments demonstrate that a variety of mechanisms or combinations of mechanisms are associated with the development of fluconazole drug resistance. Additional studies are needed to estimate the frequency and clinical impact of these mechanisms of resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1998

Induction of resistance to azole drugs in Trypanosoma cruzi.

Trypanosoma cruzi is the protozoan parasite that causes Chagas' disease, a frequently fatal illness affecting the heart and gastrointestinal systems. An estimated 16 million to 18 million people in Latin America and 50,000 to 100,000 people in the United States are infected with this pathogen. Treatment options for T. cruzi infections are suboptimal due to the toxicities and limited effectiveness of the available drugs. Azole antimicrobial agents have been discovered to have antitrypanosomal activity by inhibition of ergosterol synthesis. The triazole itraconazole was recently shown to produce a parasitologic cure rate of 53% in chronically infected patients (W. Apt et al., Am. J. Trop. Med. Hyg. 59:133-138, 1998), a result which may lead to more use of this family of drugs for the treatment of T. cruzi infections. In the experiments reported on here, resistance to azoles was induced in vitro by serial passage of mammalian-stage parasites in the presence of fluconazole for 4 months. These parasites were cross resistant to the other azoles, ketoconazole, miconazole, and itraconazole. They remained susceptible to benznidazole and amphotericin B. The azole-resistant phenotype was stable for more than 2 months of in vitro serial passage without fluconazole. In addition, the parasites resisted treatment in mice receiving ketoconazole. The rapid development of azole resistance in T. cruzi in vitro suggests that resistance to azole drugs has the potential to occur in patients and may pose an impediment to the progress being made in the treatment of T. cruzi infection.

Animals↗

PubMed1998

Clinical, cellular, and molecular factors that contribute to antifungal drug resistance.

In the past decade, the frequency of diagnosed fungal infections has risen sharply due to several factors, including the increase in the number of immunosuppressed patients resulting from the AIDS epidemic and treatments during and after organ and bone marrow transplants. Linked with the increase in fungal infections is a recent increase in the frequency with which these infections are recalcitrant to standard antifungal therapy. This review summarizes the factors that contribute to antifungal drug resistance on three levels: (i) clinical factors that result in the inability to successfully treat refractory disease; (ii) cellular factors associated with a resistant fungal strain; and (iii) molecular factors that are ultimately responsible for the resistance phenotype in the cell. Many of the clinical factors that contribute to resistance are associated with the immune status of the patient, with the pharmacology of the drugs, or with the degree or type of fungal infection present. At a cellular level, antifungal drug resistance can be the result of replacement of a susceptible strain with a more resistant strain or species or the alteration of an endogenous strain (by mutation or gene expression) to a resistant phenotype. The molecular mechanisms of resistance that have been identified to date in Candida albicans include overexpression of two types of efflux pumps, overexpression or mutation of the target enzyme, and alteration of other enzymes in the same biosynthetic pathway as the target enzyme. Since the study of antifungal drug resistance is relatively new, other factors that may also contribute to resistance are discussed.

Antifungal Agents↗

PubMed1998

Comparison of six extraction techniques for isolation of DNA from filamentous fungi.

Filamentous fungi have a sturdy cell wall which is resistant to the usual DNA extraction procedures. We determined the DNA extraction procedure with the greatest yield of high quality fungal DNA and the least predilection for cross-contamination of equipment between specimens. Each of six extraction methods was performed using Aspergillus fumigatus hyphae. The six methods were: (1) glass bead pulverization with vortexing; (2) grinding with mortar and pestle followed by glass bead pulverization; (3) glass bead pulverization using 1% hydroxyacetyl trimethyl ammonium bromide (CTAB) buffer in a water bath sonicator; (4) water bath sonication in CTAB buffer; (5) grinding followed by incubation with CTAB; and (6) lyticase enzymatic cell lysis. Genomic DNA yields were measured by spectrophotometry and by visual reading of 2% agarose gels, with shearing assessed by the migration of the DNA on the gel. Genomic fungal DNA yields were highest for Method 1, followed by Methods 5 approximately = to 2 >3 approximately = to 4 approximately = to 6. Methods 2 and 5, both of which involved grinding with mortar and pestle, led to shearing of the genomic DNA in one of two trials each. We conclude that the use of glass beads with extended vortexing is optimal for extraction of microgramme amounts of DNA from filamentous fungal cultures.

Aspergillus fumigatus↗