Date of Award

12-2013

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Microbial Pathogenesis, Immunology, and Inflammation

Research Advisor

P. David Rogers, Pharm.D., Ph.D.

Committee

Ramin Homayouni, Ph.D. Richard E. Lee, Ph.D. Bernd Meibohm, Ph.D. Todd B. Reynolds, Ph.D.

Abstract

Although many medically important Candida species are commensal to the gut or colonizers of the skin, these organisms have the propensity to cause disease in the event of a waning immune system. Clinical manifestations of infections with Candida species can range from superficial mucosal infections to deep organ involvement usually resulting from haematogenous spread of infection. Despite significant progress made in the management of patients with fungal infections, the emergence of antifungal resistant clinical isolates creates significant problem in regards to antifungal prophylaxis and empirical treatment strategies. Antifungal resistance is associated with high mortality rates and hefty medical costs. The azole-antifungal class has been the “work horse” of antifungal pharmacotherapy for the past 20 years defined by its efficacy against Candida species and paucity of side effects. As the only oral option available for systemic antifungal treatment, the azoles are the most suitable option for the long treatment periods sometimes required for antifungal prophylaxis and therapy. As the azoles are fungistatic to Candida species, the lengthy and repeated treatment courses have resulted in azole-resistant clinical isolates resulting in treatment failure and increased patient mortality.

Candida albicans is the most prevalent etiologic cause of fungal disease. High-level azole resistance in this species is a result of the interplay of several mechanisms of resistance. Overexpression of the efflux transporter genes CDR1, CDR2, and MDR1 is a common mechanism of drug resistance in C. albicans and the majority of previous investigations pertained to defining mechanisms of transcriptional regulation of efflux transporters. Alternatively to efflux transport, point mutations in the ERG11 gene, whose gene product is the target of azoles, result in reduced target binding affinity without precluding enzymatic function. In addition to point mutations, overexpression of ERG11 has also been shown to decrease fluconazole susceptibility. ERG11 gene amplification by chromosome 5 duplication or the presence of a chr5L isochromosome is known to contribute to azole resistance. Additionally, the zinc-cluster transcription factor Upc2 has been shown to regulate the expression of ERG11 and other genes involved in ergosterol biosynthesis.

In a large group of clinical C. albicans isolates enriched for azole resistance, I created a transcriptional profile defining expression of genes known to cause azole resistance such as ERG11, CDR1, CDR2 and MDR1. Not surprisingly, CDR1 and CDR2 overexpression was generally coordinately regulated and quite prevalent among these isolates. Of those isolates that did overexpress MDR1, even fewer isolates expressed MDR1 to the levels previously observed in azoleresistant isolates. ERG11 was found to be upregulated in almost three-fourths of the fluconazoleresistant isolates examined. This suggests that ERG11 overexpression is a common contributor to fluconazole resistance in C. albicans. Among the ERG11-overexpressing isolates studied here, I repeatedly recovered eight distinct single-nucleotide substitutions in UPC2. Five of these substitutions in UPC2 have not been described previously. Of the five novel mutations, four mutations resulted in increased ERG11 expression and increased resistance to fluconazole but to various degrees. Genome-wide transcriptional analysis was performed for the four strongest Upc2 amino acid substitutions (A643V, G648D, G648S, and Y642F). Genes commonly upregulated by all four mutations included those involved in ergosterol biosynthesis, in oxidoreductase activity, the major facilitator efflux pump encoded by the MDR1 gene, and the uncharacterized ATP binding cassette transporter CDR11. These findings demonstrate that gain-of-function mutations in UPC2 are more prevalent among clinical isolates than previously thought and make a significant contribution to azole antifungal resistance, but the findings do not account for ERG11 overexpression in all such isolates of C. albicans.

Although prevalent, not all ERG11-overexpression in this group of isolates could be explained by GOF mutations in Upc2. In C. albicans, the Pho-G transcription factor NDT80 has been implicated in azole resistance not only due to its regulation of CDR1 but also due to its regulation of genes involved in the ergosterol biosynthesis pathway. In the next set of experiments, NDT80 alleles for genetically matched pairs of isolates 945/1619 and 1002/3795 were sequenced. In both matched sets, the fluconazole resistant isolate overexpresses ERG11. Sequencing of the NDT80 allele of both matched sets revealed several mutations that resulted in amino acid substitutions when compared to SC5314. This analysis also showed that a loss of heterozygostiy event occurs so that the resistant counterpart was homozygous for one allele. A strain carrying the NDT80 allele derived from fluconazole-resistant isolate 1619 did not result in increased ERG11 expression and increased fluconazole resistance. The mechanism by which ERG11 is upregulated in the absence of UPC2 gain-of-function mutations is currently under investigation.

In addition to ERG11-overexpression, mutations in ERG11 that result in amino acid substitutions in lanosterol demethylase have also been associated with decreased azole susceptibility. In the third study, I examined the prevalence and variance of ERG11 mutations in the same group clinical C. albicans isolates. In this collection, I identified that 55 of the 63 isolated contained missense mutations in ERG11 that resulted in at least one amino acid substitution. From this sequencing data, a selected a group of mutant ERG11 alleles were expressed in an azole-susceptible background so I could determine the specific contribution of the mutant ERG11 allele on antifungal susceptibility. In this analysis, I was particularly interested in characterizing amino acid substitutions that occurred alone and also when accompanied by another mutation. In total, I characterized ten ERG11 alleles containing one amino acid substitution and nine alleles which carried ERG11 alleles with a combination of amino acid substitutions. Fluconazole, itraconazole and voriconazole susceptibilities for these strains were tested. Our data demonstrated many of these mutations resulted in fluconazole resistance, but most were not as significant when tested against voriconazole or itraconazole. Itraconazole, in particular seemed less effected by ERG11 mutations which produced significant resistance to fluconazole although amino acid combination Y132F and F145L resulted in increased itraconazole resistance. Specific combinations of ERG11 mutations resulted in increased azole resistance beyond single mutations. These data suggest that structural differences between azole effect activity against specific mutant ERG11 alleles.

DOI

10.21007/etd.cghs.2013.0096

Comments

Two year embargo expired December 2015