Date of Award
Doctor of Philosophy (PhD)
P. David Rogers, Pharm.D., Ph.D.
Damian J. Krysan, M.D., Ph.D. Richard E. Lee, Ph.D. Bernd Meibohm, Ph.D. Frank Park, Ph.D.
Despite the scientific and medical communities’ best efforts, the incidence of fungal infections in susceptible populations continues to rise. The most common cause of these opportunistic fungal infections is Candida. In fact, Candida is the fourth most common pathogen associated with nosocomial blood stream infections. Reported mortality rates for patients with candidemia vary, but have not decreased in the past fifteen years and are reported to be as high as 50%. Candida glabrata, second only to Candida albicans among Candida infections, expresses high rates of resistance to treatment with arguably the best class of currently available antifungals - the azoles.
Other available antifungals have associated toxicities, are not available as oral dosage forms or are cost prohibitive. As multidrug resistant C. glabrata have been reported, the need to find ways to overcome resistance to azoles is more pressing than ever. The work described here highlights our efforts to develop a better understanding of azole resistance in Candida, with a focus on C. glabrata, which can then be utilized to inform better strategies for decreasing or preventing resistance.
In C. glabrata clinical azole resistance is mediated almost exclusively by activating mutations in the zinc cluster transcription factor Pdr1, which controls the genes encoding the multidrug resistance transporters Cdr1, Pdh1, and Snq2. However, the specific relative contribution of these transporters to resistance is not known. In order to determine this, the SAT1 flipper method was used to delete CDR1, PDH1, and SNQ2 in a strain of C. glabrata engineered to carry a clinically relevant activating mutation in PDR1. Susceptibility testing was performed according to the CLSI guidelines with minor modifications and confirmed with Etest strips. Of the single transporter deletion strains, only CDR1 deletion resulted in decreased azole MIC. Deletion of PDH1 in combination with CDR1 resulted in a moderate decrease in MIC from that observed with deletion of CDR1 alone. SNQ2 deletion only decreased the MIC in the triple deletion strain in the absence of both CDR1 and PDH1. Deletion of all three transporters in combination decreased the MIC to the level observed in the PDR1 deletion strains for some, but not all of the azoles tested, which indicates additional Pdr1 targets likely play a minor role in this process. These results demonstrate that Cdr1 is the most important Pdr1-mediated multidrug resistance transporter for azole resistance in C. glabrata, suggesting that targeting this transporter alone might be sufficient to overcome this clinical problem.
Upc2 and Ecm22 in S. cerevisiae and Upc2 in C. albicans are the transcriptional regulators of ERG11, the gene encoding the target of azoles in the ergosterol biosynthesis pathway. Recently two homologs for these transcription factors, UPC2A and UPC2B, were identified in C. glabrata. One of these, UPC2A, was shown to influence azole susceptibility. We hypothesized that due to the global role for Upc2 in sterol biosynthesis in S. cerevisiae and C. albicans, disruption of UPC2A would enhance the activity of fluconazole in both azole-susceptible-dose dependent (SDD) and -resistant C. glabrata clinical isolates. To test this hypothesis, we constructed mutants disrupted for UPC2A and UPC2B alone and in combination in a matched pair of clinical azole-SDD and - resistant isolates. Disruption of UPC2A in both the SDD and resistant isolates resulted in increased susceptibility to sterol biosynthesis inhibitors, including a reduction in fluconazole minimum inhibitory concentration and minimum fungicidal concentration, enhanced azole activity by time-kill analysis, a decrease in ergosterol content, and downregulation of baseline and inducible expression of several sterol biosynthesis genes. Our results indicate that Upc2A is a key regulator of ergosterol biosynthesis and is essential for resistance to sterol biosynthesis inhibitors in C. glabrata. As such, the UPC2A pathway may represent a potential co-therapeutic target for enhancing azole activity against this organism.
The importance of Pdr1 in azole resistance in C. glabrata is well established. Our understanding of how Pdr1 is being regulated, however, is predominantly informed by regulation of similar systems in other organisms. In order to identify genes that interact with the Pdr1 transcriptional pathway, and influence the susceptibility of C. glabrata to fluconazole, we screened a collection of deletion mutants for those exhibiting increased resistance to fluconazole. Deletion of the gene coding for a protein homologous to the S. cerevisiae J protein Jjj1 resulted in decreased fluconazole susceptibility. We used the SAT1 flipper method to generate independent deletion mutants for JJJ1 in a SDD clinical isolate. Expression of both CDR1 and PDR1 was increased in the absence of JJJ1. In the absence of CDR1 or PDR1, deletion of JJJ1 had only a modest effect on fluconazole susceptibility. Transcriptional profiling using RNA-Seq revealed up-regulation of genes of the Pdr1 regulon in the absence of JJJ1. Jjj1 appears to be a negative regulator of fluconazole resistance in C. glabrata and acts primarily through up-regulation of the ABC transporter gene CDR1 via activation of the Pdr1 transcriptional pathway.
Unlike C. glabrata which has essentially one mechanism of resistance, in C. albicans clinical azole resistance can be attributed to multiple mechanisms, often in combination. The RTA3 gene, coding for a member of the Rta1p-like lipid-translocating exporter family, is coordinately upregulated with the ABC transporter genes CDR1 and CDR2 in azole-resistant clinical isolates of C. albicans that carry activating mutations in the transcription factor Tac1p. We show here that deleting RTA3 in an azole-resistant clinical isolate carrying a Tac1p activating mutation lowered fluconazole resistance by two-fold, while overexpressing RTA3 in an azole-susceptible clinical isolate resulted in enhanced fluconazole tolerance associated with trailing growth in a liquid microtiter plate assay. We also demonstrate that an Rta3p-GFP fusion protein localizes predominantly to the plasma membrane, consistent with a putative function for Rta3p as a lipid translocase.
Whaley, Sarah Garland (http://orcid.org/0000-0001-5851-0490), "Novel Determinants That Influence Azole Susceptibility in Candida glabrata and Candida albicans" (2018). Theses and Dissertations (ETD). Paper 453. http://dx.doi.org/10.21007/etd.cghs.2018.0447.