A Urine-Induced Transcriptional Program Links Biofilm Formation to Conditional Fluconazole Resistance in Candida albicans

Shane Alan Cristy, MS (Advisors: Michael C. Lorenz, PhD and Jennifer N. Walker, PhD) 

Catheter-Associated Urinary Tract Infections (CAUTIs) are the most common nosocomial infection in the United States, and Candida species, with Candida albicans being the predominant member, are the second most common cause of CAUTIs behind Escherichia coli.  CAUTIs are biofilm-based infections characterized by occlusion of the catheter, cystitis, and in severe cases, pyelonephritis and the potential for disseminated disease.  Despite the frequency of CAUTIs, little is known about the molecular basis of Candida biofilm formation in the urinary environment.

I leveraged a panel of urinary catheter-associated (CA) clinical isolates to examine biofilm phenotypes of C. albicans grown on silicone urinary catheters.  I then performed transcriptomic analysis on the C. albicans reference strain, SC5314, and two CA isolates with disparate biofilm phenotypes grown in a novel artificial urine medium (AUM).  Mature biofilms (24 hours of growth) of these three strains had a remarkable amount of transcriptional overlap, with all three upregulating genes involved in detoxification and xenobiotic efflux. 

To further probe the transcriptional response of C. albicans to growth in the urinary tract environment, I created deletion mutants of the nine master transcription factors that govern biofilm formation.  This was done in both SC5314 and a strong biofilm forming CA isolate, HUC110.  While some regulators defined in other conditions, including Rob1 and Flo8, are required for biofilm formation on silicone urinary catheters, others, such as Bcr1 and Ndt80, are not.  This suggests that there is substantial specialization of this complex regulatory network to specific environmental conditions.  Additionally, this regulatory architecture was not identical between SC5314 and HUC110, emphasizing the strain variation that exists within this species.  I then performed mRNA-sequencing on each of the SC5314 regulator mutants grown on silicone catheters in human urine, identifying the contribution of each regulator to the total urinary biofilm transcriptional program. 

I screened the Homann library of 143 transcription factor deletion mutants for defects in biofilm growth on silicone urinary catheters and found several of these regulators to be uniquely important for CAUTI biofilm development, demonstrating the niche specificity of these structures.  Hits from the screen included Rgt1, a regulator of glucose transporters, and Mrr1, a regulator extensively studied for its role in drug efflux and antifungal resistance. 

Mrr1, and therefore antifungal resistance, was implicated as being key to CAUTI biofilm development.  The Mrr1 regulome was enriched in the transcriptomes of SC5314 and the two CA isolates grown in AUM.  Loss of MRR1 reduces fitness for HUC110 grown in AUM, and all but one of the CA isolates contain variant alleles of MRR1 previously identified in fluconazole-resistant clinical isolates. I tested the panel of CA isolates for antifungal susceptibility and found that several were susceptible to fluconazole when grown in RPMI but were resistant to the drug when grown in AUM or human urine. 

Together, these findings reveal a urine-induced transcriptional program in C. albicans that couples biofilm formation to a form of fluconazole resistance only manifest under host-relevant growth conditions. 

Advisory Committee:

• Michael C. Lorenz, PhD, Chair
• Jennifer N. Walker, PhD, Co-Chair
• Shane R. Cunha, PhD
• Danielle A. Garsin, PhD
• J. Christian Perez, PhD

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