Bladder Pain-Associated Candida Exhibit Increased Virulence and Urothelial Toxicity

Objectives: Urinary fungi have been implicated in bladder pain, with higher levels seen during periods of pain flares. Our hypotheses are three-fold: (1) urinary fungal burden increases in subjects with bladder pain; (2) bladder pain-associated urinary Candida albicans isolates induce greater urothelial cytotoxicity in vitro; and (3) bladder pain-associated C. albicans are associated with increased filamentation and expression of virulence-associated genes in vitro relative to controls.

Methods: Urinary fungal communities in catheterized urine samples of subjects with interstitial cystitis/bladder pain syndrome and controls were examined using next-generation sequencing of the ITS1 locus. Levels of urinary fungi and Candida species were calculated by quantitative polymerase chain reaction (qPCR). Urinary fungal strains were then isolated from subjects with bladder pain, myofascial pelvic pain, and asymptomatic controls (determined by provider assessment and patient responses to standardized questionnaires). In vitro co-cultures of human urothelial cells and Candida albicans isolates from subjects with and without bladder pain examined Candida-induced cytotoxicity and were compared using the t-test statistic and one-way ANOVA. Filamentation potential was quantitated using phase contrast microscopy. RNA sequencing using the NGS Illumina platform was performed for each Candida albicans isolate at a coverage of 25 million paired end reads per sample. Differential expression analysis between pain-associated isolates and controls was performed using edgeR’s exact test. Reverse transcriptase qPCR (RT-qPCR) of known gene products was performed to confirm transcriptomics results.

Results: qPCR demonstrated increased fungal burden in subjects with bladder pain in comparison to controls. Using linear regression analysis, bladder pain severity was directly proportional to Candida DNA relative abundance in urine (p < 0.001). When co-cultured with human urothelial cells in vitro, Candida albicans isolates from subjects with bladder pain were associated with significantly higher rates of urothelial cell death than isolates from subjects with myofascial pelvic pain or controls (p < 0.001). Bladder pain-associated albicans isolates exhibited higher filamentation than those from subjects with myofascial pelvic pain and controls (p < 0.001). Finally, analysis of the transcriptomic profiles of pain-associated isolates versus control isolates demonstrated differential expression of fourteen genes. SCW11, a cell wall glucanase associated with Candida filamentation, demonstrated seven-fold higher expression in pain-associated isolates (p=0.02), which was confirmed with RT-qPCR (p=0.03).

Conclusions: Higher levels of urinary fungi and Candida species were observed in subjects with bladder pain. Bladder pain-associated Candida albicans isolates exhibited increased virulence, inducing higher levels of urothelial cytotoxicity and filamentation in vitro than isolates derived from subjects with myofascial pelvic pain and controls. Transcriptomic analysis demonstrated multi-fold higher expression of SCW11, a Candida albicans virulence gene associated with filamentation, in bladder pain-associated isolates. These findings confirm existing data that correlate increased fungal burden during bladder pain flares and provide pathogen-specific mechanisms to explain, at least partially, the association of urinary fungi and bladder pain.