Antifungal Drug Concentration Impacts the Spectrum of Adaptive Mutations in Candida albicans.

Invasive fungal infections are a leading global cause of human mortality. Only three major classes of antifungal drugs are widely used, and resistance to all three classes can arise rapidly. The most widely prescribed antifungal drug, fluconazole, disseminates rapidly and reaches a wide range of concentrations throughout the body. The impact of drug concentration on the spectrum and effect of mutations acquired during adaptation is not known for any fungal pathogen, and how the specific level of a given stress influences the distribution of beneficial mutations has been poorly explored in general. We evolved 144 lineages from three genetically distinct clinical isolates of Candida albicans to four concentrations of fluconazole (0, 1, 8, and 64 µg/ml) and performed comprehensive phenotypic and genomic comparisons of ancestral and evolved populations. Adaptation to different fluconazole concentrations resulted in distinct adaptive trajectories. In general, lineages evolved to drug concentrations close to their MIC50 (the level of drug that reduces growth by 50% in the ancestor) tended to rapidly evolve an increased MIC50 and acquired distinct segmental aneuploidies and copy number variations. By contrast, lineages evolved to drug concentrations above their ancestral MIC50 tended to acquire a different suite of mutational changes and increased in drug tolerance (the ability of a subpopulation of cells to grow slowly above their MIC50). This is the first evidence that different concentrations of drug can select for different genotypic and phenotypic outcomes in vitro and may explain observed in vivo drug response variation.

Porphyromonas somerae Invasion of Endometrial Cancer Cells.

Recent evidence suggests an association between endometrial cancer and the understudied bacterial species Porphyromonas somerae. This association was demonstrated in previous work that indicated a significantly enriched abundance of P. somerae in the uterine microbiome of endometrial cancer patients. Given the known associations of the Porphyromonas genus and oral cancer, we hypothesized that P. somerae may play a similar pathogenic role in endometrial cancer via intracellular activity. Before testing our hypothesis, we first characterized P. somerae biology, as current background data is limited. These novel characterizations include growth curves in liquid medium and susceptibility tests to antibiotics. We tested our hypothesis by examining growth changes in response to 17ß-estradiol, a known risk factor for endometrial cancer, followed by metabolomic profiling in the presence and absence of 17ß-estradiol. We found that P. somerae exhibits increased growth in the presence of 17ß-estradiol of various concentrations. However, we did not find significant changes in metabolite levels in response to 17ß-estradiol. To study direct host-microbe interactions, we used in vitro invasion assays under hypoxic conditions and found evidence for intracellular invasion of P. somerae in endometrial adenocarcinoma cells. We also examined these interactions in the presence of 17ß-estradiol but did not observe changes in invasion frequency. Invasion was shown using three lines of evidence including visualization via differential staining and brightfield microscopy, increased frequency of bacterial recovery after co-culturing, and in silico methods to detail relevant genomic and transcriptomic components. These results underscore potential intracellular phenotypes of P. somerae within the uterine microbiome. Furthermore, these results raise new questions pertaining to the role of P. somerae in the progression of endometrial cancer.

Fxr signaling and microbial metabolism of bile salts in the zebrafish intestine.

Bile salt synthesis, secretion into the intestinal lumen, and resorption in the ileum occur in all vertebrate classes. In mammals, bile salt composition is determined by host and microbial enzymes, affecting signaling through the bile salt-binding transcription factor farnesoid X receptor (Fxr). However, these processes in other vertebrate classes remain poorly understood. We show that key components of hepatic bile salt synthesis and ileal transport pathways are conserved and under control of Fxr in zebrafish. Zebrafish bile salts consist primarily of a C27 bile alcohol and a C24 bile acid that undergo multiple microbial modifications including bile acid deconjugation that augments Fxr activity. Using single-cell RNA sequencing, we provide a cellular atlas of the zebrafish intestinal epithelium and uncover roles for Fxr in transcriptional and differentiation programs in ileal and other cell types. These results establish zebrafish as a nonmammalian vertebrate model for studying bile salt metabolism and Fxr signaling.