Occhipinti, P. | Search bioRxiv

bioRxiv2020

Balancing positive and negative selection: in vivo evolution of Candida lusitaniae MRR1

The evolution of pathogens in response to selective pressures present during chronic infections can influence persistence, virulence, and the outcomes of antimicrobial therapy. Because subpopulations within an infection can be spatially separated and the host environment can fluctuate, an appreciation of the pathways under selection may be most easily revealed through the analysis of numerous isolates from single infections. Here, we continued our analysis of a set of clonally-derived Clavispora (Candida) lusitaniae isolates from a single chronic lung infection with a striking enrichment in the number of alleles of MRR1. Genetic and genomic analyses found evidence for repeated acquisition of gain-of-function mutations that conferred constitutive Mrr1 activity. In the same population, there were multiple alleles with both gain-of-function mutations and secondary suppressor mutations that either attenuated or abolished the constitutive activity suggesting the presence of counteracting selective pressures. Our studies demonstrated tradeoffs between high Mrr1 activity, which confers resistance to the antifungal fluconazole, host factors, and bacterial products through its regulation of MDR1, and resistance to hydrogen peroxide, a reactive oxygen species produced in the neutrophilic environment associated with this infection. This inverse correlation between high Mrr1 activity and hydrogen peroxide resistance was observed in multiple Candida species and in serial analysis of populations from this individual collected over three years. These data lead us to propose that dynamic or variable selective pressures can be reflected in population genomics and that these dynamics can complicate the drug resistance profile of the population. ImportanceUnderstanding microbial evolution within patients is critical for managing chronic infections and understanding host-pathogen interactions. Here, our analysis of multiple MRR1 alleles in isolates from a single Clavispora (Candida) lusitaniae infection revealed the selection for both high and low Mrr1 activity. Our studies reveal tradeoffs between high Mrr1 activity, which confers resistance to the commonly used antifungal fluconazole, host antimicrobial peptides and bacterial products, and resistance to hydrogen peroxide. This work suggests that spatial or temporal differences within chronic infections can support a large amount of dynamic and parallel evolution, and that Mrr1 activity is under both positive and negative selective pressure to balance different traits that are important for microbial survival.

microbiology↗

bioRxiv2020

Conditional antagonism in co-cultures of Pseudomonas aeruginosa and Candida albicans: an intersection of ethanol and phosphate signaling distilled from dual-seq transcriptomics

Pseudomonas aeruginosa and Candida albicans are opportunistic pathogens whose interactions involve the secreted products ethanol and phenazines. Here we describe the focal role of ethanol in mixed-species co-cultures by dual RNA-seq analyses. P. aeruginosa and C. albicans transcriptomes were assessed after growth in mono-culture or co-culture with either ethanol-producing C. albicans or a C. albicans mutant lacking the primary ethanol dehydrogenase, Adh1. Analyses using KEGG-pathways and the previously published eADAGE method revealed several P. aeruginosa responses to C. albicans-produced ethanol including the induction of a non-canonical low phosphate response mediated by PhoB. C. albicans wild-type, but not C. albicans adh1{Delta}/{Delta}, induces P. aeruginosa production of 5-methyl-phenazine-1-carboxylic acid (5-MPCA), which forms a red derivative within fungal cells. We first demonstrate that PhoB is required for this interaction and that PhoB hyperactivity, via deletion of pstB, leads to increased production of 5-MPCA even when phosphate concentrations are high, but only in the presence of ethanol. Second, we show that ethanol is only sufficient to promote 5-MPCA production at permissive phosphate concentrations. The intersection of ethanol and phosphate in co-culture is mirrored in C. albicans; the adh1{Delta}/{Delta} mutant had increased expression of genes regulated by Pho4, the C. albicans transcription factor that responds to low phosphate which we confirmed by showing the adh1{Delta}/{Delta} strain had elevated Pho4-dependent phosphatase activity. The dual-dependence on ethanol and phosphate concentrations for anti-fungal production highlights how environmental factors modulate microbial interactions and dictate antagonisms such as those between P. aeruginosa and C. albicans. Author SummaryPseudomonas aeruginosa and Candida albicans are opportunistic pathogens that are frequently isolated from co-infections. Using a Dual-Seq approach in combination with genetics approaches, we found that ethanol produced by C. albicans stimulates the PhoB regulon in P. aeruginosa asynchronously with activation of the Pho4 regulon in C. albicans. In doing so, we demonstrate that eADAGE-based analysis can improve the understanding of the P. aeruginosa response to ethanol-producing C. albicans as measured by transcriptomics: we identify a subset of PhoB-regulated genes as differentially expressed in response to ethanol. We validate our result by showing that PhoB is necessary for multiple roles in co-culture including the competition for phosphate and the production of 5-methyl-phenazine-1-carboxylic acid, and that the P. aeruginosa response to C. albicans-produced ethanol depends on phosphate availability. The conditional stimulation of virulence production in response to sub-inhibitory concentrations of ethanol only under phosphate limitation highlights the importance of considering nutrient concentrations in the analysis of co-culture interactions.

microbiology↗