Rapid evolutionary repair by secondary perturbation of a primary disrupted transcriptional network.

The discrete steps of transcriptional rewiring have been proposed to occur neutrally to ensure steady gene expression under stabilizing selection. A conflict-free switch of a regulon between regulators may require an immediate compensatory evolution to minimize deleterious effects. Here, we perform an evolutionary repair experiment on the Lachancea kluyveri yeast sef1Δ mutant using a suppressor development strategy. Complete loss of SEF1 forces cells to initiate a compensatory process for the pleiotropic defects arising from misexpression of TCA cycle genes. Using different selective conditions, we identify two adaptive loss-of-function mutations of IRA1 and AZF1. Subsequent analyses show that Azf1 is a weak transcriptional activator regulated by the Ras1-PKA pathway. Azf1 loss-of-function triggers extensive gene expression changes responsible for compensatory, beneficial, and trade-off phenotypes. The trade-offs can be alleviated by higher cell density. Our results not only indicate that secondary transcriptional perturbation provides rapid and adaptive mechanisms potentially stabilizing the initial stage of transcriptional rewiring but also suggest how genetic polymorphisms of pleiotropic mutations could be maintained in the population.

Prognostic Value of Body Surface Area-Adjusted Oxygen Uptake Efficiency Slope in Heart Failure Patients.

Background: COVID-19 has been associated with a higher risk of developing heart failure (HF). Among the parameters derived from cardiopulmonary exercise testing (CPET), oxygen uptake efficiency slope (OUES) has become one of the most important parameters for predicting the prognosis of HF patients. However, the clinical utilization of OUES is limited owing to its variation with patient height and weight. This study aimed to evaluate the prognostic value of body surface area-adjusted OUES (OUES/BSA) in adults with HF. Methods: Thirty-six HF patients (mean age, 57 ± 12 years; 30 men) undergoing CPET between July 2019 and May 2020 who were followed up for 12 months were enrolled. The endpoints were major cardiovascular (CV) events, including hospitalization due to acute decompensated HF, left ventricular assist device implantation, heart transplantation, and cardiovascular-related death. We analyzed the correlations between clinical/CPET variables and major CV events. Results: Among the analyzed CPET variables, OUES/BSA had better correlation with maximal oxygen uptake (VO2max) than other variables. In univariate Cox proportional analysis, OUES/BSA and peak VO2 were both significant independent prognostic factors. The cutoff value of OUES/BSA was 595 ml/min/m2 with an area under the curve of 0.929. The patients with OUES/BSA < 595 ml/min/m2 had a lower CV event-free survival rate at 12 months of follow-up compared with the other group (33.3% and 100%, respectively; log-rank test, p < 0.001). Conclusions: BSA-adjusted OUES is an effective independent predictor for prognosis in HF patients and can be an alternative to peak VO2 for risk stratification in HF patients, regardless of exercise intensity. However, further large-scale studies are required to validate our findings.

Plastic Rewiring of Sef1 Transcriptional Networks and the Potential of Nonfunctional Transcription Factor Binding in Facilitating Adaptive Evolution.

Prior and extensive plastic rewiring of a transcriptional network, followed by a functional switch of the conserved transcriptional regulator, can shape the evolution of a new network with diverged functions. The presence of three distinct iron regulatory systems in fungi that use orthologous transcriptional regulators suggests that these systems evolved in that manner. Orthologs of the transcriptional activator Sef1 are believed to be central to how iron regulatory systems developed in fungi, involving gene gain, plastic network rewiring, and switches in regulatory function. We show that, in the protoploid yeast Lachancea kluyveri, plastic rewiring of the L. kluyveri Sef1 (Lk-Sef1) network, together with a functional switch, enabled Lk-Sef1 to regulate TCA cycle genes, unlike Candida albicans Sef1 that mainly regulates iron-uptake genes. Moreover, we observed pervasive nonfunctional binding of Sef1 to its target genes. Enhancing Lk-Sef1 activity resuscitated the corresponding transcriptional network, providing immediate adaptive benefits in changing environments. Our study not only sheds light on the evolution of Sef1-centered transcriptional networks but also shows the adaptive potential of nonfunctional transcription factor binding for evolving phenotypic novelty and diversity.

Diverse Hap43-independent functions of the Candida albicans CCAAT-binding complex.

The CCAAT motif is ubiquitous in promoters of eukaryotic genomes. The CCAAT-binding complex (CBC) is conserved across a wide range of organisms, specifically recognizes the CCAAT motif, and modulates transcription directly or in cooperation with other transcription factors. In Candida albicans, CBC is known to interact with the repressor Hap43 to negatively regulate iron utilization genes in response to iron deprivation. However, the extent of additional functions of CBC is unclear. In this study, we explored new roles of CBC in C. albicans and found that CBC pleiotropically regulates many virulence traits in vitro, including negative control of genes responsible for ribosome biogenesis and translation and positive regulation of low-nitrogen-induced filamentation. In addition, C. albicans CBC is involved in utilization of host proteins as nitrogen sources and in repression of cellular flocculation and adhesin gene expression. Moreover, our epistasis analyses suggest that CBC acts as a downstream effector of Rhb1-TOR signaling and controls low-nitrogen-induced filamentation via the Mep2-Ras1-protein kinase A (PKA)/mitogen-activated protein kinase (MAPK) pathway. Importantly, the phenotypes identified here are all independent of Hap43. Finally, deletion of genes encoding CBC components slightly attenuated C. albicans virulence in both zebrafish and murine models of infection. Our results thus highlight new roles of C. albicans CBC in regulating multiple virulence traits in response to environmental perturbations and, finally, suggest potential targets for antifungal therapies as well as extending our understanding of the pathogenesis of other fungal pathogens.

Candida albicans Hap43 is a repressor induced under low-iron conditions and is essential for iron-responsive transcriptional regulation and virulence.

Candida albicans is an opportunistic fungal pathogen that exists as normal flora in healthy human bodies but causes life-threatening infections in immunocompromised patients. In addition to innate and adaptive immunities, hosts also resist microbial infections by developing a mechanism of "natural resistance" that maintains a low level of free iron to restrict the growth of invading pathogens. C. albicans must overcome this iron-deprived environment to cause infections. There are three types of iron-responsive transcriptional regulators in fungi; Aft1/Aft2 activators in yeast, GATA-type repressors in many fungi, and HapX/Php4 in Schizosaccharomyces pombe and Aspergillus species. In this study, we characterized the iron-responsive regulator Hap43, which is the C. albicans homolog of HapX/Php4 and is repressed by the GATA-type repressor Sfu1 under iron-sufficient conditions. We provide evidence that Hap43 is essential for the growth of C. albicans under low-iron conditions and for C. albicans virulence in a mouse model of infection. Hap43 was not required for iron acquisition under low-iron conditions. Instead, it was responsible for repression of genes that encode iron-dependent proteins involved in mitochondrial respiration and iron-sulfur cluster assembly. We also demonstrated that Hap43 executes its function by becoming a transcriptional repressor and accumulating in the nucleus in response to iron deprivation. Finally, we found a connection between Hap43 and the global corepressor Tup1 in low-iron-induced flavinogenesis. Taken together, our data suggest a complex interplay among Hap43, Sfu1, and Tup1 to coordinately regulate iron acquisition, iron utilization, and other iron-responsive metabolic activities.

Enforcement of Postzygotic Species Boundaries in the Fungal Kingdom.

Understanding the molecular basis of speciation is a primary goal in evolutionary biology. The formation of the postzygotic reproductive isolation that causes hybrid dysfunction, thereby reducing gene flow between diverging populations, is crucial for speciation. Using various advanced approaches, including chromosome replacement, hybrid introgression and transcriptomics, population genomics, and experimental evolution, scientists have revealed multiple mechanisms involved in postzygotic barriers in the fungal kingdom. These results illuminate both unique and general features of fungal speciation. Our review summarizes experiments on fungi exploring how Dobzhansky-Muller incompatibility, killer meiotic drive, chromosome rearrangements, and antirecombination contribute to postzygotic reproductive isolation. We also discuss possible evolutionary forces underlying different reproductive isolation mechanisms and the potential roles of the evolutionary arms race under the Red Queen hypothesis and epigenetic divergence in speciation.

Experimental evolution improves mitochondrial genome quality control in Saccharomyces cerevisiae and extends its replicative lifespan.

The mitochondrion is an ancient endosymbiotic organelle that performs many essential functions in eukaryotic cells.1-3 Mitochondrial impairment often results in physiological defects or diseases.2-8 Since most mitochondrial genes have been copied into the nuclear genome during evolution,9 the regulatory and interaction mechanisms between the mitochondrial and nuclear genomes are very complex. Multiple mechanisms, including antioxidant, DNA repair, mitophagy, and mitochondrial biogenesis pathways, have been shown to monitor the quality and quantity of mitochondria.10-12 Nonetheless, it remains unclear if these pathways can be further modified to enhance mitochondrial stability. Previously, experimental evolution has been used to adapt cells to novel growth conditions. By analyzing the resulting evolved populations, insights have been gained into the underlying molecular mechanisms.13 Here, we experimentally evolved yeast cells under conditions that selected for efficient respiration while continuously assaulting the mitochondrial genome (mtDNA) with ethidium bromide (EtBr). We found that the ability to maintain functional mtDNA was enhanced in most of the evolved lines when challenged with mtDNA-damaging reagents. We identified mutations of the mitochondrial NADH dehydrogenase NDE1 in most of the evolved lines, but other pathways are also involved. Finally, we show that cells displaying enhanced mtDNA retention also exhibit a prolonged replicative lifespan. Our work reveals potential evolutionary trajectories by which cells can maintain functional mitochondria in response to mtDNA stress, as well as the physiological implications of such adaptations.

Zebrafish as a model host for Candida albicans infection.

In this work, the zebrafish model organism was developed to obtain a minivertebrate host system for a Candida albicans infection study. We demonstrated that C. albicans can colonize and invade zebrafish at multiple anatomical sites and kill the fish in a dose-dependent manner. Inside zebrafish, we monitored the progression of the C. albicans yeast-to-hypha transition by tracking morphogenesis, and we monitored the corresponding gene expression of the pathogen and the early host immune response. We performed a zebrafish survival assay with different C. albicans strains (SC5314, ATCC 10231, an hgc1 mutant, and a cph1/efg1 double mutant) to determine each strain's virulence, and the results were similar to findings reported in previous mouse model studies. Finally, using zebrafish embryos, we monitored C. albicans infection and visualized the interaction between pathogen and host myelomonocytic cells in vivo. Taken together, the results of this work demonstrate that zebrafish can be a useful host model to study C. albicans pathogenesis, and they highlight the advantages of using the zebrafish model in future invasive fungal research.

Dynamic transcript profiling of Candida albicans infection in zebrafish: a pathogen-host interaction study.

Candida albicans is responsible for a number of life-threatening infections and causes considerable morbidity and mortality in immunocompromised patients. Previous studies of C. albicans pathogenesis have suggested several steps must occur before virulent infection, including early adhesion, invasion, and late tissue damage. However, the mechanism that triggers C. albicans transformation from yeast to hyphae form during infection has yet to be fully elucidated. This study used a systems biology approach to investigate C. albicans infection in zebrafish. The surviving fish were sampled at different post-infection time points to obtain time-lapsed, genome-wide transcriptomic data from both organisms, which were accompanied with in sync histological analyses. Principal component analysis (PCA) was used to analyze the dynamic gene expression profiles of significant variations in both C. albicans and zebrafish. The results categorized C. albicans infection into three progressing phases: adhesion, invasion, and damage. Such findings were highly supported by the corresponding histological analysis. Furthermore, the dynamic interspecies transcript profiling revealed that C. albicans activated its filamentous formation during invasion and the iron scavenging functions during the damage phases, whereas zebrafish ceased its iron homeostasis function following massive hemorrhage during the later stages of infection. Most of the immune related genes were expressed as the infection progressed from invasion to the damage phase. Such global, inter-species evidence of virulence-immune and iron competition dynamics during C. albicans infection could be crucial in understanding control fungal pathogenesis.