Microevolution of Candida albicans in Macrophages Restores Filamentation in a Nonfilamentous Mutant

et al. (2014) Microevolution of Candida albicans in Macrophages Restores Filamentation in a Nonfilamentous Mutant. PLoS Genet 10(12): e1004824. doi:10.1371/journal.pgen.1004824 Microevolution of Candida albicans in Macrophages Restores Filamentation in a Nonfilamentous Mutant Anja Wartenberg 0 Jo rg Linde 0 Ronny Martin 0 Maria Schreiner 0 Fabian Horn 0 Ilse D. Jacobsen 0 Sabrina Jenull 0 Thomas Wolf 0 Karl Kuchler 0 Reinhard Guthke 0 Oliver Kurzai 0 Anja Forche 0 Christophe d'Enfert 0 Sascha Brunke 0 Bernhard Hube 0 Geraldine Butler, University College Dublin, Ireland 0 1 Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI) , Jena, Germany , 2 Research Group Systems Biology & Bioinformatics, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI) , Jena, Germany , 3 Septomics Research Center, Friedrich Schiller University and Leibniz Institute for Natural Product Research and Infection Biology -Hans Knoell Institute , Jena, Germany , 4 Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI) , Jena, Germany, 5 Medical University Vienna , Max F. Perutz Laboratories , Department of Medical Biochemistry , Vienna , Austria , 6 Department of Biology, Bowdoin College, Brunswick, Maine, United States of America, 7 Institut Pasteur, Unite Biologie et Pathoge nicite Fongiques, De partement Ge nomes et Ge ne tique , Paris, France, 8 INRA, USC2019, Paris , France , 9 Integrated Research and Treatment Center, Sepsis und Sepsisfolgen, Center for Sepsis Control and Care (CSCC), Universita tsklinikum Jena, Germany, 10 Friedrich Schiller University , Jena , Germany Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1D/efg1D mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1D/efg1D strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure. - Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. Data were deposited at the Gene Expression Omnibus (GSE56174) and can be found in S2 Table. Funding: This work was supported by the German Federal Ministry of Education and Health (BMBF, www.bmbf.de) Germany, FKZ: 01EO1002 - Integrated Research and Treatment Center, Center for Sepsis Control and Care (CSSC, www.cscc.uniklinikum-jena.de) and the DACH program of the DFG and FWF (DFG HU 528/17-1 & FWF-I-746-B11). AW and TW were also supported by the excellence graduate school Jena School for Microbial Communication (JSMC; www.jsmc.unijena.de) and the International Leibniz Research School for Microbial and Biomolecular Interactions (ILRS, www.ilrs.hki-jena.de), respectively. JL was supported by the Deutsche Forschungsgemeinschaft (DFG, www.dfg.de) CRC/Transregio 124 FungiNet-Pathogenic fungi and their human host: Networks of interaction (www. funginet.de), subproject INF. IDJ was supported by BMBF 0314108. AF was supported by the National Institute of Allergy and Infectious Diseases, grant R15 AI090633 02 (NIAID, www.niaid.nih.gov). Cd has received funding from the French Governments Investissement dAvenir program, Laboratoire dExcellence Integrative Biology of Emerging Infectious Diseases (Grant #ANR-10-LABX-62-IBEID). Work in the KK laboratory was supported by the Austrian Science Foundation FWF by a grant from the Christian Doppler Society. SJ was additionally supported by the FWF (project P-25333-B22). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The incidence of invasive fungal infections has steadily increased within the past decades, largely because of a growing population of susceptible individuals, reflecting the progress of modern medicine in prolonging life even with severe underlying diseases and the increasing rate of immuno-deficient patients. One of the most frequently isolated fungi is Candida albicans, an ubiquitous and normally harmless commensal of the alimentary tract and mucocutaneous membranes. As an opportunistic pathogen, it can cause superficial infections like oropharyngeal candidiasis, especially in HIV patients, as well as life-threatening Pathogenic microbes often evolve complex traits to adapt to their respective hosts, and this evolution is ongoing: for example, microorganisms are developing resistance to antimicrobial compounds in the clinical setting. The ability of the common human pathogenic fungus, Candida albicans, to switch from yeast to hyphal (filamentous) growth is considered a central virulence attribute. For example, hyphal formation allows C. albicans to escape from macrophages following phagocytosis. A well-investigated signaling network integrates different environmental cues to induce and maintain hyphal growth. In fact, deletion of two central transcription factors in this network results in a mutant that is both nonfilamentous and avirulent. We used experimental evolution to study the adaptation capability of this mutant by continuous coincubation within macrophages. We found that this selection regime led to a relatively rapid re-connection (...truncated)