The NDR/LATS Kinase Cbk1 Controls the Activity of the Transcriptional Regulator Bcr1 during Biofilm Formation in Candida albicans

et al. (2012) The NDR/LATS Kinase Cbk1 Controls the Activity of the Transcriptional Regulator Bcr1 during Biofilm Formation in Candida albicans. PLoS Pathog 8(5): e1002683. doi:10.1371/journal.ppat.1002683 The NDR/LATS Kinase Cbk1 Controls the Activity of the Transcriptional Regulator Bcr1 during Biofilm Formation in Candida albicans Pilar Gutie rrez-Escribano 0 Ute Zeidler 0 M. Bele n Sua rez 0 Sophie Bachellier-Bassi 0 Andre s Clemente- Blanco 0 Julie Bonhomme 0 Carlos R. Va zquez de Aldana 0 Christophe d'Enfert 0 Jaime Correa- 0 Bordes 0 Anita Sil, UCSF, United States of America 0 1 Departamento Ciencias Biome dicas, Universidad de Extremadura , Badajoz , Spain , 2 Institut Pasteur, Unite Biologie et Pathoge nicite Fongiques, De partement Ge nomes et Ge ne tique , Paris, France, 3 INRA, USC2019, Paris , France , 4 Instituto de Biolog a Funcional y Gen o mica, CSIC-Universidad de Salamanca , Salamanca , Spain , 5 Cell Cycle Group, MRC Clinical Sciences Centre, Imperial College , London , United Kingdom In nature, many microorganisms form specialized complex, multicellular, surface-attached communities called biofilms. These communities play critical roles in microbial pathogenesis. The fungal pathogen Candida albicans is associated with catheter-based infections due to its ability to establish biofilms. The transcription factor Bcr1 is a master regulator of C. albicans biofilm development, although the full extent of its regulation remains unknown. Here, we report that Bcr1 is a phosphoprotein that physically interacts with the NDR kinase Cbk1 and undergoes Cbk1-dependent phosphorylation. Mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to alanine markedly impaired Bcr1 function during biofilm formation and virulence in a mouse model of disseminated candidiasis. Cells lacking Cbk1, or any of its upstream activators, also had reduced biofilm development. Notably, mutating the two putative Cbk1 phosphoacceptor residues in Bcr1 to glutamate in cbk1D cells upregulated the transcription of Bcr1-dependent genes and partially rescued the biofilm defects of a cbk1D strain. Therefore, our data uncovered a novel role of the NDR/LATS kinase Cbk1 in the regulation of biofilm development through the control of Bcr1. - Funding: This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2009-11251 to J. C.-B. and BFU2010-15884 to C.R.V.) and the Regional Government of Extremadura (PRI08A017, GRU09001 and GRU10008) to J. C.-B. All Spanish funding was co-sponsored by the European Union FEDER programme. P.G-E. was supported by a pre-doctoral fellowship (FPU program) from the Spanish Government. Work in the C. dE. laboratory was supported by the European Commission (FINSysB, PITN-GA-2008-214004) and Agence Nationale de la Recherche (KANJI, ANR-08-MIE-033-01). U.Z. was a recipient of post-doctoral fellowship of Institut Carnot Pasteur Maladies Infectieuses (Programme Fungi). 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. Biofilms are surface-attached microbial communities embedded in an extracellular matrix. Cells in a biofilm exhibit phenotypic properties different from those of their planktonic counterparts, including an increased resistance to the host immune system and to antimicrobial agents [13]. Several tissues, as genitourinary or oral epithelia, and biomedical devices can serve as substrates for biofilm development. In this context, biofilm formation is a key feature in microbial pathogenesis. Among the pathogenic fungi, C. albicans is one of the organisms most commonly associated with implant-related infections [2,4,5]. C. albicans is a polymorphic fungus that can change between three different forms: yeast, pseudohyphae and hyphae. Morphogenetic transitions are critical for the acquisition of proper biofilm architecture: initially, a basal layer of cells is formed when yeast cells attach to a surface followed by cell division and proliferation. In a second phase, cells differentiate into hyphal and pseudohyphal forms and produce extracellular material; the development of these forms and the increase in extracellular matrix deposition would finally arise in a dense and mature biofilm structure. Genes required for hyphal morphogenesis, cell wall remodeling, amino acid and lipid metabolism and glycolytic processes have been involved in the progression of biofilm formation in C. albicans (for a review, see [6]). Notably, biofilm development requires the activation of specific transcription programs different from those of free-living planktonic cells [7]. Tec1, a hypha-specific gene regulator; Bcr1, required for the expression of different cell wall proteins and Zap1, which governs matrix production, are examples of C. albicans biofilm transcriptional regulators [811]. In particular, Bcr1 has been shown to regulate the expression of a subset of genes encoding cell wall-anchored proteins including members of the agglutinin-like protein family, such as Als1 and Als3, and the hyphal wall protein Hwp1 [9,12]. Deletion of BCR1 results in defective biofilm formation in vivo and in vitro because of altered cell-to-cell interactions mediated by Als1, Als3 and Hwp1 [9]. The RAM signaling network is a conserved pathway that controls cell separation, polarized growth and cell integrity in yeast [1317]. In Saccharomyces cerevisiae, the central core of the pathway C. albicans infections frequently involve the formation of biofilms on implanted devices such as indwelling catheters. These complex communities of surface-associated fungal cells embedded in a matrix of extracellular polysaccharides protect C. albicans from host defences and antifungal agents. In recent years, several genes involved in the development of biofilms of C. albicans have been identified. These studies have uncovered complex regulatory networks that control the activity of several transcription factors during different steps of biofilm development. Bcr1 is a transcription factor that plays a major role in this process and yet, its regulation has not been studied extensively. Here, we show that Bcr1 function in biofilm formation and virulence requires phosphorylation of threonine 191 and serine 556 by the NDR/LATS kinase Cbk1. Moreover, given that Cbk1 is also required for the onset and maintenance of hyphal growth, our study highlights this kinase as a pivotal regulator of several developmental programs that are essential for the biology and pathogenesis of C. albicans. consists of the Cbk1 kinase, a member of the NDR/LATS kinase family, its binding partner Mob2, the scaffolding protein Tao3, and the Ste20-like kinase Kic1. The activity of Cbk1, the main effector of the RAM pathway, is regulated by phosphorylation in a Kic1- and Tao3-dependent manner [18]. While Cbk1 polarity targets still remain largely u (...truncated)