The Protein Kinase Tor1 Regulates Adhesin Gene Expression in Candida albicans

Citation: Bastidas RJ, Heitman J, Cardenas ME ( The Protein Kinase Tor1 Regulates Adhesin Gene Expression in Candida albicans Robert J. Bastidas 0 Joseph Heitman 0 Maria E. Cardenas 0 Scott G. Filler, David Geffen School of Medicine at University of California Los Angeles, United States of America 0 1 Department of Molecular Genetics and Microbiology, Duke University Medical Center , Durham , North Carolina, United States of America, 2 Department of Medicine, Duke University Medical Center , Durham , North Carolina, United States of America, 3 Department of Pharmacology and Cancer Biology, Duke University Medical Center , Durham, North Carolina , United States of America Eukaryotic cell growth is coordinated in response to nutrient availability, growth factors, and environmental stimuli, enabling cell-cell interactions that promote survival. The rapamycin-sensitive Tor1 protein kinase, which is conserved from yeasts to humans, participates in a signaling pathway central to cellular nutrient responses. To gain insight into Tormediated processes in human fungal pathogens, we have characterized Tor signaling in Candida albicans. Global transcriptional profiling revealed evolutionarily conserved roles for Tor1 in regulating the expression of genes involved in nitrogen starvation responses and ribosome biogenesis. Interestingly, we found that in C. albicans Tor1 plays a novel role in regulating the expression of several cell wall and hyphal specific genes, including adhesins and their transcriptional repressors Nrg1 and Tup1. In accord with this transcriptional profile, rapamycin induced extensive cellular aggregation in an adhesin-dependent fashion. Moreover, adhesin gene induction and cellular aggregation of rapamycin-treated cells were strongly dependent on the transactivators Bcr1 and Efg1. These findings support models in which Tor1 negatively controls cellular adhesion by governing the activities of Bcr1 and Efg1. Taken together, these results provide evidence that Tor1mediated cellular adhesion might be broadly conserved among eukaryotic organisms. - Funding: These studies were supported by RO1 grant CA114107 from the National Cancer Institute to MEC and RO1 grant AI50438 from the National Institute of Allergy and Infectious Diseases/National Institutes of Health to MEC and JH. Competing Interests: The authors have declared that no competing interests exist. Coordinated cellcell adhesion is an essential biological process widely employed by organisms throughout the tree of life. In metazoans, cellular adhesion is important for numerous processes ranging from establishment of body plans and maintenance of differentiated tissues to regulation of cancer progression (reviewed in [1,2]). Bacterial and fungal species commonly rely on cellular adhesion during mating and conjugation and for maintenance of multicellular biofilms that function as anchored shields against foreign attack by antimicrobial agents. In essence, cellular adhesion has driven important evolutionary benefits across kingdoms, ranging from the evolution of multicellularity in metazoans to drug resistance in bacteria and fungi. Adhesion plays major roles in virulence-associated traits of several fungal pathogens. In Candida albicans, the most pervasive human fungal pathogen, cellular adhesion is essential for biofilm development. C. albicans biofilms form on both biotic and abiotic surfaces (such as tissues, plastic prosthesis, dentures and catheters) [3] and function as reservoirs of infective cells that among immunocompromised individuals can cause deep seated and often fatal mycosis [4]. The typical architecture of a biofilm consists of mixed layers of intertwined yeast and hyphal cells stabilized by adhesive interactions among neighboring cells. These cellcell adhesive interactions are mediated by a set of cell surface displayed adhesins, including the Als proteins and the cell wall protein Hwp1 [5,6,7,8]. The ALS genes ALS1 and ALS3, two of eight ALS family members, are required for adherent interactions during biofilm formation in both in-vitro and in-vivo models of catheter biofilm formation and appear to have redundant functions [6]. HWP1, which codes for a cell surface glycoprotein targeted by mammalian transglutaminase that links Hwp1 to proteins on the mammalian cell surface (reviewed in [9]), surprisingly is also required for cell adhesive interactions during biofilm formation [6,7]. Notably, Als1, Als3 and Hwp1 play complementary roles during biofilm formation suggesting that they might interact to promote adhesion between adjacent cellular surfaces [8]. Adhesin regulation in C. albicans occurs primarily at the transcriptional level. During biofilm formation, expression of ALS1, ALS3 and HWP1 is regulated by the transcription factor Bcr1 [5]. Additional factors, such as the transcription factors Tec1, and the repressors Nrg1 and Tup1, have also been implicated in regulating adhesin expression [10,11,12,13,14,15,16]. Furthermore, both ALS3 and HWP1 are developmentally regulated and exclusively expressed in C. albicans hyphae [17,18]. This level of regulation falls under the domain of the cAMP-protein kinase-A signaling pathway that regulates yeast-hypha morphogenesis via the transcription factor Efg1 in response to nutritional and environmental cues [19,20]. However, aside from the cAMP signaling pathway, little is known about additional molecular pathways that transduce nutritional signals to the multiple transcriptional regulators governing adhesin expression. Living organisms have an intrinsic ability to coordinate their growth and proliferation in response to nutrient availability. In organisms ranging from yeasts to humans, the Tor1 signaling pathway responds to nutrient-derived signals and orchestrates cell growth. Accordingly, we find that in the human fungal pathogen Candida albicans, Tor1 signaling also functions to promote growth. We also uncovered a novel role for the Tor1 molecular pathway in promoting hyphal growth of C. albicans on semi-solid surfaces and in controlling cellcell adherence. Gene expression analysis and genetic manipulations implicate the known cell surface adhesins Als1 and Als3 as mediators of Tor1-regulated cellular adhesion. Further genetic analysis identified the transcriptional regulators Bcr1, Efg1, Nrg1, and Tup1 that together with Tor1 compose a regulatory network governing adhesin gene expression and cellular adhesion. Given that the Tor pathway is the target of several small molecule inhibitors including rapamycin, a versatile pharmacological drug used in medicine, there is considerable interest in Tor signaling pathways and their function. Moreover, given the potent fungicidal activity of rapamycin against C. albicans, novel antifungal therapies remain to be developed, which may also include novel antifungal therapies with less immunosuppressive rapamycin analogs. Our understanding of signaling networks regulating virulence traits in C. albica (...truncated)