Critical Role of Bcr1-Dependent Adhesins in C. albicans Biofilm Formation In Vitro and In Vivo

et al. (2006) Critical role of Bcr1-dependent adhesins in C. albicans biofilm formation in vitro and in vivo. PLoS Pathog 2(7): e63. DOI: 10.1371/journal.ppat.0020063 Critical Role of Bcr1-Dependent Adhesins in C. albicans Biofilm Formation In Vitro and In Vivo Clarissa J. Nobile 0 1 David R. Andes 0 1 Jeniel E. Nett 0 1 Frank J. Smith 0 1 Jr. 0 1 Fu Yue 0 1 Quynh-Trang Phan 0 1 John E. Edwards 0 1 Jr. 0 1 Scott G. Filler 0 1 Aaron P. Mitchell 0 1 0 Editor: Alexander Johnson, University of California San Francisco , United States of America 1 1 Department of Microbiology, Columbia University , New York , New York, United States of America, 2 Biological Sciences Program, Department of Biological Sciences, Columbia University , New York , New York, United States of America, 3 Department of Medicine, Section of Infectious Diseases, University of Wisconsin, Madison, Wisconsin, United States of America, 4 Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California, United States of America, 5 The David Geffen School of Medicine, University of California Los Angeles , Los Angeles, California , United States of America The fungal pathogen Candida albicans is frequently associated with catheter-based infections because of its ability to form resilient biofilms. Prior studies have shown that the transcription factor Bcr1 governs biofilm formation in an in vitro catheter model. However, the mechanistic role of the Bcr1 pathway and its relationship to biofilm formation in vivo are unknown. Our studies of biofilm formation in vitro indicate that the surface protein Als3, a known adhesin, is a key target under Bcr1 control. We show that an als3/als3 mutant is biofilm-defective in vitro, and that ALS3 overexpression rescues the biofilm defect of the bcr1/bcr1 mutant. We extend these findings with an in vivo venous catheter model. The bcr1/bcr1 mutant is unable to populate the catheter surface, though its virulence suggests that it has no growth defect in vivo. ALS3 overexpression rescues the bcr1/bcr1 biofilm defect in vivo, thus arguing that Als3 is a pivotal Bcr1 target in this setting. Surprisingly, the als3/als3 mutant forms a biofilm in vivo, and we suggest that additional Bcr1 targets compensate for the Als3 defect in vivo. Indeed, overexpression of Bcr1 targets ALS1, ECE1, and HWP1 partially restores biofilm formation in a bcr1/bcr1 mutant background in vitro, though these genes are not required for biofilm formation in vitro. Our findings demonstrate that the Bcr1 pathway functions in vivo to promote biofilm formation, and that Als3-mediated adherence is a fundamental property under Bcr1 control. Known adhesins Als1 and Hwp1 also contribute to biofilm formation, as does the novel protein Ece1. - Biofilms are microbial communities that are associated with solid surfaces. Most bacteria and fungi exist predominantly in such communities in nature, and they form the basis for numerous interactions that affect human health. Cells in a biofilm display phenotypes that are distinct from their free-living counterparts, including extreme resistance to many antimicrobial agents [14]. Their health impact is reflected in the fact that implanted medical devices, such as intravascular catheters, are major risk factors for bloodstream and deep tissue infection [5, 6]. These devices serve as substrates for biofilm development; the mass and intrinsic drug resistance of the biofilm limits efficacy of host defenses and antimicrobial therapy. These biofilm-based infections are estimated to cause about 50% of all nosocomial infections [5, 7]. The fungal pathogen Candida albicans is a major cause of device-associated infections [5, 8, 9]. It produces adherent biofilms on a variety of different surfaces in vitro [3, 4, 10, 11]. Biofilm formation begins with surface adherence of yeastform cells, which grow to yield a basal layer. Basal layer cells include some hyphae, or long tubular chains of cells, which extend to yield an upper layer that is almost exclusively hyphae. As the biofilm matures, it produces an extracellular matrix containing predominantly carbohydrate and protein [1, 12, 13]. C. albicans Bcr1, a C2H2 zinc finger protein, has a significant role in biofilm formation: bcr1/bcr1 insertion and deletion mutants form only rudimentary biofilms on silicone catheter material in vitro [14]. Bcr1 is required for expression of several cell wall protein genes, and we have proposed that Bcr1 is a positive regulator of adherence. Many Bcr1 target genes had been identified initially as hyphal-specific genes, and BCR1 RNA accumulation depends upon the hyphal developmental activator Tec1 [14]. Bcr1 is not required for hyphal morphogenesis, and we believe that it acts downstream of Tec1 to activate the acquisition of hyphal adherence properties. Biofilms are considerably more complex in vivo than in vitro. For example, in vivo, biofilms form on intravascular catheters under conditions of vascular flow, and are exposed to and incorporate many plasma constituents. The complexThe formation of biofilms (surface-attached microbial communities) on implanted medical devices such as catheters is a major cause of fungal and bacterial infections. Prior studies of the fungal pathogen Candida albicans have shown that the regulator Bcr1 is required for biofilm formation in vitro, but the mechanism through which it promotes biofilm formation and its significance for biofilm formation in vivo was uncertain. The authors demonstrate that Bcr1 is required for biofilm formation in vivo in a rat model of catheter-based infection. Manipulation of Bcr1 target genes through mutation and gene overexpression shows that the known surface adhesin Als3 has a pivotal role in biofilm formation and that adhesins Als1 and Hwp1 also contribute to biofilm formation. The results thus indicate that adherence is the key property regulated by Bcr1 and highlight a group of adhesins as potential therapeutic targets. ity involved in forming a biofilm in vivo underscores the question of whether the same mechanisms are required for biofilm formation in vitro as in vivo. Indeed, several fungal and bacterial mutants have medium-dependent biofilm defects in vitro [15, 16]. Thus, the functions of key regulators must be appraised in vivo in order to connect questions in developmental biology to answers in antimicrobial therapy. Recently developed animal models permit analysis of C. albicans biofilm formation in vivo. Central venous catheter infection models have been described for both rabbits [17] and rats [18]. These catheter surfaces are substrates for extensive biofilm formation, and biofilm cells on these substrates exhibit reduced antifungal susceptibility. These models further reflect the circumstances of human infection, in that the biofilm cells can lead to seeding and infection of organs [18]. In this report, we test the roles of Bcr1 target genes in biofilm formation in vitro. Our findings subst (...truncated)