Large-Scale Biochemical Profiling of the Candida albicans Biofilm Matrix: New Compositional, Structural, and Functional Insights Jose L. Lopez-Ribot Department of Biology and South Texas Center for Emerging Infectious Diseases, The University of Texas at San Antonio, San Antonio, Texas, USA
ABSTRACT Among pathogenic fungi, Candida albicans is most frequently associated with biofilm formation, a lifestyle that is
entirely different from the planktonic state. One of the distinguishing features of these biofilms is the presence of extracellular material, commonly referred to as the “biofilm matrix.” The fungal biofilm matrix embeds sessile cells within these communities and plays important structural and physiological functions, including antifungal drug resistance with important clinical repercussions. This matrix is mostly self-produced by the fungal cells themselves and is composed of different types of biopolymers. In C. albicans, the main components of the biofilm matrix are carbohydrates, proteins, lipids, and DNA, but many of them remain unidentified and/or poorly characterized. In their recent article, Zarnowski et al. [mBio 5(4):e01333-14, 2014, doi:10.1128/ mBio.01333-14] used a variety of biochemical and state-of-the-art “omic” approaches (glycomics, proteomics, and lipidomics) to identify and characterize unique biopolymers present in the C. albicans biofilm matrix. Besides generating a true “encyclopedic” catalog of individual moieties from each of the different macromolecular categories, results also provide important insights into structural and functional aspects of the fungal biofilm matrix, particularly the interaction between different components and the contribution of multiple matrix constituents to biofilm antifungal drug resistance.
C
andida albicans is a normal commensal of humans, frequently colonizing our skin and mucosae. However, as an opportunistic pathogen, it is fully capable of causing disease in an everexpanding population of immunosuppressed and medically compromised patients. Candidiasis is now the third- to fourth-mostcommon nosocomial infection in U.S. hospitals, with one of the highest mortality rates among nosocomial pathogens (1). These infections are often associated with the formation of biofilms, as different types of biomaterials used in the clinics support colonization and subsequent biofilm development by C. albicans (2). The increase in candidiasis during the last few decades has fundamentally paralleled the increase in the use of a variety of medical implant devices, most notably catheters, which serve as a substrate for biofilm formation. This in turn causes elevated levels of resistance to host defenses and antifungal agents, thereby complicating treatment and resulting in high morbidity and mortality rates. This biofilm lifestyle is the predominant mode of growth of microorganisms in nature and is radically different from the planktonic (free-living) state. Biofilms are highly structured consortia of microorganisms, attached to a substrate and encased within a matrix of hydrated exopolymeric material which is mostly produced by the organisms themselves. In fact, the presence of this extracellular matrix is one of the main defining attributes of the biofilm mode of growth: simply put, there is no “bona fide” biofilm without an extracellular matrix. Biofilm matrices consist of a conglomeration of different types of biopolymers, normally referred to as “exopolymeric substances” (EPS). Although the composition of the matrix varies among different microorganisms, in general, polysaccharides and proteins are the most common and abundant EPS components; however, other macromolecules such as lipids and nucleic acids seem to be almost universally present in biofilm matrices (3). The cohesive and adhesive forces of the matrix contribute to the architectural and mechanical stability of the biofilm by mediating stable cell-cell and cell-surface interactions, essentially acting as a glue that holds the cells together. The biofilm matrix acts as a protective barrier, preventing drugs from penetrating the biofilms, and as such, it
September/October 2014 Volume 5 Issue 5 e01781-14
represents one of the main contributing factors to resistance against anti-infective therapies. It also protects the cells within the biofilms against other environmental insults, including desiccation, radiation, oxidation, the action of predators and host immune defenses, and plays important nutritional and metabolic functions (3). The C. albicans biofilm matrix has been the focus of investigation since the late 1990s when studies pioneered by the Douglas research group described increased matrix production in biofilms formed under flow, investigated its contribution to antifungal drug resistance, and performed preliminary elemental analyses of matrix polymers (4, 5). To date, studies have identified polysaccharides, proteins, and nucleic acids as the main macromolecular components of the EPS in C. albicans (6–8). Several proteins and glycoproteins extracted from the biofilm matrix were identified using proteomic techniques (2-dimensional gel electrophoresis and mass spectrometry), but the study provided little insight into the functions of these proteinaceous materials (8). Regarding carbohydrates, in studies mostly by the Andes research group, one critical and perhaps the best-studied component of the C. albicans biofilm matrix, -1,3-glucan, has been reported to sequester antifungal molecules, basically acting as a “drug sponge” and conferring resistance to C. albicans biofilms (7, 9). More-recent studies have described the presence of extracellular DNA (eDNA) in the C. albicans biofilm EPS, which appears to play both a structural and protective role (6, 10). Despite this accumulated knowledge, the full composition and function of the C. albicans biofilm matrix still remain to be fully elucidated. Fully embracing this challenge, Published 9 September 2014 Citation Lopez-Ribot JL. 2014. Large-scale biochemical profiling of the Candida albicans biofilm matrix: new compositional, structural, and functional insights. mBio 5(5):e0178114. doi:10.1128/mBio.01781-14. Copyright © 2014 Lopez-Ribot. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to [email protected].
®
mbio.asm.org 1
Downloaded from mbio.asm.org on September 11, 2015 - Published by mbio.asm.org
crossmark
COMMENTARY
the recent article by Zarnowski et al. (11) constitutes a genuine tour de force for the comprehensive biochemical analysis of the biopolymers present in the matrix, facilitated by the use of stateof-the-art instrumentation and powerful glycomic, proteomic, and lipidomic techniques. In the process they uncover, identify, and catalog matrix components. Furthermore, their data suggest that different matrix components interact physically with each other. From a functional perspective, their findings indicate that multiple matrix constituents contribute to antifungal drug resistance and suggest that some matrix components may also play an important metabolic role in C. albicans biofilms. Isolation of the biofilm matrix can be challenging, and it is of critical importance to adapt the extraction procedure to the specific type of biofilm under investigation. For this work, Zarnowski and colleagues (11) used a gentle-sonication solubilization protocol mostly based on extraction methods previously described by others (5, 8) in order to ensure the lack of cell leakage or cell damage. Very importantly, they used electron microscopy techniques to convincingly demonstrate that their isolation methodology specifically removes matrix, leaves the fungal cells intact, and does not extract cell wall components. After purification, and consistent with previous analyses (6–8), Zarnowski et al. describe the overall composition of the C. albicans biofilm matrix, with representation of each of the four macromolecular classes, including proteins and glycoproteins (55% [wt/wt]), carbohydrates (~25% [wt/wt]), lipids (~15% [wt/wt]), and nucleic acids (mostly noncoding DNA) (~5% [wt/wt]). This study reported that proteins represented the main component, far exceeding the polysaccharide content on a mass basis (11). Using proteomic techniques, the authors identified a total of 565 different proteins in the matrix, including a few predicted to form part of the secretome, but also many secretion signal-less proteins, which suggests a noncanonical secretion pathway and/or the accumulation of proteins after cell death (12). According to functional ontology analyses, a total of 458 distinct activities and 16 different metabolic pathways were represented, with a predominance of enzymes involved in carbohydrate and amino acid metabolism. This is highly suggestive of a putative role of the matrix as an external digestive system and as a nutritional/energy source, as previously demonstrated in the case of bacterial biofilms (3). In addition, some enzymes can be potentially involved in the degradation of structural EPS to promote the dispersion of cells from biofilms. Another intriguing possibility is that some of these enzymes may act as virulence factors during biofilm-associated infections. Somewhat surprisingly, the authors did not identify cell wall-associated proteins (such as members of the agglutinin-like sequence [ALS] family) that have been shown to play important roles in cell-cell and cell-substrate interactions during biofilm growth (13). Thus, it would seem that there is a two-tier system for cohesive and adhesive interactions in C. albicans biofilms: one mediated by surface adhesins in the cell wall and a second one provided by the extracellular matrix itself. Interestingly, proteomic analysis of the matrix recovered from C. albicans biofilms grown in vivo demonstrated a very different proteinaceous composition, with the majority of proteins contributed by the host. Polysaccharides represent a relatively major fraction in most biofilm matrices, and C. albicans is no exception to this rule, with carbohydrates representing about 25% of its EPS by weight (11). The authors reported the highest degree of complexity associated with the exopolysaccharide fraction of the C. albicans biofilm ma-
2
®
mbio.asm.org
trix of all macromolecules, a not completely unexpected result. Monosaccharide analysis indicated that arabinose, mannose, glucose, and xylose constituted the four most abundant basic units of the total carbohydrate pool, although their relative abundance varied among high- and low-molecular-weight fractions prepared by size exclusion chromatography. Subsequent nuclear magnetic resonance (NMR) analysis of the different fractions detected three major polysaccharides that were similar to the main carbohydrate components of the C. albicans cell wall (14), although their relative abundance in the matrix was different. Somewhat surprisingly, -1,3-glucan, which represents the major cell wall polysaccharide and plays a major role in biofilm drug resistance comprised only a small portion of the total carbohydrate fraction of the EPS. Newly described and more abundant (about 87%) polysaccharides in the matrix included ␣-1,2 branched and ␣-1,6-mannans. Importantly, these two polysaccharides were associated with unbranched -1,6-glucans in an apparent mannan-glucan complex, thus indicating a physical association between glucan and mannan residues. Imaging techniques corroborated that the distribution of EPS polysaccharides was similar in in vitro and in vivo biofilms of C. albicans. Using lipidomics, the authors described that the matrix lipid profile included predominantly glycerolipids (99.5%) and sphingolipids (0.5%) (11). Different fatty acids were identified on these lipids, namely, oleic and linoleic acids (in the most abundant neutral glycerolipids) and palmitoleic, palmitic, and oleic acids (in the less abundant polar glycerolipids). Consistent with a previous report (15), small quantities of prostaglandin E2, a precursor of eicosanoids, were also found in the matrix, whereas ergosterol was the only sterol detected. Finally, and from a functional perspective, radiolabeling experiments and NMR studies demonstrated the interaction of the aggregate matrix with the antifungal fluconazole, further supporting the contribution of the matrix to biofilm drug resistance (11). Moreover, results clearly indicated that multiple matrix components contribute to antifungal drug resistance, which lead the authors to postulate that the C. albicans biofilm matrix displays “properties of an amalgam in which multicomponent interactions yield emergent properties” (11). The long-term challenge of studies of the biofilm matrix is colossal: to comprehensively define the identity, quantity, structure, interactions, and function(s) of complete complements of the different macromolecular components of the EPS, to understand their spatial and temporal distribution, and to characterize how they change in response to different physical, biological, and environmental conditions. Although many questions remain, the analyses and high-throughput methods described in this article represent a major step toward this end and without any doubt will serve as an excellent framework for future studies. Understanding the structure and function of the matrix components represents one auspicious path for the development of effective and much needed therapies against C. albicans biofilms. ACKNOWLEDGMENTS Work in the laboratory is supported by grants R01DE023510 and R03AI103295 from the National Institute of Dental and Craniofacial Research and National Institute of Allergy and Infectious Diseases, respectively. Additional support is provided by the Army Research Office of the Department of Defense under contract W911NF-11-1-0136. The funders had no role in study design, data collection, and analysis,
September/October 2014 Volume 5 Issue 5 e01781-14
Downloaded from mbio.asm.org on September 11, 2015 - Published by mbio.asm.org
Commentary
decision to publish, or preparation of the manuscript, and the content is solely the responsibility of the author.
9.
REFERENCES 1. Pfaller MA, Diekema DJ. 2007. Epidemiology of invasive candidiasis: a persistent public health problem. Clin. Microbiol. Rev. 20:133–163. http://dx.doi.org/10.1128/CMR.00029-06. 2. Ramage G, Martínez JP, López-Ribot JL. 2006. Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS Yeast Res. 6:979 –986. http://dx.doi.org/10.1111/j.1567-1364.2006.00117.x. 3. Flemming HC, Wingender J. 2010. The biofilm matrix. Nat. Rev. Microbiol. 8:623– 633. http://dx.doi.org/10.1038/nrmicro2415. 4. Al-Fattani MA, Douglas LJ. 2006. Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance. J. Med. Microbiol. 55:999 –1008. http://dx.doi.org/10.1099/jmm.0.46569-0. 5. Baillie GS, Douglas LJ. 2000. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J. Antimicrob. Chemother. 46:397– 403. http://dx.doi.org/10.1093/jac/46.3.397. 6. Martins M, Uppuluri P, Thomas DP, Cleary IA, Henriques M, LopezRibot JL, Oliveira R. 2010. Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms. Mycopathologia 169:323–331. http://dx.doi.org/10.1007/s11046-009-9264-y. 7. Nett J, Lincoln L, Marchillo K, Massey R, Holoyda K, Hoff B, VanHandel M, Andes D. 2007. Putative role of beta-1,3 glucans in Candida albicans biofilm resistance. Antimicrob. Agents Chemother. 51:510 –520. http://dx.doi.org/10.1128/AAC.01056-06. 8. Thomas DP, Bachmann SP, Lopez-Ribot JL. 2006. Proteomics for the
10.
11.
12. 13.
14. 15.
analysis of the Candida albicans biofilm lifestyle. Proteomics 6:5795–5804. http://dx.doi.org/10.1002/pmic.200600332. Nett JE, Crawford K, Marchillo K, Andes DR. 2010. Role of Fks1p and matrix glucan in Candida albicans biofilm resistance to an echinocandin, pyrimidine, and polyene. Antimicrob. Agents Chemother. 54:3505–3508. http://dx.doi.org/10.1128/AAC.00227-10. Martins M, Henriques M, Lopez-Ribot JL, Oliveira R. 2012. Addition of DNase improves the in vitro activity of antifungal drugs against Candida albicans biofilms. Mycoses 55:80 – 85. http://dx.doi.org/10.1111/j.14390507.2011.02047.x. Zarnowski R, Westler WM, Lacmbouh GA, Marita JM, Bothe JR, Bernhardt J, Sahraoui L-HA, Fontaine J, Sanchez H, Hatfield RD, Ntambi JM, Nett JE, Mitchell AP, Andes DR. 2014. Novel entries in a fungal biofilm matrix encyclopedia. mBio 5(4):e01333-14. http:// dx.doi.org/10.1128/mBio.01333-14. Nombela C, Gil C, Chaffin WL. 2006. Non-conventional protein secretion in yeast. Trends Microbiol. 14:15–21. http://dx.doi.org/10.1016/ j.tim.2005.11.009. Nobile CJ, Schneider HA, Nett JE, Sheppard DC, Filler SG, Andes DR, Mitchell AP. 2008. Complementary adhesin function in C. albicans biofilm formation. Curr. Biol. 18:1017–1024. http://dx.doi.org/10.1016/ j.cub.2008.09.017. Chaffin WL, López-Ribot JL, Casanova M, Gozalbo D, Martínez JP. 1998. Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol. Mol. Biol. Rev. 62:130 –180. Alem MA, Douglas LJ. 2004. Effects of aspirin and other nonsteroidal anti-inflammatory drugs on biofilms and planktonic cells of Candida albicans. Antimicrob. Agents Chemother. 48:41– 47. http://dx.doi.org/ 10.1128/AAC.48.1.41-47.2004.
The views expressed in this Commentary do not necessarily reflect the views of this journal or of ASM.
September/October 2014 Volume 5 Issue 5 e01781-14
®
mbio.asm.org 3
Downloaded from mbio.asm.org on September 11, 2015 - Published by mbio.asm.org
Commentary
ABSTRACT Among pathogenic fungi, Candida albicans is most frequently associated with biofilm formation, a lifestyle that is
entirely different from the planktonic state. One of the distinguishing features of these biofilms is the presence of extracellular material, commonly referred to as the “biofilm matrix.” The fungal biofilm matrix embeds sessile cells within these communities and plays important structural and physiological functions, including antifungal drug resistance with important clinical repercussions. This matrix is mostly self-produced by the fungal cells themselves and is composed of different types of biopolymers. In C. albicans, the main components of the biofilm matrix are carbohydrates, proteins, lipids, and DNA, but many of them remain unidentified and/or poorly characterized. In their recent article, Zarnowski et al. [mBio 5(4):e01333-14, 2014, doi:10.1128/ mBio.01333-14] used a variety of biochemical and state-of-the-art “omic” approaches (glycomics, proteomics, and lipidomics) to identify and characterize unique biopolymers present in the C. albicans biofilm matrix. Besides generating a true “encyclopedic” catalog of individual moieties from each of the different macromolecular categories, results also provide important insights into structural and functional aspects of the fungal biofilm matrix, particularly the interaction between different components and the contribution of multiple matrix constituents to biofilm antifungal drug resistance.
C
andida albicans is a normal commensal of humans, frequently colonizing our skin and mucosae. However, as an opportunistic pathogen, it is fully capable of causing disease in an everexpanding population of immunosuppressed and medically compromised patients. Candidiasis is now the third- to fourth-mostcommon nosocomial infection in U.S. hospitals, with one of the highest mortality rates among nosocomial pathogens (1). These infections are often associated with the formation of biofilms, as different types of biomaterials used in the clinics support colonization and subsequent biofilm development by C. albicans (2). The increase in candidiasis during the last few decades has fundamentally paralleled the increase in the use of a variety of medical implant devices, most notably catheters, which serve as a substrate for biofilm formation. This in turn causes elevated levels of resistance to host defenses and antifungal agents, thereby complicating treatment and resulting in high morbidity and mortality rates. This biofilm lifestyle is the predominant mode of growth of microorganisms in nature and is radically different from the planktonic (free-living) state. Biofilms are highly structured consortia of microorganisms, attached to a substrate and encased within a matrix of hydrated exopolymeric material which is mostly produced by the organisms themselves. In fact, the presence of this extracellular matrix is one of the main defining attributes of the biofilm mode of growth: simply put, there is no “bona fide” biofilm without an extracellular matrix. Biofilm matrices consist of a conglomeration of different types of biopolymers, normally referred to as “exopolymeric substances” (EPS). Although the composition of the matrix varies among different microorganisms, in general, polysaccharides and proteins are the most common and abundant EPS components; however, other macromolecules such as lipids and nucleic acids seem to be almost universally present in biofilm matrices (3). The cohesive and adhesive forces of the matrix contribute to the architectural and mechanical stability of the biofilm by mediating stable cell-cell and cell-surface interactions, essentially acting as a glue that holds the cells together. The biofilm matrix acts as a protective barrier, preventing drugs from penetrating the biofilms, and as such, it
September/October 2014 Volume 5 Issue 5 e01781-14
represents one of the main contributing factors to resistance against anti-infective therapies. It also protects the cells within the biofilms against other environmental insults, including desiccation, radiation, oxidation, the action of predators and host immune defenses, and plays important nutritional and metabolic functions (3). The C. albicans biofilm matrix has been the focus of investigation since the late 1990s when studies pioneered by the Douglas research group described increased matrix production in biofilms formed under flow, investigated its contribution to antifungal drug resistance, and performed preliminary elemental analyses of matrix polymers (4, 5). To date, studies have identified polysaccharides, proteins, and nucleic acids as the main macromolecular components of the EPS in C. albicans (6–8). Several proteins and glycoproteins extracted from the biofilm matrix were identified using proteomic techniques (2-dimensional gel electrophoresis and mass spectrometry), but the study provided little insight into the functions of these proteinaceous materials (8). Regarding carbohydrates, in studies mostly by the Andes research group, one critical and perhaps the best-studied component of the C. albicans biofilm matrix, -1,3-glucan, has been reported to sequester antifungal molecules, basically acting as a “drug sponge” and conferring resistance to C. albicans biofilms (7, 9). More-recent studies have described the presence of extracellular DNA (eDNA) in the C. albicans biofilm EPS, which appears to play both a structural and protective role (6, 10). Despite this accumulated knowledge, the full composition and function of the C. albicans biofilm matrix still remain to be fully elucidated. Fully embracing this challenge, Published 9 September 2014 Citation Lopez-Ribot JL. 2014. Large-scale biochemical profiling of the Candida albicans biofilm matrix: new compositional, structural, and functional insights. mBio 5(5):e0178114. doi:10.1128/mBio.01781-14. Copyright © 2014 Lopez-Ribot. This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited. Address correspondence to [email protected].
®
mbio.asm.org 1
Downloaded from mbio.asm.org on September 11, 2015 - Published by mbio.asm.org
crossmark
COMMENTARY
the recent article by Zarnowski et al. (11) constitutes a genuine tour de force for the comprehensive biochemical analysis of the biopolymers present in the matrix, facilitated by the use of stateof-the-art instrumentation and powerful glycomic, proteomic, and lipidomic techniques. In the process they uncover, identify, and catalog matrix components. Furthermore, their data suggest that different matrix components interact physically with each other. From a functional perspective, their findings indicate that multiple matrix constituents contribute to antifungal drug resistance and suggest that some matrix components may also play an important metabolic role in C. albicans biofilms. Isolation of the biofilm matrix can be challenging, and it is of critical importance to adapt the extraction procedure to the specific type of biofilm under investigation. For this work, Zarnowski and colleagues (11) used a gentle-sonication solubilization protocol mostly based on extraction methods previously described by others (5, 8) in order to ensure the lack of cell leakage or cell damage. Very importantly, they used electron microscopy techniques to convincingly demonstrate that their isolation methodology specifically removes matrix, leaves the fungal cells intact, and does not extract cell wall components. After purification, and consistent with previous analyses (6–8), Zarnowski et al. describe the overall composition of the C. albicans biofilm matrix, with representation of each of the four macromolecular classes, including proteins and glycoproteins (55% [wt/wt]), carbohydrates (~25% [wt/wt]), lipids (~15% [wt/wt]), and nucleic acids (mostly noncoding DNA) (~5% [wt/wt]). This study reported that proteins represented the main component, far exceeding the polysaccharide content on a mass basis (11). Using proteomic techniques, the authors identified a total of 565 different proteins in the matrix, including a few predicted to form part of the secretome, but also many secretion signal-less proteins, which suggests a noncanonical secretion pathway and/or the accumulation of proteins after cell death (12). According to functional ontology analyses, a total of 458 distinct activities and 16 different metabolic pathways were represented, with a predominance of enzymes involved in carbohydrate and amino acid metabolism. This is highly suggestive of a putative role of the matrix as an external digestive system and as a nutritional/energy source, as previously demonstrated in the case of bacterial biofilms (3). In addition, some enzymes can be potentially involved in the degradation of structural EPS to promote the dispersion of cells from biofilms. Another intriguing possibility is that some of these enzymes may act as virulence factors during biofilm-associated infections. Somewhat surprisingly, the authors did not identify cell wall-associated proteins (such as members of the agglutinin-like sequence [ALS] family) that have been shown to play important roles in cell-cell and cell-substrate interactions during biofilm growth (13). Thus, it would seem that there is a two-tier system for cohesive and adhesive interactions in C. albicans biofilms: one mediated by surface adhesins in the cell wall and a second one provided by the extracellular matrix itself. Interestingly, proteomic analysis of the matrix recovered from C. albicans biofilms grown in vivo demonstrated a very different proteinaceous composition, with the majority of proteins contributed by the host. Polysaccharides represent a relatively major fraction in most biofilm matrices, and C. albicans is no exception to this rule, with carbohydrates representing about 25% of its EPS by weight (11). The authors reported the highest degree of complexity associated with the exopolysaccharide fraction of the C. albicans biofilm ma-
2
®
mbio.asm.org
trix of all macromolecules, a not completely unexpected result. Monosaccharide analysis indicated that arabinose, mannose, glucose, and xylose constituted the four most abundant basic units of the total carbohydrate pool, although their relative abundance varied among high- and low-molecular-weight fractions prepared by size exclusion chromatography. Subsequent nuclear magnetic resonance (NMR) analysis of the different fractions detected three major polysaccharides that were similar to the main carbohydrate components of the C. albicans cell wall (14), although their relative abundance in the matrix was different. Somewhat surprisingly, -1,3-glucan, which represents the major cell wall polysaccharide and plays a major role in biofilm drug resistance comprised only a small portion of the total carbohydrate fraction of the EPS. Newly described and more abundant (about 87%) polysaccharides in the matrix included ␣-1,2 branched and ␣-1,6-mannans. Importantly, these two polysaccharides were associated with unbranched -1,6-glucans in an apparent mannan-glucan complex, thus indicating a physical association between glucan and mannan residues. Imaging techniques corroborated that the distribution of EPS polysaccharides was similar in in vitro and in vivo biofilms of C. albicans. Using lipidomics, the authors described that the matrix lipid profile included predominantly glycerolipids (99.5%) and sphingolipids (0.5%) (11). Different fatty acids were identified on these lipids, namely, oleic and linoleic acids (in the most abundant neutral glycerolipids) and palmitoleic, palmitic, and oleic acids (in the less abundant polar glycerolipids). Consistent with a previous report (15), small quantities of prostaglandin E2, a precursor of eicosanoids, were also found in the matrix, whereas ergosterol was the only sterol detected. Finally, and from a functional perspective, radiolabeling experiments and NMR studies demonstrated the interaction of the aggregate matrix with the antifungal fluconazole, further supporting the contribution of the matrix to biofilm drug resistance (11). Moreover, results clearly indicated that multiple matrix components contribute to antifungal drug resistance, which lead the authors to postulate that the C. albicans biofilm matrix displays “properties of an amalgam in which multicomponent interactions yield emergent properties” (11). The long-term challenge of studies of the biofilm matrix is colossal: to comprehensively define the identity, quantity, structure, interactions, and function(s) of complete complements of the different macromolecular components of the EPS, to understand their spatial and temporal distribution, and to characterize how they change in response to different physical, biological, and environmental conditions. Although many questions remain, the analyses and high-throughput methods described in this article represent a major step toward this end and without any doubt will serve as an excellent framework for future studies. Understanding the structure and function of the matrix components represents one auspicious path for the development of effective and much needed therapies against C. albicans biofilms. ACKNOWLEDGMENTS Work in the laboratory is supported by grants R01DE023510 and R03AI103295 from the National Institute of Dental and Craniofacial Research and National Institute of Allergy and Infectious Diseases, respectively. Additional support is provided by the Army Research Office of the Department of Defense under contract W911NF-11-1-0136. The funders had no role in study design, data collection, and analysis,
September/October 2014 Volume 5 Issue 5 e01781-14
Downloaded from mbio.asm.org on September 11, 2015 - Published by mbio.asm.org
Commentary
decision to publish, or preparation of the manuscript, and the content is solely the responsibility of the author.
9.
REFERENCES 1. Pfaller MA, Diekema DJ. 2007. Epidemiology of invasive candidiasis: a persistent public health problem. Clin. Microbiol. Rev. 20:133–163. http://dx.doi.org/10.1128/CMR.00029-06. 2. Ramage G, Martínez JP, López-Ribot JL. 2006. Candida biofilms on implanted biomaterials: a clinically significant problem. FEMS Yeast Res. 6:979 –986. http://dx.doi.org/10.1111/j.1567-1364.2006.00117.x. 3. Flemming HC, Wingender J. 2010. The biofilm matrix. Nat. Rev. Microbiol. 8:623– 633. http://dx.doi.org/10.1038/nrmicro2415. 4. Al-Fattani MA, Douglas LJ. 2006. Biofilm matrix of Candida albicans and Candida tropicalis: chemical composition and role in drug resistance. J. Med. Microbiol. 55:999 –1008. http://dx.doi.org/10.1099/jmm.0.46569-0. 5. Baillie GS, Douglas LJ. 2000. Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents. J. Antimicrob. Chemother. 46:397– 403. http://dx.doi.org/10.1093/jac/46.3.397. 6. Martins M, Uppuluri P, Thomas DP, Cleary IA, Henriques M, LopezRibot JL, Oliveira R. 2010. Presence of extracellular DNA in the Candida albicans biofilm matrix and its contribution to biofilms. Mycopathologia 169:323–331. http://dx.doi.org/10.1007/s11046-009-9264-y. 7. Nett J, Lincoln L, Marchillo K, Massey R, Holoyda K, Hoff B, VanHandel M, Andes D. 2007. Putative role of beta-1,3 glucans in Candida albicans biofilm resistance. Antimicrob. Agents Chemother. 51:510 –520. http://dx.doi.org/10.1128/AAC.01056-06. 8. Thomas DP, Bachmann SP, Lopez-Ribot JL. 2006. Proteomics for the
10.
11.
12. 13.
14. 15.
analysis of the Candida albicans biofilm lifestyle. Proteomics 6:5795–5804. http://dx.doi.org/10.1002/pmic.200600332. Nett JE, Crawford K, Marchillo K, Andes DR. 2010. Role of Fks1p and matrix glucan in Candida albicans biofilm resistance to an echinocandin, pyrimidine, and polyene. Antimicrob. Agents Chemother. 54:3505–3508. http://dx.doi.org/10.1128/AAC.00227-10. Martins M, Henriques M, Lopez-Ribot JL, Oliveira R. 2012. Addition of DNase improves the in vitro activity of antifungal drugs against Candida albicans biofilms. Mycoses 55:80 – 85. http://dx.doi.org/10.1111/j.14390507.2011.02047.x. Zarnowski R, Westler WM, Lacmbouh GA, Marita JM, Bothe JR, Bernhardt J, Sahraoui L-HA, Fontaine J, Sanchez H, Hatfield RD, Ntambi JM, Nett JE, Mitchell AP, Andes DR. 2014. Novel entries in a fungal biofilm matrix encyclopedia. mBio 5(4):e01333-14. http:// dx.doi.org/10.1128/mBio.01333-14. Nombela C, Gil C, Chaffin WL. 2006. Non-conventional protein secretion in yeast. Trends Microbiol. 14:15–21. http://dx.doi.org/10.1016/ j.tim.2005.11.009. Nobile CJ, Schneider HA, Nett JE, Sheppard DC, Filler SG, Andes DR, Mitchell AP. 2008. Complementary adhesin function in C. albicans biofilm formation. Curr. Biol. 18:1017–1024. http://dx.doi.org/10.1016/ j.cub.2008.09.017. Chaffin WL, López-Ribot JL, Casanova M, Gozalbo D, Martínez JP. 1998. Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol. Mol. Biol. Rev. 62:130 –180. Alem MA, Douglas LJ. 2004. Effects of aspirin and other nonsteroidal anti-inflammatory drugs on biofilms and planktonic cells of Candida albicans. Antimicrob. Agents Chemother. 48:41– 47. http://dx.doi.org/ 10.1128/AAC.48.1.41-47.2004.
The views expressed in this Commentary do not necessarily reflect the views of this journal or of ASM.
September/October 2014 Volume 5 Issue 5 e01781-14
®
mbio.asm.org 3
Downloaded from mbio.asm.org on September 11, 2015 - Published by mbio.asm.org
Commentary
