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Targeting Fibronectin To Disrupt In Vivo Candida albicans Biofilms Jeniel E. Nett,a,b Jonathan Cabezas-Olcoz,a,b Karen Marchillo,a,b Deane F. Mosher,a,c David R. Andesa,b Departments of Medicine,a Medical Microbiology and Immunology,b and Biomolecular Chemistry,c University of Wisconsin, Madison, Wisconsin, USA
New drug targets are of great interest for the treatment of fungal biofilms, which are routinely resistant to antifungal therapies. We theorized that the interaction of Candida albicans with matricellular host proteins would provide a novel target. Here, we show that an inhibitory protein (FUD) targeting Candida-fibronectin interactions disrupts biofilm formation in vitro and in vivo in a rat venous catheter model. The peptide appears to act by blocking the surface adhesion of Candida, halting biofilm formation.
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andida albicans, the most common nosocomial fungal pathogen, adheres to medical devices and proliferates as a biofilm (1). Vascular catheters, urinary catheters, and dentures are commonly infected devices. Biofilms are routinely resistant to antifungal therapies, often withstanding drug concentrations 1,000-fold higher than those needed to kill free-floating cells (2, 3). Given the multidrug-resistant phenotype of biofilm infections, new drugs with antibiofilm activity are desperately needed. Recent investigation of the host-biofilm interface showed that the vast majority of the extracellular matrix components surrounding in vivo biofilms are actually of host origin (4). For example, in a rat venous catheter biofilm, ⬎95% of proteins identified by mass spectrometry were of rat origin. One of the intriguing categories of biofilm-associated proteins is a group of host matricellular proteins. Fibronectin, vitronectin, and fibrinogen were found to deposit on a variety of infected and uninfected medical devices, including a vascular catheter and urinary catheter, and denture devices (4). This finding prompted examination of the potential of disrupting these interactions for development of a novel antibiofilm therapeutic agent. We adapted an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay to examine the impact of matricellular proteins on biofilm formation (5). Fibronectin, vitronectin, and fibrinogen were selected for study based on their conserved presence in Candida biofilms with albumin as a control (4). Human fibrinogen and albumin purchased from Sigma and purified human fibronectin and vitronectin were used at concentrations based on their estimated concentrations in 6.25 to 12.5% plasma (6–9). C. albicans (strain K1) was used for all studies and was propagated overnight in yeast-peptonedextrose with uridine at 30°C at 200 rpm and resuspended in RPMI medium-MOPS (morpholinepropanesulfonic acid) at 5 ⫻ 105 (10). For adherence assays, C. albicans was seeded in the wells of microtiter plates with host components diluted in phosphatebuffered saline (PBS). After 1 h (37°C), nonadherent cells were removed, and an XTT assay was performed to estimate adherent Candida. For maturation assays, C. albicans was incubated for 1 h (37°C), nonadherent cells were removed, and host proteins in RPMI medium-MOPS were added for 18 h prior to the XTT assay. For the blocking experiments, FUD was included at 500 nM (11, 12). This peptide sequence is based on a Streptococcus pyogenes adhesin domain, which competitively binds the fibronectin type 1 module, inhibiting fibrillogenesis (11, 12).
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A rat venous catheter model was used for in vivo C. albicans biofilm studies (13, 14). Animals were maintained in accordance with the American Association for Accreditation of Laboratory Care criteria, and all studies were approved by the institutional animal care committee. Briefly, 24 h after implantation, the jugular venous catheters were inoculated (106 cells/ml), flushed after 6 h, and collected after 24 h for microbiological enumeration or imaging by scanning election microscopy (SEM) (13). FUD peptide was included at 500 nM (11, 15). The results of the adhesion experiments demonstrated that plasma augmented C. albicans adhesion to plastic by approximately 2-fold (Fig. 1A). However, among the host matricellular proteins selected for study, only fibronectin significantly promoted C. albicans adhesion to a similar degree. Although increased adherence was observed with fibrinogen, this did not reach statistical significance. In this model system, the influence of fibronectin was limited to the initial adhesion phase with no effect during maturation (Fig. 1B). However, plasma did augment biofilm maturation, indicating that alternative plasma factors were responsible for this phenomenon. We examined the impact of combining the matricellular proteins but did not identify synergistic enhancement of biofilm maturation (data not shown). To further examine the interaction of fibronectin and Candida biofilms, we utilized a peptide (FUD) which inhibits fibronectin assembly by competitively binding the fibronectin type 1 module that has been shown to interact with Gram-positive bacteria (11, 15). In vitro, addition of this peptide counteracted the positive effect of fibronectin on adherence (Fig. 2A). It also inhibited the increased plasma-promoting effects on Candida adherence. The inhibitory effect of the FUD peptide was significantly more pronounced in vivo. Administration of the peptide along with C. albicans decreased the viable catheter burden by nearly an entire order of magnitude (1 log) of growth (Fig. 2B). Visualization of the catheters by SEM qualitatively confirmed a sub-
Received 24 December 2015 Returned for modification 22 January 2016 Accepted 14 February 2016 Accepted manuscript posted online 22 February 2016 Citation Nett JE, Cabezas-Olcoz J, Marchillo K, Mosher DF, Andes DR. 2016. Targeting fibronectin to disrupt in vivo Candida albicans biofilms. Antimicrob Agents Chemother 60:3152–3155. doi:10.1128/AAC.03094-15. Address correspondence to David R. Andes, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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FIG 1 Impact of various host factors on Candida adherence and biofilm formation. (A) When added during the 1-h adhesion phase, plasma and fibronectin significantly promoted C. albicans adhesion. (B) When added during the 18-h biofilm maturation phase, only plasma promoted biofilm formation. Concentrations were 100 g/ml fibrinogen, 2 g/ml vitronectin, 100 g/ml albumin, 6.25% plasma, and 25 g/ml fibronectin. Viable burdens were estimated by an XTT assay following washing to remove nonadherent cells. Error bars indicate standard deviations about the means. Experiments were performed in triplicate on two occasions. An analysis of variance (ANOVA) with pairwise comparisons using the Holm-Sidak method was used for statistical analysis. *, P ⬍ 0.05.
stantial decrease in biofilm formation in the presence of FUD (Fig. 2C). We suspect that much of the inhibitory activity of FUD may occur during the initial adherence period. Although the safety and tolerability of FUD have not been extensively studied, the ani-
mals tolerated the peptide well. The finding that inhibition of the fibronectin-Candida interaction disrupts biofilm formation suggests that targeting the biofilm-host interaction may be a viable option for preventing or treating biofilm infections.
FIG 2 Fibronectin peptide inhibitor FUD impairs C. albicans biofilm formation in vitro and in vivo. C. albicans was incubated with fibronectin (25 g/ml) or human plasma (12.5%) with or without FUD peptide for 1 h. (A) Viable burdens were estimated by an XTT assay following washing to remove nonadherent cells. Error bars indicate standard deviations about the means. Experiments were performed in triplicate on two occasions. C. albicans was inoculated into a jugular venous rat catheter with or without FUD peptide. Catheters were collected at 24 h and or analyzed for viable burdens following biofilm disruption (B) or imaged by SEM (C). Microbiological counts were performed in triplicate. For SEM, images were obtained at ⫻75 magnification, and measurement bars represent 500 m. The addition of FUD impaired biofilm formation. The Student t test was used for comparison. *, P ⬍ 0.05.
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Matricellular proteins, including vitronectin, fibronectin, and fibrinogen, have previously been shown to bind to C. albicans, likely augmenting invasion of various tissues (16–23). These proteins consistently associate with biofilms of medical devices and have been studied as host-conditioning proteins prior to biofilm initiation (24–29). In this study, we demonstrate that fibronectin is influential for adhesion to plastic and that targeting this interaction reduces biofilm formation. Interestingly, the impact of the fibronectin inhibitory peptide was far more pronounced in vivo. There are several factors which may account for this phenomenon. First, Candida cells adherent to a rat vascular catheter are likely subjected to higher shear stress in the form of the flushing and blood flow; thus, poorly adhesive cells may be more easily removed. Second, C. albicans has been shown to express multiple receptors for fibronectin with differing binding affinities, including Als1p, Als5p, and two inducible receptors (30–33). One of these receptors is induced by hemoglobin, a protein which uniformly associates with in vivo biofilms (4, 31). The expression levels of the individual fibronectin receptors may vary profoundly with the experimental conditions. A role for fibronectin binding in Candida pathogenesis has been previously proposed for fungal endocarditis (34). This was based on a correlation between fibronectin binding and the propensity of clinical isolates to induce endocarditis in a rabbit model. The findings suggest that Candida binds the fibronectin in valvular clots. A similar process may also be involved in the adherence of Candida to devices. Targeting this interaction is a potential avenue for antibiofilm therapy.
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ACKNOWLEDGMENTS This work was supported by the National Institutes of Health (R01 AI073289-01 and K08 AI108727) and the Burroughs Wellcome Fund (1012299). We acknowledge the use of instrumentation supported by the UW MRSEC (DMR-1121288) and the UW NSEC (DMR-0832760).
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FUNDING INFORMATION
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This work, including the efforts of Jeniel E. Nett, was funded by Burroughs Wellcome Fund (BWF) (1012299). This work, including the efforts of David R. Andes, was funded by HHS | NIH | NIH Clinical Center (NIH CC) (R01 AI073289-01). This work, including the efforts of Jeniel E. Nett, was funded by HHS | NIH | NIH Clinical Center (NIH CC) (K08 AI108727).
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in plasma of severely ill patients with disseminated intravascular coagulation. J Lab Clin Med 91:729 –735. Hayman EG, Pierschbacher MD, Ohgren Y, Ruoslahti E. 1983. Serum spreading factor (vitronectin) is present at the cell surface and in tissues. Proc Natl Acad Sci U S A 80:4003– 4007. http://dx.doi.org/10.1073/pnas .80.13.4003. Mosher DF, Johnson RB. 1983. In vitro formation of disulfide-bonded fibronectin multimers. J Biol Chem 258:6595– 6601. Bittorf SV, Williams EC, Mosher DF. 1993. Alteration of vitronectin. Characterization of changes induced by treatment with urea. J Biol Chem 268:24838 –24846. Andes D, van Ogtrop M. 1999. Characterization and quantitation of the pharmacodynamics of fluconazole in a neutropenic murine disseminated candidiasis infection model. Antimicrob Agents Chemother 43:2116 – 2120. Tomasini-Johansson BR, Kaufman NR, Ensenberger MG, Ozeri V, Hanski E, Mosher DF. 2001. A 49-residue peptide from adhesin F1 of Streptococcus pyogenes inhibits fibronectin matrix assembly. J Biol Chem 276:23430 –23439. http://dx.doi.org/10.1074/jbc.M103467200. Ma W, Ma H, Fogerty FJ, Mosher DF. 2014. Bivalent ligation of the collagen-binding modules of fibronectin by SFS, a non-anchored bacterial protein of Streptococcus equi. J Biol Chem 290:4866⫺4876. http://dx.doi .org/10.1074/jbc.M114.612259. Nett JE, Marchillo K, Andes DR. 2012. Modeling of fungal biofilms using a rat central vein catheter. Methods Mol Biol 845:547–556. http://dx.doi .org/10.1007/978-1-61779-539-8_40. Andes D, Nett J, Oschel P, Albrecht R, Marchillo K, Pitula A. 2004. Development and characterization of an in vivo central venous catheter Candida albicans biofilm model. Infect Immun 72:6023– 6031. http://dx .doi.org/10.1128/IAI.72.10.6023-6031.2004. Maurer LM, Tomasini-Johansson BR, Ma W, Annis DS, Eickstaedt NL, Ensenberger MG, Satyshur KA, Mosher DF. 2010. Extended binding site on fibronectin for the functional upstream domain of protein F1 of Streptococcus pyogenes. J Biol Chem 285:41087– 41099. http://dx.doi.org/10 .1074/jbc.M110.153692. Jakab E, Paulsson M, Ascencio F, Ljungh A. 1993. Expression of vitronectin and fibronectin binding by Candida albicans yeast cells. APMIS 101:187–193. http://dx.doi.org/10.1111/j.1699-0463.1993.tb00100.x. Bouali A, Robert R, Tronchin G, Senet JM. 1987. Characterization of binding of human fibrinogen to the surface of germ-tubes and mycelium of Candida albicans. J Gen Microbiol 133:545–551. Saville SP, Thomas DP, Lopez Ribot JL. 2006. A role for Efg1p in Candida albicans interactions with extracellular matrices. FEMS Microbiol Lett 256: 151–158. http://dx.doi.org/10.1111/j.1574-6968.2006.00109.x. Sturtevant J, Calderone R. 1997. Candida albicans adhesins: Biochemical aspects and virulence. Rev Iberoam Micol 14:90 –97. Lopez CM, Wallich R, Riesbeck K, Skerka C, Zipfel PF. 2014. Candida albicans uses the surface protein Gpm1 to attach to human endothelial cells and to keratinocytes via the adhesive protein vitronectin. PLoS One 9:e90796. http://dx.doi.org/10.1371/journal.pone.0090796. Pendrak ML, Krutzsch HC, Roberts DD. 2000. Structural requirements for hemoglobin to induce fibronectin receptor expression in Candida albicans. Biochemistry 39:16110 –16118. http://dx.doi.org/10 .1021/bi0012585. López-Ribot JL, Monteagudo C, Sepulveda P, Casanova M, Martinez JP, Chaffin WL. 1996. Expression of the fibrinogen binding mannoprotein and the laminin receptor of Candida albicans in vitro and in infected tissues. FEMS Microbiol Lett 142:117–122. Santoni G, Spreghini E, Lucciarini R, Amantini C, Piccoli M. 2001. Involvement of alpha(v)beta3 integrin-like receptor and glycosaminoglycans in Candida albicans germ tube adhesion to vitronectin and to a human endothelial cell line. Microb Pathog 31:159 –172. http://dx.doi.org /10.1006/mpat.2001.0459. Nikawa H, Nishimura H, Makihira S, Hamada T, Sadamori S, Samaranayake LP. 2000. Effect of serum concentration on Candida biofilm formation on acrylic surfaces. Mycoses 43:139 –143. http://dx.doi.org/10 .1046/j.1439-0507.2000.00564.x. Nikawa H, Nishimura H, Hamada T, Yamashiro H, Samaranayake LP. 1999. Effects of modified pellicles on Candida biofilm formation on acrylic surfaces. Mycoses 42:37– 40. http://dx.doi.org/10.1046/j.1439-0507.1999 .00270.x. Frade JP, Arthington-Skaggs BA. 2011. Effect of serum and surface char-
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31. Yan S, Rodrigues RG, Cahn-Hidalgo D, Walsh TJ, Roberts DD. 1998. Hemoglobin induces binding of several extracellular matrix proteins to Candida albicans. Identification of a common receptor for fibronectin, fibrinogen, and laminin. J Biol Chem 273:5638 –5644. 32. Pendrak ML, Rodrigues RG, Roberts DD. 2007. Induction of a high affinity fibronectin receptor in Candida albicans by caspofungin: requirements for beta (1,6) glucans and the developmental regulator Hbr1p. Med Mycol 45:157–168. http://dx.doi.org/10.1080/13693780601164314. 33. Gaur NK, Smith RL, Klotz SA. 2002. Candida albicans and Saccharomyces cerevisiae expressing ALA1/ALS5 adhere to accessible threonine, serine, or alanine patches. Cell Commun Adhes 9:45–57. http://dx.doi.org/10.1080 /15419060212187. 34. Calderone RA, Scheld WM. 1987. Role of fibronectin in the pathogenesis of candidal infections. Rev Infect Dis 9(Suppl 4):S400 –S403. http://dx.doi .org/10.1093/clinids/9.Supplement_4.S400.
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Targeting Fibronectin To Disrupt In Vivo Candida albicans Biofilms Jeniel E. Nett,a,b Jonathan Cabezas-Olcoz,a,b Karen Marchillo,a,b Deane F. Mosher,a,c David R. Andesa,b Departments of Medicine,a Medical Microbiology and Immunology,b and Biomolecular Chemistry,c University of Wisconsin, Madison, Wisconsin, USA
New drug targets are of great interest for the treatment of fungal biofilms, which are routinely resistant to antifungal therapies. We theorized that the interaction of Candida albicans with matricellular host proteins would provide a novel target. Here, we show that an inhibitory protein (FUD) targeting Candida-fibronectin interactions disrupts biofilm formation in vitro and in vivo in a rat venous catheter model. The peptide appears to act by blocking the surface adhesion of Candida, halting biofilm formation.
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andida albicans, the most common nosocomial fungal pathogen, adheres to medical devices and proliferates as a biofilm (1). Vascular catheters, urinary catheters, and dentures are commonly infected devices. Biofilms are routinely resistant to antifungal therapies, often withstanding drug concentrations 1,000-fold higher than those needed to kill free-floating cells (2, 3). Given the multidrug-resistant phenotype of biofilm infections, new drugs with antibiofilm activity are desperately needed. Recent investigation of the host-biofilm interface showed that the vast majority of the extracellular matrix components surrounding in vivo biofilms are actually of host origin (4). For example, in a rat venous catheter biofilm, ⬎95% of proteins identified by mass spectrometry were of rat origin. One of the intriguing categories of biofilm-associated proteins is a group of host matricellular proteins. Fibronectin, vitronectin, and fibrinogen were found to deposit on a variety of infected and uninfected medical devices, including a vascular catheter and urinary catheter, and denture devices (4). This finding prompted examination of the potential of disrupting these interactions for development of a novel antibiofilm therapeutic agent. We adapted an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] assay to examine the impact of matricellular proteins on biofilm formation (5). Fibronectin, vitronectin, and fibrinogen were selected for study based on their conserved presence in Candida biofilms with albumin as a control (4). Human fibrinogen and albumin purchased from Sigma and purified human fibronectin and vitronectin were used at concentrations based on their estimated concentrations in 6.25 to 12.5% plasma (6–9). C. albicans (strain K1) was used for all studies and was propagated overnight in yeast-peptonedextrose with uridine at 30°C at 200 rpm and resuspended in RPMI medium-MOPS (morpholinepropanesulfonic acid) at 5 ⫻ 105 (10). For adherence assays, C. albicans was seeded in the wells of microtiter plates with host components diluted in phosphatebuffered saline (PBS). After 1 h (37°C), nonadherent cells were removed, and an XTT assay was performed to estimate adherent Candida. For maturation assays, C. albicans was incubated for 1 h (37°C), nonadherent cells were removed, and host proteins in RPMI medium-MOPS were added for 18 h prior to the XTT assay. For the blocking experiments, FUD was included at 500 nM (11, 12). This peptide sequence is based on a Streptococcus pyogenes adhesin domain, which competitively binds the fibronectin type 1 module, inhibiting fibrillogenesis (11, 12).
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A rat venous catheter model was used for in vivo C. albicans biofilm studies (13, 14). Animals were maintained in accordance with the American Association for Accreditation of Laboratory Care criteria, and all studies were approved by the institutional animal care committee. Briefly, 24 h after implantation, the jugular venous catheters were inoculated (106 cells/ml), flushed after 6 h, and collected after 24 h for microbiological enumeration or imaging by scanning election microscopy (SEM) (13). FUD peptide was included at 500 nM (11, 15). The results of the adhesion experiments demonstrated that plasma augmented C. albicans adhesion to plastic by approximately 2-fold (Fig. 1A). However, among the host matricellular proteins selected for study, only fibronectin significantly promoted C. albicans adhesion to a similar degree. Although increased adherence was observed with fibrinogen, this did not reach statistical significance. In this model system, the influence of fibronectin was limited to the initial adhesion phase with no effect during maturation (Fig. 1B). However, plasma did augment biofilm maturation, indicating that alternative plasma factors were responsible for this phenomenon. We examined the impact of combining the matricellular proteins but did not identify synergistic enhancement of biofilm maturation (data not shown). To further examine the interaction of fibronectin and Candida biofilms, we utilized a peptide (FUD) which inhibits fibronectin assembly by competitively binding the fibronectin type 1 module that has been shown to interact with Gram-positive bacteria (11, 15). In vitro, addition of this peptide counteracted the positive effect of fibronectin on adherence (Fig. 2A). It also inhibited the increased plasma-promoting effects on Candida adherence. The inhibitory effect of the FUD peptide was significantly more pronounced in vivo. Administration of the peptide along with C. albicans decreased the viable catheter burden by nearly an entire order of magnitude (1 log) of growth (Fig. 2B). Visualization of the catheters by SEM qualitatively confirmed a sub-
Received 24 December 2015 Returned for modification 22 January 2016 Accepted 14 February 2016 Accepted manuscript posted online 22 February 2016 Citation Nett JE, Cabezas-Olcoz J, Marchillo K, Mosher DF, Andes DR. 2016. Targeting fibronectin to disrupt in vivo Candida albicans biofilms. Antimicrob Agents Chemother 60:3152–3155. doi:10.1128/AAC.03094-15. Address correspondence to David R. Andes, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
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FIG 1 Impact of various host factors on Candida adherence and biofilm formation. (A) When added during the 1-h adhesion phase, plasma and fibronectin significantly promoted C. albicans adhesion. (B) When added during the 18-h biofilm maturation phase, only plasma promoted biofilm formation. Concentrations were 100 g/ml fibrinogen, 2 g/ml vitronectin, 100 g/ml albumin, 6.25% plasma, and 25 g/ml fibronectin. Viable burdens were estimated by an XTT assay following washing to remove nonadherent cells. Error bars indicate standard deviations about the means. Experiments were performed in triplicate on two occasions. An analysis of variance (ANOVA) with pairwise comparisons using the Holm-Sidak method was used for statistical analysis. *, P ⬍ 0.05.
stantial decrease in biofilm formation in the presence of FUD (Fig. 2C). We suspect that much of the inhibitory activity of FUD may occur during the initial adherence period. Although the safety and tolerability of FUD have not been extensively studied, the ani-
mals tolerated the peptide well. The finding that inhibition of the fibronectin-Candida interaction disrupts biofilm formation suggests that targeting the biofilm-host interaction may be a viable option for preventing or treating biofilm infections.
FIG 2 Fibronectin peptide inhibitor FUD impairs C. albicans biofilm formation in vitro and in vivo. C. albicans was incubated with fibronectin (25 g/ml) or human plasma (12.5%) with or without FUD peptide for 1 h. (A) Viable burdens were estimated by an XTT assay following washing to remove nonadherent cells. Error bars indicate standard deviations about the means. Experiments were performed in triplicate on two occasions. C. albicans was inoculated into a jugular venous rat catheter with or without FUD peptide. Catheters were collected at 24 h and or analyzed for viable burdens following biofilm disruption (B) or imaged by SEM (C). Microbiological counts were performed in triplicate. For SEM, images were obtained at ⫻75 magnification, and measurement bars represent 500 m. The addition of FUD impaired biofilm formation. The Student t test was used for comparison. *, P ⬍ 0.05.
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Matricellular proteins, including vitronectin, fibronectin, and fibrinogen, have previously been shown to bind to C. albicans, likely augmenting invasion of various tissues (16–23). These proteins consistently associate with biofilms of medical devices and have been studied as host-conditioning proteins prior to biofilm initiation (24–29). In this study, we demonstrate that fibronectin is influential for adhesion to plastic and that targeting this interaction reduces biofilm formation. Interestingly, the impact of the fibronectin inhibitory peptide was far more pronounced in vivo. There are several factors which may account for this phenomenon. First, Candida cells adherent to a rat vascular catheter are likely subjected to higher shear stress in the form of the flushing and blood flow; thus, poorly adhesive cells may be more easily removed. Second, C. albicans has been shown to express multiple receptors for fibronectin with differing binding affinities, including Als1p, Als5p, and two inducible receptors (30–33). One of these receptors is induced by hemoglobin, a protein which uniformly associates with in vivo biofilms (4, 31). The expression levels of the individual fibronectin receptors may vary profoundly with the experimental conditions. A role for fibronectin binding in Candida pathogenesis has been previously proposed for fungal endocarditis (34). This was based on a correlation between fibronectin binding and the propensity of clinical isolates to induce endocarditis in a rabbit model. The findings suggest that Candida binds the fibronectin in valvular clots. A similar process may also be involved in the adherence of Candida to devices. Targeting this interaction is a potential avenue for antibiofilm therapy.
7.
8. 9. 10.
11.
12.
13. 14.
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ACKNOWLEDGMENTS This work was supported by the National Institutes of Health (R01 AI073289-01 and K08 AI108727) and the Burroughs Wellcome Fund (1012299). We acknowledge the use of instrumentation supported by the UW MRSEC (DMR-1121288) and the UW NSEC (DMR-0832760).
17.
FUNDING INFORMATION
19.
This work, including the efforts of Jeniel E. Nett, was funded by Burroughs Wellcome Fund (BWF) (1012299). This work, including the efforts of David R. Andes, was funded by HHS | NIH | NIH Clinical Center (NIH CC) (R01 AI073289-01). This work, including the efforts of Jeniel E. Nett, was funded by HHS | NIH | NIH Clinical Center (NIH CC) (K08 AI108727).
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REFERENCES 1. Kojic EM, Darouiche RO. 2004. Candida infections of medical devices. Clin Microbiol Rev 17:255–267. http://dx.doi.org/10.1128/CMR.17.2.255 -267.2004. 2. Ramage G, Rajendran R, Sherry L, Williams C. 2012. Fungal biofilm resistance. Int J Microbiol 2012:528521. http://dx.doi.org/10.1155/2012 /528521. 3. LaFleur MD, Kumamoto CA, Lewis K. 2006. Candida albicans biofilms produce antifungal-tolerant persister cells. Antimicrob Agents Chemother 50:3839 –3846. http://dx.doi.org/10.1128/AAC.00684-06. 4. Nett JE, Zarnowski R, Cabezas-Olcoz J, Brooks EG, Bernhardt J, Marchillo K, Mosher DF, Andes DR. 2015. Host contributions to construction of three device-associated Candida albicans biofilms. Infect Immun 83:4630 – 4638. http://dx.doi.org/10.1128/IAI.00931-15. 5. Nett JE, Cain MT, Crawford K, Andes DR. 2011. Optimizing a Candida biofilm microtiter plate model for measurement of antifungal susceptibility by tetrazolium salt assay. J Clin Microbiol 49:1426 –1433. http://dx.doi .org/10.1128/JCM.02273-10. 6. Mosher DF, Williams EM. 1978. Fibronectin concentration is decreased
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