Putative Role of β-1,3 Glucans in Candida albicans Biofilm Resistance

Abstract

Biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface. Nearly all device-associated infections involve growth in the biofilm life style. Biofilm communities have characteristic architecture and distinct phenotypic properties. The most clinically important phenotype involves extraordinary resistance to antimicrobial therapy, making biofilm infections very difficulty to cure without device removal. The current studies examine drug resistance in Candida albicans biofilms. Similar to previous reports, we observed marked fluconazole and amphotericin B resistance in a C. albicans biofilm both in vitro and in vivo. We identified biofilm-associated cell wall architectural changes and increased ␤-1,3 glucan content in C. albicans cell walls from a biofilm compared to planktonic organisms. Elevated ␤-1,3 glucan levels were also found in the surrounding biofilm milieu and as part of the matrix both from in vitro and in vivo biofilm models. We thus investigated the possible contribution of ␤-glucans to antimicrobial resistance in Candida albicans biofilms. Initial studies examined the ability of cell wall and cell supernatant from biofilm and planktonic C. albicans to bind fluconazole. The cell walls from both environmental conditions bound fluconazole; however, four-to fivefold more compound was bound to the biofilm cell walls. Culture supernatant from the biofilm, but not planktonic cells, bound a measurable amount of this antifungal agent. We next investigated the effect of enzymatic modification of ␤-1,3 glucans on biofilm cell viability and the susceptibility of biofilm cells to fluconazole and amphotericin B. We observed a dose-dependent killing of in vitro biofilm cells in the presence of three different ␤-glucanase preparations. These same concentrations had no impact on planktonic cell viability. ␤-1,3 Glucanase markedly enhanced the activity of both fluconazole and amphotericin B. These observations were corroborated with an in vivo biofilm model. Exogenous biofilm matrix and commercial ␤-1,3 glucan reduced the activity of fluconazole against planktonic C. albicans in vitro. In sum, the current investigation identified glucan changes associated with C. albicans biofilm cells, demonstrated preferential binding of these biofilm cell components to antifungals, and showed a positive impact of the modification of biofilm ␤-1,3 glucans on drug susceptibility. These results provide indirect evidence suggesting a role for glucans in biofilm resistance and present a strong rationale for further molecular dissection of this resistance mechanism to identify new drug targets to treat biofilm infections.

Fluconazole impacts the extracellular matrix of fluconazole-susceptible and -resistant Candida albicans and Candida glabrata biofilms

“…The ASPs, such as β-1,3 glucans, sequestrate antifungals preventing them from diffusing through the ECMs of C. albicans [22,52–55] and non- albicans biofilms [56]. It has been demonstrated that β-1,3 glucan has a role in antifungal resistance as it binds to FLZ avoiding this drug to reach its targets [22,52–56]. Thus, ASP’s significant reduction in the ECM of CaS biofilms demonstrates that the biofilms of this specific strain become more vulnerable when FLZ is present.…”

“…Antifungal resistance of Candida biofilms is multifactorial and involves the stimulation of drug efflux pumps, the physiological state of cells, and the protection exerted by the ECM via β-glucans that bind to FLZ and amphotericin B [22], which limits diffusion of these antifungals through biofilms [23]. C. albicans biofilms that grow under constant flow produce higher quantities of ECM than statically cultured biofilms.…”

Inactivation of genes TEC1 and EFG1 in Candida albicans influences extracellular matrix composition and biofilm morphology

“…Antifungal resistance in Candida biofilms is multifactorial and is associated with the physiological state of the cells, the activation of drug efflux pumps, and the protective effect of the ECM performed by β-glucans [alkali-soluble polysaccharides (ASPs)], which bind to fluconazole [13] and amphotericin B, preventing the penetration of drugs into the biofilm [14]. In addition to the protective effects of the ECM due to β-glucan, it has been shown that the extracellular DNA (eDNA) is another key component contributing to the structural integrity of C. albicans biofilms [15].…”

Biofilm Exopolysaccharides of Pathogenic Fungi: Lessons from Bacteria

“…A similar role in suppressing oxidative burst and neutrophil killing has been reported for the P. aeruginosa exopolysaccharide PslG, through reducing complement binding and opsonization of bacteria within biofilms (66). ␤-1,3-Glucan directly enhances antifungal resistance by the binding and sequestration of fluconazole, preventing its intracellular penetration and antifungal activity (5,67,68). Sequestration of antimicrobials by bacterial exopolysaccharides has also been reported in P. aeruginosa.…”

“…No studies in animal models evaluating the effects of DNase on antifungal susceptibility have been reported to date. However, ␤-glucanase therapy enhanced the activity of neutrophils and antifungals against C. albicans biofilms both in vitro and in a rat catheter model of infection (6,68), thus providing evidence that biofilm-degrading enzymes can be effective in vivo. The use of hydrolytic enzymes targeting GAG has not yet been explored as an anti-biofilm strategy against A. fumigatus; however, Sph3 has been demonstrated to hydrolyze both purified and cell wall-associated GAG (15), suggesting that this may be a promising avenue to explore in the future.…”