Extracellular DNA release confers heterogeneity in Candida albicans biofilm formation

Ranjith Rajendran 0 Leighann Sherry 0 David F Lappin 0 Chris J Nile 0 Karen Smith 2 Craig Williams 2 Carol A Munro 1 Gordon Ramage 0 0 Infection and Immunity Research Group, Glasgow Dental School, School of Medicine, College of Medical, Veterinary and Life Sciences, University of Glasgow , 378 Sauchiehall Street, Glasgow G2 3JZ , UK 1 Aberdeen Fungal Group, Institute of Medical Sciences , Foresterhill , University of Aberdeen , Aberdeen AB25 2ZD , UK 2 Institute of Healthcare Associated Infection, School of Health , Nursing and Midwifery , University of the West of Scotland , Paisley , UK Background: Biofilm formation by Candida albicans has shown to be highly variable and is directly associated with pathogenicity and poor clinical outcomes in patients at risk. The aim of this study was to test the hypotheses that the extracellular DNA release by C. albicans is strain dependent and is associated with biofilm heterogeneity. Results: Initially, biofilm formed by C. albicans high biofilm formers (HBF) or low biofilm formers (LBF) were treated with DNase to find whether eDNA play a role in their biofilm formation. Digestion of biofilm eDNA significantly reduced the HBF biofilm biomass by five fold compared to untreated controls. In addition, quantification of eDNA over the period of biofilm formation by SYBR green assay demonstrate a significantly higher level of 2 to 6 fold in HBF compared to LBF. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase genes, a marker of autolysis, were upregulated in 24 h biofilm formation by HBF compared to LBF, indicating autolysis pathway possibly involved in causing variation. The biofilm biomass and eDNA release by single (cht2, cht3) and double knockout (cht2/cht3) chitinase mutants were significantly less compared to their parental strain CA14, confirming the role of chitinases in eDNA release and biofilm formation. Correlation analysis found a positive correlation between chitinases and HWP1, suggesting eDNA may release during the hyphal growth. Finally, we showed a combinational treatment of biofilms with DNase or chitinase inhibitor (acetazolamide) plus amphotericin B significantly improved antifungal susceptibility by 2 to 8 fold. Conclusions: Collectively, these data show that eDNA release by C. albicans clinical isolates is variable and is associated with differential biofilm formation. Digestion of biofilm eDNA by DNase may provide a novel therapeutic strategies to destabilise biofilm growth and improves antifungal sensitivity. - Background Candida albicans is a dimorphic fungal pathogen that causes both superficial and systemic candidiasis [1]. Invasive systemic forms of the disease affect at-risk patients and can lead to mortality rates as high as 63% in some instances [2]. The disease is multifactorial [3], yet the capacity of C. albicans to form biofilms has been identified as a pivotal factor to patient outcomes [4,5]. These structures are found frequently associated with indwelling medical devices, as well as biological surfaces such as the mucosa [6], and are clinically important due to their high recalcitrance to antifungal treatment [7]. However, there is increasing evidence in the literature that C. albicans biofilm formation is heterogeneous, which has a direct impact upon treatment and pathogenicity [4,8-10]. Studies have indicated that depending on the ability to form a biofilm, or not, has a bearing on patient outcomes, though the conclusions from some of these studies are diametrically opposed [4,9]. Therefore, rather than defining biofilm formation as an absolute parameter we have recently described a series of clinical isolates with a differential ability to form biofilms, in which high biofilm forming (HBF) isolates displayed a greater pathogenic capacity and lower antifungal sensitivity when compared to low biofilm forming (LBF) isolates [10]. However, the mechanisms underlying the difference in biofilm formation are not yet fully understood. Extracellular matrix (ECM) is an important and defining characteristic of biofilms, providing a structural scaffold whilst coincidentally facilitating protection from external factors, including antifungal agents [7,11]. Typical fungal biofilm ECM is a heterogeneous substance consisting of exopolysaccharides, proteins, surfactants, lipids and water [12,13], though recent studies have shown the presence of another important component extracellular DNA in fungal biofilm matrix [13,14]. These studies demonstrated that eDNA plays a significant role with respect to structural stability and as a consequence antifungal sensitivity. In both C. albicans and A. fumigatus it was shown that addition of exogenous DNA significantly improved biofilm formation, and that depletion of biofilm eDNA through the addition of DNase adversely affects the biomass [13,14]. It can be inferred from bacterial biofilm studies that eDNA has a multifactorial purpose, namely as a nutrient source [15], facilitator of genetic information exchange [16], contributor to biofilm stability and dispersal [17-20], and as an antimicrobial resistance factor [21,22]. The mechanism of eDNA release in biofilm environment is not yet fully understood, though studies in bacterial species suggests various mechanisms that are potentially responsible for this process, including cell lysis [23,24]. We recently demonstrated the association of the chitinase regulated autolytic pathway with eDNA release in A. fumigatus [14], however, chitinase activity in C. albicans biofilms with respect to eDNA has yet to be studied. Given that eDNA contributes to ECM integrity, and ECM is a key factor in promoting biofilm formation, we hypothesised that the capacity to release eDNA through differential chitinase activity may be an underlying mechanism supporting biofilm heterogeneity. Methods Isolates and maintenance C. albicans SC5314 and a series of routine patient anonymised clinical bloodstream isolates (n = 6) were collected under the approval of the NHS Scotland Caldicott Gaurdians from the Royal Hospital for Sick Children (Yorkhill Division), Glasgow, UK, as part of candidaemia epidemiology surveillance study were used throughout this study. Furthermore, chitinase mutants cht2, cht3, cht2/cht3, including the parental strain CA14 were also used. Isolates were stored on Sabouraud dextrose agar (Oxoid, Basingstoke, UK) at 4C. All C. albicans strains were grown on YPD at 37C overnight. Cells were then washed and resuspended in appropriate media to the desired concentration, as described previously [25]. All procedures were carried out in a laminar flow cabinet (Hera Safe laminate flow cabinet, Kendro, model K515). Assessment of biofilm formation The growth rate of the clinical isolates was first assessed. Each isolate was standardised to 1 104 cells/mL in YPD dispensed into each well of a 96 well round-bottom plate and incubated at 37C for 24 h. The absorbance was measured at (...truncated)