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May 30, 2010 - Ranjith Rajendran • Douglas P. Robertson •. Penny J. Hodge • David F. Lappin •. Gordon Ramage. Received: 22 March 2010 / Accepted: 7 May ...
Mycopathologia (2010) 170:229–235 DOI 10.1007/s11046-010-9319-0

Hydrolytic Enzyme Production is Associated with Candida Albicans Biofilm Formation from Patients with Type 1 Diabetes Ranjith Rajendran • Douglas P. Robertson Penny J. Hodge • David F. Lappin • Gordon Ramage



Received: 22 March 2010 / Accepted: 7 May 2010 / Published online: 30 May 2010 Ó Springer Science+Business Media B.V. 2010

Abstract Oral candidosis is common in patients with diabetes mellitus, as yeasts, particularly Candida albicans, have the propensity to colonise, form biofilms and release hydrolytic enzymes which cause inflammation. This study aimed to investigate these characteristics in isolates from three groups of patients with type 1 diabetes: individuals with better controlled diabetes (BCD; C6\8%), individuals with poorly controlled diabetes (PCD; C8%) and nondiabetics (ND; HbA1c \5.9%). The biomass (Bm), phospholipase (Pz), haemolysin (Hz) and proteinase (Prz) were assessed using a microtitre biofilm assay and agar-based hydrolytic enzyme assays. Biofilm formation was significantly increased in the PCD group compared to ND and BCD groups (P \ 0.05). No significant differences in Pz levels were observed between groups, whereas both Hz and Prz were significantly greater in the diabetes groups than in the healthy control group (P \ 0.05). Statistically significant correlations were found to exist between the HbA1c levels of the patients and the Bm (R = 0.384; P = 0.033), haemolysin activity (R = -0.455; P = 0.010) and proteinase activity (R = -0.531; P = 0.002). There was no apparent correlation between

R. Rajendran  D. P. Robertson  P. J. Hodge  D. F. Lappin  G. Ramage (&) Infection and Immunity Research Group, Glasgow Dental School, Faculty of Medicine, University of Glasgow, 378 Sauchiehall Street, Glasgow G2 3JZ, UK e-mail: [email protected]

the Bm and Pz activity (R = -0.305; P = 0.053) or Hz activity (R = -0.100; P = 0.296). However, a negative correlation was found between Bm and Prz values (R = -0.343; P = 0.030). These data suggest that biofilm formation is likely to play a role in the pathogenicity of oral candidosis, and in patients with diabetes, this may be due to the ability of C. albicans to adapt to the altered physiological environment. The production of hydrolytic enzymes is independently associated with this growth modality. Keywords Candida  Biofilm  Type 1 diabetes mellitus  Proteinase  Phospholipase  Haemolysin

Introduction Diabetes mellitus is a persistent and chronic metabolic disease characterised by a relative or absolute deficiency of insulin secretion and/or associated resistance to the metabolic action of insulin on target tissues [14]. Diabetes mellitus is associated with significant morbidity and mortality which results from various complications, including atherosclerosis, retinopathy, neuropathy, nephropathy, delayed healing and periodontal disease [3, 20]. In addition, an increased carriage of oral yeasts has been reported to be associated with diabetes mellitus [2, 29]. Previous studies reported that 77% of patients with diabetes are colonised by Candida species which

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form biofilm on both mucosal and prosthetic surfaces within the oral cavity and are associated with numerous oral complications, including oral candidosis and burning mouth syndrome [4, 32]. Oral candidosis is typified by inflammation and hyperplasia of the gingival and palatal tissue, which can in immunocompromised patients cause discomfort, pain and disseminating infection [5]. C. albicans is the predominant biofilm forming yeast pathogen due to its ability to form hyphae, a key determinant of these complex consortia [26]. Other species including C. glabrata and C. krusei are also implicated in these diseases, but to a lesser extent [5]. C. albicans biofilms are highly resistant to antimicrobial agents, contributing to their pathogenic potential [24]. Nevertheless, the primary armamentarium of C. albicans is its ability to produce hydrolytic enzymes, important secreted proteins utilised in the process of host tissue invasion and liberation of nutrients [31]. These enzymes are secreted extracellularly and include secreted aspartyl proteinase, haemolysin and phospholipase [9, 13, 17]. A recent study of C. albicans strains isolated from patients with type 2 diabetes demonstrated that hydrolytic extracellular enzyme production was increased in comparison with strains from healthy controls [31]. These data imply that the higher reported incidence of oral candidosis in this patient group may be due to the biological properties of their resident C. albicans. To our knowledge, no research group has yet examined the propensity of C. albicans strains from patients with type 1 diabetes to exhibit more pathogenic tendencies than strains from healthy individuals. This study aimed at assessing how groups of clinical isolates of C. albicans from patients with better controlled diabetes, patients with poorly controlled diabetes and healthy controls are compared with respect to their ability to form biofilms, and how this related to their expression of phospholipase, haemolysin and proteinases.

Materials and Methods

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and Clyde). Patients were recruited from local outpatient diabetes and physiotherapy clinics in Greater Glasgow and Clyde Health Board. Subjects were aged between 20 and 55 (mean = 30.93) and had been non-smokers for a minimum of 5 years. The following exclusion criteria were applied: pregnancy; immunosuppression; medication with side effects affecting the oral cavity; patients prescribed antibiotics or anti-inflammatory drugs within the previous 6 weeks and patients with less than 20 teeth. Patients with type 1 diabetes had been diagnosed with diabetes for a minimum of 5 years. After giving informed consent, patients attended Glasgow Dental Hospital for sampling and clinical examination. Blood samples were taken from control subjects to confirm that the patients were definitely controls and not in a pre-diabetic state (glycosylated haemoglobin (HbA1c) \5.9%). HbA1c levels for the patients with diabetes were determined by averaging the HbA1c measurements for each patient over the previous 2 years. Isolate Collection and Identification All patients included in this study were carriers of C. albicans with no overt signs of oral candidosis observed. Each patient was given 10 ml of 0.1 M PBS, pH 7.3, in a sterile universal container and instructed to rinse the mouth for 60 s. Denturewearing patients did not remove their prostheses. After the oral rinse was expectorated into the container, the sample was transferred immediately to the laboratory and processed within 30 min of collection. Specimens (50 ll) were spiral plated to dispense the suspension onto various media, including Sabouraud (SAB) agar and CHROMAgarTM, and incubated at 37°C for 48 h. A tentative identification of specific Candida species was determined by colony colour on the CHROMAgarTM medium and by the germ tube test from growth at 45°C. Formal identifications of single morphotypes were performed using the API 32C biochemical assimilation testing panel, as per the manufacturer’s instructions (bioMe´rieux UK Ltd, Basingstoke).

Patients Preparation of Test Isolates The protocol for the study was approved by the local research ethics committee (Glasgow Royal Infirmary Ethics Committee, NHS Greater Glasgow

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The clinical isolates were subcultured and transferred to MicroBead storage tubes (ProLab Diagnostics) and

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stored at -80°C for subsequent analysis. C. albicans strains selected for use in this study were maintained on SAB slopes and stored at 4°C prior to the investigations described below. A single colony of C. albicans was inoculated onto a SAB agar plate, which was then incubated at 37°C overnight. A loopful of colony was then taken and emulsified in sterile PBS and standardised to approximately 1 9 108 cells/ml using a haemocytometer.

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agar supplemented with 7% (v/v) horse blood and 3% (w/v) glucose. A drop containing 5 9 105 cells/ml of test isolates was carefully spotted onto the blood agar plate and incubated at 37°C in 5% CO2 for 48 h, after which the diameter of both the C. albicans colony and the zone of haemolysis was measured. Hz was calculated as the proportion of colony diameter compared to the diameter of the haemolysis zone. Proteinase Activity

Biofilm Formation Biofilm growth was produced in the commercially available pre-sterilised, polystyrene, flat-bottomed 96-well microtitre plates prepared, as described previously [21, 25]. Briefly, C. albicans was propagated overnight in yeast peptone dextrose (YPD) broth, the cells washed by centrifugation in PBS and a standardised suspension of 1 9 106 cells/ml prepared with RPMI. This was transferred (200 ll) into each well of a microtitre plate and incubated at 37°C for 24 h. The biomass (Bm) of the resultant biofilms was then assessed using a crystal violet assay, as previously described [15]. The absorbance was read at 550 nm (Ab550) in a FluoStar Optima (BMG Labtech). Phospholipase Activity The phospholipase activity (Pz) of each C. albicans isolate was examined using an egg yolk agar (EYA) assay, as described previously [10]. Media was prepared by the addition of 10% (v/v) of egg yolk (Oxoid) to cooled, pre-autoclaved base media (0.11 M dextrose, 1 M NaCl, 1 M CaCl2, 1% [w/v] peptone, 0.5% [w/v] yeast extract, and 2% [w/v] agarose). A drop containing 5 9 105 cells/ml of test isolates was carefully spotted onto the 10% EYA plate and incubated at 37°C for 3 days, after which the diameter of both the C. albicans colony and the zone of precipitation was measured. Pz was calculated as the proportion of colony diameter compared to the diameter of the precipitation zone. Haemolysin Activity Determination of haemolysin (Hz) production was performed according to Manns and colleagues (1994) [12]. The test medium consisted of 2% (w/v) SAB

Proteinase activity (Prz) was assayed using a bovine serum albumin (BSA) assay, as previously described [7, 31]. This test medium consisted of a solution containing 0.2% BSA, 1.17% dextrose and 0.01% yeast extract, which was adjusted to a pH of 5.0 by adding 10 M hydrochloric acid and sterilised by filtration, which was mixed with molten agar to achieve a final concentration of 2% (w/v) BSA agar. A drop containing 5 9 105 cells/ml of test isolates was carefully spotted onto the BSA medium and incubated at 37°C for 48 h. Plates were then stained with amido black (0.25% [w/v], glacial acetic acid 49.75% [v/v]) and immediately washed with distilled H2O, after which the diameter of both the C. albicans colony and the zone of precipitation was measured. Prz was calculated as the proportion of colony diameter compared to the diameter of haemolysis zone. Data Analysis Statistical analysis was carried out using SPSS (version 15). Patients with type 1 diabetes were subdivided into two equal-sized groups based on the median HbA1c = 8.0%. The Kolgomerov–Smirnov test was used to test the distribution of the data, e.g. for enzyme activity and biofilm Bm and indicated that the null hypothesis, ‘the data are normally distributed’, could not be rejected. Proportional data (e.g., HbA1c) were transformed utilising an angular transformation. Analysis of variance ANOVA and post hoc Bonferroni test were carried out to identify statistically significant differences between the patient groups. The Pearson correlation coefficient (R) was determined to investigate the relationship between the parameters measured. A Bonferroniadjusted p value of 0.05 was considered significant. Graphs were prepared by using software GraphPad Prism (version 5.0).

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Results

Hydrolytic Enzyme Production

Clinical Isolates

The Pz levels ranged from 0.47 to 0.64 for BCD and 0.47 to 0.68 for PCD (Fig. 2). For ND, the range was 0.47 to 0.57. There were no statistically significant differences between the groups (P [ 0.05). The Hz values of isolates ranged from 0.42 to 0.64 for the BCD group, 0.51–0.60 for PCD and 0.43–0.88 for ND groups. The haemolysin activity of PCD strains was higher when compared to BCD strains and ND strains. ANOVA indicated statistically significant differences in Hz values between the patient groups (P = 0.017). Post hoc analysis indicated that the statistically significant differences in Hz were between ND and BCD (P = 0.041) and ND and PCD (P = 0.039). The Prz values of isolates ranged from 0.29 to 0.39 for BCD group, 0.20–0.39 for PCD and 0.30–0.64 for ND group. The proteinase activity of PCD strains was higher when compared to BCD strains and ND strains. ANOVA indicated statistically significant differences in Prz values between the patient groups (P = 0.0018). Post hoc analysis indicated that the statistically significant differences in Prz were between ND and BCD (P = 0.043) and ND and PCD (P = 0.002).

Thirty C. albicans clinical isolates were separated into three defined cohorts based on their HbA1C levels, and one type strain ATCC 3153A was added to the non-diabetic control group, which was defined as follows: individuals with better controlled diabetes (BCD; C6 \8% [n = 10]), individuals with poorly controlled diabetes (PCD; C8% [n = 10]) and non-diabetics (ND; HbA1c \5.9% [n = 10 ? 1]). Biofilm Formation The Bm values of isolates range from 0.29 to 1.32 for PCD strains, 0.24–1.16 for BCD strains and 0.15–0.76 for ND strains. By comparing the Bm values of the three cohorts, the PCD strains appear to have a greater Bm than the other two cohorts (Fig. 1). ANOVA indicated statistically significant differences in the Bm of the strains between patient groups (P = 0.032). Post hoc analysis indicated that the statistically significant differences in Bm were between ND and PCD strains (P = 0.037). The difference in Bm between the ND and BCD strains was not statistically significant (P = 0.155).

Absorbance @550nm

1.50

*

1.25 1.00 0.75 0.50 0.25 0.00 ND

BCD

PCD

Bm Fig. 1 Biomass production of Candida albicans isolated from PCD, BCD and ND individuals. The Bm was assessed using a crystal violet assay from which the Ab550 was used as a means of quantification. Strains from the PCD indicated statistically significant differences in the Bm compared to BCD and ND groups (P = 0.037). The difference in Bm between the ND and BCD strains was not statistically significant (P = 0.155)

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Correlations Statistically significant correlations were found to exist between the HbA1c levels of the patients and the Bm (R = 0.384; P = 0.033), Hz activity (R = -0.455; P = 0.010) and Prz activity (R = -0.531; P = 0.002). The hypothesis that the Bm and enzyme activity was correlated (that is Bm and Pz, Hz and Prz values, respectively, were inversely related) was tested. It was found that there was no apparent correlation between the Bm and Pz (R = -0.305; P = 0.053) or Hz activity (R = -0.100; P = 0.296). However, a negative correlation was found between Bm and Prz values (R = -0.343; P = 0.030), which in reality is a positive correlation between Bm and Prz enzyme activity. All correlations are shown in Table 1.

Discussion This study has shown that biofilm formation is positively associated with secretion of hydrolytic

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A

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B

1.00

0.75

Prz

Pz

0.75

1.00

0.50 0.25

0.50

*

**

BCD

PCD

0.25

0.00

0.00 ND

BCD

PCD

ND

Prz

Pz 1.00

C

Hz

0.75

*

*

BCD

PCD

0.50 0.25 0.00 ND

Hz Fig. 2 Comparison of hydrolytic enzyme production of Candida albicans isolated from PCD, BCD and ND individuals. Pz, Hz and Prz levels were determined using agar-based assays. Values represent a ratio of the diameter of colony divided by the diameter of the zone of clearing on each respective agar, i.e. the lower the value, the greater the enzyme activity. No significant differences were observed between

either group for Pz activity (P [ 0.05). Statistically significant differences in haemolysin activity (Hz) were observed between ND and BCD (P = 0.041) and ND and PCD (P = 0.039). Statistically significant differences were also observed for Prz between ND and BCD (P = 0.043) and ND and PCD (P = 0.002)

enzymes involved in proteolysis. The clinical isolates isolated from individuals with poorly controlled type 1 diabetes were those that tended to exhibit these pathogenic characteristics. It is therefore plausible that individuals who harbour these clinical isolates are more frequently prone to developing clinical

manifestations of oral candidosis when an opportunity arises. To our knowledge, this study is the first to show an association between C. albicans biofilm formation and the pathogenic potential of strains isolated from patients with type 1 diabetes. In the study described herein, it was shown that no significant differences were detected for phospholipase production between the groups, and there was no association between phospholipase and biofilm formation. Previous studies have shown that phospholipase production is not associated with the production of other virulence factors and that its regulation is controlled by the growth milieu [27, 28]. The lack of association from these in vitro observations does not suggest that phospholipase is not important in these patient groups, but rather its regulation is dependent on the hostile oral environment, which is high in glucose and low with respect to salivary flow [29]. In comparison, haemolysin activity was shown to be significantly higher in C. albicans from the BCD and PCD groups (P \ 0.05) than the healthy

Table 1 Correlation analysis between HbA1C, Pz, HZ and PrZ BIOMASS HBA1C Pz Hz Prz

Prz

Hz

Pz

R*

0.384

20.531

20.455

-0.030

P#

0.033

0.002

0.010

0.877

R

-0.305

0.071

0.056

P

0.053

0.714

0.772

R

-0.100

0.249

P

0.296

0.177

R

20.343

P

0.030

R* = Pearson correlation coefficient P# = Probability value based on post hoc Bonferroni test. P \ 0.05 denotes significance

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individuals. Tsang and colleagues (2007) similarly demonstrated significantly higher haemolysin production from C. albicans isolated from patients with type 2 diabetes than healthy controls (P \ 0.0001) [31]. Our study also showed that HbA1C levels were positively correlated with haemolysin and biofilm production. Tanaka and colleagues (1997) reported that hyphal cells were able to bind and utilise haemoglobin and expressed higher levels of receptor compared to yeast cells [30]. Hyphae are pivotal for biofilm formation; therefore, within a biofilm, C. albicans may be more able to utilise haemoglobin. This may explain the higher levels observed here, as these strains have adapted to the altered physiological oral environment associated with type 1 diabetes. Proteinase production is a key hydrolytic enzyme involved in oral candidosis, and it has a reported association with biofilm formation [16, 19]. In our study, proteinase production was shown to be significantly higher in C. albicans from the BCD (P \ 0.05) and PCD (P \ 0.01) groups than the healthy individuals and that it was correlated with biofilm production. Tsang and colleagues (2007) also reported that proteinase production was significantly greater from C. albicans isolated from patients with type 2 diabetes when compared to healthy controls (P \ 0.0027) [31]. However, an earlier study had shown that proteinase production was greater in isolates from type 2 compared to that from patients with type 1 diabetes [11, 31]. Interestingly, this same study reported a higher incidence of adhesive capacity in isolates from patients with diabetes compared to controls. This increased ability to colonise relates to the ability of C. albicans to form biofilms and is promoted by adhesins such as agglutinin-like sequences [6]. It is likely that the high density of cells within the biofilm co-ordinates their proteolytic activities through a quorum-sensing network, and the cells collectively release the proteinase as a means of liberating essential nutrients. However, recent studies on aspartyl proteinase mRNA abundance have indicated that the contribution of secreted aspartyl proteinases is low and that hyphal-specific tissue destruction is the predominant cause of tissue damage [18]. The ability to form complex consortia is integrally related to the capacity of C. albicans to cause disease and is principally associated with its filamentous architecture, a requisite of biofilm development [23].

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Biofilm formation is a defining characteristic of oral candidosis, whether associated with soft tissue (mucosa) or hard surfaces (enamel or acrylic prostheses) [22]. Recent studies from our group and others have demonstrated that C. albicans biofilms readily produce hydrolytic enzymes, including secreted aspartyl proteinases [1, 4]. The correlation in this study between HbA1C and biofilm formation suggests that these isolates have adapted to high glucose levels and a hyposalivary environment that is common to patients with diabetes [29]. This may both enhance adhesion and reduce clearance, respectively. The selective advantage of this growth modality may be independently associated with the co-ordinated release of hydrolytic enzymes, which may explain the higher incidence of oral candidosis that is observed in patients with diabetes [8]. In this study, none of the strains examined were isolated directly from biofilms between the palate and the denture because of the sampling procedure, and none of the patients were infected. Nevertheless, this study indicates that in the poorly controlled individuals, there is a higher likelihood for these isolates to succeed within a biofilm consortium and that these strains have an enhanced pathogenic potential as a result. Further studies are required to investigate C. albicans biofilms as pathogenic entities in their own right. Acknowledgments The clinical arm of this study was funded by a grant from the Chief Scientist Office, Scottish Government Health Directorates.

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