Identification of proteins involved in the adhesionof Candida species to different medical devices

Identification of proteins involved in the adhesionof Candida species to different medical devices

Accepted Manuscript Identification of proteins involved in the adhesion of Candida species to different medical devices Arianna Núñez-Beltrán, Everard...

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Accepted Manuscript Identification of proteins involved in the adhesion of Candida species to different medical devices Arianna Núñez-Beltrán, Everardo López-Romero, Mayra Cuéllar-Cruz PII:

S0882-4010(17)30109-2

DOI:

10.1016/j.micpath.2017.04.009

Reference:

YMPAT 2211

To appear in:

Microbial Pathogenesis

Received Date: 5 February 2017 Revised Date:

4 March 2017

Accepted Date: 6 April 2017

Please cite this article as: Núñez-Beltrán A, López-Romero E, Cuéllar-Cruz M, Identification of proteins involved in the adhesion of Candida species to different medical devices, Microbial Pathogenesis (2017), doi: 10.1016/j.micpath.2017.04.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Identification of proteins involved in the adhesion of Candida species to different

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medical devices

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Arianna Núñez-Beltrán, Everardo López-Romero and Mayra Cuéllar-Cruz*

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Departamento de Biología, División de Ciencias Naturales y Exactas, Campus Guanajuato,

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Universidad de Guanajuato, Guanajuato, México.

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Corresponding author:

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Mayra Cuéllar-Cruz. Mailing address: Departamento de Biología, División de Ciencias

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Naturales y Exactas, Campus Guanajuato, Universidad de Guanajuato. Noria Alta S/N, C.P.

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36050, Guanajuato, Guanajuato, México. Phone: (+52) 473 73 20006 Exts. 1455, 8159. E-

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mail: [email protected] or [email protected].

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†Dedication: To Consuelo Eugenia Velazco-Guzmán, in memoriam.

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Abstract

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Adhesion is the first step for Candida species to form biofilms on medical devices

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implanted in the human host. Both the physicochemical nature of the biomaterial and cell

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wall proteins (CWP) of the pathogen play a determinant role in the process. While it is true

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that some CWP have been identified in vitro, little is known about the CWP of pathogenic

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species of Candida involved in adhesion. On this background, we considered it important

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to investigate the potential role of CWP of C. albicans, C. glabrata, C. krusei and C.

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parapsilosis in adhesion to different medical devices. Our results indicate that the four

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species strongly adher to polyvinyl chloride (PVC) devices, followed by polyurethane and

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finally by silicone. It was interesting to identify fructose-bisphosphate aldolase (Fba1) and

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enolase 1 (Eno1) as the CWP involved in adhesion of C. albicans, C. glabrata and C.

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krusei to PVC devices whereas phosphoglycerate kinase (Pgk) and Eno1 allow C.

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parapsilosis to adher to silicone-made implants. Results presented here suggest that these

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CWP participate in the initial event of adhesion and are probably followed by other proteins

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that covalently bind to the biomaterial thus providing conditions for biofilm formation and

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eventually the onset of infection.

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Keywords: Candida species; biofilms; cell wall proteins; adhesion; biomaterials

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chloride and silicone medical devices.

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Fba1 and Eno1 as the CWP involved in adhesion of C. albicans, C. glabrata and C. krusei to PVC devices.

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Moonlight-like proteins are involved in adhesion of Candida species to polyvinyl

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Pgk and Eno1 allow C. parapsilosis to adher to silicone-made implants.

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Introduction

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The high rates of nosocomial morbility and mortality due to invasive fungal infections (IFI)

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are mainly due to Candida species [1-5], with biofilm formation playing a determinant role

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in virulence [6-11]. Candida can form biofilms on human cells as well as on any

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biomaterial implanted in the human host [12-15], and pathogenic species are commonly

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isolated from medical implants such as central venous, peritoneal, vascular and urinary

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catheters, orthopedic and valve prothesis, parenteral nutrition catheters, contact lenses and

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dental devices [4, 5, 16-18]. The physicochemical properties of the dispositive play a

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central role in adhesion of the pathogen [19]. Accordingly, it has been described that

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Candida species preferentially adher to high rugosity and porosity biomaterials [5, 20]. C.

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albicans is the species with the highest ability to adher to practically any surface and the

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ensuing formation of biofilms [20] and therefore it is considered as the main etiological

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agent of mortality by disseminated candidiasis. Other non-C. albicans (NCAC) species

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with the ability to form biofilms such as C. glabrata, C. krusei, C. parapsilosis and C.

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tropicalis have been isolated from medical dispositives with different physicochemical

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properties [21] and are considered as the cause of 50% of fatal fungal infections as

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compared with strains unable to form biofilms [22, 23]. Apart from the chemical nature of

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the biomaterial, another determinant factor for Candida adherence are cell wall proteins

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(CWP). In this regard, some reports indicate that Als proteins are the main CWP implicated

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in adherence and biofilm formation by C. albicans [24-27]. Several genes coding for

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adhesins of the Als family are differentially expressed in diverse materials during biofilm

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formation [28-31]. In the same line, 23 homologous genes known as EPA that code for

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putative CWP have been identified in the genome of C. glabrata [24-27]. When the

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expression of these genes was evaluated during biofilm formation in different culture

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media, it was found that in YPD medium the expression of EPA3 increases while that of the

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EPA7 decreases and in SdmYg medium the expression of EPA1, EPA3, EPA7 and EPA22

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genes was induced [32]. Epa6 is another adhesin involved in biofilm formation by C.

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glabrata [33]. Five orthologs of ALS genes and six GPI-anchored proteins that may be

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involved in biofilm formation have been identified in C. parapsilosis [34]. Other proteins

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are present in the CW but they are not covalently bound to this structure [35]. These

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components are known as moonlighting proteins [36, 37]. One such a protein is Enolase 1

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(Eno1) which forms complexes with kinin protein precursors and high molecular weight

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kininogen (HK) in C. albicans. It has also been shown that Eno1 interacts with the CW

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glucan [38] which facilitates its secretion to the medium [39]. To get an inside into the

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mechanisms of adhesion and biofilm formation by these and other pathogens, it is essential

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to learn on the biomaterials used in medical dispositives and to identify the CWP involved

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in these processes. Here, we evaluated the ability of C. albicans, C. glabrata, C. krusei and

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C. parapsilosis to adher to different medical devices of common use in hospitalized

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patients and identified some CWP potentially involved in this process.

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Materials and methods

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Strains and culture conditions

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The four species of Candida used in this study are described previously [40]. Yeast strains

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were grown in yeast peptone (YP; yeast extract, 1%; peptone, 2%, Sigma-Aldrich, USA) or

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aminoacid-free yeast nitrogen base (YNB, Sigma-Aldrich, USA). YP and YNB media were

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supplemented (YPD/2.0%, YNB/2%) or not (YP) with 2% glucose [40], and 2% agar was

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added to solidify the media [41].

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Biomaterials

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Medical devices used in this study were made of PVC [pediatric intravenous infusion

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(Industrias Plásticas Médicas, México) and adult (NB Surgical Flebotek) patients,

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nasogastric feeding tube (América Médica Asociados, México) and gastrointestinal

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nutrition (Fabricur)], silicone [foley catheter (Esteriflex) and enteral feeding tube for

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premature infants (Kortex)] or polyurethane [adult intravenous catether (Leukomed)].

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Biofilm formation

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The four Candida species were first grown at 28°C in YP medium supplemented with 2%

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glucose. After 48 h, yeasts were harvested by centrifugation and the OD600nm of the cell

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suspension was adjusted to 1.0 in 100 or 500 µL of the following media: YP (C. albicans),

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YPD/2% glucose (C. krusei and C. parapsilosis) and YNB/2% glucose (C. glabrata). Equal

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aliquots (100 or 500 µL) of each suspension were placed in wells of flat-bottomed 96-well 4

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microtiter plates (Nunc, Nalgene) containing the same amount (0.5 cm long) of previously

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scissor-cutted pieces of the different medical devices that were sterilized by exposure to

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UV radiation, and incubated at 37°C. After 0, 6, 18, 24, 30, 36 or 42 h, planktonic cells

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were discarded by washing the wells thrice with sterile phosphate-buffered saline, pH 7.2

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(PBS, Sigma-Aldrich) containing 10 mM calcium chloride. Metabolic activity of sessile

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cells adhered to the plastic surface (biofilm) was measured with XTT to quantify adhesion.

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The same XTT assay was used to determine the viability of sessile cells after exposure to

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different concentrations of H2O2: 0, 50 and 150 mM for C. albicans and C. glabrata and 0,

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300 and 1500 mM for C. krusei and C. parapsilosis, and four antifungals (fluconazole,

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ketoconazole, itraconazole and miconazole, Sigma-Aldrich) at concentrations in the range

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of 0 to 256 µg/mL. Results are the average of three independent experiments.

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XTT assay

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Metabolic activity of sessile cells adhered to medical devices was measured using XTT

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[2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)- Accordingly, 100 ??L of XTT-menadione (0.1

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mg/mL XTT, 2H-tetrazolium-5-carboxanilide] (Sigma-Aldrich), which is reduced by

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mitochondrial dehydrogenase to a water- 1 ??M menadione, Sigma-Aldrich) was added to

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each well soluble formazan that results in a colorimetric change [33], and plates were

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incubated at 37 ºC. After 105 min, the XTT- derived formazan was measured at 492 nm

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using a microtiter plate reader (Varioskan Flash, Thermo Scientific). The same assay was

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used to determine the viability of cells exposed to antifungals and oxidative stress [33].

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Scanning electron microscopy (SEM)

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To form biofilms, pieces (0.5 cm long) cut from the tested medical devices were placed

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under sterile conditions in wells of 24-well tissue culture plates (Corning, USA) and each

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well received 500 µL of a cell suspension (OD600nm 1.0) of each Candida species. Plates

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were gently shaken a 37°C for 48 h and non-adhered (planktonic) cells were removed by

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washing the wells thrice with sterile 10 mM CaCl2 10 mM. Biofilms formed on these

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medical devices were fixed with 4.0% (v/v) glutaraldehide in 0.1 N Na-cacodylate buffer,

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pH 7.4 (Sigma-Aldrich) for 3 h at room temperature and fixed cells were then dried for 6 h

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in a Tousimis Auto Samdri 815 Critical Point Dryer. Dried cells were placed on aluminum

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supports with the help of copper tape, covered with coloidal gold and observed by SEM in

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a EVO HD15 model, high definition ZEISS® microscope. Images were photographed with

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the SE detector of normal electrode at 10 kV under high-vacuum conditions and at a

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working distance of 4 mm.

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Assay of antifungal susceptibility

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Susceptibility to antifungals was carried out in sessile cells of all four Candida species. To

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this purpose, 500 µl containing increasing concentrations of each antifungal dissolved in

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DMSO in the range of 0 to 256 µg/ml, were added to wells containing the biomaterial with

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the biofilm, and the plate was gently shaken 37°C. In all assays, the concentration of

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DMSO did not exceed 1%. After 90 min, antifungals and planktonic cells were removed

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and viability of sessile cells was determined with XTT as described above. Results shown

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are the average of three independent experiments.

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Assay of H2O2 susceptibility

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Pieces of biomaterials carrying the biofilm formed by each Candida species were exposed

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to the indicated concentrations of H2O2 (0, 50 and 150 mM for C. albicans and C. glabrata

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and 0, 300 and 1500 mM for C. krusei and C. parapsilosis) and shaken at 28°C for 90 min.

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Concentrations of H2O2 were used according to previous studies where we demonstrated a

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different susceptibility of Candida species to the oxidant in terms of cell viability as

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determined with XTT [40].

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Statistical analysis

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In order to maximize statistical power (minimum number of comparisons), all temporal

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data were summarized by using the area under the curve (AUC). This was computed for

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each set of data corresponding to all time points of one trial. Area under the curve (AUC) of

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the amount of viable cells. Bars represent the mean value (±SD) of the area under the curve

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(trapezoidal rule), which was computed for the values of eight consecutive time points (0,

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15, 30, 45, 60, 75, 90, and 105 min) (ODxTime) at intervals of 6, 12, 18, 24, 30, 36 and 48

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h. The AUC data were analysed with a two-way ANOVA test, followed by a Bonferroni

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posthoc test (α = 0.05, *:P<0.05, **:P<0.01, ***:P<0.001). The AUC computation

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(trapezoid rule) and the statistical analysis were performed with the GraphPad Prism

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software (Graphpad, USA).

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Cell adhesion assays

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To isolate the CWP potentially involved in cell adhesion, several 4.0 cm-long pieces of

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biomaterials were placed in 90 x 15 mm Petri dishes (Interlux, Klinicus) under sterile

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conditions, mixed with 15 mL of a cell suspension (OD600nm 1.0) of each Candida species

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and gently shaken at 37°C. After 2 h, the biomaterial with the adhered cells was carefully

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removed with the help of tweezers, cells were mechanically detached with a sterile spatula

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and used for extraction of CWP.

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Extraction of CWP

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Cells of the four Candida species detached from the biomaterials as described above were

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centrifuged at 1300 x g for 5 min. The supernatant was discarded and the cell pellet was

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resuspended in 2 mL of cold sterile water containing 1 mM phenylmethylsulfonyl fluoride

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(PMSF), an inhibitor of serine proteases and acetylcholinesterase [42, 43]. Cells were

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disrupted with an ultrasonic homogenizer (Fisher Scientific) set at 80% pulser and 30 W for

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a total time of 3 min in a salt ice-water bath. Sonication was stopped every 30 s to allow

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cells to cool down for 30 s. The lysate was centrifuged at 4°C for 10 min at 18000 x g, the

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supernatant was discarded and the CW pellet was washed exhaustively with cold sterile

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water containing 1 mM PMSF until a clear supernatant was obtained [42, 44]. To extract

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the CWP, the washed walls were resuspended in 2% SDS and 5% β-mercaptoethanol and

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boiled for 4 min. After removing the insoluble material by centrifugation at 6800 x g for 10

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min, the supernatant containing the soluble proteins was saved and cleaned with the

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ReadyPrep 2-D cleanup kit (Bio-Rad). After cleaning, protein concentration was

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determined by the DC method (Bio-Rad).

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Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)

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CWP (200 µg) of the four Candida species were solubilized in lysis buffer [7 M urea, 2 M

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thiourea, 2% CHAPS, 65 mM dithiothreitol (DTT), 2% ampholytes pH 3-10, and

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bromophenol blue] and loaded on 7 cm, pH 4-7 strips (Bio-Rad). Isoelectric focusing was 7

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done using an Ettan IPGphor (GE Healtcare) under the following conditions: passive

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hydration for 16 h without electric current, 500 V for 1.5 h, 800 V for 1.0 h, 7000 V for 2.5

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h and 2200 V for 1.5 h. Later, the strips were reduced (2% DTT) and alkylated (2.5%

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iodoacetamide) in equilibrium buffer (6 M urea, 50 mM Tris-HCl, pH 6.8, 30% glycerol

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and 2% SDS). After separation by isoelectric point, the strip was equilibrated with DTT (10

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mg/mL) and the CWPs were separated in the second dimension in a 12% SDS

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polyacrylamide gel using a Mini-PROTEAN Tetra Cell (Bio-Rad). Finally, the CWP

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profile was detected by staining the 2D-PAGE gels with colloidal Coomassie Blue [42].

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Image analysis of the 2D-PAGE gels and protein identification

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Analysis and image identification of the 2D-PAGE gels was carried out using a PDQuest

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7.0 software (Bio-Rad). Comparisons of the gels were done using a synthetic image

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containing all the protein spots of all analyzed gels. The intensity of the spots was

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normalized and validated in the master gel. A unique identification number was assigned to

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each protein spot. A spot was considered relevant when there was a minimum of two-fold

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difference in its intensity as compared with the corresponding spot obtained from the mock,

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untreated sample [42].

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Protein digestion with trypsin and LC-MS/MS Analysis

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The in-gel digestion protocol is based on the results obtained by Havlis et al. (2003) [45].

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All peptide extracts were pooled into the 96-well plate and then completely dried in a

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vacuum centrifuge. The plate was sealed and stored at 20ºC for LC-MS/MS analysis [46].

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The LC-MS/MS analysis were performed as described previously [46].

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Protein identification

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Protein database searches were performed using Mascot 2.3 (Matrix Science) against

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Candida [47].

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Results

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Evaluation of adhesion of Candida species to different medical devices

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The ability of Candida species to adhere to seven medical dispositives of different

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physicochemical nature was evaluated after eight time intervals. As shown in Fig. 1A, after

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6 h of incubation, C. albicans preferentially adheres to the pedriatic intravenous infusion

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(PII), followed by the catether for nasogastric feeding tube (NFT), intravenous

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administration for adult (INA), adult gastrointestinal catether (CGI), catether for premature

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enteral nutrition (EFT), intravenous catether (INC) and foley catether for adult (FOC). As

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in the case of C. albicans, C. glabrata and C. parapsilosis preferentially adhered to PII and

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to a lower extent to FOC. On its part, C. krusei adhered mainly to CGI and to a lower

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extent to FOC. After 12 h, the four Candida species exhibited the highest avidity for PII,

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followed by NFT, CGI, EFT, INC, INA and FOC (Figure 1B). After 18 h, it was observed

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that C. albicans and C. glabrata adhered mainly to PII and CGI, C. krusei to PII and C.

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parapsilosis to NFT (Figure 1C). After this time, as it occurred after 6 and 12 h, the four

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Candida species poorly adhered to FOC. Interestingly, after 24 and 48 h, all four species

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exhibited the highest adherence to PII, followed by NFT, CGI, INA, INC, EFT and FOC

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(Figures 1D-1G). Taken together, these data show that the four species adhere better to

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PVC-made medical devices than those made with other materials such as silicone and

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polyurethane.

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Physical evaluation of biomaterials by SEM

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The physical characteristics of PII (PVC) and FOC (silicone) to which the four Candida

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species presented the highest and lowest adherence, respectively, were evaluated by

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scanning electron microscopy. As illustrated in Fig. 2A, PII exhibits a rugose, porous

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surface with hollows, in contrast to FOC which shows a uniform, smooth, hollowless

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surface (Fig. 2B). C. albicans adheres uniformly to PII (Fig. 2C), but only to a small part of

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the FOC surface leaving most of it free of cells (Fig. 2D). The same behaviour holds true

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for C. glabrata, C. krusei and C. parapsilosis (Figs. 2E-2J). These observations

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demonstrate that the physical properties of devices implanted in patients noticeably

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influence the processes of adhesion and the ensuing biofilm formation by Candida and

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other pathogens no matter of the infecting species.

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Influence of the physicochemical composition of biomaterials on the response of

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adhered cells to azole-based antifungals and oxidative stress

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After it was demonstrated that the physical and chemical characteristics of medical devices

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influence Candida adhesion, it was important to determine whether these properties also

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affected the response of sessile cells to antifungals and OS, which were used at the

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concentrations indicated in Materials and Methods. Results for antifungals are depicted in

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Fig. 3. As observed in Fig. 3A and 3B, cell viability of sessile cells of C. albicans

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decreased as a function of increasing concentrations of antifungals on either PVC or FOC.

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However, sessile cells formed on PVC showed significant differences at 0.18 µg/mL

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fluconazole e itraconazole, 2.1 µg/mL fluconazole and 6.9 µg/mL itraconazole and at 0.18

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µg/mL itraconazole and ketoconazole. On silicone, significant differences in viability were

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observed at 2.1 µg/mL fluconazole and miconazole and at the same concentration of

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ketoconazole and miconazole (Figure 3B). Sessile cells of C. glabrata formed on PII

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exhibited significant differences at 6.9 µg/mL fluconazole and itraconazole, 0.05 µg/mL

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itraconazole and ketoconazole, and 6.9 µg/mL ketoconazole and miconazole (Figure 3C),

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in contrast to those formed on FOC which showed significant differences at concentrations

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of fluconazole and miconazole of 6.9 and 23.0 µg/mL, respectively (Figure 3D). The

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responses of C. krusei sessile cells were different to those of C. albicans and C. glabrata,

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exhibiting significant differences with all antifungals (Figure 3E). Accordingly, when

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adhered to PVC, variations in viability were observed with fluconazole and miconazole at

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0.05, 0.62 and 2.1 µg/mL, and with fluconazole, miconazole and itraconazole at 2.1 µg/mL

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(Figure 3E). In contrast, sessile cells on FOC were not affected by none of the drugs at all

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tested concentrations (Figure 3F). In the case of C. parapsilosis, differences were observed

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in the response of biofilm cells on PVC to fluconazole and miconazole at 78.6 µg/mL

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(Figure 3G). On silicone, viability varied between itraconazole and miconazole at 256

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µg/mL (Figure 3G). While it is true that, though significant, differences in cell viability for

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the two biomaterials are small, it is also true that sessile cells of all Candida species formed

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on PVC exhibited significant differences to all antifungals whereas those adhered to FOC

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showed differences preferentially to miconazole.

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Regarding the effect of H2O2, the viability of sessile cells of the four Candida species

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formed on either PVC or FOC was not affected by this oxidant (Figure 4).

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Identification of CWP involved in adhesion to medical devices

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To identify the CWP potentially involved in adhesion of Candida to medical devices, cells

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of the four species were allowed to interact with PII or FOC in the conditions described

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above. After 2h, cells were separated and used to extract the CWP. As shown in Fig. 5,

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proteins from cells adhered to either material were separated as well defined, integral bands

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and were thus suitable for further proteomic analysis by 2D-PAGE and LC-MS/MS.

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Accordingly, bands were separated by 2D-PAGE as indicated in Materials and Methods.

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PPC obtained from planktonic cells were used as control. As illustrated below, two spots (1

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and 2) were differentially expressed in the cell wall of C. albicans adhered to PVC (Fig.

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6B) as compared to the control (Fig. 6A) and with cells adhered to silicone (Figure 6C) or

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FOC. Spots 1 and 2 were observed in the cell wall of yeasts adhered to PVC (Fig. 6B) but

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not in those adhered to silicone (Figure 6C). Essentially the same results were obtained

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with C. glabrata and C. krusei adhered to PII (Figs. 6E, 6H) and FOC (Figs. 6F, 6I) and, as

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it occurred with C. albicans, the PPC identified in cells adhered to PII (Figs. 6E, 6H) were

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not expressed in control cells (Figs. 6D, 6G). On the contrary, in the case of C.

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parapsilosis, two differential proteins (spots 7 and 8) were observed in cells adhered to

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FOC (Fig. 6L) but not in planktonic cells (Fig. 6J) nor those adhered to PII (Fig. 6K).

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All differential spots were cut out from the protein gels, digested with trypsin and the

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resulting peptide fragments were analyzed by LC-MS/MS. This technique revealed

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molecular masses and isoelectric points of the proteins in the ranges from 39 to 47 kDa and

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5.8 to 6.3, respectively (Fig. 6, Table 1). Differentially expressed CWP from C. albicans,

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C. glabrata and C. krusei adhered to PVC corresponded to fructose-bisphosphate aldolase

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(Fba1; spots 1, 3, 5) and enolase I (Eno1; spots 2, 4, 6). Those expressed on FOC by C.

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parapsilosis were identified as phosphoglycerate kinase (Pgk; spot 7) and enolase (spot 8).

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Discussion

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Results presented here indicate that the chemical and physical composition of the

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biomaterial influence adhesion and biofilm formation by the four species of Candida,

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which exhibit a stronger avidity for those made of PVC followed by polyurethane and

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finally by silicone. These findings are in accord with other reports that indicate that these 11

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pathogens preferentially adhere to PVC than polyurethane [48] and may be due to the fact

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that PVC exhibits a more rugose surface than teflon and polystyrene [20], polyurethane and

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silicone with a more smooth texture, as well as a high hydrophobicity. The combination of

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chemical composition and surface topography make PVC an ideal material for

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microorganisms to adhere to pores and hollows thus colonizing the whole surface.

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Adherence is based on a phenomenon known as superhydrophobicity which is defined as a

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surface with an angle of contact with water of about 115º a 150º, and an autocleaning

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surface defined as a rugose and hydrophobic region in which a drop of water mantains an

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air layer below that effectively reduces the number of contact points between the drop and

349

the surface [49]. On this background, it seems clear that autocleaning of an implanted

350

device surface can not occur as Candida cells will not slip over as a water drop because

351

CWP will act as adherents to the surface. Thus, it is important to develop biomaterials that

352

allow the pathogens to slip like a water drop thus decreasing their adhesion. To this

353

purpose, biomaterials of common use have been modified by chemical and physical

354

treatments including nano-coating, yet most widely used commercially available devices

355

continue to be those made of PVC, polyurethane, polycarbonate, stainless steel, among

356

others [16, 20, 50-52].

357

In terms of adhesion of Candida cells to biomaterials, our results are comparable with other

358

reports; however, it was interesting to observe that the four Candida species preferentially

359

adher to PVC-made biomaterials, followed by polyurethane and silicone and also that they

360

form the same amount of biofilm when this is measured in same medical dispositive. The

361

latter results are in contrast with other reports that claim that biofilms formed by Candida

362

on different devices vary in thickness and adherence [53]. This difference may be due to the

363

fact that culture media used in this study are more appropriate for adhesion and biofilm

364

formation [40], with the physicochemical nature of the biomaterial being the only variable.

365

In other studies, the same culture medium is used for all Candida species [20, 48, 51].

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The physicochemical nature of the biomaterial does not influence the response of

368

sessile cells to antifungals and oxidative stress

369

Knowledge of the physicochemical properties of medical devices is important in the

370

understanding of the mechanisms involved in the interaction with the cell wall of Candida 12

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and other pathogens. Their surfaces exhibit a particular reactivity which is necessarily

372

different to the internal one. Biomaterials may or may not release toxic substances to the

373

medium in response to the biological surrounding. For this reason, the fine control of the

374

surface is crucial in the design of these dispositives. In this concern, a number of properties

375

such as hydrophobicity/hydrophilicity, the electrical charge of the surface, some specific

376

functional groups, surface receptors and rugosity, among others, have been considered.

377

Despite the efforts to correlate these properties with the responses of biomaterials to factors

378

in the external medium, there exist only a limited information on this issue [54], because

379

biological reactions may depend on a combination of several properties. Very commonly,

380

the surface exhibits some degree of mobility such that atoms and molecules close to it may

381

reorganize in response to the external milieu. Here, the presence of antifungals and H2O2

382

did not affect the biofilm formation suggesting that PVC and silicone failed to release a

383

toxic factor. In any case, if a release occurred, this may have happened after Candida

384

adhered and was therefore irrelevant. These findings demonstrate that the physicochemical

385

nature of the biomaterial does not affect the response of sessile cells to antifungals and

386

oxidative stress.

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The CWP Fba1, Pgk and Eno1 participate in adhesion of Candida species to PVC,

389

silicone and both biomaterials, respectively

390

Here, the potential correlation between CWP and the physicochemical nature of the

391

biomaterial was evaluated in the four Candida species. The presence of different types of

392

proteins in the cell wall such as adhesins, GPI-anchored proteins, PIR proteins [55, 56] and

393

others not covalently bound to this structure is a well documented observation [35].

394

Proteins with dual localization are known as moonlighting proteins [36, 37] and those

395

identified in this study belong to this group as they are found in the cell wall and also

396

playing specific functions in the cytoplasm. Here, Fba1, a glycolytic enzyme detected in C.

397

albicans biofilms [36], was found to favour the adhesion de C. albicans, C. glabrata and C.

398

krusei to PVC dispositives. Moreover, it has been shown that recombinant antigens of this

399

protein from C. albicans react with sera of candidemia patients infected with this and other

400

species of Candida such as C. tropicalis, C. parapsilosis, C. glabrata and C. guilliermondii

401

[57]. Pgk, like Fba1, has also been identified in C. albicans biofilms [36], in the cell wall

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and in patients with IC due to C. albicans [58]. In this study, this protein was found to be

403

involved in adhesion of C. parapsilosis to silicone. In contrast to Pgk and Fba1 that are

404

seemingly specie-specific proteins that allow adhesion to one or the other device, Eno1,

405

also a glycolytic enzyme, participate in adhesion of the four Candida species to medical

406

devices of different composition. This behaviour may be underlied by the same mechanism

407

reported for C. albicans, where it has been shown the presence of kinin protein precursors

408

and high molecular weight kininogen (HK) that trigger the kinin-forming cascade on the

409

cell surface.

410

Absorption studies of CWP of HK-couples have demonstrated that Als3 adhesin, Eno1,

411

phosphoglycerate mutase and triosephosphate isomerase are the major HK receptors.

412

Moreover, Eno1 associates with the cell wall glucan [38], which facilitates secretion of this

413

protein to the medium [39]. These findings are in accord with the fact that Eno1 has been

414

identified in the sera of IC patients [59-61], a finding that has been used to produce

415

candidate vaccines against C. albicans [62]. Our results suggest that Pgk and Fba1 function

416

essentially as Eno1; however, Fba1 and Pgk, but not Eno1, are specie-specific which may

417

explain why Eno1 and not other glycolytic enzymes, is usually identified in sera of IC

418

patients [38, 39]. A plausible explanation for the observation that CWP moonlighting

419

proteins are early identified during adhesion of Candida to medical devices is sustained by

420

the fact that the properties of the surface are determinants for protein adsorption. This event

421

occurs a few seconds after the dispositive contacts the biological medium [63] and before

422

cells reach the substrate domain [64]. In general, proteins prefer an aqueous environment;

423

however, when a protein solution is mixed with another phase, they tend to accumulate in

424

the interphase. In a solid interphase, proteins interact with the surface through electrostatic,

425

polar and hydrophobic forces. Thus, when CWP such as Eno1, Fba1 and Pgk are secreted

426

to the medium, they facilitate Candida adhesion to the device thus allowing that other CWP

427

such as adhesins (Als, Epa, Hwp1, among others) covalently bind to the biomaterial to

428

establish a strong contact with the pathogen.

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429 430

Conclusions

431

Adhesion of Candida cells to medical devices is strongly influenced by the

432

physicochemical nature of the implant and CWP of the pathogen. Our results indicate that 14

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this process is much more complicated than previously believed and proteins such enolase

434

1, fructose-bisphosphate aldolase and phosphoglycerate kinase are likely to represent the

435

first point of contact between the organism and the dispositive, followed by other CWP that

436

prevent the detachment of cells from the implant. These results can serve as the basis to

437

design new biomaterials that consider not only the GPI-anchored adhesins but also CWP as

438

well.

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Competing interests

441

The authors declare that there were no conflicts of interest with any organization or entity

442

with a financial interest or financial conflict with the material discussed in this work.

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Acknowledgements

445

This work was carried out with the financial support granted to Dr. M. Cuéllar-Cruz by

446

Proyecto-Institucional-831/2016 (Universidad de Guanajuato, México). We are grateful to

447

Universidad de Guanajuato for grant 89/2016 to Departamento de Biología. We thank Dr.

448

M.A. Martínez-Rivera from the Departamento de Microbiología, ENCB-IPN, México, for

449

kindly providing the clinical isolates of Candida species used in this study. We also thank

450

Prof. Denis Faubert from the Proteomics Discovery Platform, IRCM, Montréal (Québec),

451

Canada, for the facilities and technical assistance in the LC-MS/MS analysis, and Dr. Mario

452

Ávila-Rodríguez, Dr. Ricardo Navarro, Dr. Ramón Zárraga-Núñez and MSc Paulina

453

Lozano-Sotomayor (Universidad de Guanajuato, México) for their help with the SEM

454

photographs. Finally, we thank the technical assistance of Dr. Arturo Vega-González (DCI,

455

Universidad de Guanajuato) in the statistic analysis.

456 457

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Figure legends

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Figure 1. Determination of adherence of Candida species cells to different medical

644

devices as measured by the XTT assay. Bars represent the mean value (± SEM) of the

645

area under the curve, which was computed for the OD values of eigth consecutive time

646

points (0, 15, 30, 45, 60, 75, 90 and 105 min) at eight different elapsed intervals of 6, 12, 21

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18, 24, 30, 36, 42 and 48 h. The AUC data were analyzed with a two-way ANOVA test,

648

followed by a Bonferroni posthoc test (α = 0.05, *:P<0.05, **:P<0.01).

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Figure 2. SEM images of adherence of the four Candida species to PVC (PII) and

651

Silicone (FOC). (A), PII; (B), FOC. (C), C. albicans in PII; (D) C. albicans in FOC; (E),

652

C. glabrata in PII; (F), C. glabrata in FOC; (G), C. krusei in PII; (H), C. krusei en FOC;

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(I), C. parapsilosis en PII, and (J), C. parapsilosis in FOC. Samples were observed with a

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scanning, high definition ZEISS® microscope, model EVO HD15. Images were taken using

655

the Secondary Electron Detector (SE1) at 15 kV under high vacuum and at a working

656

distance of 4 mm. The scale bar is indicated for each microphotography.

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Figure 3. Influence of the physicochemical composition of PVC (PII) and Silicone

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(FOC) on the effect of azole-derived antifungals on sessile cells of the four Candida

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species. The effect of four antifungals on sessile cells (biofilm) of the Candida species was

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measured as described under Materials and Methods. (A), C. albicans en PII; (B), C.

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albicans in FOC; (C), C. glabrata in PII; (D), C. glabrata in FOC; (E), C. krusei en PII;

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(F), C. krusei in FOC; (G), C. parapsilosis en PII, and (H), C. parapsilosis en FOC. Bars

664

represent the mean value (± SEM), of the area under the curve of eigth consecutive time

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points. The AUC data were analysed with a two-way ANOVA test, followed by a

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Bonferroni posthoc test (α = 0.05, *:P<0.05, **:P<0.01).

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Figure 4. Representative graphs of the response of sessile cells to oxidative stress

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(H2O2). The effect of increasing concentrations of H2O2 on sessile cells C. glabrata (A) and

670

C. krusei (B) is shown. The same results were obtained for C. albicans and C. parapsilosis

671

(data not shown). Bars represent the mean value (± SEM), of the area under the curve,

672

which was computed for the OD values of eigth consecutive time points. The AUC data

673

were analysed with a two-way ANOVA test, followed by a Bonferroni posthoc test (α =

674

0.05, *:P<0.05, ***:P<0.001).

AC C

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675 676

Figure 5. Separation of CWP of the four Candida species in unidimensional gels.

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CWP extracted from sessile cells of the four Candida species were analyzed in SDS-gels as 22

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described under Materials and Methods and gels were stained with Coomasie Blue.

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Asterics refer to differentially expressed bands. MWM, Molecular weight markers.

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Figure 6. 2D-PAGE analysis shows that Fba1 favours adhesion to PVC, Pgk to

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Silicone and Eno1 to both biomaterials. 2D-PAGE maps of (A) planktonic cells of C.

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albicans; (B) C. albicans in PII; (C) C. albicans in FOC; (D) planktonic cells of C.

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glabrata; (E) C. glabrata in PII; (F) C. glabrata in FOC; (G) planktonic cells of C. krusei;

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(H) C. krusei in PII; (I) C. krusei in FOC; (J) planktonic cells of C. parapsilosis; (K) C.

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parapsilosis en PII, and (L) C. parapsilosis in FOC. Circled spots correspond to up- and

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down-regulated proteins with respect to both biomateriales (PVC and Silicone). Sequence

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information from mass spectrometry analysis was performed using Mascot 2.3 (Matrix

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Science) against Candida. MWM, molecular weight markers.

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Table 1. CWP identified in sessile cells of Candida species separated by 2D-PAGE and

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analyzed by LC-MS/MS.

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23

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2

Mass

Fructose-bisphosphate aldolase (Fba1)

Phosphopyruvate hydrate (Eno2)

pI

47

5.7

69

6.2

Organism/reference

Candida albicans

Candida albicans

Fructose-bisphosphate aldolase (Fba1)

47

5.7

Candida glabrata

4

Enolase 1 (Eno1)

69

6.2

Candida glabrata

6

Enolase 1 (Eno1)

69

Sequence coverage (%)

Upregulated

Glycolytic process Interaction with host Induction by symbiont of host defense response

193

95

Gluconeogenesis Regulation of vacuole fusion, non-autophagic Glycolytic process

381

95

Glycolytic process Interaction with host Induction by symbiont of host defense response

72

95

Upregulated

Interaction whit the host Glycolytic process Response to stress

28

95

57

95

Upregulated

Upregulated

5.7

Candida krusei

upregulated

Glycolytic process Interaction with host Induction by symbiont of host defense response

6.2

Candida krusei

upregulated

Interaction whit the host Glycolytic process Response to stress

30

95

312

95

35

95

TE D

47

Peptide matching

EP

Fructose-bisphosphate aldolase (Fba1)

Function

M AN U

3

5

Expression

RI PT

1

Protein identity

SC

Spot

Phosphoglycerato kinase (Pgk)

8

Enolase 1 (Eno1)

Table 1.

47

5.7

Candida parapsilosis

upregulated

69

6.2

Candida parapsilosis

upregulated

Interaction whit the host Glycolytic process Response to stress

AC C

7

Cell wall organization Induction by symbiont of host defense response Glycolytic process, Interaction with host

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AC C

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TE D

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AC C

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TE D

M AN U

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AC C

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TE D

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Highlights: •

Moonlight-like proteins are involved in adhesion of Candida species to polyvinyl chloride and silicone medical devices.



Fba1 and Eno1 as the CWP involved in adhesion of C. albicans, C. glabrata and C.

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Pgk and Eno1 allow C. parapsilosis to adher to silicone-made implants.

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krusei to PVC devices.