World J Microbiol Biotechnol (2015) 31:11–21 DOI 10.1007/s11274-014-1760-7

ORIGINAL PAPER

A monoclonal antibody against 47.2 kDa cell surface antigen prevents adherence and affects biofilm formation of Candida albicans Nripendra Nath Mishra • Shakir Ali Praveen K. Shukla

•

Received: 12 September 2014 / Accepted: 14 October 2014 / Published online: 18 October 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract Candida albicans is an opportunistic dimorphic pathogen that exists in both planktonic and biofilm phases causing deep-rooted infections in mainly immunocompromised patients. Antibodies are believed to play anti-Candida activity by different mechanisms, like inhibition of adhesion and neutralization of virulence-related antigens. Inhibition of adhesion is one of the important strategies to prevent Candida infections and biofilm formation. In this study, monoclonal antibody (MAb 7D7) against C. albicans biofilm cell surface antigen (47.2 kDa) was generated to determine the changes in adherence and viability of C. albicans. In this regard XTT assay was carried out in 30, 60, 90 min and 48 h (maturation time) time points using MAb 7D7 and it (MAb 7D7) was found to be effective against adhesion and the formation of C. albicans biofilm on polystyrene as well as monolayer of human epithelial cells (HeLa). This result may also prove to be a valuable addition to the reagents available to study C. albicans cell surface dynamics and interaction of the fungus with host cells. Keywords Biofilm  Candida albicans  Cell surface protein  Hela cells  Monoclonal antibody

N. N. Mishra  P. K. Shukla (&) Medical Mycology Lab, Division of Microbiology, CSIR-Central Drug Research Institute, Sitapur Road, Lucknow 226 031, India e-mail: [email protected] S. Ali Department of Biochemistry, Jamia Hamdard, New Delhi, India

Introduction Candida albicans is the most common fungal pathogen that is frequently found in normal microbiota of human and which expedites their confrontation with implant biomaterials and immunocompromised hosts (Harriott and Noverr 2011; Kumamoto 2011; Macfarlane and Dillon 2007). For the past few years transplantation procedures, use of chronic medical devices, dental infections and prolonged intensive care unit stays have increased the prevalence of fungal infection and biofilm formation (Ramage et al. 2009; Zijnge et al. 2010). Candida infections and biofilm formation primarily begin with adherence and colonization on the surface such as polystyrene/silicone or a biotic host and recently, it has been reported that almost 65 % of all human microbial infections involve biofilms (Zijnge et al. 2010; Potera 1999). The modern medical technology allows the use of a wider and newer variety of devices in the immunocompromised patients and the growing numbers of insertion devices may be a fatal combination to escalate the complications particularly those involving the bloodstream and urinary tract infections (Jarvis 1995; Richards et al. 1999). The cell wall of C. albicans is a dynamic structure that protects from host response and adjusts itself according to that (Chaffin 2008). The cell surface is also the site that mediates adherence to host surfaces, ligands and interacts with host defenses. During the C. albicans infection, high propensity to cause candidiasis and biofilm formation is due to the expression of many cell surface proteins, which are highly immunogenic and able to act as virulence factor (Pietrella et al. 2006). Recently, it has been observed that antibodies in human sera that recognize surface proteins of C. albicans may have an important role in diagnosis and therapy (Pitarch et al. 2004, 2006). Many novel antifungals

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therapies target cell-surface, cell wall, and secreted proteins; moreover cell-surface proteins in different conditions may prove to be a powerful tool to study as potential targets against Candida biofilm. At present, limited works are available that deal with possible ways to reduce biofilm formation through inhibition of C. albicans adherence using antifungals or antibodies (Francolini and Donelli 2010; Cateau et al. 2007; Maza et al. 2009). To eliminate biofilm from the host aimed mainly via antifungals, however some research have published on nano particles, but their limited efficacy draw the attention to need of new therapy against Candida infection and its biofilm formation. In earlier studies, it has been reported that various known Candida proteins found as circulating antigens in blood, elicit an immune response in candidiasis patient during infection (Walsh et al. 1991; Sentandreu et al. 1995). Thus, any antibody against a cell surface protein of C. albicans biofilm can be effective to prevent adherence and biofilm formation. The objective of the present study was to generate monoclonal antibodies against cell surface protein(s) of C. albicans biofilm and investigate its inhibitory effect against the adherence and biofilm formation. This study was focused on two aspects: (1) can blocking the surface protein of C. albicans with the generated MAb 7D7 significantly contribute to a reduction in adherence during biofilm formation? and (2) can a decrease in adherence of Candida cells affect the quantity of mature biofilm?.

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(1 9 106 cells/ml) in YNB with amino acids (Difco, BD) supplemented with 0.9 % D-glucose (Sigma, Aldrich) and kept at 37 °C for 90 min (adhesion phase) under shaking condition (75 rpm). Further, the cells were washed with 19 phosphate buffered saline (PBS) to remove non-adherent cells and incubated for 48 h with fresh YNB medium. To determine the expression of C. albicans biofilm cell surface antigen by ELISA method biofilm formation was performed in 96-well culture plates (flat bottom, Griener, USA). C. albicans biofilm cell surface protein isolation and generation of monoclonal antibodies Isolation of proteins Biofilms were collected from the polystyrene surfaces with the help of a cell scraper (Griener, USA). The collected cells were washed three times with chilled Triple distilled water (TDW), mixed with ammonium bicarbonate (1.89 g/ l) and 1 % b merceptoethanol, and incubated for 1 h on a rocker at 37 °C for extraction of cell surface proteins (Videyappan et al. 2000). After incubation the cells were centrifuged and the collected supernatant was filtered through 0.22 lm membrane and dialyzed against 5 mM Tris–Cl buffer (pH7.4) for 24 h at 4 °C. The protein contents were determined by using 2D-Quant kit (Amersham Biosciences, USA). Monoclonal antibody generation

Materials and methods Organisms and media All Candida strains were maintained on Sabouraud dextrose agar slants and yeast cells were grown in YPD (Yeast extract, Peptone and Dextrose) medium for further study. Two strains of C. albicans, ATCC-10231(standard strain) and ATCC-14053 and four patient isolates PK-9, PK-30, PK-31, and PK-32 were used during this study. Yeast nitrogen base (YNB) medium supplemented with amino acids and 0.9 % glucose was used for biofilm formation. The identity of patient isolates was confirmed through PCR targeting internal transcribed spacer regions and singlestranded conformation polymorphism analysis in our lab (Kumar and Shukla 2006; Masaki et al. 2011). Biofilm formation Candida albicans biofilm formation was carried out according to the protocol of Li et al. (2003) with minor modifications. Briefly, 12 well tissue culture plates (polystyrene) were dispensed with 500 ll of cell suspension

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Female BALB/c mice were immunized subcutaneously with 100 ll of C. albicans ATCC-10231 biofilm cell surface antigens (100 lg/dose) in Freund’s complete adjuvant (1:1) followed by five booster doses with incomplete Freund’s adjuvant. Fusion of the splenocytes from immunized mice with Sp2/O (ATCC-CRL-1581) myeloma cells was carried out in 50 % polyethylene glycol (Hybridoma tested from Sigma, USA). This suspension was then mixed with RPMI 1640-HEPES modified medium supplemented with 10 % fetal bovine serum (Sigma, USA) and dispensed in 96-well tissue culture plates (Greiner, Bio One GmbH). The plates were incubated at 37 °C in a 5 % CO2 atmosphere and positive hybrid clones were selected by ELISA after 7 days of incubation followed by single cell cloning (Chaturvedi et al. 2005; Bakshi et al. 2007). Antibodies were produced in serum-free medium from where the supernatant were precipitated with 50 % ammonium sulfate, dialyzed against Dulbecco’s phosphate-buffered saline (DPBS), and stored at -80 °C. The antibodies preparation was quantified with 2D Quant kit (Amersham Bioscience, USA) and the percentage of immunoglobulin were determined in a protein gel by image analysis using

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Quantity 1 software (Bio-Rad) as per User Manual Version 4.2.1. Purification and identification of C. albicans biofilm cell surface protein To purify the C. albicans biofilm cell surface antigen by immunoaffinity chromatography, production of monoclonal antibody 7D7 (MAb 7D7) was accomplished by development of ascites in BALB/c mice. The mice were first primed with Freund’s incomplete adjuvant (0.5 ml each) for 7 days and implanted with 0.5 ml of 7D7 hybridoma cell suspension (2 9 106 cells/ml) in PBS through intraperitoneal route. The ascites fluid was collected and purification of the antigen recognized by the MAb 7D7 was carried out using Protein A column (GE Health Care, USA) as per instructions of manufacturer as follows. Briefly, ascites fluid (MAb 7D7) was diluted with binding buffer (50 mM sodium borate buffer of pH 9.0 to final volume of 10 ml and final concentration of NaCl was 3 M). Sepharose (5 ml) coupled protein A beads (GENEI) were added to this solution and incubated at RT for 1 h with gentle shaking. Antibody coupled beads were then washed with binding buffer and re-suspended in 200 mM sodium borate buffer (pH 9.0). Dimethylpimelimidate (covalent coupling reagent) was added to this suspension to a final concentration of 20 mM and incubated at RT for 30 min with gentle shaking and the reaction was stopped by washing the beads with 0.2 M ethanolamine (pH 8.0). Finally the beads were washed thrice with PBS (pH 7.4) and MAb 7D7 coupled beads were packed in a glass column (Sigma). C. albicans biofilm cell surface proteins were applied onto the column (flow rate 1 ml/min) and flow through was collected. Elution of the bound antigen was carried out by using elution buffer (0.1 M glycine–HCl, pH 2.7) and collected in microfuge tubes containing neutralizing buffer (1 M Tris–HCl, pH 9.0). The purified protein was separated by SDS PAGE and transblotted to perform Western blotting for confirmation of the purified protein. MAb 7D7 recognized protein was cut from the SDS-PAGE gel and processed with acetonitrile and ammonium bicarbonate buffer. The processed protein was digested with trypsin (Huynh et al. 2009) and identified by MALDI-TOF. PMF and MS/MS ion data obtained by MALDI-TOF MS analysis was used for identification of protein by interrogating NCBI protein database for all entries using MASCOT search program (Matrix Sciences, London, UK). The individual ion scores [59 indicated identity or extensive homology (p \ 0.05) and therefore, the mascot ion score above 59 was taken as significant. Immunofluorescence assay and ELISA The epitope localization of MAb 7D7 was demonstrated by immunofluorescence assay against all the test strains. The

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strains were subjected to biofilm formation in 6 well tissue culture plates and adherent cells were fixed with paraformaldehyde for 4 min and washed subsequently with 19 PBS. The biofilm cell surface antigens were blocked with 1 % gelatin and after blocking, the cells were incubated with MAb 7D7 for 90 min on ice. The cells were washed thrice with 1X PBS and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (1:200) in PBS-Tween 20 at 37 °C for 90 min in a moist chamber (Chaturvedi et al. 2005). The biofilm cells were examined under a fluorescence microscope (Lieca DM2500). To evaluate the expression level of cell surface antigen by ELISA, the biofilm of C. albicans test strains was performed in 96 well plates (Bujdakova et al. 2010). The biofilms were washed thrice with 200 ll of 19 PBS and the surface antigens were blocked with 1 % gelatin (w/v in 19 PBS) for 90 min at 37 °C. The biofilms were washed with 19 PBS and incubated with 100 ll of MAb 7D7 (diluted 1:100 in PBS) and 1C9 MAb (as control) for 1 h, on ice. After incubation biofilms were washed with 19 PBS (0.05 % v/v Tween 20) followed by centrifugation to remove unbound antibody. One hundred ll of secondary antibody goat anti-mouse immunoglobulin G (IgG) was added to each well of 96 well plates and incubated at 37 °C for 90 min. The plates were then washed finally with 19 PBS and ortho phenylene diamine dihydrochloride in substrate buffer was added and quantified with the help of a spectrophotometer at 405 nm. Blocking of surface proteins by MAb 7D7 and determination of viability in adherence phase and mature biofilm Candida albicans cells (107 cells/ml) were grown overnight in YPD medium at 35 °C, centrifuged, and washed three times with 19 PBS. The appropriate amount of the cell pellet was used to block the surface antigens with 1 % gelatin (in 19 PBS) on ice for 1 h with shaking (Bujdakova et al. 2010). The cells were washed with 19 PBS, centrifuged, and the pellet was re-suspended in 100 ll solution of MAb 7D7 in PBS (1:100) whereas the control samples were suspended in PBS only. After 1 h incubation on ice, unbound antibodies were removed by centrifugation and the cells were re-suspended in a precise volume of YNB with amino acids containing 0.9 % D-glucose (cell concentration, 106 cells/ml). A 100 ll aliquot of this suspension was dispensed in 96-well plates to undergo the adherence phase for 30, 60, and 90 min at 37 °C. At these time points, nonadherent cells were removed by washing with 19 PBS and the adherent ones washed thrice with 19 PBS. The viability of biofilm cells was evaluated by 2,3bis(2-methoxy-4-nitro-5-sulfophenyl)- 2H-tetrazolium-5carboxanilide (XTT) sodium salt (Sigma-Aldrich) in 96

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well plates (Bujdakova et al. 2010). The parallel experiments were continued after the adherence phase (90 min), in addition the adherent cells were washed with 19 PBS and overlaid with 200 ll of the fresh YNB medium followed by incubation at 37 °C for 48 h (San Millan et al. 1996). The viability of mature biofilm was also evaluated by XTT assay. Every experiment was performed in three parallel wells and performed thrice and the results were expressed as mean ± SD. Adhesion assay The monolayer adherence assay was performed on HeLa cells (ATCC-CCL2), following the method of Samaranayake and MacFarlane (1981 and 1982) with some modifications. HeLa cells were detached from tissue culture flasks by trypsinization and resuspended in RPMI 1640 medium with 10 % FBS, followed by incubation for 48 h at 37 °C. To quantify the adhesion of C. albicans cells over the monolayer of HeLa cells, the monolayers were washed thrice with DPBS. Furthermore, C. albicans cells were incubated with 1 % gelatine in PBS for 90 min on ice and then after cells were incubated with or without MAb 7D7 in PBS (1:100) for 1 h on ice. After incubation the cells were washed with 19 PBS and incubated over HeLa monolayer in RPMI medium without FBS for 90 min. The total C. albicans cells were quantified under phase contrast microscope (409) in four different fields (Romeo et al. 2009). This was followed by a PBS wash to remove non adherent cells and quantified in same field of microscope. The inhibition in adhesion was expressed as percentage of adhering cells.

Fig. 1 Characteristic profile of C. albicans (ATCC-10231) biofilm cell surface proteins on SDS-PAGE, Strips (left to right): M standard molecular mass marker, P complete protein profile on gel stained with Coomassie blue, IM immunogenic proteins of C. albicans biofilm cell surface as detected by using sera of immunized mice, MAb C. albicans biofilm cell surface antigen probed with MAb 7D7 and G, ConA blotting of protein, demonstrating it to be a glycoprotein

Statistical analysis In this manuscript results were calculated as average ± SD and the significance of result was calculated by using Student’s t test. In this study p value of \0.05 was considered significant, and \0.005 highly significant.

Results In this study, C. albicans (ATCC-10231) biofilm cell surface proteins were isolated to generate antibodies and protein content was estimated to be 2.4 mg/ml. Biofilm cell surface proteins isolated from C. albicans ATCC-14053 and 4 other clinical isolates were estimated to be 1.5–0.32 mg/ml. The SDS PAGE profile of cell surface proteins from C. albicans ATCC-10231 have shown in Fig. 1 and protein profile of patient isolates in Fig. 2a, which depicted nearly similar profile (20–90 kDa). To generate monoclonal antibodies (MAbs), the fusion of

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Fig. 2 SDS-PAGE profile and immunoblotting analysis of patient isolates biofilm cell surface proteins. a C. albicans patients isolates (M-Molecular weight marker, 1-C. albicans ATCC14053, 2-PK-9, 3-PK-30, 4-PK-31, and 5-PK-32) cell surface proteins profile stained with Coomassie brilliant blue R250. b Western blotting of C. albicans patient isolates biofilm cell surface proteins (M-Molecular weight marker, 1-C. albicans ATCC14053, 2-PK-9, 3-PK-30, 4-PK-31, and 5-PK-32) probed with MAb7D7

spleen cells from immunized mice with Sp2/O myeloma cells was performed and resulted in production of hybrid cells. A total of 7 hybridoma clones producing MAbs against C. albicans (ATCC-10231) cell surface proteins identified by ELISA were subjected to single cell cloning and cryopreserved in liquid nitrogen for further studies. Indirect immunofluorescence study and Western blotting

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Fig. 3 SDS PAGE of the purified cell surface antigen stained with Coomassie brilliant blue, western blot of this purified antigen probed with MAb 7D7 and identification of this protein by the help of MALDI-TOF. a SDS-PAGE of purified antigen (47.2 kDa) stained with Coomassie brilliant blue. b Western blot of purified protein with MAb7D7 and single band was appeared. c MALDI-TOF score graph data. d Mascot matrix analysis of MALDI-TOF data reveals that this hypothetical protein (47.2 kDa) shows 38 % sequence coverage (in red) with enolase1 of C. albicans SC5314. (Color figure online)

b

a

c

1 51 101 151 201 251 301 351 401

MSYATKIHAR RDGDKSKWLG TPNKSKLGAN PFQNVLNGGS KKYGQSAGNV SSEFYKDGKY AEDDWDAWVH NQIGTLTESI TGAPARSERL

YVYDSRGNPT KGVLKAVANV AILGVSLAAA HAGGALAFQE GDEGGVAPDI DLDFKNPESD FFERVGDKIQ QAANDSYAAG AKLNQILRIE

VEVDFTTDKG NDIIAPALIK NAAAAAQGIP FMIAPTGVST KTPKEALDLI PSKWLSGPQL IVGDDLTVTN WGVMVSHRSG EELGSEAIYA

LFRSIVPSGA AKIDVVDQAK LYKHIANISN FSEALRIGSE MDAIDKAGYK ADLYEQLISE PTRIKTAIEK ETEDTFIADL GKDFQKASQL

STGVHEALEL IDEFLLSLDG AKKGKFVLPV VYHNLKSLTK GKVGIAMDVA YPIVSIEDPF KAANALLLKV SVGLRSGQIK

d

revealed that out of these hybridomas only one monoclonal antibody (MAb 7D7) which had a strong capacity to bind to surface antigen of C. albicans biofilm cells. Identification of C. albicans biofilm cell surface purified antigen The cell surface antigen of C. albicans biofilm recognized by MAb 7D7 was purified using Sepharose (5 ml) coupled

protein A beads to identify with MALDI-TOF analysis. The protein could finally be purified under high salt conditions (1 M NaCl), eluted at low pH conditions (pH 2.7) to avoid any permanent structural changes and checked for purity by SDS PAGE (Fig. 3a). Western blotting was also performed with this purified antigen to confirm further that it is the same protein that was recognized by MAb 7D7 (Fig. 3b). For identification of purified protein the protein spot from the gel (SDS PAGE) was processed for MALDI-

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Table 1 Identification of antigen recognized by MAb 7D7 NCBI ID

Protein

MW/PI

Score

Sequence coverage

Matches

Description

3646484

Hypothetical proteina

47.2/5.54

507

38 %

11

C. albicans Enolase 1

a

This protein show similarity with C. albicans Enolase; major cell-surface antigen of C. albicans biofilm; binds host plasmin/plasminogen; immunoprotective; glycolysis and gluconeogenesis; flow model biofilm induced

Fig. 4 Expression of the surface antigen of C. albicans (ATCC10231 and ATCC-14053) and patient isolates (PK-9, PK-30, PK-31 and PK-32) detected by ELISA in adhesion phase (90 min) as well as in mature (48 h) biofilm using MAb 7D7 as primary antibody. From left to right C.a (Candida albicans ATCC-10231), C.a17 (C. albicans

ATCC-14053) and patient isolates (PK-9, PK-30, PK-31and PK-32). No significant reaction was detected for the strains incubated with MAb1C9 (irrelevant antibody). a The expression level in adhesion phase (90 min) and b the expression level in mature biofilm (48 h)

TOF analysis. The mass spectrometric data were generated using the reflector positive mode. The peptide mass fingerprinting and peptide fragment ion data were analyzed for the search of proteins in all the entries of the NCBI database with the help of Mascot software (Matrix Science) online. The protein databank search revealed that purified surface protein was of 47.2 kDa and this protein has 38 % sequence coverage with enolase 1(Eno) of the yeast C. albicans SC5314 (Fig. 3d, Table 1). Data also suggested that this protein (47.2 kDa) is present on the cell surface of C. albicans biofilm, enzymatic in nature and biofilm induced (Table 1).

as a non specific antibody (control antibody). In this experiment, the surface protein (47.2 kDa) was detected in the adherence phase (90 min) as well as in the mature (48 h) biofilm. During the adherence phase surface protein (47.2 kDa) expression was higher in C. albicans ATCC10231 with compared to other used strains (Fig. 4a), and it was also found to be markedly higher in the mature biofilm because of the presence of the hyphal morphological form (Fig. 4b). The surface antigen expression was found almost equal in C. albicans ATCC-14053 and patient isolates (PK30, PK-32) in both adherence and biofilm phase (Fig. 4a, b). In contrast, the patient isolates PK-9 and PK-31 showed lower expression of the protein (47.2 kDa) in both adherence and mature biofilm phase. These results indicate that, the expression of this antigen is strain dependent and it needs to be further investigated. In the other experiment, strong immunofluorescence was detected after incubation of test strains with MAb 7D7. In this experiment MAb 7D7 was used as primary antibody to detect surface epitope, during immunofluorescence microscopy with fluorescein isothiocyanate-conjugated secondary anti-mouse IgG. The result exhibited that the epitope of antigen is uniformly distributed over the entire surface of all C. albicans adherent cells (Fig. 5a–f) and C. albicans ATCC-10231 biofilm cells (Fig. 5a) exhibited strong fluorescence compared to other test strains (Fig. 4a, b). The immunofluorescence result of C. albicans 14,053 cells

Expression analysis and immunofluorescence assay of C. albicans surface antigen in biofilm Candida albicans biofilm cell surface protein (47.2 kDa) expression and its epitope localization recognized by MAb 7D7 was carried out to assess the distribution of this antigen on the surface of the pathogen in both adhesion phase and mature biofilm. Two experiments were carried out to confirm the expression identification of surface antigen in the adhesion phase as well as in mature biofilm. In the first one quantification of surface antigen of C. albicans biofilm was carried out by ELISA (In both standard and patients isolates), where MAb 7D7 was used to detect surface protein (47.2 kDa) expression and MAb1C9

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exhibited slightly more fluorescence with compare to patient isolates (Fig. 5b–d, f). C. albicans surface protein (47.2 kDa) was manifested in both phases of the biofilm, where its expression was markedly higher in the mature biofilm, thus it may be possible that surface protein (47.2 kDa) expression is increasing from adherence phase to mature biofilm phase (Fig. 4a, b). Reduction in adherence of C. albicans and biofilm (48 h) formation The main focus of this study was to assess the effect of MAb 7D7 (anti-47.2 kDa) on the reduction of biofilm formation and the results are summarized in Fig. 6a–c, where it is clearly shown that the adhesion of C. albicans cells was reduced after 90 min adhesion phase, although this process appeared to be strain dependent. In C. albicans ATCC-10231 strain, the percentage of reduction in adherence using MAb 7D7 compared to control was found to be significant (p \ .005) with regard to time points: 69.0 % ± 2.2, 72.7 % ± 3.0, and 54.4 % ± 2.8 respectively, at 30, 60, and 90 min, as indicated in Fig. 6a–c. In case of patient isolates the reduction in adherence (50.2 % ± 5.3, 35.0 % ± 1.9, 41.8 % ± 1.1, and 34.8 % ± 1.6 respectively) caused by monoclonal antibody MAb 7D7 at 60 min was found to be less as compared to the standard isolates (72.7 % ± 3.0 and 59.1 % ± 3.2) used in this study whereas there was no significant difference in any of the isolates at 90 min (Fig. 6b, c and Table 2). The monoclonal antibody MAb 7D7 was also capable of reducing the mature biofilm formation up to 49.2 % ± 1.6 and 35.4 % ± 1.3 against C. albicans ATCC-10231 and C. albicans ATCC-14053 respectively (p \ .005). However this effect was less prominent in case of patient isolates PK-31, PK-9, PK-30, and PK-32 (26.4 % ± 1.1, 24.7 % ± 1.2, 22.0 % ± 1.0, and 21.8 % ± 1.1 respectively) in decreasing order (Fig. 6d; Table 2). The reduction in the adherence capability of test strains observed is apparently due to blocking of 47.2 kDa surface antigen which also effectively decreased the biofilm formation (Fig. 6a–c). Our findings also confirm that any changes in adherence of C. albicans to a surface affect the formation and further revitalization of biofilm. Further, modest but significant ability of MAb7D7 to inhibit adhesion of C. albicans to HeLa cells was also evident in this study. The effect of MAb 7D7 on C. albicans adherence to HeLa monolayer was performed in 6 well culture plates. C. albicans adherent cells were quantified by phase contrast microscope in four different fields and inhibition in adhesion was expressed as percentage of adhering cells at 90 min time point (adhesion phase). When compared to the control (without incubation with MAb), MAb 7D7 caused a 47.2 % ± 2.1 inhibition in the

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adherence of C. albicans ATCC-10231 (Fig. 7a, a0 ) and 45.6 % ± 1.8 reduction in case of C. albicans ATCC14053 (Fig. 7b, b0 ) to the monolayer of HeLa cells. In the case of patient isolates, inhibition was maximum in PK-30 (41.5 % ± 1.2) and minimum in PK-9 (31.2 % ± 1.4) with comparison to control (Fig. 7d, d0 and c, c0 respectively). The above experiment of monolayer adhesion assay was performed in triplicate and results were calculated as average ± SD.

Discussion Candidiasis is the most common fungal infection; during infection the host generates a wide range of defensive responses. It has been observed that the antibody formation is one of the responses in protection against candidiasis and biofilm formation by the host. Evidences demonstrate that antibodies with defined specificities show different degrees of protection against systemic and mucosal candidiasis (Bugli et al. 2013). Likewise, identification of a selective antibody directed against specific epitopes may be protective against local and systemic infection and may form the basis for immunotherapy. Hence, antibody production has been shown to be important for protection against candidiasis, as demonstrated by therapeutic use of whole immune serum, or its IgG-enriched fraction. Similarly some studies have also been oriented to decrease the adhesion and biofilm formation of C. albicans using various approaches including monoclonal antibodies. In our study, MAb7D7 was assessed as anti-biofilm antibody against C. albicans test strains and results support this hypothesis. Con A blot analysis of biofilm cell surface proteins of C. albicans ATCC-10231 on PVDF membrane resulted in confirmation of carbohydrate residues of glycoproteins in the form of sharp band (Fig. 1). In Western blot analysis the protein binding with MAb 7D7 has a molecular mass of *47 kDa and was identified as a glycoprotein, moreover the epitope on C. albicans ATCC10231 biofilm cell surface recognized by MAb 7D7 was proteinaceous in nature. In contrast, Western blot analysis of C. albicans patient isolates cell surface proteins with MAb 7D7 exhibited additional bands in the range of 36–50 kDa (Fig. 2b). Although, the antibodies against the 45–47 kDa protein are found in the sera of candidiasis patients, and these antigens are similar to enolase1 and highly immunogenic in nature (Franklyn et al. 1990; Sandini et al. 1999), our result confirms and expand this observation concerning the anti-biofilm activity of MAb 7D7 directed against 47.2 kDa C. albicans biofilm surface antigen. The immunoisotyping of MAb indicated that the MAb 7D7 belonged to class IgG and subclass G3 with kappa

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Fig. 5 Indirect immunofluorescence of the cell surface antigen expressed in C. albicans ATCC-10231 (a), C. albicans ATCC14053 (b) and patient isolates (PK-9, c; PK-30, d; PK-31, e; and PK-

32, f) during adhesion phase (90 min), probed with MAb7D7 and detected with FITC labeled secondary antibody. Photographs were captured by fluorescence microscope

light chain. Likewise, the ability of IgG purified from rabbit serum immunized with C. albicans cytoplasmic extract has been reported to have capability to reduce the adherence of C. albicans to polystyrene (Rodier et al. 2003) also emphasize our result. In recent studies, it has

been also proved that use of anti-glucan antibodies of the IgG class, conferred as therapeutic agents against both systemic and mucosal candidiasis (Bujdakova et al. 2008; Potera 1999). Furthermore, the role of saliva, polyclonal antisera, and MAbs against the adhesins of C. albicans has

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Fig. 6 The kinetics of adherence during biofilm formation in C.a (C. albicans ATCC-10231), C.a17 (C. albicans ATCC-14053) and patients isolates (PK-9.PK-30, PK-31 and PK-32) after incubation with MAb 7D7 (Blue bar) at different time points (30, 60, 90 min). C. albicans strains were incubated in parallel without MAb7D7 at mentioned time points (a, b and c) as control (Red bar). d The inhibition of mature biofilm (48 h) formed by C.a, C.a17 and patients isolate (PK-9, PK-30, PK-31, PK-32) compared with control (C. albicans strains were incubated without MAb7D7). (Color figure online)

Table 2 Inhibition (in %) of C. albicans biofilm adherence to polystyrene surface

Percent inhibition of adherence and biofilm formation in Candida strain Strains

30 min

60 min

90 min

C. albicans ATCC-10231

69.0 ± 2.2*

72.7 ± 3.0£

54.4 ± 2.8*

49.2 ± 1.6£

31.8 ± 6.6*

59.1 ± 3.2*

£

35.4 ± 1.3£

¥

£

£

C. albicans ATCC-14053

£ ¥

p \ 0.005; * p \ 0.05; p = 0.13

48 h

44.0 ± 1.6

PK-9

41.6 ± 2.2

50.2 ± 5.3

48.2 ± 1.1

24.7 ± 1.2£

PK-30 PK-31

36.0 ± 4.1* 25.8 ± 3.6*

35.0 ± 1.9* 41.8 ± 1.1£

40.7 ± 1.1£ 32.3 ± 4.6*

22.0 ± 1.0* 26.4 ± 1.1£

PK-32

24.9 ± 2.3*

34.8 ± 1.6£

37.5 ± 1.4£

21.8 ± 1.1*

been demonstrated as blocking agent to host surface (San Millan et al. 2000; Umazume et al. 1995). Although, enolase (Eno) from C. albicans cell wall was specific for the genus Candida (Lopez-Villar et al.2006) and anti-Eno antibodies exhibited diagnostic value against candidiasis (Li et al. 2013; Franklyn et al. 1990), the assessment of anti- Eno antibodies against C. albicans biofilm has not been demonstrated. Indeed, our result indicates that MAb 7D7 is against 47.2 kDa surface protein and it contains 38 % similarity with C. albicans enolase 1 (Eno1) in proteomic analysis result. In this study the MAb 7D7 was evaluated for its therapeutic use, chiefly a decrease in adherence of C. albicans cells to the polystyrene surface and consequently the less biofilm formation was observed by the mediation of MAb 7D7 (Fig. 4a, b; Table 2). This antibody against C. albicans biofilm has potential in future study.

However, the kinetic adhesion are strain dependent (Fig. 5a–c), in this phenomena C. albicans cells begin to express surface antigen promoting adhesion and start to form biofilm. At this stage the process of biofilm formation was inhibited (24.9–72.7 %) by MAb 7D7 significantly (p \ 0.005). Moreover, the inhibitory effect of MAb 7D7 is in conformity with the earlier studies where blocking of the surface antigen has been reported to decrease the biofilm formation (Bujdakova et al. 2008). The adhesion assay of C. albicans was performed on HeLa monolayer and it demonstrated that MAb 7D7 is also effective against adherence of C. albicans to mammalian cell. The ability of MAb 7D7 to inhibit the germination of C. albicans may be attributed to the limited exposure of the epitope which is proteinic in nature and seems to be hidden in the sugar residues of the antigenic glycoprotein. The result of adhesion assay shows that the MAb 7D7 reacts with C.

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Fig. 7 Adhesion assay of C. albicans cells to monolayer of human epithelial (HeLa) cells was visualized (409) by crystal violet staining and evaluated under the microscope. In a-f arrows indicate adhered

C. albicans cells to monolayer of HeLa cells without incubation with MAb7D7 and a’–f’ show decrease in adherence of C. albicans cells labeled with MAb7D7 to monolayer of HeLa cells

albicans cell surface antigen exerts anti-adhesion activity to HeLa cells and demonstrated by the reduction in adhesion of all test strains (47.2–31.2 %). As such, we cannot make definitive conclusions about humoral responses against C. albicans biofilm surface antigens. Nevertheless, we demonstrated that MAb 7D7 against 47.2 kDa protein can effectively decrease the adhesion and biofilm formation. In conclusion, the present study supports the hypothesis that monoclonal antibody 7D7 against cell surface protein (47.2 kDa) of C. albicans biofilm inhibits the adherence of C. albicans and possibly also of C. albicans patient isolates to the polystyrene surface and a monolayer of human epithelial cells (HeLa). This inhibitory effect can be mimicked by MAb 7D7 directed against antigens expressed on the cell wall/cell surface of the C. albicans. Further, it

may also be concluded that C. albicans biofilm cell surface protein (47.2 kDa) is the target of MAb 7D7 (anti47.2 kDa,) and this property may be exploited for the treatment of Candida biofilm infections either alone or in combination with current antifungals. This MAb 7D7 may also be used for diagnosis purpose against candidiasis because 47.2 kDa antigen is highly immunogenic and detected in Candida patients isolates also.

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Acknowledgments The authors are thankful to the Director CSIR CDRI, Lucknow India for providing facilities. NNM thanks ICMR, New Delhi for a Senior Research Fellowship. CDRI Communication No. 8819. Conflict of interest interest.

The authors declare to have no conflict of

World J Microbiol Biotechnol (2015) 31:11–21

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