ZEBRA cell-penetrating peptide as an efficient delivery system in Candida albicans.

Biotechnology Journal

Biotechnol. J. 2014, 9, 1088–1094

DOI 10.1002/biot.201300505

www.biotechnology-journal.com

Technical report

ZEBRA cell-penetrating peptide as an efficient delivery system in Candida albicans Roberta Marchione1,*, David Daydé1,*, Jean-Luc Lenormand1 and Muriel Cornet1,2 1 TIMC-IMAG 2 Laboratoire

TheREx, UMR 5525 CNRS-UJF, Université Joseph Fourier, La Tronche, France de Parasitologie-Mycologie, Centre Hospitalier Universitaire, Grenoble, France

There is increasing interest in drug delivery systems, such as nanoparticles, liposomes, and cellpenetrating peptides, for the development of new antimicrobial treatments. In this study, we investigated the transduction capacity of a carrier peptide derived from the Epstein–Barr virus ZEBRA protein in the pathogenic fungus Candida albicans. ZEBRA-minimal domain (MD) was able to cross the cell wall and cell membrane, delivering eGFP to the cytoplasm. Uptake into up to 70% of the cells was observed within two hours, without toxicity. This new delivery system could be used in C. albicans as a carrier for different biological molecules including peptides, proteins, and nucleic acids. Thereby, in antifungal therapy, MD may carry promising bioactive fungal inhibitors that otherwise penetrate poorly into the cells. Furthermore, MD will be of interest for deciphering molecular pathways involving cell-cycle control in yeast or signaling pathways. Short interfering peptides could be internalized using MD, providing new tools for the inhibition of metabolic or signaling cascades essential for the growth and virulence of C. albicans, such as yeast-to-hyphae transition, cell wall remodeling, stress signaling and antifungal resistance. These findings create new possibilities for the internalization of cargo molecules, with applications for both treatment and functional analyses.

Received Revised Accepted Accepted article online

28 NOV 2013 28 NOV 2013 17 JAN 2014 22 JAN 2014

Keywords: Antifungal therapy · Candida albicans · Cargo delivery · Fungal pathogenesis · Internalization

1 Introduction Invasive fungal infections have become a major public health problem, with up to two million cases occurring worldwide each year. It is estimated that at least as many people die from invasive fungal infections as from tuberculosis or malaria. These diseases are opportunistic and are generally considered to be healthcare-associated. As such, their incidence is growing. Despite the availability

Correspondence: Prof. Muriel Cornet, TIMC-IMAG TheREx (UMR 5525 CNRS-UJF), Université Joseph Fourier, Domaine de la Merci, 38706 La Tronche, France E-mail: [email protected] Abbreviations: CFU, colony forming unit; CPP, cell-penetrating peptide; eGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; MD, minimal domain; OD, optical density; PBS, phosphate buffered saline; YPD, yeast extract peptone dextrose

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of new antifungal agents, mortality remains unacceptably high. In developed countries, the yeast Candida albicans remains the predominant fungal pathogen identified, causing disseminated candidiasis in immunocompromised patients [1, 2]. Candidiasis treatment is currently based on only four classes of drugs: polyenes (mainly represented by amphotericin B), azoles, echinocandins and pyrimidines. The development of molecular resistance is a matter of great concern, due to the growing number of patients and the limited number of drugs available to treat them [1, 3–7]. In addition, most of these drugs have potentially major toxic effects on humans, because of the genetic proximity of fungal and human cells, which makes it more difficult to develop targeted treatments. In addition to the discovery of new inhibitors of specific targets in fungal cells, one of the major challenges in the development of new antifungal strategies is the

*

These authors contributed equally to this work.

© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim


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design of convenient and effective delivery systems. In recent years, the development of liposomal formulations of amphotericin B has been a key advance to alleviate drug toxicity and to improve drug distribution [8]. Another approach for improving the delivery of drugs or bioactive molecules is based on the use of cell-penetrating peptides (CPPs). These cationic, and often amphipathic, peptides penetrate mammalian cells and promote the intracellular transport of cargo molecules, including nucleic acids and proteins. Some CPPs, such as penetratin [9], pVEC [9], TP10 [10], and TAT [11], have been shown to enter C. albicans cells, delivering reporter proteins such as green fluorescent protein (GFP) or other fluorescent dyes. The possibility of using such CPPs to deliver antifungal agents has excited considerable attention. Rothe et al. have shown that the minimal domain (MD) derived from the Epstein–Barr virus ZEBRA protein is an efficient delivery system for proteins, such as enhanced GFP (eGFP) or β-galactosidase, in mammalian cells [12]. This 43-amino acid peptide transduces cells with the fused cargo protein by direct, non-endocytosis-dependent translocation across the lipid bilayer of the plasma membrane, without toxicity [12]. In this study, we investigated the ability of this new carrier system to cross the C. albicans cell wall and membrane and to deliver the eGFP reporter protein into cells. With its efficient uptake and its lack of toxicity in C. albicans, the MD system has potential for use as a vehicle for delivering non-penetrating proteins or peptides. These findings open up new possibilities for both treatment and functional studies in C. albicans.

2 Materials and methods 2.1 Expression and purification of the recombinant eGFP and MD-eGFP proteins The molecular cloning of eGFP and MD-eGFP was carried out as described by Rothe et al. [13]. For protein production, culture of E. coli BL21 (DE3) was induced at an optical density (OD600 nm) of 0.8, by adding 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG; Euromedex, Souffelweyersheim, France) and incubating for a further 18 h at 16°C. The culture was then centrifuged to obtain cell pellets containing the recombinant proteins, which were resuspended in 20 mM Tris/HCl pH 7.0, 500 mM NaCl, 10% glycerol, 2 mM dithiothreitol (DTT), 10 mM imidazole supplemented with a complete protease inhibitor cocktail (Roche, Basel, Switzerland). After sonication, the soluble fractions were purified on HisGraviTrap columns (GE Healthcare, Little Chalfont, United Kingdom) and the proteins were eluted in 20 mM Tris/HCl pH 7.0, 500 mM NaCl, 75 mM KCl, 10% glycerol, by increasing the concentration of imidazole in a stepwise manner, from 100 to 500 mM.


Proteins were separated by SDS–PAGE in a 15% polyacrylamide gel and analyzed by Coomassie Blue staining and immunoblotting. The yield of purified His-tagged proteins was quantified with the bicinchoninic acid (BCA Protein Assay Kit, according to the manufacturer’s instructions (Thermo Scientific, Rockford, IL). Before use in cellular uptake experiments, the purified eGFP and MD-eGFP proteins were dialyzed against phosphate buffered saline (PBS) and 25 mM Hepes/KOH pH 7.0, 150 mM NaCl, 10% glycerol, respectively.

2.2 Yeast strain and growth conditions The C. albicans reference strain SC5314 was grown in 5 mL of yeast extract peptone dextrose (YPD) medium (10 g/L yeast extract, 20 g/L peptone, and 20 g/L glucose) at 30°C, with shaking at 200 rpm, for 16 h. The preculture was centrifuged at 3000 g for 10 min at 4°C and diluted in fresh YPD medium to obtain an inoculum of 5 × 106 cells/mL. Cell density was determined by counting under a microscope, with Kova slides (Sordalab, Etampes, France).

2.3 Confocal microscopy For microscopic analysis, 5 × 105 cells (5 × 106 cells/mL) were incubated with 2.5 µM of each recombinant protein for 120 min in 100 µL of YPD medium at 30°C, with continuous shaking (200 rpm). The cells were then washed with PBS. For microscopic observation, cells were transferred to an eight-well LabTek chambered coverslip (Dutscher, Brumath, France). Living cell preparations were observed with a LSM 710 confocal scanning laser microscope (Carl Zeiss, Jena, Germany) equipped with a 63×, NA 1.2, C-apochromatic water-immersion objective (Carl Zeiss). We used a wavelength of 488 nm for excitation and fluorescence was collected with a 510–560 nm filter. We captured 25 successive optical slices along the cell z axis, in 0.5 µm steps.

2.4 Intracellular uptake and fluorescence-activated cell sorting (FACS) analysis The intracellular uptake of recombinant proteins was assessed by flow cytometry. A total of 5 × 105 cells (5 × 106 cells/mL) in 100 µL of YPD medium were incubated with various concentrations of the eGFP and MD-eGFP fusion proteins (1, 1.5, 2, and 2.5 µM), at 30°C, for 30, 60, or 120 min, with continuous shaking (200 rpm). Cells were harvested by centrifugation at 3000g for 10 min at 4°C and rinsed with PBS. The proteins bound extracellularly to the cells were removed by incubating the cells twice, for 5 min each, with 0.01% trypsin at 37°C. The trypsin was then neutralized by adding YPD medium supplemented with 10% fetal bovine serum (PAA, GE Healthcare). The cells were then washed twice with PBS before

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fluorescence analysis. Flow cytometry was carried out with a FACSCalibur (BD Biosciences, Franklin Lakes, NJ) equipped with a 488 nm argon laser and CellQuestPro Software.

2.5 Cell viability Cell viability was investigated in 5 × 105 yeast cells suspended in 100 µL of YPD medium (5 × 106 cells/mL) and treated with 2.5 µM of each recombinant protein for 30, 60, or 120 min. The growth of a suspension of 103 treated cells/mL was evaluated by both the counting of colony forming units (CFU) and OD600 nm measurements. CFU were counted by spreading cells previously incubated with eGFP and MD-eGFP proteins on YPD agar plates and incubating for 24 h at 30°C. Growth at 30°C over a 24-h period was also assessed by OD600 nm measurements. As toxicity controls, 5 × 105 cells were incubated for 120 min at 30°C in 100 µL of YPD medium (5 × 106 cells/mL) supplemented with either 2.7 µM amphotericin B (Gilead Sciences, Foster City, CA, USA) or 50 mM Tris–HCl, pH 7.4, 100 mM EDTA, 1% Triton X-100, 2% SDS.

3 Results and discussion We investigated MD-mediated internalization, using a recombinant fusion protein (MD-eGFP) in which the MD sequence was fused to the N-terminal end of eGFP (Fig. 1A). The His-tagged eGFP and MD-eGFP proteins were produced in an E. coli expression system and purified by nickel affinity chromatography. The purity of the eGFP and MD-eGFP proteins was analyzed by Coomassie Blue staining and Western blotting with an anti-his antibody (Fig. 1B). A 29 kDa band corresponding to eGFP and a 34 kDa band corresponding to MD-eGFP were detected. Western blotting also revealed the presence of an MD-eGFP dimer (68 kDa band) due to the presence of a leucine zipper region in the MD sequence.

3.1 Intracellular uptake of MD We evaluated transduction efficiency, by incubating for 120 min the yeast cells with the recombinant eGFP and MD-eGFP proteins and then examining them by confocal microscopy. Optical sectioning was performed along the z axis of living cells. Optical slices corresponding to the central sections of the cells incubated with the MD-eGFP fusion protein are shown in Fig. 2A. The pattern of fluorescence clearly indicates diffuse cytoplasmic delivery of the cargo molecule (see white arrows in Fig. 2A) with an accumulation at the cell periphery. No fluorescent signal was observed in cells incubated with free eGFP (data not shown). This confirms that MD is able to cross the C. albicans cell wall and membrane, rapidly transferring eGFP to the cytoplasm.

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Figure 1. Recombinant eGFP and MD-eGFP protein production. (A) Schematic representation of the His-tagged eGFP and MD-eGFP proteins used in this study. The MD-eGFP recombinant protein was engineered by fusing the MD sequence to N-terminal end of eGFP. (B) Coomassie blue staining and Western blot analysis of the eGFP and MD-eGFP recombinant proteins after purification by nickel affinity chromatography. The two proteins were immunodetected with an anti-His antibody.

We then characterized this uptake and quantified the proportion of cells containing MD-eGFP, by carrying out a dose and time-course analysis (Fig. 2B and 2C). We monitored eGFP internalization by flow cytometry on living cells. The extracellularly bound proteins were removed by trypsin digestion before FACS analysis. Yeast cells were incubated with various concentrations (1, 1.5, 2, and 2.5 µM) of MD-eGFP and uptake was measured after 30, 60, and 120 min. Up to 67.8 ± 4.5% of the cells were transduced in the presence of 2.5 µM MD-eGFP, after 120 min of incubation. The 2.5 µM MD-eGFP dose corresponds to the maximum concentration testable in yeast cells, due to limitations relating to the solubility of the reporter protein used. Low background levels of fluorescence (5.9 ± 1.1%) were detected in cells incubated with free eGFP at the same concentration for the same time (Fig. 2B and 2C). These results indicate that MD can mediate with high efficiency the internalization of eGFP into the pathogenic yeast C. albicans. Furthermore, the fluorescence of transduced cells increased in parallel with protein concentration at the three time points tested. Thus, the uptake of



Biotechnol. J. 2014, 9, 1088–1094



Figure 2. Intracellular distribution (A) and fluorescence-activated cell sorting (FACS) quantification (B, C) of the uptake of MD-eGFP in C. albicans cells. (A) After 120 min of incubation with 2.5 µM recombinant MD-eGFP, cells were observed under a confocal scanning laser microscope. Nine successive optical slices, corresponding to the central sections of yeast cells, are shown on the left. The central images are magnified and the corresponding z-stack positions are displayed on the right. The white arrows indicate recombinant protein in the central area of the yeasts. FACS quantification of the uptake (B) Time-course and (C) dose-course study of protein uptake. C. albicans cells were incubated for 30, 60, and 120 min with 1, 1.5, 2, and 2.5 µM solutions of each recombinant protein. The values reported are the means of four experiments.


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MD-eGFP in C. albicans is a dose- and time-dependent process. We still observed a persistent fluorescent cytoplasmic signal 2 h after the beginning of MD-eGFP internalization (Fig. 2A). It has been demonstrated that GFP fluorescence responds to pH changes [14] and that the acidity of the endosome or lysosome compartment triggers protein quenching [15]. In yeasts, some CPPs, like penetratin or (KFF)3K, are totally or partially degraded 20 min after their uptake. However, this intracellular degradation differs between the yeast species. (KFF)3K is stable in S. cerevisiae but not in C. albicans whereas penetratin shows an opposite pattern [9]. The persistence of the fluorescent signal observed in our study suggests that MD may penetrate in C. albicans by an endocytosis-independent mechanism. This observation is consistent with our previous study in mammalian cells showing the MD ability to cross directly the plasma membrane, without the need for endocytosis [12]. However, further investigations are required to define the MD-mediated mechanism of uptake in C. albicans more accurately.

3.2 Cell viability after MD uptake We checked that the uptake observed was not due to damage into the cell wall and membrane or to cell toxici-


ty. We evaluated the effect of the treatment with eGFP and MD-eGFP by monitoring cell growth rates. The analysis was performed on yeasts incubated with the highest concentration of the protein tested (2.5 µM) and which showed efficient internalization after 30, 60, and 120 min of incubation. As toxicity controls, yeasts were incubated with amphotericin B or Triton X-100/SDS buffer. The effect of the recombinant proteins on yeast growth was evaluated by counting CFU and determining OD600 nm. Cell viability after treatment with eGFP or MD-eGFP was similar to that of untreated cells. We also detected no effect of the duration of incubation with MD-eGFP (30, 60, or 120 min) on C. albicans viability. By contrast, amphotericin B and Triton X-100-SDS buffer were rapidly toxic, with no cells survival after 120 min of treatment (Fig. 3A). Similar results were obtained for OD600 nm measurements after 24 h of incubation, following treatment with the two proteins (Fig. 3B). The cells treated with eGFP or MDeGFP had growth rates similar to that of untreated cells, whereas much lower OD600 nm values were obtained after treatment with amphotericin B or Triton–SDS buffer (Fig. 3B). The use of CPPs as an approach to overcome the limitations of antifungal compounds has not yet been fully explored, despite the ability of these peptides to carry

Figure 3. C. albicans viability after the uptake of eGFP and MD-eGFP. Yeast cells were exposed to 2.5 µM solutions of each recombinant protein for 30, 60, and 120 min. The effects of eGFP (black bars) and MD-eGFP (gray bars) on yeast growth were analyzed by (A) CFU counting and (B) OD600 nm measurements. As positive toxic controls, yeast cells were incubated with 2.7 µM amphotericin B or Triton X-100-SDS buffer. The values shown are the means of four experiments.

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bioactive macromolecules into fungal cells. Only a few reports have described the use of CPPs to deliver cargo proteins into yeasts [16, 17]. The major difficulties encountered with this delivery approach concern peptide degradation after internalization [9], transduction via an endocytosis-dependent mechanism [16–18], and the requirement of high concentrations to achieve either penetration or significant antifungal effects [11, 19–22]. The delivery properties of the MD system in C. albicans could be useful for future applications in treatment. For instance, MD could be used to carry promising bioactive fungal inhibitors that otherwise penetrate cells poorly, such as nikkomycin or anti-Hsp90 antibodies [23]. Short interfering peptides could also be internalized by this system, providing new tools for the inhibition of metabolic or signalling cascades essential for the growth and virulence of C. albicans. Short interfering peptides derived from the Als3 protein have been shown to block the Als3-mediated adhesion and invasion of human cells and biofilm formation in vitro [24]. MD may enhance this peptide-based strategy and other treatments targeting proteins involved in key cellular functions relating to fungal virulence, such as yeast-to-hyphae transition, cell wall remodeling, stress signaling, and antifungal resistance. The delivery properties of the MD system in C. albicans could be useful for future applications in functional analyses for deciphering molecular mechanisms involving cell-cycle control or signaling pathways. Short interfering peptides could be internalized by this system, providing new tools for the inhibition of metabolic or signaling cascades involved in C. albicans pathogenesis. The MD transduction capacity into Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are commonly used as model yeasts, still remain to be investigated.

4 Concluding remarks We have shown that MD can penetrate cells and deliver the active biomolecule GFP to the cytoplasm of C. albicans. This CPP has several remarkable advantages supporting its use as a new transduction system in this pathogenic yeast. MD (i) is taken up efficiently and rapidly (in up to 70% of the cells within 2 h; (ii) is not toxic to either C. albicans or mammalian cells [12]; (iii) can carry large macromolecules, such as GFP; and (iv) may deliver cargo bioactive proteins, peptides or nucleic acids to the cytoplasm of C. albicans. In addition, its delivery properties and its lack of toxicity open up new possibilities for functional research applications and progress in our understanding of fungal pathogenesis.

We thank Delphine Aldebert and Bastien Touquet (highcontent screening platform, Laboratoire adaptation et pathogénie des microorganismes, University Joseph Fou-


rier) for the FACS analysis, and Yves Usson (TIMC-IMAG DyCTiM) for confocal microscopy. This work was supported by the Pôle de recherche Chimie, Sciences du Vivant et de la Santé, Bio-ingénierie (CSVSB) of the Université Joseph Fourier, Grenoble. The study sponsor had no role in study design and completion, or in the interpretation of the results, or the writing and submission of the manuscript. The authors declare no financial or commercial conflict of interest.

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[21] Garibotto, F. M., Garro, A. D., Rodríguez, A. M., Raimondi. M. et al., Penetratin analogues acting as antifungal agents. Eur. J. Med. Chem. 2011, 46, 370–377. [22] Masman, M. F., Rodríguez, A. M., Raimondi, M., Zacchino, S. A. et al., Penetratin and derivatives acting as antifungal agents. Eur. J. Med. Chem. 2009, 44, 212–228. [23] Ostrosky-Zeichner, L., Casadevall, A., Galgiani, J. N., Odds, F. C. et al., An insight into the antifungal pipeline: Selected new molecules and beyond. Nat. Rev. Drug Discov. 2010, 9, 719–727. [24] Kucharikova, S., Fiori, A., Wachtler, B. et al., Induced aggregation of the Candida albicans Als3 protein by Als3-specific peptides reduces mature biofilm development, adhesion and invasion of human epithelial cells. In: HFP Fifth FEBS Adv. Lect., 2013, pp. 25–31.


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Systems & Synthetic Biology · Nanobiotech · Medicine

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