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. 2020 Nov 17;10(12):538. doi: 10.1007/s13205-020-02515-8

Expression, purification, and characterization of phospholipase B1 from Candida albicans in Escherichia coli

Zigang Zhao 1,✉,#, Yaguang Zhou 1, Rui Wang 3,#, Fang Xie 3, Zhengyuan Zhai 2
PMCID: PMC7672135  PMID: 33224707

Abstract

Candida albicans is an important fungal pathogen that causes a wide variety of human infections, ranging from mucocutaneous infections to life-threatening systemic infections. Phospholipase B1 (PLB1) has been reported to be directly responsible for C. albicans pathogenicity and is likely to be involved in the early steps of host invasion. Therefore, PLB1 could be a potential marker for diagnosis of C. albicans infection. In this study, PLB1 was expressed using an Escherichia coli expression system. Recombinant PLB1 is found in inclusion bodies and constitutes up to 38.4% of total insoluble protein. After refolding in a GSH/GSSG redox system, GST-tagged PLB1 was purified by GST-sepharose 4B affinity chromatography and then cleaved with thrombin to remove the GST-tag. The recombinant PLB1 was further purified by anion-exchange chromatography and reverse phase HPLC. The final yield of purified PLB1 was approximately 15.6 mg from 100 mL of bacterial cell culture, and its concentration was 784 μg/μL. The recombinant PLB1 could form a white precipitation zone on egg yolk agar plate, suggesting its phospholipase activity. Moreover, the maximum activity of PLB1 was 68 IU/mg at pH 6.0, 37 °C. Therefore, recombinant PLB1 has potential application in structural analytical studies, or diagnosis of C. albicans infection.

Keywords: Candida albicans, Phospholipase, GST-tagged PLB1, Refolding, PLB1 activity

Introduction

Candida albicans is a normally harmless organism that can be isolated from the gastrointestinal tract, and the oral and vaginal mucosa of many healthy individuals (Kim and Sudbery 2011; Chin et al. 2016). C. albicans is also an opportunistic pathogen in some immunocompromised people (Williams et al. 2013). It can infect the bloodstream (candidemia) or cause local mucosal infections, such as oropharyngeal candidiasis and vulvar vaginitis. C. albicans also causes invasive local infections including urethritis, endophthalmitis, and peritonitis pericarditis in immunodeficient or immunosuppressed patients (Kim and Sudbery 2011). In recent years, infection rates of C. albicans have increased due to interventional therapies, surgeries, cancer chemotherapy, and extensive use of various high-efficiency broad-spectrum antibiotics, immunosuppressants, and glucocorticoids (Pierce and Lopez-Ribot 2013). The increasingly high frequency of C. albicans infection constitutes a serious clinical problem worldwide. Systemic candidiasis is a serious disease and has a mortality rate of approximately 40% (Gunsalus et al. 2016; Dadar et al. 2018). The high mortality of C. albicans-related infections in immunocompromised patients is a persistent problem exacerbated by difficulties in diagnosis and treatment (Chin et al. 2016). Thus, there is a dire need for sensitive and specific diagnostic methods, and appropriate treatment strategies for candidiasis. Achievement of these goals could be fostered by better understanding of the virulence factors and pathogenic mechanisms of C. albicans.

Candida albicans virulence factors include host recognition biomolecules (adhesins), morphogenesis (the reversible transition between unicellular yeast cells and filamentous, growth forms), and invasion enzymes (secreted aspartyl proteases and phospholipases) (Calderone and Fonzi 2001). The secreted aspartyl proteinases (SAP) and phospholipases (PL) are two rather large families of C. albicans enzymes, some of which have been associated with invasion. In C. albicans, the phospholipase family includes different subclasses: PLA, PLB, PLC, and PLD (Calderone and Fonzi 2001). However, only PLB1 has been shown to be required for virulence in an animal model of candidiasis (Ghannoum 2000). The virulence of C. albicans was significantly attenuated by the disruption of the PLB1 gene and restored by its reintroduction (Mukherjee et al. 2001; Ying and Chunyang 2012). In addition, PLB1 activity has recently been detected in hyphal tips during tissue invasion (Ghannoum 2000). Therefore, phospholipase B1 plays a critical role in host cell adherence and invasion.

PLB1 is a glycoprotein that has both hydrolase and lysophospholipase–transacylase activity (Hoover et al. 1998). This enzyme can hydrolyse fatty acids esterified at the sn-1 or sn-2 position of a phospholipid (Borrelli and Trono 2015). Thus, PLB1 can degrade host cell membrane phospholipids, resulting in increased membrane permeability and impaired integrity, further promoting invasion by C. albicans, especially in early stage infections. In addition, C. albicans phospholipase may increase C. albicans pathogenicity through other mechanisms, such as stimulating the host cell to release cytokines and causing an inflammatory response (Songer 1997; Mukherjee et al. 2001). Therefore, PLB1 is a potential diagnostic marker for C. albicans infection, or target for developing anti-C. albicans drugs. PLB1 has been purified from C. albicans strain 16,240 by DEAE-cellulose column and sequential chromatography on hydroxyapatite HCA-100S, TSK-gel 3000 HPLC, and Mono Q HPLC columns. (Mirbod et al. 1995; Leidich et al. 1998). The purified PLB1 from C. albicans showed phospholipase activity. However, PLB1 has not been produced by heterologous expression system yet. In this study, we attempt to produce the recombinant PLB1 using an E. coli expression system and determine the phospholipase activity of recombinant PLB1. These results will provide the basis for exploring the mechanism of C. albicans infection, and the development of antifungal drugs.

Materials and methods

Materials

Escherichia coli Top10 and BL21 (DE3) were purchased from Invitrogen (Carlsbad, CA, USA). The vector pGEX-4T-1, which allows gene insertion in frame with a glutathione S-transferase (GST)-tag, was purchased from GE Healthcare (Waukesha, WI, USA). Agarose, ampicillin, acrylamide, bis-acrylamide, TEMED, and isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Sigma (St. Louis, MO, USA). Pyrobest DNA polymerase R005A, restriction endonucleases, and T4 DNA ligase were purchased from Takara (Beijing, China). A yeast genomic DNA kit was obtained from Tiangen (Beijing, China). The QIAprep spin kit for Mini-prep isolation of E. coli plasmids was purchased from Qiagen Inc. (Valencia, CA, USA). All primers used in this study were designed using PRIMER V5 software (PREMIER Biosoft International, Palo Alto, CA, USA) and synthesized by Sangon Biotech (Beijing, China). Thrombin for GST-tag cleavage was purchased from GE Healthcare.

Bacterial strains, plasmids, and growth conditions

Bacterial strains and plasmids used in this study are listed in Table 1. The clinical Candida albicans wild-type strain SC5314 was cultured in yeast extract peptone-dextrose (YEPD) broth at 37 °C. Escherichia coli cells were aerobically propagated in Luria–Bertani (LB) broth with shaking at 250 rpm at 37 °C. When required, LB medium was supplemented with 100 μg/mL ampicillin for E. coli strains.

Table 1.

Bacterial strains and plasmids used in this study

Strain or plasmid Relevant phenotype or genotypea Source or reference
Bacterial strains
 E. coli Top10 F−, mcrA, Δ(mrr-hsdRMS-mcrBC), Φ80lacZΔM15, ΔlacX74, recA1, araD139, Δ(ara-leu)7697, galU, galK, rpsL, (StrR), endA1, nupG λ− Invitrogen
 E. coli BL21 (DE3) F−, ompT, hsdSB, (rB−mB−), gal, dcm, (DE3) Invitrogen
Candida albicans SC5314 Wild-type strain Laboratory strain
E. coli Top10-PLB1 E. coli Top10 harbouring pGEX-PLB1 This work
E. coli CK E. coli BL21(DE3) harbouring pGEX-4T-1 This work
 E. coli PLB1 E. coli BL21(DE3) harbouring pGEX-PLB1 This work
Plasmids
 pGEX-4T-1 Ampr, tac promoter, GST fusion vector GE
 pGEX-PLB1 pGEX-4T-1 derivative containing PLB1 gene This work
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aAmpr ampicillin resistance, GST glutathione S-transferase

DNA manipulation techniques

Genomic DNA of C. albicans was extracted using TIANamp Yeast DNA Kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. Mini-prep isolation of E. coli plasmids was performed using the QIAprep spin kit. Standard PCR with Pyrobest DNA Polymerase, restriction endonuclease digestions, and DNA ligations were performed following the manufacturer’s instructions, respectively. Ligation mixtures or purified plasmids were introduced into E. coli using standard heat shock transformation (Hanahan 1983). DNA sequencing was performed by Sangon Biotech. The results were further analysed with the DNAMAN software package (Version 5.2.2; Lynnon Biosoftware, Vaudreuil, Quebec, Canada).

Construction of the pGEX-PLB1 vector and the recombinant E. coli strain PLB1

PLB1 was amplified by PCR from the chromosomal DNA of C. albicans SC5314 using the primers (PLB1F: 5′-CGGAATTCATGATTTTGCATCATTTG-3′ and PLB1R: 5′-CCGCTCGAGTTATGCCTTTTTTAAAAT-3′), which were designed according to the DNA sequence (NCBI Gene ID: 3,644,561). Restriction sites EcoRI and XhoI used for subsequent cloning are underlined. The PCR product was digested with EcoRI and XhoI and inserted into the pGEX-4T-1 expression vector. The ligation mixture was transformed into E. coli Top10, and transformants were selected on LB agar with ampicillin. The recombinant plasmid, designated pGEX-PLB1, was sequenced and further analysed with the DNAMAN software package. Subsequently, the vector pGEX-PLB1 was transformed into E. coli BL21 (DE3) by heat shock transformation. Positive transformant was screened on LB agar with ampicillin and then named as E. coli strain PLB1. The recombinant plasmid pGEX-PLB1 in E. coli PLB1 was further verified by DNA sequencing.

Expression of phospholipase B PLB1 in E. coli

Overnight culture of E. coli PLB1 was inoculated (2% v/v) into 100 mL of fresh LB broth supplemented with ampicillin. When cell density reached an OD600 nm of 0.6, IPTG was added to a final concentration of 1 mM to induce fusion protein expression for 4 h at 37 °C as described previously (Tian et al. 2006; Hao et al. 2008). Cell pellets were collected by centrifugation at 6000×g for 5 min at 4 °C. Subsequently, cell pellets were suspended in 1 mL phosphate-buffered saline (PBS; pH 7.4) and subjected to sonication in an ice bath at 300 W with 10 s on/off cycles for a total time of 6 min. Total protein was partitioned into soluble and insoluble fractions by centrifugation at 12,000×g for 10 min at 4 °C. To check expression and solubility of the fusion protein, both soluble and insoluble fractions were analysed by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE).

Refolding and purification of recombinant PLB1

Refolding and purification of recombinant PLB1 were performed as previously described (Hao et al. 2008) with some modifications. Briefly, inclusion bodies were washed twice with washing buffer containing 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 10 mM EDTA, and 0.5% (v/v) Triton X-100. Washed inclusion bodies were further dissolved in buffer containing 20 mM sodium phosphate, 0.5 M NaCl, 20 mM imidazole, 20 mM 2-mercaptoethanol, and 8.0 M urea (pH 7.4). After solubilization, recombinant protein was refolded by urea gradient dialysis at 4 °C in PBS buffer containing 1 mM EDTA, 1% (w/v) glycine, 5% (v/v) glycerol, 0.9 mM reduced glutathione (GSH), and 0.1 mM oxidized glutathione (GSSG) (Hao et al. 2008). Urea was added into the PBS buffer every 12 h in decreasing concentrations (6, 4, 2, 1, 0.5 and 0 M). The renatured recombinant protein was purified by affinity chromatography. A GST-sepharose 4B affinity chromatography column (GE Healthcare, Little Chalfont, UK) was packed and equilibrated with 10 mM PBS (pH 7.5), 1 mM EDTA. The target protein was eluted with elution buffer containing 10 mM PBS (pH 7.5) and 50 mM Tris–Cl (pH 8.0). Then, the GST-fusion protein was cleaved by thrombin to remove the GST-tag.

The target protein was then purified on a pre-packed Mono P 5/50 GL ion exchange column, followed by reverse phase HPLC (Janson 2012). The separation was carried out on a Resource RPC column (3 ml; GE healthcare, Uppsala, Sweden). The elution was performed using the linear gradient of acetonitrile (90 min, concentration of acetonitrile 10% to 60% v/v), with the addition of trifluoroacetic acid (TFA, 0.1% v/v) at the flow rate of 1 ml/min. Progress of the elution was monitored by UV absorbance at wavelengths of 280 nm. The eluted fractions, corresponding to the absorbance peaks, were collected and freeze-dried prior to further analysis. The concentration of purified PLB1 was measured by the Bradford method.

Phospholipase activity assay by egg yolk agar plate

The biological activity of recombinant PLB1 was determined in vitro using the egg yolk agar plate method of Price et al. (Price et al. 1982). Oxford cups were placed on the agar. Excess purified recombinant PLB1 and misfolded PLB1 were added into different cups. Petri dishes were incubated at 37 °C, and precipitation zones were checked after 24 h incubation.

Effects of pH and temperature on recombinant PLB1 activity

The phospholipase activity of PLB1 was determined by measuring the release of free fatty acids from egg lecithin as described previously (Yang and Roberts 2002). To investigate the effect of pH on refolded recombinant phospholipase B activity, PLB1 and yolk lecithin mixtures were incubated for 15 min (37 °C) at different pH’s ranging from 3 to 10. To determine the effect of temperature on refolded PLB1 activity, PLB1 and yolk lecithin mixtures were incubated for 15 min (pH 7.0) at different temperatures, ranging from 20 to 80 °C. PLB1 activity was measured by pH–stat technique as previously described (Park et al. 2020). The amount of free fatty acids released from a lecithin solution can be quantified by adding 0.01 M NaOH to maintain a constant pH of 7.0 using an automatic pH–stat (Metrohm, Switzerland). Phospholipase activity is shown in international units (IU) per milligram of enzyme. One international unit = 1 μmol of fatty acid released per minute.

Results and discussion

Construction of fusion protein expression vector pGEX-PLB1

The gene PLB1, encoding phospholipase B1, was obtained by PCR with specific primers. Genomic DNA from C. albicans SC5314 was used as template, since the PLB1 gene contains no intron. The expected 1.8 kb PCR product was purified and cloned into expression vector pGEX-4T-1 to generate a recombinant plasmid, designated pGEX-PLB1 (Fig. 1). DNA sequencing verified that the full length of the amplified PLB1 gene was 1818 bp. The sequence displayed 99% homology with the published sequence of C. albicans phospholipase PLB1 gene (Fekete‐Forgács et al. 2000). Only one mutation at position 897 (T to A) was observed in the amplified PLB1 gene. This mutation did not result in a change in amino acid sequence. The recombinant pGEX-PLB1 vector was transformed by heat shock into E. coli BL21 (DE3) for expression.

Fig. 1.

Fig. 1

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The confirmation of recombinant vector pGEX-PLB1 by restriction enzyme of EcoRI and XhoI. M, D15000 DNA Marker; Lane 1, the empty vector pGEX-4T-1 digested by EcoRI and XhoI; Lane 2, the recombinant vector pGEX-PLB1 digested by EcoRI and XhoI; Lane 3, the PCR production of gene PLB1

Heterologous expression of GST-tagged PLB1 in E. coli

The phospholipase PLB1 from C. albicans SC5314 consists of 605 amino acids. Thus, the theoretical molecular weight of GST-tagged PLB1 is approximately 92 kDa. In this study, SDS-PAGE analysis revealed the production of a protein with molecular mass less than 97.2 kDa in E. coli PLB1 after IPTG induction (Fig. 2, Lane 6), suggesting successful expression of GST-tagged PLB1. Notably, this GST-tagged PLB1 was only found in the insoluble fraction, suggesting that it was expressed in the form of inclusion bodies. SDS–PAGE analysis showed that GST-tagged PLB1 can constitute as much as 38.4% of total insoluble protein (Fig. 2).

Fig. 2.

Fig. 2

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SDS-PAGE analysis of recombinant GST-tagged PLB1 protein expressed in E. coli. Lane M, Pre-stained low molecular weight protein marker (Takara); Lane 1, the induced soluble fraction of E. coli CK; Lane 2, the uninduced soluble fraction of E. coli PLB1; Lane 3, the induced soluble fraction of E. coli PLB1; Lane 4, the induced insoluble fraction of E. coli CK; Lane 5, the uninduced insoluble fraction of E. coli PLB1; Lane 6, the induced insoluble fraction of E. coli PLB1

Refolding and purification of recombinant phospholipase PLB1

In many cases, production of recombinant proteins in E. coli results in incomplete folding and protein accumulation as insoluble aggregates, known as inclusion bodies (de Groot and Ventura 2006). To decrease formation of inclusion bodies and improve solubility of recombinant proteins, E. coli strains are usually grown at lower temperatures, such as 16 °C, 25 °C, or 30 °C. This will provide greater time for protein refolding since rates of transcription and translation will substantially decrease at lower temperatures (Makrides 1996; Hao et al. 2008). In this study, E. coli PLB1 was grown at 16 or 25 °C, but the solubility of GST-tagged PLB1 was not improved (data not shown). To obtain high-level production of recombinant PLB1, E. coli PLB1 was then grown at 37 °C allowing inclusion bodies to form. The recombinant PLB1 in the insoluble fraction was further refolded in a buffered glutathione redox system (Wang et al. 2008) and purified by GST-sepharose 4B affinity chromatography. Insoluble GST-tagged PLB1 was completely solubilized in buffer containing 8 M urea and then refolded by dialysis at 4 °C in buffers with gradually decreased urea concentrations.

Several studies have revealed that reduced and oxidized forms of low-molecular weight thiols such as GSH/GSSG and cystamine/cysteamine play an important role in increasing the electron potential for disulphide bond formation, and reshuffling of incorrect disulphide bridges during substrate folding (Dharmatti et al. 2018; Huth et al. 1994). Precise disulphide bond formation during refolding of denatured GST-tagged PLB1 might be required to obtain active phospholipase B. Therefore, 0.9 mM GSH and 0.1 mM GSSG were used during inclusion body refolding. After GST-sepharose 4B affinity chromatography and thrombin digestion, SDS-PAGE analysis revealed recombinant PLB1 with a molecular mass of approximately 84 kDa (Fig. 3). To remove impurities, including the GST-tag, the target protein was synergistically further purified by anion-exchange chromatography and reverse phase HPLC. The yield of purified PLB1 from 100 mL of bacterial cell culture was approximately 15.6 mg with a purity of 99%, and its concentration was 784 μg/μL (Fig. 3).

Fig. 3.

Fig. 3

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SDS-PAGE analysis of purified PLB1 protein. a Lane M, Pre-stained low molecular weight protein marker (Takara); Lane 1, the induced insoluble fraction of E. coli CK; Lane 2, the GST-fusion protein treated by thrombin; Lane 3, the purified recombinant PLB1

Effects of pH and temperature on recombinant PLB1 activity

To determine if the recombinant PLB1 was correctly refolded, in vitro PLB1 activity was firstly tested on egg yolk agar plate. Purified PLB1 refolded in PBS buffer containing 1 mM EDTA, 1% glycine, and 5% glycerol showed no biological activity (Fig. 4). However, when recombinant PLB1 was refolded in this refolding buffer supplemented with 0.9 mM GSH and 0.1 mM GSSG, the purified PLB1 could efficiently hydrolyse egg lecithin to form white zones of precipitation (Fig. 4). Therefore, reducing and oxidizing agents (GSH/GSSH) are essential for refolding of active recombinant PLB1 expressed in E. coli.

Fig. 4.

Fig. 4

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Activity measurement of recombinant PLB1 using the egg yolk agar plate method. 1: PBS buffer as a negative control; 2: misfolded PLB1; 3: refolded purified PLB1

To investigate the effect of pH on recombinant PLB1 activity, hydrolysis of egg lecithin was monitored by pH–stat technique at different pH ranging from 3 to 10. The highest PLB1 activity was 68 IU/mg at pH 6.0 (Fig. 5). At pH values below 4.0 or above 9.0, more than 55% of maximum activity was preserved. These results are in agreement with previous studies that phospholipase B form C. albicans showed a maximum activity at pH 6.0 (Mirbod et al. 1995; Leidich et al. 1998). In addition, recombinant PLB1 activity was monitored at different temperatures, ranging from 20 °C to 80 °C. Recombinant PLB1 efficiently hydrolysed egg lecithin between 30 °C and 40 °C, but its activity rapidly decreased from 40 °C to 80 °C (Fig. 5).

Fig. 5.

Fig. 5

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Effects of pH and temperature on recombinant PLB1 activity. Phospholipase specific activities are given as international units (IU) per milligram of PLB1. One international unit = 1 μmol of fatty acid released per minute

Conclusion

Phospholipase B1 (PLB1) from C. albicans SC5413 has been over-expressed in E. coli. The GST-tagged PLB1 protein was expressed in the form of inclusion bodies. After solubilizing in 8 M urea and refolding in a GSH/GSSG redox system, the recombinant protein was purified by GST-sepharose 4B affinity chromatography and then cleaved with thrombin to remove the GST-tag. The recombinant PLB1 was further purified by anion-exchange chromatography and reverse phase HPLC. The concentration of purified recombinant PLB1 was 784 μg/μL. It is notable that only recombinant PLB1 refolded in buffer supplemented with 0.9 mM GSH and 0.1 mM GSSG can efficiently hydrolyse egg lecithin to form white zones of precipitation. Therefore, reducing and oxidizing agents (GSH/GSSH) are essential for refolding of active recombinant PLB1 expressed in E. coli. The highest activity of PLB1 was 68 IU/mg, which was observed at pH 6.0, 37 °C. To our knowledge, this is the first time that PLB1 from C. albicans was successfully expressed using an E. coli expression system. This recombinant PLB1 has potential application to subsequent studies of PLB1 structure, or development of diagnostics for C. albicans infection.

Acknowledgments

This work was supported by the Sanya Medical and Health Technology Innovation Project (2017YW15). We thank Professor Yanling Hao (China Agricultural University) for helpful comments and revisions.

Author contributions

Conceptualization was contributed by ZGZ, YGZ, and ZYZ; Methodology was contributed by RW and FX; Formal analysis and investigation were contributed by ZGZ, YGZ, and RW; Writing—original draft preparation, was contributed by ZGZ and ZYZ; Writing—review and editing, was contributed by all authors.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest in the publication.

Ethics approval and consent to participate

Not applicable.

Footnotes

*Zigang Zhao, Yaguang Zhou and, Rui Wang contributed equally to this work.

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