Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum

Accepted Manuscript Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum Suibin He, Jingjing Qi, Shengqing Yu, Yuncong Yin, Lei Tan, Shijun Bao, Xvsheng Qiu, Xiaolan Wang, Rongmei Fei, Chan Ding PII:

S0882-4010(15)00165-5

DOI:

10.1016/j.micpath.2015.10.005

Reference:

YMPAT 1680

To appear in:

Microbial Pathogenesis

Received Date: 29 April 2014 Revised Date:

24 September 2015

Accepted Date: 4 October 2015

Please cite this article as: He S, Qi J, Yu S, Yin Y, Tan L, Bao S, Qiu X, Wang X, Fei R, Ding C, Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum, Microbial Pathogenesis (2015), doi: 10.1016/j.micpath.2015.10.005. 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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Expression and immunological characteristics of the surface-localized pyruvate kinase in Mycoplasma gallisepticum

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Suibin Hea, b, †, Jingjing Qia, †, Shengqing Yua, Yuncong Yinb, Lei Tana, Shijun Baoa, Xvsheng Qiua, Xiaolan Wang, Rongmei Feib, * and Chan Dinga, c *

Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural

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a

Sciences (CAAS), 518 Ziyue Road, Shanghai 200241, PR China

Key Laboratory of Animal Disease Diagnosis & Immunology, College of Veterinary

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b

Medicine, Nanjing Agricultural University, No. 1 Weigang, Nanjing 210095, PR China c

Jiangsu Co-innovation Center for Prevention and Control of Important Animal

Infectious Diseases and Zoonoses, 88 South University Ave., Yangzhou, 225009, P. R.

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China

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†Theses authors contributed equally to this work.

Corresponding Authors

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Chan Ding, Tel.: +86-21-34293441; Fax: +86-21-58081818. E-mail address: [email protected] (C. Ding) Rongmei Fei, Tel.: +86-25-84398606; Fax: +86-25-84398606. E-mail address: [email protected] (R.M. Fei)

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Abstract The widespread avian pathogen Mycoplasma gallisepticum is a causative agent of respiratory disease. The wall-less prokaryotes lack some tricarboxylic acid cycle

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enzymes, therefore, the glycolysis metabolic pathway is of great importance to these organisms. Pyruvate kinase (PK) is one of the key enzymes of the glycolytic pathway,

and its immunological characteristics in Mycoplasma are not well known. In this study,

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the M. gallisepticum pyruvate kinase fusion protein (PykF) was expressed in a pET system. The full-length of the gene was subcloned into the expression vector

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pET28a(+) to construct the pET28a-rMGPykF plasmid, which was then transformed into Escherichia coli strain BL21 (DE3) cells. The expression of the 62 kDa recombinant protein of rMGPykF in E. coli strain BL21 (DE3) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Coomassie blue staining. Purified rMGPykF exhibited PK catalytic activity, which could reflect the

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conversion of NADH to NAD+. Mouse anti-PykF antibodies were generated by immunization of mice with rMGPykF. Immunoblot and immunoelectron microscopy assays identified PykF as an immunogenic protein expressed on the surface of M.

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gallisepticum cells. Bactericidal assay showed that anti-rMGPykF antiserum killed 70.55% of M. gallisepticum cells, suggesting the protective potential of PykF.

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Adherence inhibition assay on immortalized chicken fibroblasts (DF-1) cells revealed more than 39.31% inhibition of adhesion in the presence of anti-rMGPykF antiserum, suggesting that PykF of M. gallisepticum participates in bacterial adhesion to DF-1 cells.

Keywords: Mycoplasma gallisepticum; pyruvate kinase; adhesion; immunological characteristics.

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1. Introduction Mycoplasma gallisepticum (MG) induces severe chronic respiratory disease in

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chickens and infectious sinusitis in turkeys. These diseases are globally prevalent and affect feed efficiency, resulting in significant economic losses in the poultry industry worldwide [1]. These bacteria belong to the class Mollicutes, which are characterized

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by an extreme reductive evolution in which genomes are reduced to the smallest sizes that can allow independent life. Moreover, individuals of class Mollicutes lack cell

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walls and most metabolic pathways, as they obtain the building blocks for their cellular macromolecules from host tissue [2].

Pyruvate kinase (EC 2.7.1.40, PK) is one of the key enzymes of the glycolytic pathway and carbon metabolism in general. This enzyme catalyzes the transphosphorylation of phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP)

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to pyruvate and adenosine triphosphate (ATP) under physiological conditions [3, 4]. This is an essentially irreversible reaction and is the second ATP-generating step of the glycolytic pathway. It is of particular importance in energy production during

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anaerobic glycolysis, and it produces a yield of nearly 50% of the total ATP [5]. The resultant pyruvate product feeds into many metabolic pathways; thus placing this

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enzyme at a primary metabolic intersection. Although PK from different sources has been extensively studied for many years,

its immunological characteristics have been rarely reported. The reactivity of two glycolytic enzymes of Candida albicans, namely, PK and alcohol dehydrogenase,

with the antisera of patients suffering from superficial candidiasis, suggests that glycolytic enzymes may be presented on the cell surface and function as an immunogenic protein [6]. In addition, three glycolytic enzymes, namely, 3

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6-phosphofructokinase, aldolase, and PK have been reported to be presented in the culture supernatant of group A streptococcal (GAS) type M1 strain by two-dimensional gel electrophoresis [7]. Thus far, however, no studies have

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investigated the roles of PK in Mycoplasmas, making it meaningful to research this topic. In this study, we investigated the function of surface-localized PK in M.

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gallisepticum strain Rlow (PykF, NP_853261.1).

2. Materials and methods

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2.1. Bacterial strains and growth conditions

M. gallisepticum strain Rlow was provided by the Chinese Veterinary Culture Collection Center (CVCC, Beijing, China). The strain was grown for less than five passages in complete medium as previously described [8]. Escherichia coli DH5a (Invitrogen, Carlsbad, CA, USA) cells were used as the host strain for DNA

LB

broth

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manipulation. The cells were cultured at 37°C on Luria-Bertani (LB) agar plates or in supplemented

with

100

µg·mL–1

ampicillin

or

0.5

mM

isopropyl-β-D-thiogalactopyranoside (IPTG), as required. E. coli BL21 (DE3) RIL

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(Invitrogen) cells were cultured at 37°C in LB broth and used as the host strain for recombinant protein expression. A continuous cell line of chicken embryo fibroblasts,

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DF-1, which was certified to be free of Mycoplasma contamination, was obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). These cells were grown in RPMI-1640 medium (10% fetal bovine serum, 2 mM L-glutamine, 100 IU·mL–1 penicillin, 100 mg·mL–1 streptomycin, and 10 mM HEPES buffer) at 37°C

and 5% CO2. Cell culture reagents were obtained from Gibco. SPF chicken embryos (Merial, Beijing, China) were used to hatch chickens for infection of M. gallisepticum. All animal experiments were performed according to the relevant laws and were 4

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approved by an ethics committee. 2.2. Expression and purification of the recombinant protein (rMGPykF) M. gallisepticum strain Rlow was cultured to the mid-logarithmic phase,

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centrifuged at 10,000 × g for 10 min, washed, and suspended in phosphate-buffered saline (PBS, pH 7.4) for genomic DNA extraction, which was performed as described previously [9]. The full-length of pykF gene in M. gallisepticum was amplified by

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PCR using the specific primers PpykF-F and PpykF-R (Table 1). The resultant PCR products were purified by agarose gel electrophoresis and subcloned into a pET28a(+)

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vector (Promega, Madison, WI, USA) at the BamHI and EcoRI sites to construct the recombinant plasmid pET28a-rMGPykF. DNA sequence analysis of the recombinant plasmid was conducted. For rMGPykF expression, E. coli BL21 (DE3) was transformed with pET28a-rMGPykF and induced by incubation with 0.5 mM IPTG at 37°C for 6 h.

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The expression product was further purified using a His-Bind Purification kit (Novagen, Madison, WI, USA) according to the manufacturer’s instructions, and assessed by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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(SDS-PAGE) with Coomassie blue staining. Proteins were quantified using a BCA protein assay kit (Thermo Scientific-Pierce, Rockford, IL, USA).

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To construct a PykF-green fluorescent protein (GFP) fusion protein (rMGpE), the EGFP-N1 open reading frame from the plasmid vector pEGFP-N1 (Clontech, Mountain View, CA, USA) was amplified using primers PEGFP-F and PEGFP-R (Table 1). Another fragment of PykF with a 12-base pair (bp) linker (pykF-12L) was

amplified from the recombinant plasmid pET28a-rMGPykF by PCR using primers PpykF-FL and PpykF-RL (Table 1). In the reverse primer PpykF-RL, a 12 bp linker (ACCAGAACCACC) was added, and two restriction sites (Pst I, CTGCAG; Sac I, 5

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GAGCTC) was designed for gfp gene insertion. The fragment of pykF-12L was cut with restriction enzymes BamH I and Sac I, and ligated into the plasmid pET28a(+) to get a intermediate plasmid pET28a-pykF-12L. The EGFP-N1 fragment was ligated

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into the plasmid pET28a-pykF-12L at the restriction sites between Pst I and Sac I, and then transformed into E. coli BL21 (DE3) cells. EGFP-N1 alone was ligated into

pET28a(+) and transformed into E. coli BL21 (DE3) cells as a control. The E. coli

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transformants were named E. coli (pET28a-pykF-gfp) and E. coli (pET28a-gfp), respectively.

PK

activity

was

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2.3. Enzymatic activity determined

by

a

lactate

dehydrogenase-coupled

spectrophotometric assay [10, 11], and NADH oxidation due to pyruvate reduction was measured at 340 nm with a UV-1600 spectrophotometer (Shimadzu Co., Kyoto, Japan). For the assay of rMGPykF activity, the standard reaction mixture contained 82

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mM triethanolamine-HCl buffer (pH 7.6), 10 mM MgCl2, 0.2 mM NADH (Roche, Rotkreuz, Switzerland), 10 mM KOH, 5.0 mM ADP (Sigma, St. Louis, MO, USA), 1 mM phosphoenolpyruvate (PEP, Sigma), 15 U·mL–1 L-lactate dehydrogenase (Sigma),

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and the sample enzyme, making a final volume of 1 mL. Unless otherwise stated, the reaction was initiated after 30 min of incubation by the addition of purified rMGPykF.

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The OD340 was measured for 8 min at 30 s intervals. All assays were performed in triplicate at 25°C. PK from Bacillus stearothermophilus (Sigma) and the reaction

buffer without PK were used as the positive and negative controls, respectively. 2.4. Mouse antiserum to rMGPykF and antibody reagents Female BALB/c mice (SLAC, Shanghai, China) were subcutaneously injected

with 100 µg of purified rMGPykF or M. gallisepticum strain Rlow mixed with Freund’s complete adjuvant or Freund’s incomplete adjuvant (Sigma), respectively. The 6

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injection was administered three times at two-week intervals. Blood samples were collected five days after the third injection. Sera were mixed, and titers were determined by Western blot analysis and immunofluorescence assay. A titer greater

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than 1:1000 qualified the antiserum for use in future bactericidal and adherence inhibition assays. Qualified antisera and normal mice sera were inactivated at 56°C for 30 min and then stored at -20°C until analysis.

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M. gallisepticum-positive serum (anti-MG serum) was purchased from Synbiotics (San Diego, CA, USA) for the immunogenic test of PykF.

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2.5. Purification of membrane proteins

Membrane proteins of M. gallisepticum, E. coli (pET28a-pykF-gfp), and E. coli (pET28a-gfp) were extracted using the ReadyPrep protein extraction kit (membrane I, Bio-Rad, Hercules, CA, USA), according to the manufacturer’s protocol. Proteins were quantified using a BCA protein assay kit (Pierce), as described by the

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manufacturer. 2.6. Immunoblot analysis

SDS-PAGE and immunoblot analyses were performed using nitrocellulose

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membranes (Bio-Rad) according to the procedure as described by Chen et al. (2011). Immunoblots were probed with anti-MG serum or antiserum from immunized mice.

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The purity of the E. coli membrane fractions were tested using a mouse monoclonal antibody to the RNA polymerase beta (RpoB) subunit of E. coli (Abcam, Cambridge, UK; 1:1000). 2.7. Immunogold transmission electron microscopy The M. gallisepticum Rlow strain was cultured to the mid-logarithmic phase, centrifuged at 3,000 × g for 10 min at 4°C, washed twice, suspended in PBS, fixed in 4% paraformaldehyde, and embedded in “LR White” resin (London Resin Co. Ltd, 7

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Basingstoke, England). Grids with ultrathin sections were incubated with rMGPykF or normal mouse serum at 4°C overnight in a moist chamber. The sections were rinsed with 0.5% bovine serum albumin (BSA) and 0.1% gelatin in PBS, and incubated with

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10 nm anti-mouse IgG gold conjugate (1:100; Sigma, Buchs, Switzerland) for 2 h. After fixing with 1% glutaraldehyde in PBS for 15 min and staining with 3% (w/v)

uranyl acetate for 5 min, the dried grids were visualized using a transmission electron

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microscope (TEM, Philips CM120) at 80 kV [12]. 2.8. Bactericidal assay

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Inactivated antisera against rMGPykF and M. gallisepticum were used to assay the bactericidal activity against M. gallisepticum Rlow using a complement-mediated bactericidal assay described previously, with some modifications [13, 14]. Specifically, 40 µL of mouse antiserum or normal mouse serum was added to the wells of a sterile 96-well plate, to which 120 µL of bacterial suspension (5 × 103 colony-forming units

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[CFU]·mL–1 in Dulbecco’s PBS containing 0.90 mM·L–1 calcium, 0.49 mM·L–1 magnesium, and 0.1% gelatin; DPBSG) and 40 µL of guinea pig complement sera (1:5 in DPBSG; CVCC) were added. After the plate was incubated at 37°C for 60 min,

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100 µL of the mixture was plated onto preheated (37°C) agar plates (60 mm). The plates were incubated at 37°C with 5% CO2 for 5-7 days, and the colonies were

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counted. The percentage of killed cells was obtained by calculating the mean value of [1 – (CFU following antiserum treatment/CFU in normal mouse serum control)] × 100 from three independent trials performed in triplicate. 2.9. Fluorescence assay The fluorescence assay was used to determine the adhesion of E. coli

(pET28a-pykF-gfp) to DF-1 cells [12, 15]. Briefly, DF-1 cells grown on coverslips were washed with PBS and fixed in 4% (w/v) formaldehyde for 20 min at room 8

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temperature. After washing five times with PBS, the cells were infected with E. coli (pET28a-pykF-gfp) or E. coli (pET28a-gfp) at 37°C for 2 h. Adhesion was visualized under an Olympus BX40 fluorescence microscope. The fluorescence images were

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captured using Nikon Eclipse 80i (Japan). 2.10. Adherence inhibition assay

The inhibition of bacterial adhesion by anti-rMGPykF antiserum was evaluated

described previously with some modifications [8].

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by testing the adherence of anti-rMGPykF-treated M. gallisepticum to DF-1 cells as

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M. gallisepticum strain Rlow was cultured to the mid-logarithmic phase, centrifuged at 3,000 g for 10 min at 4°C, washed twice, suspended in PBS (pH 7.4), and incubated with mouse anti-rMGPykF antiserum or normal mouse serum for 1 h at 37°C. During incubation, DF-1 monolayers, which had been cultured in 60-mm dishes, were washed three times with PBS to remove the serum from the medium. The

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monolayers were then infected with strain Rlow at a multiplicity of infection (MOI) of 500 and incubated with 5% CO2 at 37°C for 2 h. After washing with PBS to remove non-adherent Mycoplasma, the cells were detached with 0.05% trypsin at 37°C for

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3–5 min. Serial dilutions of the detached cells were plated onto ATCC medium 243, containing 1% Noble agar (BD) solid medium and incubated at 37°C with 5% CO2 for

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5–7 days. The Mycoplasma colonies were then counted to calculate the adherence frequency. Percentage inhibition was calculated using the following equation: [1 – (CFU following antiserum treatment/CFU in normal mouse serum control)] × 100.

Experiments were performed in triplicate. 2.11. Statistical analysis Bactericidal assay and adherence inhibition frequency were expressed as the mean ± standard deviation of n independent values. The statistical significance of the 9

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differences between the mean values was determined using the Student’s t test (P < 0.05). 3. Results

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3.1. Expression and purification of the recombinant M. gallisepticum PykF The pykF gene is 1,527 nucleotides in length and encodes a protein of 508 amino acid residues. The full-length sequence of M. gallisepticum pykF was obtained by

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PCR amplification and then cloned into a pET28a(+) vector. The overexpression of

the M. gallisepticum PK (rMGPykF) in E. coli BL21 (DE3) resulted in migration of a

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62-kDa protein on Coomassie blue-stained SDS-PAGE gels. The observed molecular mass was consistant with the size of PykF (MGA_0156) containing a hexahistidine (His) tag at its N-terminus (Fig. 1A, Lane 2). SDS-PAGE of the purified rMGPykF protein revealed a single protein band on the membrane (Fig. 1A, Lane 4). Homology analysis using the BLAST program search of the National Center for

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Biotechnology Information (NCBI) database revealed only moderate homology of the protein obtained in this study with known PKs of other Mycoplasma species, ranging from the highest identity of 55% (Mycoplasma penetrans HF-2, Accession. No.

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NP_757464.1), to the lowest identity of 40% (Mycoplasma conjunctivae HRC/581, YP_002960708.1; data not shown). None of these proteins has been functionally

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characterized.

3.2. PK activity

The PK activity of rMGPykF was measured in a coupled enzyme assay. A

schematic of the glycolytic cycle involving PK catalysis is shown in Fig. 2A. The rMGPykF protein exhibited similar catalytic activity to the positive control of PK

from B. stearothermophilus, leading to the conversion of NADH to NAD+, whereas the reaction buffer without PK or rMGPykF (negative control) exhibited no activity 10

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change (Fig. 2B). This indicated that rMGPykF exhibited PK activity in vitro in catalyzing the conversion of PEP to pyruvate, which was a precursor step before the conversion of NADH to NAD+ that is detected in this assay.

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3.3. Localization of PK in M. gallisepticum The protein preparations from whole-cell, cytoplasmic, and membrane of M.

gallisepticum were analyzed to identify the localization of PK in M. gallisepticum.

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The hyperimmune antiserum specifically reacted with the 58 kDa PK proteins in all three preparations (Fig. 1C, Lanes 7–9), indicating that PK was presented in the

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membrane as well as in the cytoplasm of M. gallisepticum.

Furthermore, TEM analysis of immunogold-stained M. gallisepticum showed that M. gallisepticum PK was gold-labeled on the cell surface (Fig. 3). 3.4. Immunogenicity analysis

Whole-cell proteins of the transformants E. coli (pET28a-rMGpykF) and E. coli

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(pET28a) were transferred to a nitrocellulose membrane and incubated with anti-MG serum (1:10,000) for the immunogenicity analysis. E. coli (pET28a-rMGpykF), but not E. coli (pET28a), showed a positive band at 62 kDa, suggesting that PykF plays

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an immunogenic role in M. gallisepticum (Fig. 1B). 3.5. Bactericidal assay

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Complement-mediated bactericidal assay was performed to estimate the immunological protective effect of M. gallisepticum PykF. Anti-MG serum presented a 93.84% killing of the M. gallisepticum Rlow bacteria. Unexpectedly, the

anti-rMGPykF antiserum killed 70.55% of M. gallisepticum Rlow cells, which showed

significant difference when compared with normal mouse serum control (Table 2), suggesting that PykF plays an important role in the specific immunity against M. gallisepticum in host cells. 11

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3.6. Localization of rMGpE in E. coli Immunoblot analysis was carried out to analyze the subcellular localization of rMGpE in E. coli (pET28a-pykF-gfp) transformants. E. coli cytoplasm and cell

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membrane preparations and anti-rMGPykF antibodies were used for this assay. As shown in Fig. 4A, an 87 kDa band corresponding to rMGpE appeared in both purified

cytoplasm and cell membrane components of E. coli (pET28a-pykF-gfp). Furthermore,

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IPTG-induced E. coli (pET28a-pykF-gfp) cells showed specific reactions with

polyclonal anti-rMGPykF antibodies (Fig. 4A), indicating that rMGpE or GFP was

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expressed on the surface of these transformants. In contrast, the induced E. coli (pET28a-gfp) did not react with rMGPykF. The purity of the membrane fraction was demonstrated by immunoblot analysis using monoclonal antibodies against RpoB of E. coli. A reaction was observed in the cytoplasmic fraction, while no reaction could be found in the membrane preparation (Fig. 4B).

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3.7. E. coli transformants adhere to DF-1cells

Adhesion of E. coli (pET28a-pykF-gfp) was visualized by fluorescence microscopy. Specific adhesion of E. coli (pET28a-pykF-gfp) to DF-1 cells was clearly

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visible (Fig. 5A). On the contrary, control E. coli (pET28a-gfp) cells (lacking PK) showed no adhesion to DF-1 cells (Fig. 5B).

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3.8. Adherence inhibition assay Mycoplasma-free DF-1 cells were used for the adherence inhibition assay. As

shown in Table 3, mouse anti-rMGPykF antiserum inhibited significantly the adherence of M. gallisepticum Rlow bacteria to DF-1 cells at the rate of 39.31% (P <

0.01). 4. Discussion

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PK has been purified and characterized from various sources. It has a number of other functions apart from essential catalytic roles in metabolism for the purpose of survival. Recent study has strongly suggested that PK may be included in the group of

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biological membrance-associated enzymes, by binding to integral membrane proteins [16]. PK and other glycolytic enzymes constitute a potentially excellent target for

chemotherapy in cases of infection with protozoan parasites such as Leishmania and

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Trypanosoma [17]. The monomeric form of mammalian muscle-type PK (p58-M2) is a thyroid hormone-binding protein that plays a critical role in the regulation of the

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transcriptional responses of the thyroid hormone nuclear receptors (TRs). PK M2 (M2-PK or PKM2) serves as a key regulator of the metabolic budget system in tumor cells and as a metabolic sensor that regulates cell proliferation, cell growth, apoptosis and cell death in a glucose supply-dependent manner [18, 19]. PK M2 is a target of tumor-suppressive microRNA-326 and regulates the survival of glioma cells,

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suggesting that it can be inhibited to treat glioblastoma, with the potential for minimal toxicity to the brain [20]. Finally, PK has been identified as an antigen associated with Tourette syndrome [21].

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Several other metabolic enzymes have also been reported as surface localized proteins and to enhance virulence for pathogens [22]. For instance, the

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glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been demonstrated to be presented on the surface of many pathogens such as streptococci [23-25], mycobacteria [26], and neisseria [27], and has also been thought to play roles in bacteria-host cell interaction. Furthermore, phosphoglycerate kinase (PGK) [28], triose-phosphate isomerase (TPI) [29], and α-enolase have been shown as the surface proteins [30-32]. The GAPDH-like protein MSG1 of Mycoplasma suis has been shown to be surface localized and involved in the adhesion of this organism to 13

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erythrocytes [12]. Other studies have reported that alpha-enolase is localized on the cell surface of Mycoplasma fermentans, M. gallisepticum, and M. suis. Furthermore, this enzyme has been shown to mediate adherence by binding to plasminogen [8, 33,

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34]. It is noteworthy that all of these five enzymes (GAPDH, PGK, PGM, enolase, LDH) are sequential enzymes in the second half of the Embden-Meyerhof-Parnas (EMP) pathway. Pancholi and Chhatwal (2003) reported that PK and other glycolytic

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enzymes that belong to the first half of the EMP glycolytic pathway are not found on the surface of any Gram-positive bacteria. However, no studies have determined

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whether PK is displayed on the surface of other Gram-negative bacteria. Therefore, in the present study, we aimed to localize and characterize the PykF protein in M. gallisepticum. In doing so, we cloned the complete pykF gene of M. gallisepticum strain Rlow and successfully expressed the recombinant protein rMGPykF in E. coli BL21 (DE3), as verified by SDS-PAGE with Coomassie blue staining. The purified

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rMGPykF exhibited similar enzyme activity in vitro compared to the PK enzyme of B. stearothermophilus. The purified membrane proteins from M. gallisepticum were blotted onto nitrocellulose membranes and treated with mouse anti-rMGPykF

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antibodies, and a positive band was detected. Together with the transmission electron microscopic analysis of immunogold-stained cells, PykF was found to be a membrane

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component of M. gallisepticum. Our immunological data (Fig. 1B and Table 2) demonstrated that M.

gallisepticum PykF exhibited strong immunogenicity. In our study, the recombinant protein in E. coli BL21 (DE3) specifically reacted with the serum of M. gallisepticum-infected chickens. Similar to these results, cattle naturally infected with M. bovis develop an immune response against the GAPDH protein combined with the antigenic conservation of the protein, suggesting that the M. bovis GAPDH protein 14

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could be a potential target for the development of a more effective vaccine against all M. bovis isolates [35]. In another previous study, enolase and PK of Mycoplasma synoviae were identified as two major immunogenic proteins [36]. Two other

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recombinant proteins of M. suis, i.e., MSG1 and α-enolase, have been evaluated as diagnostic and immunogenic antigens [12, 34]. Unexpectedly, fructose-bisphosphate aldolase class II, TPI, PGK, GAPDH, phosphoglyceromutase (GPM), enolase, L-lactate

dehydrogenase of Mycoplasma

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glucose-6-phosphate isomerase, and

mycoides subsp. mycoides small colony type (MmmSC) were identified as novel

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immunogenic proteins by immunoblot analysis with sera from MmmSC-infected animals and MALDI-ToF mass spectrometry [37]. Furthermore, the bactericidal assay using mouse antiserum killed 70.55% of M. gallisepticum Rlow cells. This result suggests that PykF of M. gallisepticum could be used to develop a more sensitive and specific serological diagnostic method for M. gallisepticum infections, and also could

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be used as a potentially effective DNA vaccine candidate in the poultry industry. Due to the surface expression of M. gallisepticum PykF, further immunological experiments could be carried out to determine whether this protein had more functions

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in addition to its common glycolytic function. In pathogens, glycolytic enzymes would evolve by virtue of their location to function differently and more appropriately

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to benefit the organism. Therefore, it is possible that glycolytic enzymes, when expressed on the cell surface, could exhibit an important role in microbial virulence. From the published data it can be assumed that, after secretion, glycolytic enzymes become re-associated on the surface of the pathogen and play important roles in pathogenesis by binding to host components such as fibronectin and plasminogen. By binding to host components, these glycolytic enzymes, which are presented on the cell surface, could enable the pathogen to adhere to and invade host cells, survive and 15

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persist intracellularly, and degrade and invade host tissue [22]. Mycoplasma species differ from other bacteria in two general aspects. First, they have the smallest genomes as a result of the fact that their coding capacity is limited, making them

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“minimal cells” that are dependent on the supply of nutrients from their host environments, and they exhibit apparent under-representation of recognizable components that regulate gene expression in response to environmental changes.

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Second, they lack cell wall, emphasizing the role of membrane surface proteins as key components for a variety of functions involved in host interactions [38]. In some

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Mycoplasma species, MSG1, GAPDH, and α-enolase, as mentioned above, and elongation factor Tu (EF-Tu) together with pyruvate dehydrogenase E1 β subunit [39], which are surface-expressed proteins, are all involved in adhesion to host cells as virulence factors.

Adherence of Mycoplasma to the mucosal epithelium is an essential first stage

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of infection and an absolute requirement for successful colonization, which is dictated by the need to avoid rapid clearance by host innate immune mechanisms, physical removal through mucociliary clearance, and for acquisition of nutrients and refuge.

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Adhesion to host cells is currently considered to be the major virulence factor of Mycoplasma, leading to pathogenesis [40, 41]. Bacterial adhesion to the extracellular

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matrix (ECM; such as fibronectin, heparin, collagen, laminin, and vitronectin) of avian respiratory epithelial cells is a common and essential step in tissue adherence, invasion, and colonization [42, 43]. In this study, a fluorescence assay was carried out to determine whether the M. gallisepticum PykF is involved in adhesion. As M. gallisepticum is very small in size, it could barely be visualized by the fluorescence microscope; therefore, E. coli transformants were used as a model system to estimate the additional non-enzymatic functions of PykF. In E. coli, rMGpE was expressed in 16

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the cytoplasm as well as the membrane, as verified by Western blot analysis. Membrane localization and the surface accessibility of PykF may suggest that this protein has additional functions in M. gallisepticum. Fluorescence assays showed that

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only E. coli (pET28a- pykF -gfp), but not the negative control, could adhere to DF-1 cells, indicating that PykF can mediate adherence of M. gallisepticum to host cells by a hitherto unknown mechanism. This was confirmed again by the adherence inhibition

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assay. This is the first study to report that PK is at the cell surface of M. gallisepticum.

and PykF might contribute to the virulence of Mycoplasma. Further studies are

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required including using M. gallisepticum PykF as a potential vaccine candidate to develop a vaccine against avian Mycoplasma infections and determining the mechanism by which PykF facilitates adhesion to the host epithelium.

Conclusion

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In conclusion, we determined that M. gallisepticum PK is localized at the cell surface of M. gallisepticum and confers immunogenic protection, suggesting that it could probably be useful for developing additional immunodiagnostic applications.

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This is the first study to report immunogenic protection induced by PK in pathogenic microorganisms. Moreover, the adherence inhibition assay demonstrated that this

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protein may play an additional role on the bacterial adhesion to avian cells. The superior bactericidal activity, together with the percentage of adherence inhibition by anti-PykF antiserum, makes this protein a potential candidate for vaccine development to prevent the diseases caused by M. gallisepticum.

Acknowledgments We are grateful to the Key Open Laboratory of Animal Parasitology at the 17

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Ministry of Agriculture of China for providing key laboratory equipment. We also thank Xinyu Chen of the School of Medicine, Shanghai Jiao Tong University for her assistance with transmission electron microscopic technique. This research was

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supported by the National Natural Science Foundation of China (30871883 and 31001077), and Chinese Special Fund for Agro-scientific Research in the Public

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Interest (201303044).

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Fig. 1 Immunoblot analysis of M. gallisepticum PykF. (A) SDS-PAGE analysis of the recombinant M. gallisepticum PykF(rMGPykF). M: Molecular weight marker; Lane 1: Negative control, E. coli BL21(DE3) transformed with plasmid pET28a(+) with IPTG

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induction; Lane 2: rMGPykF was expressed in E. coli BL21(DE3) transformed with recombinant plasmid pET28a-rMGPykF with IPTG induction; Lane 3: Supernatant of

E. coli (pET28a-rMGPykF) expression products with IPTG induction; Lane 4:

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Purified rMGPykF. (B) Immunogenicity analysis of PykF with M. gallisepticum positive serum. Lane 5: negative control, E. coli BL21(DE3) transformed with

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plasmid pET28a(+); Lane 6: E. coli BL21(DE3) transformed with recombinant plasmid pET28a-rMGPykF. (C) Cellular localization of PykF in M. gallisepticum by immunoblot analysis with rMGPykF-specific mouse antiserum. Lane 7: M. gallisepticum whole cell proteins; Lane 8: M. gallisepticum cytoplasma proteins; Lane 9: M. gallisepticum membrane proteins. Lane 10 : negative control, E. coli BL21(DE3)

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whole cell proteins.

Fig. 2 Enzymatic activity of rMGPykF. (A) Enzymatic reactions of pyruvate kinase

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involved in the glycolytic cycle. (B) Each 10 mg/mL of purified rMGPykF or Bacillus stearothermophilus pyruvate kinase (positive control) was added to the reaction buffer

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to measure the enzymatic kinetics. OD340 values decreased similarly with time for both rMGPykF and Bacillus stearothermophilus pyruvate kinase, whereas no

absorbance change was observed in the reaction buffer without PK (negative control). The reaction buffer without NADH was used as the blank control.

Fig.

3

Immunogold

transmission

electron

microscopy

demonstrated

the

surface-accessibility of M. gallisepticum PykF. (A) Specific accumulation of 10 nm

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gold particles bound to PykF at the surface of M. gallisepticum (indicated by arrows). (B) Negative control with normal mouse serum showed no gold particles.

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Fig. 4 Immunoblot analysis of the sub cellular localization of rMGPykF in E. coli. (A) rMGPykF could be detected in the cytoplasma (cyto+) and membrane fraction (mem+) of IPTG-induced E. coli (pET28a-pykF-gfp). No rMGPykF was detected in the

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cytoplasma (cyto-) and membrane fraction (mem-) of IPTG-induced E. coli

(pET28a-gfp) transformants. (B) Purity of the membrane fractions was confirmed by

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using a monoclonal antibody against E. coli RpoB. The E. coli cytoplasmic fractions (cyto+, cyto-) reacted with the antibody, the E. coli membrane fractions (mem+, mem-) showed no reaction.

Fig. 5 Analysis of E. coli transformants adhering to DF-1 cells using fluorescence

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microscopy. (A) Cells incubated with GFP-expressing E. coli (pET28a-pykF-gfp) transformants, which coexpressing PykF and GFP as fusion protein adhere to DF-1 cells. (B) Cells incubated with GFP-expressing E. coli (pET28a-gfp) transformants,

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which expressing GFP alone, showed no adhesion.

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Table 1 Oligonucleotides used in this study Sequence (5’→3’)

PpykF-F

CCGGGATCCaATGAATAAGAAACATAA

PpykF-R

CGGAATTCTTTTAGTAATACAAA

PpykF-FL

CCGGGATCCATGAATAAGAAACATAAAGA

PpykF-RL

ATAGAGCTC CTGCAGACCAGAACCACC bTTTTAGTAATACAAA

PEGFP-F

ATACTGCAGATGGTGAGCAAGGGCGAG

PEGFP-R

CCGGAGCTCTTACTTGTACAGCTCGTCC

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The restriction enzyme sites of BamH I (GGATCC), EcoR I (GAATTC), Pst I

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a

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Primers

(CTGCAG) and Sac I (GAGCTC) were underlined. b

A 12 bp linker which designed for co-expressing of the PykF and GFP fusion protein

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Table 2 Bactericidal assay Killing percent (%)

Anti-MG serum

9 ± 0.09

93.84**

Anti-rMGPykF serum

43 ± 0.05

Normal mouse serum

146 ± 0.09

70.55** -

P< 0.01, compared with normal mouse serum control.

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**

Geometric mean CFU ± SD

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Sera

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Geometric mean CFU± SD)

Inhibition (% )

Anti-rMGPykF serum

352 ± 0.29

39.31**

Normal mouse serum

580 ± 0.65

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Table 3 Adherence inhibition assay Sera

P< 0.01, compared with normal mice serum.

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Highlights

•M. gallisepticum pyruvate kinase fusion protein (PykF) was expressed in a pET

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system

•Recombinant M. gallisepticum PykF catalyzed the conversion of NADH to NAD+

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•PykF was identified as an immunogenic protein on the M. gallisepticum cell surface

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•M. gallisepticum PykF was identified as a potential protective antigen

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•M. gallisepticum PykF participated in the bacterial adhesion to DF-1 cells