1 Primary Structure and Antibacterial Activity Characterization of Chicken Bone-marrow 1 Derived ...

31 Pages·2009·0.86 MB·English

AAC Accepts, published online ahead of print on 8 September 2009
Antimicrob. Agents Chemother. doi:10.1128/AAC.00301-09
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
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1 Primary Structure and Antibacterial Activity Characterization of Chicken Bone-marrow
2 Fgtkxgf"(cid:533)-defensins
3 Chrystelle Derache,1 Valérie Labas,2 Vincent Aucagne,3 Hervé Meudal,3 Céline Landon,3 Agnès F.
4 Delmas,3 Thierry Magallon,2 and Anne-Christine Lalmanach1*
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6 INRA, UR 1282 Infectiologie Animale et Santé Publique,1 UMR INRA 85 Î CNRS 6175 Î D
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7 Université François Rabelais, Physiologie de la Reproduction et des Comportements, Plate-forme n
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8 de Protéomique Analytique et Fonctionnelle,2 F-37380 Nouzilly, and Centre de Biophysique d
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9 Moléculaire CNRS UPR4301 affiliated to the University of Orléans and Inserm, rue Charles Sadron f
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10 45071 Orléans cedex 2,3 France
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11 * corresponding author : Anne-Christine Lalmanach, INRA, UR1282 Infectiologie Animale et Santé :
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12 Publique, F-37380 Nouzilly, France, phone number : [phone], fax number : +332 47 42 c
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13 77 74, Email address : [email protected] m
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14 g
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15 Abstract<"Vjtgg"dkqnqikecnn{"cevkxg"(cid:533)-defensins were purified by chromatography from chicken
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16 bone marrow extract: AvBD1, AvBD2 and the newly isolated AvBD7. Mass spectrometry analyses e
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17 showed that bone marrow derived AvBD1, -2 and -7 peptides were present as mature peptides and e
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18 revealed post-translational modifications for AvBD1 and AvBD7 as compared to their in silico 9
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19 predicted amino acid sequences. MS/MS analysis using nanoESI-Q-TOF showed N-terminal 1
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20 glutaminyl cyclization of mature AvBD7 and C-terminal amidation of mature AvBD1 peptide, while y

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21 post-translational modifications were absent in bone marrow derived mature AvBD2 peptide. e
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22 Futhermore, mass spectrometry analysis performed on intact cells confirmed the presence of these
23 vjtgg"rgrvkfgu"kp"ocvwtg"jgvgtqrjknu0"Kp"cffkvkqp."vjg"cpvkdcevgtkcn"cevkxkv{"qh"vjg"vjtgg"(cid:533)-defensins
24 was assessed against a large panel of Gram positive and negative bacteria. While the three defensins
25 displayed a similar antibacterial spectrum against Gram positive strains, AvBD1 and AvBD7
26 exhibited the strongest activity against Gram negative strains compared to AvBD2.
27
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28 INTRODUCTION
29 Defensins belong to the large family of antimicrobial peptides which are important
30 components of innate and adaptive immunity (23). They are 30 to 50 amino acids long, rich in
31 cationic residues and their tertiary structure is maintained by three disulfide bridges. Depending on
32 their disulfide motif and the position of conserved cysteines, defensins are divided in three major
33 itqwru"kp"xgtvgdtcvgu<"g-."(cid:533)- cpf"(cid:537)-defensins0"Vjg"nctiguv"hcoknkgu"eqortkug"g- cpf"(cid:533)-defensins D
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34 which are produced by leukocytes and epithelial cells. The role of both defensin families is not n
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35 restricted to antimicrobial effectors at host-pathogen interfaces. In mammals they have been shown d
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36 to modulate adaptive immunity by attracting antigen presenting cells and lymphocytes (45). f
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37 Interest in defensins as therapeutic drugs is growing because they may constitute an
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38 alternative to the controversial use of antibiotics. Moreover, they may be used as an additive in food :
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39 preservation. Bird defensins have a double economic interest: besides their interesting antimicrobial c
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40 properties, the poultry industry may represent an important source of production. The only known m
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41 dktf"fghgpukpu"dgnqpi"vq"vjg"(cid:533)-defensin family ((40) for review). The 3-dimensional structure and g
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42 broad antimicrobial activity spectrum have been studied only for king penguin AvBD103b
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43 (Spheniscin-2) and gave new insights into the structure-activity relationship of avian (cid:533)-defensins e
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44 (11, 36). (cid:533)-defensins have also been isolated from ostrich heterophils (AvBD1, 2, 4, 7 and 8), the e
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45 so-called ostricacins, which are highly similar to chicken defensins. The antimicrobial activity of 9
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46 these ostrich peptides is sensitive to cations (35) and their targets seem to be intracellular (34). In 1
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47 ejkemgp."vjg"(cid:533)-defensin family encompasses fourteen genes that have been mostly identified from y

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48 nucleic acid sequence data (40). The mature peptide of avian -defensins 1 and 2 (AvBD1 and e
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49 AvBD2), formerly called gallinacin 1 and gallinacin 2, were the only ones isolated from heterophil
50 granulocytes (7, 8). In these previous studies, the resolution of the mass spectrometry techniques
51 used allowed the amino acid sequence identification from average masses of these two peptides but
52 not post-translational modification characterization. Therefore, in order to further characterize the
53 ugswgpeg"cpf"vjg"cpvkoketqdkcn"cevkxkv{"qh"cxkcp"(cid:533)-defensins 1 and 2, larger amounts of defensins
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54 were required. Bone marrow, in which defensin genes have been shown to be highly expressed (15),
55 should be a relevant source of natural peptides. It is the active site of leukopoiesis and contains the
56 heterophil precursors, AvBD1 and AvBD2 producing cells. These cell precursors are present in
57 larger numbers in bone marrow than mature heterophils in blood (25).
58 In the present work, we describe the extraction of AvBD1 and AvBD2 from bone marrow
59 and their fine characterization. Using modern proteomic analyses, the primary structure of AvBDs D
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60 has been refined as well as the N- and C-terminal cleavage site of mature defensin. Besides AvBD1 n
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61 and AvBD2, we characterize for the first time AvBD7, whose mature primary sequence was only d
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62 known as bioinformatic prediction (15, 43). The specific expression of AvBD1, AvBD2 and AvBD7 f
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63 in mature effector cells has been confirmed using Intact Cells Matrix-Assisted Laser Desorption
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64 Ionization-Time Of Flight (MALDI-TOF). Finally, the antimicrobial activity of the three bone- :
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65 marrow extracted defensins has been characterized and compared against a large panel of bacterial c
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66 strains including zoonotic agents. m
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67 g
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68 MATERIALS & METHODS
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69 Sample preparation from bone marrow and extraction. Bone marrow was obtained from femur e
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70 and tibia of ten two-month old PA12 chickens. The bones were cut longitudinally and the marrow e
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71 was collected by several Phosphate Buffered Saline (PBS, pH 7.4) rinses. The PBS solution 9
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72 containing bone marrow was centrifuged at 1,500 x g for 10 min. The pellet was then resuspended 1
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73 in 5% acetic acid. The mixture obtained was sonicated on ice for 5 min (6 s at a power of 40 W y

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74 (Vibra-Cell 75455, Bioblock) separated by 1 s resting) and stirred for 16 h at 4°C. Finally, acid- e
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75 insoluble material was removed by ultracentrifugation at 70,000 x g for 30 min at 4°C and the
76 supernatant was lyophilized.
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79 Purification of bone marrow-extracted peptides. Bone marrow lyophilized extract was dissolved
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80 in 5% acetic acid and fractionated on a 2- by 100-cm Bio-Gel P10 column (Bio-Rad) equilibrated in
81 5% acetic acid. The column effluent at a flow rate of 0.5 mL/min was monitored at 280 nm. The
82 fractions (6 mL) corresponding to the major gel filtration peaks were screened by MALDI-TOF
83 mass spectrometry. The peptides characterized on the basis of their intact mass were further purified
84 by semi-preparative C18 reverse phase HPLC (Nucleosil® C18, 300 Å, 5µm, 250x10mm, Merck)
85 using a linear acetonitrile gradient with 0.1% trifluoracetic acid (TFA) at a constant flow rate of D
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86 3 ml/min. The TFA counter anions of the purified peptides were finally exchanged by evaporating n
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87 acetonitrile, loading on a Sep-Pak C18 6ml column (Waters) followed by washings with 5% acetic d
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88 acid, and eluted by 10% acetic acid in 50% acetonitrile and lyophilized. The powder was dissolved f
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89 in ultrapure water at 500 µg/mL concentration.
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90 :
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91 Heterophils collection from peritoneal exudates. A suspension (20 mL) of Sephadex G75 c
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92 (Sigma) (3% in 0.15 M NaCl) was injected in the peritoneal cavity of three five-week old PA12 m
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93 chickens. The animals were sacrificed twelve hours after the injection. Peritoneal cavities were g
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94 washed with 50 oN"qh"JcpmÓu"Dwhhgtgf"Ucnv"Uqnwvkqp"*JDUU."Ukioc+"vq"eqnngev"the cells. Each cell
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95 suspension was centrifuged at 300 x g for 10 min at 4°C and the cell pellets were suspended in fresh e
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96 HBSS. Peritoneal exudates gave approximately 108 cells (95% heterophils) per chicken. Cells were be
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97 finally centrifuged in 1.5 mL tubes and the cell pellets were frozen until Intact Cells MALDI-TOF 9
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98 Mass Spectrometry (ICM-MS) analysis was carried out. 1
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99 y

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100 MALDI-TOF Mass Spectrometry. All mass spectra were generated on a M@LDI LR (Waters, e
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101 Micromass Ltd, Manchester, UK) MALDI-TOF mass spectrometer, operating in positive linear
102 mode. External mass calibration was performed with a mixture containing Glu-fibrinopeptide B,
103 ACTH (18-39 clip), insulin, ubiquitin, all at 1 pmol/µL, cytochrome C at 2 pmol/µL, myoglobin at
104 4 pmol/µL and trypsinogen at 8 pmol/µL. Mass spectra were recorded in the mass range
105 2,000-20,000 m/z, acquiring 10 shots per spectrum at a laser firing rate of 10 Hz. Data processing
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106 was performed using MassLynxTM 4.0 software. The spectrum result for each sample well was
107 background substracted using a polynomial order of 10% below the curve and smoothed with the
108 minimum peak width at half height set to 15 channels. Smoothing was performed twice using the
109 Savitzky Golay algorithm. All the spectra were processed using the same parameters.
110 For gel filtration eluted peptides mass fingerprint analysis, 1µL of each fraction and the
111 matrix (1:1, v/v) were loaded on the target using the dried droplet method. The matrix used was D
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112 sinapinic acid at 20 mg/mL dissolved in 50% acetonitrile / 50% H20 / TFA 0.1%. n
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113 For Intact Cells MALDI-TOF Mass spectrometry (ICM-MS) analysis, the frozen whole cell d
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114 pellets were resuspended with a 10 µL aliquot of a sinapinic acid matrix solution prepared by f
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115 dissolving 20 mg of the solid crystals in 1 mL of 50% acetonitrile / 50% H20 / TFA 1% reagent. The h
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116 matrix/sample was spotted onto the MALDI sample probe and dried at room temperature. To obtain :
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117 a mass accuracy of around 0.06%, internal calibration was accomplished using the major identified c
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118 peak of AvBD2 as lock mass (average mass : 3316.66 Da) for all spectra. m
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119 g
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120 NanoLC-MS/MS mass spectrometry: bottom-up analysis. Identification by bottom-up proteomic
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121 analysis was conducted on peptides from gel filtration fractions. Aliquots were dried and e
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122 resuspended in 20 µL of 50 mM NH4HCO3 then reduced with 5 mM dithiothreitol, alkylated with e
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123 12.5 mM iodoacetamide and incubated overnight at 37°C with 0.3 µg of bovine trypsin (Sequencing 9
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124 Grade, Roche, Paris). The tryptic digests were acidified with 5 µL of formic acid 5% and sonicated 1
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125 for 10 min. Nanoscale capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) y

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126 analysis of the digested peptides was performed using a CapLC system coupled to a hybrid e
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127 quadrupole time-of-flight mass spectrometer (Q-TOF UltimaGlobal, Waters, Manchester, UK) fitted
128 with a Z-spray ion source. The capillary voltage was set at 3200 V, the cone voltage at 100 V and
129 the microchannel plate (MCP) voltage at 2300 V. Samples were desalted and concentrated using an
130 on line precolumn (Monolithic Trap column, 200 m i.d x 5 mm, LC Packings, Dionex). Peptide
131 separations were conducted on a C18 column (AtlantisTM dC18, 75 m I.D x 150 mm NanoEaseTM,
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132 Waters) running with a 200 nL/min flow. The gradient profile consisted of a gradient from 100% A
133 (0.1% formic acid / 2% acetonitrile / 98% H O, v/v) to 15% B (0.1% formic acid / 20% H O / 80%
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134 acetonitrile, v/v) in 2 min and 15% to 55% B in 60 min. Mass data were acquired using automatic
135 switching between MS and MS/MS (fragmentation) modes: one MS survey scan was followed by
136 MS/MS scans on the three most intense peptide ions detected. Only doubly and triply charged ions
137 were allowed to be selected as precursors over a mass range of 400-1300 m/z. The collision energy D
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138 was selected depending on the precursor ion mass and charge. The mass spectrometer was n
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139 calibrated using the fragmentation spectrum of Glu-fibrinopeptide B at 500 fmol/ l in 50% water / d
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140 50% acetonitrile / 0.1% formic acid. Data were processed using a ProteinLynx Global server 2.2 fr
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141 (Waters). The peptide and fragment masses obtained were matched automatically to proteins in a h
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142 non-redundant database (SwissProt 56.5) and decoy database using the MS/MS ion search option of :/
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143 the MASCOT software (Matrix Science, UK, http://www.matrixscience.com) against the all taxa c
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144 section (as of 15th Dec 2008 with 402482 sequences). Enzyme specificity was set to semitrypsin m
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145 with 2 missed cleavages using carbamidomethylcysteine and methionine oxidation as variable g
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146 modifications. The tolerance of the ions was set to 0.3 Da for both parent and fragment ion matches.
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147 All hits with a P value < 0.05 were manually verified and proteins detected by one peptide were e
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148 considered positively identified with 5 consecutive fragment ions and with a false discovery rate at e
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149 0% (peptide matches above the identity threshold). 9
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150 1
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151 NanoElectrospray mass spectrometry: top-down proteomic analysis. HPLC fractions were y

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152 diluted with a mixture of water / formic acid / acetonitrile (45 : 5 : 50) and were analyzed by MS e
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153 and MS/MS on a nanoESI Q-TOF UltimaGlobal mass spectrometer (Waters, Manchester, UK). The
154 samples were loaded into nanoES spray capillaries (Proxeon, Odense, Denmark). The capillary
155 voltage was set at 1200 V, the cone voltage at 100 V, the microchannel plate (MCP) voltage at 2300
156 V and collision energies on the order of 20-50 eV. Argon was used as the collision gas. Data
157 ceswkukvkqp"cpf"cpcn{uku"ygtg"rgthqtogf"wukpi"OcuuN{pzª"x602"uqhvyctg"*Ycvgtu."Ocpejguvgt."
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158 UK). Multicharged precursor ions were selected to m/z 900.99 (5+), 979.23 (4+) and 892.27 (6+)
159 respectively for AvBD1, AvBD2 and AvB7 for the fragmentation of intact molecular species.
160
161 Bacterial strains. To test the antimicrobial activity, the following strains were used : Bacillus
162 subtilis, Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 29740, Staphylococcus
163 saprophyticus ATCC 19701, Staphylococcus haemolyticus DSM 20263, Listeria monocytogenes D
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164 strain EGD, Enterobacter cloacae ATCC 13047, Klebsiella pneumoniae ATCC 13882, n
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165 Pseudomonas aeruginosa ATCC 25010, Salmonella enterica serovar Enteritidis ATCC 13076, d
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166 Salmonella enterica serovar Enteritidis LA5 and Salmonella enterica serovar Typhimurium f
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167 ATCC 14028. Salmonella enterica serovar Enteritidis LA5 is a wild-type strain (nalidixic acid
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168 resistant at 20 µg mlÎ1) isolated from natural chicken infections (1). Enterobacter cloacae :/
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169 ATCC 13047, Klebsiella pneumoniae ATCC 13882 and Pseudomonas aeruginosa ATCC 25010 c
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170 were purchased from the Centre International de Ressources Microbiennes-Bactéries Pathogènes m
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171 (CIRM-BP, INRA, Nouzilly, France). Salmonella enterica serovar Enteritidis ATCC 13076 and g
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172 Salmonella enterica serovar Typhimurium ATCC 14028 were purchased from the Centre de
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173 Tguuqwtegu" Dkqnqikswgu" fg" nÓKpuvkvwv" Rcuvgwt" *ETDKR." Kpuvkvwv" Rcuvgwt." Rctku." Htcpeg+0" e
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174 Staphylococcus strains were kindly provided by Dr Pascal Rainard (INRA, UR1282, Nouzilly, e
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175 France). Bacillus strains, Listeria monocytogenes and Salmonella strain LA5 were kindly provided 9
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176 by Dr Philippe Velge (INRA, UR1282, Nouzilly, France). 1
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177 y

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178 Antimicrobial activity test. The antibacterial activities of peptides were measured by radial e
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179 diffusion assay according to the method described by Lehrer and colleagues (12, 31). Bacteria
180 incubated overnight were diluted in Trypticase Soy Broth (TSB) or Brain Heart Infusion (BHI) at an
181 absorbance of 0.02 and were incubated 2.5 to 4 hours according to the bacterial strain at 37°C to
182 obtain a mid-logarithmic-phase culture for which bacterial concentration was determined by plating
183 serial ten-fold dilutions of bacterial suspension on Trypticase Soy Agar (TSA) plates and by
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184 counting colony forming units (cfu) after 24 hours incubation at 37°C. A volume containing bacteria
185 at 1.107 cfu was centrifuged at 900 g for 5 min at 4°C and bacteria were washed once with cold 10
186 mM sodium phosphate buffer (pH 7.4), resuspended in a small volume of cold sodium phosphate
187 buffer and mixed to 25 ml of previously autoclaved, warm (42°C) 10 mM sodium phosphate buffer
188 containing 3.0 mg of TSB medium, 1% (w/v) low-electroendosmosis-type agarose (Sigma), and
189 0.02% Tween 20. The agarose solution containing bacteria was poured into a square Petri dish (120 D
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190 x 120 x 15 mm) to form a 1mm deep uniform layer. A 2.5-mm-diameter gel punch was used to n
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191 make 36 evenly spaced wells. In each well, 5 µL of peptide dilutions or control solutions were d
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192 added. MSI-94 (a broad antimicrobial spectrum linear amphipathic magainin variant) was used as a f
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193 positive control and was a kind gift from Dr Philippe Bulet (CNRS UMR 5525, Archamps, France).
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194 Peptide and control solutions were allowed to diffuse in the gel containing bacteria by incubating :
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195 the plates for 3 h at 37°C. The gel was then overlaid with 25 ml of agar consisting of a double- c
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196 strength (6% [w/v]) solution of TSB containing 1% (w/v) agarose. After overnight incubation at m
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197 37°C, the diameter of the clear zone surrounding each well was measured. For each bacterial strain, g
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198 three identical independent measurements of the antibacterial activity were performed. The
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199 minimum inhibitory concentration (MIC) of each peptide was determined according to Lehrer and e
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200 colleagues (31) from a graph constructed by plotting the Log10 peptide concentration against the e
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201 diameter of the clear zone on the plate minus the diameter of the well as a function of the log 9
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202 peptide concentration. The best-fit straight-line was determined using linear regression with 1
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203 GraphPad Prism 5 software (GraphPad Software). The MIC was calculated by finding the y

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204 intersection of the line with the x axis, indicating the lowest peptide concentration from which no e
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205 clear zone is obtained.
206
207 RESULTS
208 AvBD purification and identification from bone marrow extract. AvBD1, AvBD2 and AvBD7
209 were identified in the bone marrow extract. As a first purification step, a size exclusion
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210 chromatography was run on P10 gel (Fig.1). MALDI-TOF MS analysis of fractions revealed three
211 main peptides corresponding to average molecular masses ([M+H]+) of 4504, 5353 and 3916 Da in
212 peaks A, B and C, respectively (Fig.1). The gel filtration fractions containing the three main
213 peptides were subjected to trypsin digestion, and the proteolyzed peptides were analyzed by
214 nanoLC-MS/MS. AvBD1, AvBD2 and AvBD7 were identified (Table 1). The fractions containing
215 each AvBD, selected by MALDI-TOF MS measurements, were pooled. An additional step of RP- D
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216 HPLC was necessary to purify AvBD1 in fractions from peak A (Fig. 2A) and AvBD7 in fractions n
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217 from peak B (Fig. 2B). The AvBD2 RP-HPLC chromatogram showed that gel filtration pooled d
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218 elution fractions corresponding to AvBD2 peak were quite pure (Fig.2C). After the final step of f
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219 HPLC purification, the amount of recovered peptides was between one and two milligrams of
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220 defensin from ten chickens. :
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221 c
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222 Structural characterization of AvBDs using top-down analysis. To complete the characterization m
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223 of the primary structure sequence and post-translational modifications and to sequence the N- and g
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224 C-termini, a top-down mass spectrometry strategy was applied on intact proteins. An aliquot of
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225 HPLC-purified intact AvBDs was subjected to a top-down MS and MS/MS analysis using nanoESI- e
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226 Q-TOF without any prior reduction or alkylation steps. The m/z value of the precursor multiple e
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227 charged monoisotopic ions was measured with a mass accuracy of 100 ppm affording m/z 901.04 (z 9
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228 = 5) for AvBD1 ([M+H]+ = 4501.2 Da), m/z 979.23 (z = 4) for AvBD2 ([M+H]+ = 3913.9) and m/z 01
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229 1070.84 (z = 5) for AvBD7 ([M+H]+ = 5350.2). Bone marrow AvBD2 exhibited the expected by

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230 monoisotopic mass when compared to AvBD2 extracted from heterophils (8) with three disulfide e
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231 dqpfu" *(cid:507)ocuu" ?" -6 Da) (Table 2). Bone marrow AvBD1 presented a monoisotopic mass
232 corresponding to an amidated C-terminus with three disulfide bonds (Table 2). Fragment ions y
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233 and y in the MS/MS spectrum of the five-fold charged precursor ion m/z 901 (Fig.3) confirmed this
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234 post-translational modification. Moreover, the double loss of NH from the precursor with low
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235 collision energy confirmed the presence of an amidated C-terminal tryptophan (16). The
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236 monoisotopic mass of AvBD7 corresponded to the mass of the AvBD7 propeptide predicted by Xiao
237 et al (2004) with three disulfide bonds (43) and with the N-terminal Gln being cyclized as a
238 pyroglutamic acid residue (see Table 2 for comparison). The presence of this modified amino acid
239 at the N-terminus explained the failure of the AvBD7 sequence determination using Edman
240 degradation (data not shown). Fragmentation of the intact AvBD7 six-fold charged precursor ion
241 (m/z 892.53) corresponded to the N-terminal sequence of the propeptide AvBD7 predicted by Xiao D
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242 et al. with the pyroglutamic acid at the first position (Fig. 4). n
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243 d
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244 Characterization of mature AvBDs in heterophils. In order to strengthen the finding that the three f
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245 AvBD sequences determined above correspond to the mature peptide sequences exported out from
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246 the bone marrow, spectral profiling by MALDI-TOF MS was performed on intact heterophils :
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247 isolated from peritoneal exudates (Fig.5). This ICM-MS experiment demonstrated that the observed c
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248 average masses in whole heterophils correspond to the masses observed by nanoESI-MS for m
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249 AvBD1, AvBD2 and AvBD7 in bone marrow. g
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250
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251 Antimicrobial activity and Minimum Inhibitory Concentration (MIC). The antimicrobial e
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252 activity of each purified AvBD was evaluated against a large panel of bacterial strains in radial e
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253 diffusion assay. Clearing units (U) were calculated by the diameter in mm of the clear zone 9
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254 surrounding the well minus the diameter in mm of the well, and were plotted as a function of log 1
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255 (peptide concentration) for each bacterial strain, as illustrated on figure 6 for AvBD2 against E. coli. y

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256 The various MIC obtained in M are shown on Table 3. In comparison to control antimicrobial e
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257 peptide MSI-94, similar MIC were observed for AvBD1, AvBD2 and AvBD7 against Gram positive
258 bacterial strains. However, AvBD1 displayed a particularly low MIC of 80 nM against
259 Staphyloccocus aureus. Indicative of a high antibacterial effect, the MIC of AvBD1 and AvBD7
260 remained low against the Gram negative bacteria tested as well as against Gram positive ones. In
261 contrast the antimicrobial activity of AvBD2 was 2 to more than 100 times less effective against