Characterization of Acp, a peptidoglycan hydrolase of Clostridium perfringens with N ...

47 Pages·2010·1.88 MB·English

JB Accepts, published online ahead of print on 26 February 2010
J. Bacteriol. doi:10.1128/JB.01546-09
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
1 Characterization of Acp, a peptidoglycan hydrolase of Clostridium perfringens
2 with N-acetylglucosaminidase activity,
3 implicated in cell separation and stress-induced autolysis
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5 Emilie Camiade1, 2, Johann Peltier1, Ingrid Bourgeois1, Evelyne Couture-Tosi2, Pascal Courtin3, ow
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6 Ana Antunes2, Marie-Pierre Chapot-Chartier3, Bruno Dupuy2 and Jean-Louis Pons1*. lo
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8 1 Laboratoire G.R.A.M., EA 2656 IFR 23, Rouen University Hospital, University of Rouen, 22 m

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9 Boulevard Gambetta, 76183 Rouen Cedex, France; 2 Unité des Toxines et Pathogénie tt
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10 Bactérienne, Institut Pasteur, 25 Rue du Docteur Roux, 75015 Paris, France; 3 INRA UMR1319 /jb
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11 Micalis, Domaine de Vilvert, F-78352 Jouy-en-Josas, France. m
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13 Running Title: Acp, a N-acetylglucosaminidase of C. perfringens. n

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15 Key Words: C. perfringens, N-acetylglucosaminidase, cell separation, stress-induced autolysis b
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17 Corresponding Author: Jean-Louis Pons, Groupe de Recherche sur les Antimicrobiens et les 0
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18 Micro-organismes (UPRES EA 2656, IFR 23), Université de Rouen, 22 Boulevard Gambetta, F- b
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19 76183 Rouen Cedex, France. Tel: [phone] E-mail: [email protected] u
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21 The GenBank accession number for the acp sequence reported in this paper is GU192369.
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22 Abstract
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24 This work reports the characterization of the first known peptidoglycan hydrolase (Acp) mainly
25 produced during vegetative growth of C. perfringens. Acp has a modular structure with three
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26 domains: a signal peptide domain, a N-terminal domain with repeated sequences and a C- o
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27 terminal catalytic domain. The purified recombinant catalytic domain of Acp displayed a lytic lo
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28 activity on the cell walls of several Gram positive bacterial species. Its hydrolytic specificity was e
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29 established by analyzing the Bacillus subtilis peptidoglycan digestion products by coupling RP- ro
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30 HPLC and MALDI-TOF MS analysis, which displayed a N-acetylglucosaminidase activity. The h
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31 study of acp expression showed a constant expression during growth, which suggested an //
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32 important role of Acp in growth of C. perfringens. Furthermore, cell fractionation and indirect s
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33 immunofluorescence staining using anti-Acp antibodies revealed that Acp is located at the septal o
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34 peptidoglycan of vegetative cells during exponential growth phase, indicating a role in cell o
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35 separation or division of C. perfringens. A knockout acp mutant strain was obtained by using the e
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36 insertion of mobile Group II intron strategy (ClosTron). The microscopic examination indicated a m
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37 lack of vegetative cell separation in the acp mutant strain, as well as the wild type strain r
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38 incubated with anti-Acp antibodies, demonstrating the critical role of Acp in cell separation. The 2
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39 comparative responses of wild type and acp mutant strains to stresses induced by Triton X-100, 8

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40 bile salts and vancomycin revealed an implication of Acp in autolysis induced by these stresses. g
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41 Overall, Acp appears as a major cell wall N-acetylglucosaminidase implicated in both vegetative s
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42 growth and stress-induced autolysis.
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43 Introduction
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45 Autolysins are endogenous peptidoglycan hydrolases (PGHs) that can break covalent bonds in
46 the bacterial cell wall peptidoglycan (16, 58). Various PGHs are distinguished on the basis of
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47 their specific cleavage site in the peptidoglycan: N-acetylmuramidases, N- o
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48 acetylglucosaminidases, N-acetylmuramoyl-L-alanine amidases and endopeptidases. PGHs are lo
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49 involved in different physiological functions that require cell wall remodelling such as cell-wall e
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50 expansion, peptidoglycan turnover, daughter cell separation or sporulation (53, 54, 60). These ro
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51 enzymes may also be implicated in antibiotic-induced lysis (39), and may contribute to bacterial h
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52 pathogenesis by generating inflammatory cell-wall degradation products (32, 40), by releasing //
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53 virulence factors (4) or by mediating bacterial adherence (1, 20, 21). The roles of PGHs in s
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54 bacterial physiology, and probably in bacterial pathogenicity, further reinforce the importance of o
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55 understanding bacterial autolysis. o
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56 Autolytic systems of several Gram-positive low G+C bacteria have been studied (5, 13, 34, 43, e
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57 54, 55). Belonging to this phylum, Clostridium perfringens is a common agent of food poisoning, m
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58 is implicated in infectious diseases initiating from the digestive tract (peritonitis, bacteraemia…), r
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59 and is the most common cause of clostridial gas gangrene in humans. Two PGHs have been 2
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60 described as implicated in the sporulation and germination of C. perfringens, an amidase (SleC) 8

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61 (37, 51) and a muramidase (SleM) (9), which are both produced at the early stage of sporulation, g
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62 located outside the cortex in the dormant spore (9, 37, 38, 51), and involved in peptidoglycan s
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63 cortex hydrolysis during germination (45). However, PGHs implicated in the vegetative growth
64 of C. perfringens have never been characterized. In addition, the implication of PGHs in
65 antibiotic-induced lysis of C. perfringens has never been studied.
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66 In the present study, we identified and characterized Acp, the first known autolysin of C.
67 perfringens produced by vegetative cells and displaying N-acetylglucosaminidase activity.
68 Furthermore, we localized Acp at the cell septum during vegetative cell growth, and constructed
69 a knockout mutant of the acp gene to demonstrate that Acp is involved in daughter cell separation
70 during vegetative growth. Finally, we studied the implication of Acp in autolysis induced by D
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71 stresses such as bile salts and cell wall targeting antibiotics. n
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72 Materials and methods
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74 Bacterial strains and culture conditions. C. perfringens strain 13 (52) was used in all
75 experiments of cloning, Acp characterization and construction of the acp mutant and was
76 cultivated in Brain Heart Infusion (BHI) broth under anaerobic conditions at 37°C. D
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77 Escherichia coli strain BL21 harbouring DE3-RIL (Promega), which constitutively n
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78 expresses the Lac repressor protein encoded by the lacI gene, was used as a recipient for d
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79 expression of the catalytic domain of Acp. E. coli TOP10 (chemo-competent cells, Invitrogen) fr
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80 was used to construct the pMTL007 derivated plasmid containing the retargeted intron of the acp
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81 gene. E. coli strains were respectively cultivated in 2×YT broth (Difco) and LB broth (Difco). :/
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82 When required, chloramphenicol (25 µg/ml), kanamycin (25 µg/ml) (Sigma), and IPTG (1 mM) a
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83 (Sigma) were added. .o
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84 Bacillus subtilis 168 HR (14) was used as a substrate to establish Acp hydrolytic activity o
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85 and was cultivated in LB broth (Difco) at 37°C with shaking.
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86 Spore counting. Spore counting from cultures of C. perfringens was performed as follows: m
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87 culture samples were incubated in ethanol 95° (vol/vol) for 30 min in order to kill vegetative r

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88 cells, then aliquots of various dilutions were plated onto blood agar plates and the plates were ,
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89 incubated at 37°C anaerobically for 24 h. 8

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90 General DNA techniques. Chromosomal DNA from C. perfringens culture was extracted by
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91 phenol-chloroform. DNA fragments used in the cloning procedures and PCR products were s
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92 isolated from agarose gels with the Geneclean II kit (Promega), according to the manufacturer’s
93 instructions. Plasmid DNA from E. coli was isolated and purified with the QIAprep Spin
94 Miniprep Kit (Qiagen). PCRs were performed on a PTC-100 Programmable Thermal
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95 Controller (MJ Research, inc.) in a final volume of 50 µl containing 0.5 µM each primer, 200 µM
96 each deoxynucleoside triphosphate and 1 U LA Taq DNA polymerase (Takara) in a 1X cloned
97 LA taq DNA polymerase reaction buffer [20 mM Tris/HCl, pH 8.8, 10 µM KCl, 2 µM MgSO ,
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98 10 µM (NH ) SO ]. The PCR mixtures were denatured (2 min at 94°C), and the amplification
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99 procedure followed, consisting of 30 s at 94°C, annealing for 30 sec at 55°C and ending with an D
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100 extension step at 72°C for 1 min, for a total of 35 cycles. DNA sequences were determined with a n
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101 3100 genetic Analyser (Applied biosystem) sequencer using an ABI-PRISM Big Dye Terminator d
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102 Sequencing kit (Perkin Elmer). fr
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103 Cloning, expression and purification of Acp-His-tagged fusion protein in E. coli. Acp-His-
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104 tagged protein was expressed in E. coli BL21 codon plus (DE3)-RIL as an Acp-His-tagged fusion :
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105 protein using the expression vector pET28b (Stratagen). Primers (MWG-Biotech, Invitrogen) 790 a
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106 F and 790 R (see supplemental material, Table 1) were used to amplify DNA fragment encoding .o
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107 the catalytic domain of Acp (780bp) from C. perfringens strain 13 total DNA. After
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108 amplification, PCR products were digested with BamHI and EcoRI and cloned in the pET28 D
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109 vector, digested by the same restriction enzymes. This construction created a translational fusion m
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110 adding 10 N-terminal histidine codons to acp coding sequence and placed it under the control of r

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111 the T7 promoter. ,

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112 E. coli BL21 codon plus (DE3)-RIL electro-competent cells were transformed with the resultant
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113 plasmid (pCD470) by electroporation (200 Ω; 2.5 kV; 25 µF). Nucleotide sequencing of plasmids
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114 from recombinant clones confirmed the insertion of 780 bp fragment encoding the catalytic s
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115 domain of Acp. E. coli recombinant strain was grown at 22°C overnight in 2×YT medium
116 containing selective agents. Protein expression was achieved by induction of cells with 1 mM
117 IPTG followed by subsequent incubation during 5 hours at 22°C to avoid formation of inclusion
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118 bodies. Acp-His tag protein was purified by affinity chromatography on Ni-NTA columns
119 (Qiagen) under native conditions. Purity of the His-tagged protein was checked by SDS-PAGE
120 and then dialysed against sodium phosphate buffer (1X, pH 8.0).
121 Detection of cell wall lytic enzymes in SDS-PAGE renaturing gel. Proteins were extracted
122 from bacteria with an SDS treatment as described by Leclerc and Asselin (31). Briefly, the D
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123 bacterial pellet of 100 ml of C. perfringens strain 13 cell culture was resuspended in 25 ml of 4% n
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124 (wt/vol) SDS solution. The suspension was shaken for 120 min and sonicated twice on ice for 1 d
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125 min. The extract was heated at 90°C for 15 min, centrifuged at 9,500 × g for 20 min, and the fr
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126 supernatant was stored at -20°C. Lytic activity was detected by using SDS-polyacrylamide gels
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127 (31) containing 0.2% (wt/vol) Micrococcus lysodeikticus ATCC 4698 (Sigma), B. subtilis 168 :
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128 HR (14), C. difficile 630 and C. perfringens strain 13 lyophilized or autoclaved cells (121°C, 20 a
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129 min). SDS-PAGE was performed as described by Laemmli (30) with 15% polyacrylamide. After .o
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130 electrophoresis, gel was gently shaken at 37°C for 16 h in 50 ml of 25 mM Tris-HCl (pH 8.0)
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131 solution containing 1% (vol/vol) Triton X-100 to allow protein renaturation. Clear bands
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132 resulting from lytic activity were visualized after staining with 1% (wt/vol) methylene blue m
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133 (Sigma) in 0.01% (wt/vol) KOH and subsequent destaining with distilled water. r

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134 Determination of the hydrolytic bond specificity of Acp on peptidoglycan. Peptidoglycan from ,

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135 B. subtilis 168 HR vegetative cells was prepared with the protocol described previously for
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136 Lactococcus lactis (36) with some modifications. Briefly, pelleted cells were resuspended in 10% y
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137 (w/v) SDS and boiled for 25 min. Insoluble material was recovered by centrifugation (20,000 × g, s
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138 10 min, 20°C) and boiled again in 4% (w/v) SDS for 15 min after resuspension. The resulting
139 insoluble wall preparation was then washed with hot distilled water (60°C) six times to remove
140 SDS. The covalently attached proteins were removed by treatment with pronase (2 mg/ml) for 90
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141 min at 60°C, then by trypsin (200 mg/ml) for 16 h at 37°C. The walls were then recovered by
142 centrifugation (20,000 × g, 10 min, 20°C), washed once in distilled water and resuspended in
143 hydrofluoric acid (HF) (48%, v/v, solution); the mixture was incubated at 4°C for 24 h. The
144 insoluble material was collected by centrifugation (20,000 × g, 10 min, 20°C) and washed
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145 repeatedly by centrifugation and resuspension twice with Tris/HCl buffer (250 mM, pH 8.0) and
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146 four times with distilled water until the pH reached 5.0. The material was lyophilized and then lo
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147 stored at -20 °C. Peptidoglycan extract (2 mg) was incubated overnight at 37°C with purified Acp- e
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148 His recombinant protein (160 µg) in a final volume of 250 µl of sodium phosphate buffer (100 ro
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149 mM, pH 8.0). Samples were boiled for 3 min to stop the reaction, and the insoluble material was h
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150 removed by centrifugation at 14,000 × g for 15 min. Half of the soluble muropeptide fraction was //
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151 further digested with mutanolysin (2,500 U/ml) (Sigma). The soluble muropeptides obtained after s
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152 digestion were reduced with sodium borohydride and the reduced muropeptides were then o
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153 separated by RP-HPLC with an LC Module I system (Waters) and a Hypersyl ODS C18 column o
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154 (250 × 4.6 mm, particle size 5 µm) (ThermoHypersil-Keystone) at 50°C using ammonium e
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155 phosphate buffer and methanol linear gradient (11). Muropeptides were analyzed without desalting m
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156 by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF- r
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157 MS) using a Voyager-DE STR mass spectrometer (Applied Biosystems) as reported previously 2
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158 (11). 8

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159 Preparation of anti-Acp polyclonal antibodies. Polyclonal antibodies were obtained by Balb/C g
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160 mice immunization (AgroBio, agreement number: B 41-245-4) consisting of 3 injections with 75 s
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161 µg of the Acp purified catalytic domain.
162 Western blot analysis. Proteins separated by SDS-PAGE were electroblotted onto Hybond-
163 ECLTM nitrocellulose membranes (4°C, 1 hour, 100 V) (Amersham Biosciences). Filters were
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164 probed first with autolysin mouse antiserum (or control serum) used at 1/5000 dilution, and then
165 with goat anti-mouse immunoglobulin G conjugated to horseradish peroxidase (GE Healthcare)
166 diluted at 1/5000. Immunodetection of protein was performed with the SuperSignal® West
167 Femto Kit (Thermo Scientific) according to the manufacturer’s recommendations.
168 Cell microscopy analysis. For electron microscopy analysis, bacterial colonies were suspended D
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169 in 0.1 M of sodium Cacodylate buffer. The cells were fixed in 2.5% glutaraldehyde / 0.1 M n
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170 sodium Cacodylate buffer overnight at 4°C. The resulting pellets were washed twice with 0.1 M d
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171 sodium Cacodylate buffer and the cells were let to adhere on poly-lysine pre-coated coverslips. fr
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172 The specimen were post-fixed in 1% Osmium teroxyde / 0.1 M sodium CaCodylate buffer for
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173 one hour at room temperature, dehydrated in graded series of ethanol and followed by a critical :
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174 point drying with CO in a CPD BALTEC apparatus. The dried specimen were mounted on stubs a
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175 with carbon tape and ions sputtered with 15 nm of platin / carbone using a high-resolution ion .o
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176 beam coater, Gatan Modele 681. Analysis of Secondary Electron Images (SEI) was performed
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177 with a JEOL JSM-6700F scanning microscope with a field emission gun operating at 5 kV.
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178 For immunofluorescence assays, C. perfringens strain 13 and C. perfringens strain 13 m
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179 acp::erm were grown upon end exponential phase (3 h, 37°C, anaerobic atmosphere) in BHI r

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180 broth. Samples were fixed aerobically for 1 hour at 4°C in 2% paraformaldehyde (PFA). The ,

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181 fixative was removed, the pellets were resuspended in 400 µL of PFA and 30 µl of the samples 1
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182 were adsorbed on a poly-lysine pre-coated slide during 30 min at room temperature. Free
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183 aldehyde groups were blocked with 30 µl of NH Cl (50 mM) during 15 min at room temperature
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184 and washed twice with 0.5% gelatine / PBS. Pre-immune and immunoserum were depleted
185 during 1 hour at 37°C with mid exponential acp mutant culture (1:5 dilution). The samples were
186 then incubated with depleted anti-Acp mouse polyclonal antibodies (final dilution of 1:10 in BHI)
187 for 30 min at room temperature, washed twice with BHI, and incubated with donkey anti-mouse
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188 IgG (1:200 dilution in BHI) conjugated to Alexa Fluor 488 (Molecular Probes) for 30 min at
189 room temperature. After two washes with BHI to remove unbound antibodies, nuclear staining
190 was performed with To-Pro-3 (1:500) 10 min, rinced twice in milliQ water, and finally a drop of
191 Vectashield mounting medium was added to cover the sample. Samples were visualized on an
192 Inverted microscope Zeiss Axiovert 200M, piloted by Zeiss Axiovision 4.4 software (Carl Zeiss, D
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193 Inc.), operating a black and white CoolSNAP HQ charge-coupled device camera (Photometrics). n
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194 Cell fractionation. Cell fractions were prepared as described by Candela and Fouet (8) with d
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195 some modifications. Mid-exponential (2 h) and late stationary (24 h) phase cultures of C. fr
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196 perfringens strain 13 were centrifuged and the resulting pellet was suspended in 50 mM Tris-HCl
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197 (pH 7.4), and then sonicated (3 × 20 sec) to disrupt cells. Cell envelope components were :/
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198 separated by centrifugation (8,000 × g, 20 min, 4°C), the pellet was resuspended in 50 mM Tris- a
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199 HCl (pH 7.4) containing 5 mM EDTA and 1% Triton X-100, incubated during 1 hour at 4°C and .o
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200 centrifuged again (20,000 × g) for 1 hour at 4°C in order to separate the membrane (supernatant) o
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201 and the cell-wall (pellet) components.
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202 RNA isolation and quantitative reverse transcription real time PCR. 20 ml of RNA m
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203 protection solution (acetone-ethanol 1:1) were immediately added to 20 ml-samples of C. r

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204 perfringens strain 13 taken at various times points of cell growth, and stored at -80°C before its ,
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205 use. After centrifugation, the pellet was washed with Tris-EDTA (10–1 mM, pH 8.0) buffer and 8

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206 lysed mechanically with glass beads. The samples were further purified with RNeasy Mini kit
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207 (Qiagen) in succeeding steps with spin columns, and samples were then treated first with DNAseI s
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208 (Sigma) and after with TURBO DNA-free kit (Ambion) according to the manufacturer’s
209 recommendations. cDNA was synthesized from two micrograms of total RNA using the
210 Omniscript enzyme (Qiagen) and random fifteen’s mer primers (MWG). 6 ng of cDNA were
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