Characterization of Antibacterial Action of Polyphosphate on Porphyromonas 1 gingivalis 2 3 Ji-Hoi

39 Pages·2010·0.67 MB·English

AAC Accepts, published online ahead of print on 22 November 2010
Antimicrob. Agents Chemother. doi:10.1128/AAC.01014-10
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

1 Characterization of Antibacterial Action of Polyphosphate on Porphyromonas
2 gingivalis
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4 Ji-Hoi Moon1,3, Jae-Hong Park2,3, Jin-Yong Lee1,3,* o
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6 Department of Maxillofacial Biomedical Engineering1 and Pediatric Dentistry2, School f
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7 of Dentistry, and Institute of Oral Biology3, Kyung Hee University, Seoul, Republic of h
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8 Korea a
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10 * Corresponding author. Mailing address: Department of Maxillofacial g/

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11 Biomedical Engineering, School of Dentistry, Kyung Hee University, 1 Hoegi-dong, A
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12 Dongdaemun-gu, Seoul 130-701, Republic of Korea. Phone: [phone]. Fax: ,
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13 [phone]. E-mail: [email protected].
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15 Running title: Antibacterial action of polyP on P. gingivalis
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18 Polyphosphate (polyP) has antibacterial activity against various gram-positive
19 bacteria. In contrast, gram-negative bacteria are generally resistant to polyP. Here we
20 describe antibacterial characterization of polyP against a gram-negative
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21 periodontopathogen Porphyromonas gingivalis. The minimum inhibitory o
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22 concentrations (MICs) of pyrophosphate (Na4P2O7) and all polyP (Nan+2PnO3n+1; n = 3- a
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23 75) tested for the bacterium by agar dilution method were 0.24% and 0.06%, f
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24 respectively. Orthophosphate (Na HPO ) failed to inhibit the bacterial growth. polyP75 h
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25 was chosen for further study. In liquid medium, 0.03% polyP75 was bactericidal a
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26 against P. gingivalis irrespective of growth phase and inoculum size ranging from 105 sm
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27 to109 cells/ml. UV-visible spectra of the pigments from P. gingivalis grown on blood g
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28 agar with or without polyP75 showed that polyP75 reduced formation of µ-oxo
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29 bisheme by the bacterium. polyP75 increased hemin accumulation on P. gingivalis ,
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30 surface and decreased energy-driven uptake of hemin by the bacterium. The expression
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31 of the genes encoding hemagglutinins, gingipains, hemin uptake loci, chromosome u
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32 replication, and energy production was down-regulated while that of the genes related
33 to iron storage and oxidative stress was up-regulated by polyP75. The transmission
34 electron microscope showed morphologically atypical cells of electron-dense granules
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35 and condensed nucleoid in the cytoplasm. Collectively, polyP is bactericidal against P.
36 gingivalis in which hemin/heme-utilization is disturbed and oxidative stress is
37 increased by polyP.
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40 Key words: f
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41 Porphyromonas gingivalis h
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42 polyphosphate a
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43 bactericidal m
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44 hemin
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45 INTRODUCTION
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47 Inorganic polyphosphate (polyP) is a ubiquitous compound found in bacteria, fungi,
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48 algae, plant and animals. polyP found in the organisms is a chain of few or many o
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49 hundreds of phosphate (Pi) residues linked by high-energy phosphoanhydride. It a
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50 performs varied functions in bacteria such as follows: can serve as an ATP source and f
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51 substitute; is a strong chelator of metal ions and thus can regulate levels of the ions in h
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52 the cells; a channel for DNA entry; and a regulator which contributes to bacterial a
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53 resistance and survival against stress and stringent condition (18). Therefore, m
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54 intracellular polyP is considered as a virulence factor of microorganisms.
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55 In contrast, exogenous polyP has attracted considerable attention as an
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56 antimicrobial agent since it can prevent spoilage of food (29, 32) and is listed as GRAS ,
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57 (generally recognized as safe) food additive by FDA. polyP inhibits the growth of
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58 various gram-positive bacteria such as Staphylococcus aureus (14, 17, 22, 35, 52), u
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59 Listeria monocytogenes (37, 52), Sarcina lutea (35), Bacillus cereus, Lactobacillus and
60 fungi such as Aspergillus flavus (17, 28). Concerning oral bacteria, mutans streptococci
61 were firstly found to be inhibited by condensed phosphate, resulting in decrease of
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62 plaque formation and dental caries (5, 39). The ability of polyP to chelate divalent
63 cations is regarded as being relevant to antibacterial effects of polyP, contributing to
64 cell division inhibition and loss of cell wall integrity (17, 24, 28, 35). Therefore,
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65 relatively little attention has been directed towards the effect of polyP on gram- o
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66 negative bacteria in which divalent cation is considered less important for the a
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67 membrane stability. In fact, gram-negative bacteria are generally more resistant than f
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68 gram-positive organisms to polyP; large numbers of gram-negative bacteria including h
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69 Escherichia coli and Salmonella typhimurium are capable of growing in higher a
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70 concentrations, even up to 10% of polyP (17, 34, 35). m
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71 polyP does not generally exert any adverse effect on the body, as used locally and
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72 orally, within the range of MICs determined for various bacteria (20). Besides, polyP
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73 can stimulate bone formation (11). Thus, polyP seems to be a promising substance for ,
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74 treatment of periodontal diseases, promoting bone regeneration. Before we can come
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75 to clinical application of polyP as a controlling agent for periodontal diseases, the u
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76 effect of polyP on periodontopathogens must be defined.
77 Porphyromonas gingivalis is a gram-negative, black-pigmented anaerobe associated
78 with several periodontal diseases (9). Iron is a nutrient that is indispensible for the
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79 growth of almost all living organisms including P. gingivalis and plays a crucial role in
80 the establishment and progression of an infection (40). P. gingivalis lacks members of
81 the protoporphyrin IX synthetic pathway but yet requires hemin (Fe3+-protoporphyrin
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82 IX, also known as heme, Fe2+-protoporphyrin IX, depending upon the oxidation state of o
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83 the iron atom in the center of the molecule) as a cofactor for fumarate reductase and a
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84 cytochromes, so the bacterium must acquire this nutrient from the environment (1, 6). P. f
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85 gingivalis derives hemin via hemagglutination, hemolysis, and proteolysis of the h
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86 hemoglobin (4, 42) and stores hemin on the cell surface in µ-oxo dimeric form (µ-oxo a
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87 bisheme, [Fe(III)PPIX2]O).This surface-bound µ-oxo bisheme serves not only as a m
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88 scavenger of hemin that in high concentrations (10-20 µg/ml) has been shown to have
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89 antibacterial effects on P. gingivalis, but also binds free oxygen and thereby reduces the
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90 hemin-mediated oxygen radical cell damage as well as protects from reactive oxidants ,
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91 generated by neutrophils (25, 44). P. gingivalis encodes a family of hemagglutinins
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92 (HagA, HagB, HagC) and gingipains (Arg-specific gingipain A and B [RgpA, RgpB], u
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93 Lys-specific gingipain [Kgp]) which play a crucial role in the hemin-acquisition process
94 (7, 15, 19). The surface-accumulated hemin is transported into a bacterial cell to be
95 utilized and serves as a source of iron under iron-depleted conditions (38). Since hemin
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96 is too large to diffuse freely through the bacterial membranes, P. gingivalis requires
97 transport of hemin across two membranes by a process that requires energy (41). Three
98 multigenic clusters encoding proteins thought to be involved in the hemin uptake have
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99 been detected in the genome of P. gingivalis W83 (26): IhtABCDE (iron-heme o
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100 transport), Tlr-htrABCD (hemin uptake), and HmuYRSTUV (hemin uptake). a
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101 In a preliminary study, we observed that the pellet of P. gingivalis W83 cells grown f
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102 in brucella broth supplemented with hemin appeared darker in the presence of polyP h
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103 likely due to P. gingivalis cell surface that bound more hemin (unpublished data). Hence a
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104 we hypothesized that polyP may affect the hemin utilization of P. gingivalis. Here, we m
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105 present antimicrobial activity of polyP against P. gingivalis, disturbing hemin/heme-
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106 utilization.
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107 ,
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108 MATERIALS AND METHODS
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110 Chemicals. Orthophosphate (Pi; Na HPO ), pyrophosphate (PPi; Na P O ) and
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111 polyphosphate (polyP, Na P O ; n = the number of phosphorus atoms in the chain)
n+2 n 3n+1
112 with different linear phosphorus (Pi) chain lengths (3-75) were purchased from Sigma
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113 Chemical Co.(St Louis, MO). Calgon (Sigma), a cyclic polyphosphate (sodium
114 hexametaphosphate, Na P O ; n= 12-13), was also used. These phosphates were
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115 dissolved in water to 10% (w/v), sterilized using 0.22-µm filter and stored at -20oC until
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116 use. Stock solutions of hemin (Sigma) were prepared in 0.02 N NaOH the same day that o
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117 it was used. Carbonyl cyanide m-chlorophenylhydrazone (CCCP, Sigma) was dissolved a
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118 in 20% dimethyl sulfoxide (DMSO) and used as an inhibitor of energy-driven transport f
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119 activities (3). Deferoxamine mesylate (DFO, Novartis Pharma Stein AG, Stein, h
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120 Switzerland), an iron chelator, was dissolved in water to 20% (w/v). a
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121 m
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122 Antimicrobial assays. The minimum inhibitory concentration (MIC) of polyP for P. /

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123 gingivalis W83 (kindly supplied by Dr. Koji Nakayama, Nagasaki University Graduate
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124 School of Biomedical Sciences) was obtained by agar dilution method according to ,
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125 CLSI guidelines (8). Briefly, the 2-fold serial dilutions of Pi, PPi, and polyP with
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126 various chain lengths (3-75) were prepared and added to brucella agar (Difco u
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127 Laboratories, Detroit, MI) containing 5% laked sheep blood, 5 µg/ml of hemin, and 1
128 µg/ml of vitamin K . Final concentrations of Pi, PPi, and polyP ranged from 0.015 to
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129 0.96% (w/v). The agar plates were inoculated with an inoculum of 105 CFU/spot and
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130 incubated at 37°C for 48 h anaerobically (85% N , 10% H , and 5% CO ). The MIC was
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131 defined as the lowest concentration that inhibited bacterial growth on the plate.
132 Time-kill experiments were performed in brucella broth containing hemin and
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133 vitamin K . polyP with a chain length of 75 (polyP75) was tested at concentrations of o
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134 1/4-4 MIC. Aliquots were removed from the cultures at 0, 4, 8, 12, and 24 h, and viable a
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135 cells were enumerated by plating on blood agar. In order to assess the effects of f
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136 inoculum density and growth phase on killing effect of polyP75, cells of P. gingivalis h
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137 W83 grown to exponential or stationary phase were treated with 1/4-4 MIC polyP75 a
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138 using inocula of 105-109 CFU/ml. Viable cells were enumerated following 24 h- sm
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139 incubation.
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140
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141 UV-visible spectroscopy of heme pigments. Heme pigments from P. gingivalis ,
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142 were obtained by a modification of the procedure described previously (47). Briefly, 5%
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143 sheep blood agar plates containing polyP at sub-MIC concentrations were prepared. u
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144 Lawn growths were cultured by heavy inoculation over the whole surface of the plates
145 and these were incubated at 37°C anaerobically. After 4 days, the cells were gently
146 scraped from the plates, suspended in 500 µl of NaCl/Tris buffer (0.14 M NaCl, 0.1 M
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147 Tris-HCl [pH 7.5]) and sonicated using a Vibra Cell VC600 (Sonics Material, Newtown,
148 CT) tip sonicator for 2 min to wash the pigment from the cells. The suspensions were
149 centrifuged at 11,000 × g for 10 min at 20oC and 200 µl of the supernatant was used for
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150 UV-visible spectroscopy. UV-visible spectra of the extracted heme pigments were o
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151 recorded between 340 and 700 nm using UV transparent microplates (UV-Star®, Greiner a
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152 Bio-One, Monroe, NC) on Benchmark Plus Microplate Spectrophotometer (Bio-Rad f
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153 Laboratories, Hercules, CA). h
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154 a
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155 Accumulation and active uptake of hemin. The amount of hemin associated with m
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156 P. gingivalis cell was measured by a modification of the procedure described
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157 previously (10). Briefly, P. gingivalis cells were adjusted to OD =0.1 and one aliquot
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158 of the cells was left untreated while the other aliquot was treated with 100 µM CCCP. ,
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159 After 90-min incubation, each culture was centrifuged, washed twice, resuspended, and
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160 adjusted to OD =0.2 in a new broth with or without polyP75 and DFO at the u
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161 indicated concentration. Hemin was then added to give a final concentration of 5 µM.
162 Following incubation at 37°C for 2 h anaerobically, 1.0-ml aliquot from each culture
163 was removed and centrifuged, and the supernatant was assayed spectrophotometrically
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