1 Department of Veterinary Pathobiology, Royal Veterinary and Agricultural University (KVL), Stigbøjlen 4, DK-1870 Frederiksberg C, Denmark
2 Department of Microbiology and National Food Biotechnology Centre, University College Cork, Cork, Ireland
3 Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
Correspondence
Hanne Ingmer
hi{at}kvl.dk
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ABSTRACT |
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INTRODUCTION |
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In order to avoid the production of OH, bacteria store iron in a manner that prevents the participation of iron in these reactions. Two major families of proteins are involved in sequestering iron: the haem-containing bacterioferritins and the non-haem ferritins (Andrews, 1998). In addition, members of the Dps protein family resemble the ferritins as they form spherical protein complexes with the ability to bind iron (Bozzi et al., 1997
; Grant et al., 1998
; Ilari et al., 2000
; Tonello et al., 1999
; Yamamoto et al., 2002
; Zhao et al., 2002
). While the ferritins consist of 24 subunits binding more than 4000 iron atoms (Andrews, 1998
), the Dps homologues form dodecamers that can bind approximately 500 iron molecules (Bozzi et al., 1997
; Ishikawa et al., 2003
; Tonello et al., 1999
; Yamamoto et al., 2002
; Zhao et al., 2002
). Dps was originally identified in Escherichia coli as one of the major proteins induced in late stationary growth phase (Altuvia et al., 1994
). During exponential growth, it is induced by carbon and nitrogen starvation, osmotic stress or the presence of H2O2. The key regulatory elements include RpoS, IHF and OxyR (Altuvia et al., 1994
; Lomovskaya et al., 1994
; Michán et al., 1999
). Mutants lacking dps are sensitive to H2O2 in stationary growth phase, and have an increased rate of some base substitutions caused by oxidative agents (Martinez & Kolter, 1997
). In vitro and in situ studies have revealed that Dps binds DNA in a non-sequence-specific manner, and it has been proposed that Dps has a dual role in protecting cells against oxidative stress, either by binding directly to DNA (Altuvia et al., 1994
; Martinez & Kolter, 1997
; Wolf et al, 1999
), or by sequestering iron, thus avoiding the oxidative damage mediated by Fenton chemistry (Zhao et al., 2002
).
Dps homologues are found in several Gram-negative and Gram-positive bacteria. While most of these are able to bind iron, some members appear unable to bind DNA, including the Helicobacter pylori NapA, the Listeria innocua ferritin-like protein, the Streptococcus mutans Dpr, and Dlp-1/Dlp-2 from Bacillus anthracis (Bozzi et al., 1997; Papinutto et al., 2002
; Tonello et al., 1999
; Yamamoto et al., 2002
). In addition to the E. coli Dps, DNA binding has been demonstrated for the Bacillus subtilis MrgA, the Synechococcus DpsA and the Mycobacterium smegmatis Dps (Chen & Helmann, 1995
; Gupta & Chatterji, 2003
; Gupta et al., 2002
; Peña & Bullerjahn, 1995
). In a recent report, it was suggested that DNA binding requires either an extended C-terminus, or positively charged residues in the N-terminus of Dps homologues, which allow a disordered and flexible structure (Ceci et al., 2003
). L. monocytogenes also contains a homologue of Dps named Flp (Hébraud & Guzzo, 2000
) or Fri (Glaser et al., 2001
; Polidoro et al., 2002
). We have chosen to use the latter designation to avoid confusion with the Flp recombinase system (Sadowski, 1986
). Previous studies have revealed that fri expression is induced by a number of stress conditions, including heat and cold shock, and to a lesser extent SDS, ethanol and deoxycholate (Hébraud & Guzzo, 2000
; Liu et al., 2002
; Phan-Thanh & Gormon, 1995
). Northern blot analysis showed that induction by heat and cold occurred at the transcriptional level, and that the Fri protein is expressed from a monocistronic mRNA (Hébraud & Guzzo, 2000
). Furthermore, the Fri amino acid sequence is 98 % identical to the L. innocua Dps homologue that binds iron (Bozzi et al., 1997
), and is induced by iron-limiting conditions (Polidoro et al., 2002
). Although the mechanisms by which L. monocytogenes acquires iron are obscure (Brown & Holden, 2002
; Cowart & Foster, 1985
), several studies have shown that iron availability plays an important role in virulence (Bockmann et al., 1996
; Conte et al., 1996
, 2000
; Coulanges et al., 1996
; Sword, 1966
). To determine if Fri contributes to virulence, we have deleted the corresponding gene in L. monocytogenes EGD, and analysed the resulting mutant.
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METHODS |
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Stress tolerance assays.
For investigation of the ability to form c.f.u. when subjected to iron deprivation, L. monocytogenes strains were grown in BHI until stationary phase, and transferred to BHI agar treated with 50 mg ml1 Chelex 100 (Sigma-Aldrich). The ability to grow in iron-deprived broth was investigated by growing the strains to exponential phase in BHI, and shifting the cells to improved minimal medium containing 0 or 335 mM ferric citrate.
Survival during oxidative stress was examined by growing cultures to either stationary or exponential phase, washing once in saline (5000 g, 5 min), and resuspending in saline containing H2O2 at concentrations of 50 mM (stationary phase) or 20 mM (exponential phase). The cells were incubated at 37 °C, and the samples were collected at different time points, serially diluted, and plated onto BHI agar. The numbers of c.f.u. were counted after 24 h incubation at 37 °C.
Survival during long-term growth was examined by daily withdrawing samples for determination of c.f.u. from cultures grown to stationary growth phase in BHI, and then left with shaking at 37 °C for a period of 11 days.
DNA manipulations.
Extraction of plasmid DNA from E. coli was performed using the QIAprep Spin Miniprep Kit (Qiagen), as recommended by the manufacturer. E. coli was transformed as described by Cohen et al. (1972), and electrotransformation of L. monocytogenes was achieved as described elsewhere (Dramsi et al., 1995
). PCR reagents (Taq polymerase and deoxynucleoside triphosphates) were purchased from Roche, and used according to the manufacturer's instructions.
Primer extension analysis.
Total RNA was extracted using the Nucleospin RNA extraction kit (Macherey-Nagel) according to the manufacturer's directions. The bacterial cells were lysed by incubation with 10 mg lysozyme ml1 and 200 µl glass beads (106 µm and finer) (Sigma-Aldrich). Three times during the 10 min incubation period, the cell suspension was mixed by vortexing for 30 s. Primer extension analysis was performed as described by Michán et al. (1999) using 3 mg total RNA per reaction. A 5' 32P-labelled primer, dps-B (see below), was used for detection of fri transcription start sites.
Construction of the fri deletion mutant.
A L. monocytogenes mutant with an internal 381 bp deletion in the fri gene was constructed by the splicing by overlap extension PCR procedure (Horton et al., 1990). Primers were designed to amplify two fragments (
600 and
300 bp, respectively), one comprising the 5' end of fri, which was amplified by primers dps-a (5'-TGC TCT AGA CCC AAA ACG ACC AAT AAA GTC ATT GG-3') and dps-b (5'-CGC TAC TTG GTG ATT C-3'), and the other comprising the 3' end of the gene, which was amplified by primers dps-c (5'-GAA TCA CCA AGT AGC GGA CAA ACA TAT CTG GAT G-3') and dps-d (5'-TAT CCC AAG CTT CAT CAT GAA ACC ATT TAT TTG CTT GGT CCG-3').
The resulting products were gel extracted, mixed in a 1 : 1 ratio, and reamplified using the dps-a and dps-d primers. The amplified 948 bp product was digested with XbaI and HindIII, and inserted into the temperature-sensitive shuttle vector pAUL-A (Schaferkordt & Chakraborty, 1995). The resultant plasmid was electroporated into L. monocytogenes, and forced chromosomal integration of the plasmid was achieved by growing transformed strains at 42 °C in the presence of erythromycin. In order to allow allelic exchange between the intact gene and the truncated gene to take place, as well as subsequent loss of the plasmid, cells containing the integrated plasmid were grown in BHI at 30 °C in the absence of erythromycin. Finally, the presence of the truncated gene at the correct locus was confirmed by PCR.
Macrophage assay.
The murine-macrophage-like cell line J774.A1 was cultured in Dulbecco's modified Eagle's medium (DMEM) containing Glutamax (Gibco) supplemented with 10 % heat-inactivated fetal bovine serum (Gibco). Cells were maintained in 5 % CO2 at 37 °C, and were seeded at a density of approximately 5x105 cells ml1 per well in 24-well tissue culture plates for invasion assays. Monolayers produced after 24 h incubation in 5 % CO2 at 37 °C were used for infection studies. Bacteria grown in BHI broth to stationary phase or exponential phase were pelleted by centrifugation, washed once with PBS (0·02 M sodium phosphate buffer with 0·15 M sodium chloride, pH 7·4), and adjusted to a concentration of 5x107 c.f.u. ml1. The bacteria were then opsonized with 10 % heat-inactivated fetal bovine serum for 30 min at 37 °C, and subsequently washed with PBS. Macrophages were infected at an m.o.i. of 10 bacteria per cell in DMEM supplemented with 10 % heat-inactivated fetal bovine serum (time zero of the assay). Contact between bacteria and cells was facilitated by centrifugation (150 g, 5 min). After a 1 h incubation, the cell monolayer was washed with Hanks' balanced salt solution (HBSS) containing 10 mM HEPES (Gibco), and then overlaid with fresh cell culture medium containing 100 µg gentamicin ml1 (Gibco) to kill extracellular bacteria. After 1 h, the cell monolayer was washed with HBSS containing 10 mM HEPES, and 1 ml fresh medium containing 25 µg gentamicin ml1 was added to each well. Thereafter, at defined time points, the cell monolayers were washed with HBSS, and subsequently lysed with 1 ml 0·1 % Triton X-100 (Sigma). Lysates were serially diluted 10-fold, plated on BHI agar, and c.f.u. were determined after 24 h incubation at 37 °C. The experiments were performed twice, with duplicate determinations per time point.
Mouse virulence assay (intraperitoneal).
Five 6-week-old female BALB/c mice were inoculated intraperitoneally with either 100 µl L. monocytogenes grown in 50 ml BHI for 18 h at 37 °C, or 100 µl L. monocytogenes grown to the exponential phase; both cultures were diluted in PBS, pH 7·2, to approximately 2x105 ml1. Numbers of c.f.u. in the inocula were determined by plate-spreading on BHI agar. After 3 days, mice were killed by cervical dislocation, and livers and spleens were removed and homogenized in 0·15 M NaCl. Tenfold serial dilutions were plated in duplicate on BHI agar to determine the numbers of c.f.u. in the organs. The experiments were performed twice. All mice were treated in accordance with institutional guidelines for treatment of animals. The data were analysed statistically by analysis of variance, examining the effects of the strain and the run (experiment). In order to achieve a uniform variance, the bacterial counts were log transformed before the data were analysed. Measurement of a strain consisted of five mice per run and two runs. Exponential- and stationary-phase cultures were analysed separately. The variance analysis was carried out by the GLM procedure in SAS (SAS Institute). There were no significant interactions between the strain and the run.
Mouse virulence assay (intravenous).
Five 8-week-old female BALB-c mice were inoculated intravenously with 50 µl L. monocytogenes, which had been grown in 50 ml BHI for 18 h at 37 °C, and then diluted 25-fold in PBS, pH 7·2. Numbers of c.f.u. in the inocula were determined by plate-spreading on BHI agar. After 1, 6 and 48 h, mice were killed by cervical dislocation, and livers were removed and homogenized in 0·15 M NaCl. Tenfold serial dilutions were plated in duplicate on BHI agar. All mice were treated in accordance with institutional guidelines for treatment of animals.
Lecithinase and haemolysin activity.
Using charcoal-supplemented egg-yolk agar plates, PlcB acitivity was estimated as the size of a halo of precipitation around the bacterial colony, essentially as described by Ermolaeva et al. (2003). The concentration of activated charcoal was 0·2 %. Ten per cent of an egg-yolk suspension, prepared by adding one fresh egg yolk to 25 ml sterile saline, was added to molten BHI agar. Inoculated egg-yolk medium was incubated at 37 °C for 2 days. Listeriolysin O activity in culture supernatants of exponential and stationary cultures grown in BHI, or BHI supplemented with 0·2 % activated charcoal (Merck), was assayed by quantification of cell lysis activity in serial dilutions of supernatants using calf blood, essentially as described by Leimeister-Wachter & Chakraborty (1989)
.
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RESULTS |
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In the wild-type strain, we also observed a faint band corresponding to a transcript initiated from the B promoter element (SigB, Fig. 1C
). This transcript was absent in the sigB mutant strain (Fig. 1C
, lane 2), and the amount was increased by heat, which is known to induce the general stress response controlled by
B (Fig. 1C
, lane 5). Interestingly the amount of
B-dependent transcript was also increased in the absence of PerR (Fig. 1C
, lanes 3 and 4), indicating that PerR blocks transcription initiated from the
B promoter. Upstream of the
B promoter, we identified a third transcript initiated 149 bp upstream of the translation start site, which corresponded to a
A-type promoter element indicated as SigA2 in Fig. 1(B)
. Thus, fri expression in L. monocytogenes is initiated from three promoters, and it is controlled by PerR and
B.
Fri improves survival of cells exposed to H2O2
With the aim of determining if Fri plays a protective role against peroxide stress in L. monocytogenes, we constructed an in-frame deletion of fri by removing a DNA fragment encompassing an internal 381 bp of the gene using the temperature-sensitive plasmid pAUL-A. Since Fri is expressed from a monocistronic transcript, it is highly unlikely that the in-frame fri deletion mutation will have polar effects on expression of other genes. Despite this notion, we still attempted to complement the mutant strain by introducing the fri gene in trans; however, numerous attempts proved unsuccessful in cloning the intact fri gene and promoter region in a plasmid vector when using either E. coli or L. monocytogenes. While the reason for the lack of success remains obscure, a previous study also reported difficulties when attempting to clone the fri gene (Polidoro et al., 2002). However, when we examined growth of the fri deletion mutant, we found that it was identical to the wild-type strain in both rich growth medium and improved minimal medium (data not shown). In contrast, when cells grown to either exponential or stationary growth phase were exposed to H2O2 at 20 mM (Fig. 2A
) or 50 mM (Fig. 2B
), respectively, the survival of fri mutant cells was greatly reduced when compared with wild-type cells. Notably, the fri mutant cells appeared to be more sensitive to oxidative stress during the exponential growth phase compared with cells present in stationary growth phase.
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DISCUSSION |
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Members of the Dps protein family have been shown to protect cells entering stationary phase (Martinez & Kolter, 1997). When we followed viability during prolonged incubation in rich growth medium, we observed a greater loss of viability of fri mutant cells compared with the wild-type after 6 days of incubation. Generally, we observed a dramatic decrease in viability for both cell types during the first 79 days of incubation, which subsequently stabilized during the rest of the experiment. This profile is in accordance with similar studies regarding long-term survival of L. monocytogenes (Herbert & Foster, 2001
) and Staphylococcus aureus (Watson et al., 1998
). The apparent difficulty of the fri mutant in maintaining viability compared to the wild-type may be the result of the inability to cope with oxidative damage inflicted by reactive oxygen species produced during stationary phase (Dukan & Nystrom, 1998
; Kolter et al., 1993
), as fri mutant cells also proved more sensitive to oxidative stress than wild-type cells.
The expression of fri has been shown to be only marginally induced by entry into stationary phase (Polidoro et al., 2002). In general, expression of members of the Dps family of proteins appears to fall into two classes: those induced by entry into stationary phase, such as Dps of E. coli and B. subtilis, and those that are relatively unaffected by growth phase, namely Dps of C. jejuni (Ishikawa et al., 2003
), Dpr of Streptococcus suis (Pulliainen et al., 2003
) and Fri. However, despite the lack of stationary-phase induction of fri expression, the promoter region carries a consensus sequence of the general stress
factor SigB, which in B. subtilis is responsible for the stationary-phase induction of Dps (Antelmann et al., 1997
). Primer extension analysis revealed that fri is expressed from three promoters, namely two SigA-type and one SigB-type promoter elements. In addition, the fri promoter region carries a consensus binding site for the peroxide regulator PerR, and in the absence of PerR, expression from the fri proximal promoter was strongly increased. Interestingly, the amount of
B-dependent transcript was also increased in the absence of PerR, indicating that PerR is controlling expression initiated from both the SigA1 and the SigB promoter. Despite the involvement of PerR in controlling fri expression, we failed to see an induction of expression by H2O2, suggesting that fri belongs to the subclass of PerR-controlled genes that is not part of the peroxide regulon (Fuangthong et al., 2002
).
To determine if Fri is of importance to virulence, we infected BALB/c mice intraperitoneally with wild-type and fri mutant cells, and found that the lack of Fri decreased the ability of L. monocytogenes to multiply in the organs. A major defence of the human body against invading pathogens is the bactericidal activity of the macrophages. When examining uptake and viability in the macrophage-like cell line J774.A1, we found that the absence of Fri reduced the ability of L. monocytogenes to multiply intracellularly, resulting in a 10-fold reduction in recovery of fri mutant cells compared with wild-type cells 6 h after infection. The antimicrobial activities of macrophages include the production of reactive oxygen metabolites (Forbes & Gros, 2001), and these compounds may be responsible for the accelerated killing of fri mutant cells inside macrophages. Indeed, L. monocytogenes cells lacking Fri were significantly more sensitive to H2O2 than wild-type cells, as shown for other members of the Dps protein family (Chen & Helmann, 1995
; Ishikawa et al., 2003
; Martinez & Kolter, 1997
; Nair & Finkel, 2004
). Since the amino acid sequence of Fri from L. monocytogenes only differs at two positions from the iron-binding Fri of L. innocua (Bozzi et al., 1997
), the mechanism by which this protection occurs is likely to involve sequestration of iron away from processes generating toxic hydroxyl radicals in combination with H2O2 (Almirón et al., 1992
; Ishikawa et al., 2003
; Martinez & Kolter, 1997
; Zhao et al., 2002
). Curiously, when using the mouse model, we only observed a significant difference in virulence between mutant and wild-type if the bacterial cells were in stationary growth phase, and injected in the peritoneum, whereas in the macrophage assay, the virulence of fri mutant cells was reduced independently of the growth phase of the bacteria. Although we are currently unable to explain the reason for this difference, it may reflect that Fri is only required for proliferation of L. monocytogenes in specific host cells, or compartments that are only reached during some types of infections. In a recent study of the Gram-negative dental pathogen Porphyromonas gingivalis, the inactivation of a Dps-like protein reduced survival in human umbilical vein endothelial cells (Ueshima et al., 2003
), and in Salmonella enterica serovar Typhimurium, Dps promoted survival in murine macrophages as well as in organs of infected mice (Halsey et al., 2004
). These reports, together with our findings, provide evidence that Dps-like proteins are important for virulence of distantly related bacterial pathogens.
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ACKNOWLEDGEMENTS |
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Received 9 August 2004;
revised 30 November 2004;
accepted 16 December 2004.
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