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Journal of Bacteriology, September 2006, p. 6235-6244, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00635-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Compensatory Functions of Two Alkyl Hydroperoxide Reductases in the Oxidative Defense System of Legionella pneumophila{dagger}

Jason J. LeBlanc,1 Ross J. Davidson,1,2,3 and Paul S. Hoffman1,2,4,5*

Departments of Microbiology and Immunology,1 Medicine, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7,2 Department of Pathology and Laboratory Medicine, Queen Elizabeth II Health Sciences Center, Halifax, Nova Scotia, Canada,3 Department of Internal Medicine, Division of Infectious Diseases and International Health,4 Department of Microbiology, University of Virginia School of Medicine, Charlottesville, Virginia 22908-13405

Received 4 May 2006/ Accepted 16 June 2006


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ABSTRACT
 
Legionella pneumophila expresses two catalase-peroxidase enzymes that exhibit strong peroxidatic but weak catalatic activities, suggesting that other enzymes participate in decomposition of hydrogen peroxide (H2O2). Comparative genomics revealed that L. pneumophila and its close relative Coxiella burnetii each contain two peroxide-scavenging alkyl hydroperoxide reductase (AhpC) systems: AhpC1, which is similar to the Helicobacter pylori AhpC system, and AhpC2 AhpD (AhpC2D), which is similar to the AhpC AhpD system of Mycobacterium tuberculosis. To establish a catalatic function for these two systems, we expressed L. pneumophila ahpC1 or ahpC2 in a catalase/peroxidase mutant of Escherichia coli and demonstrated restoration of H2O2 resistance by a disk diffusion assay. ahpC1::Km and ahpC2D::Km chromosomal deletion mutants were two- to eightfold more sensitive to H2O2, tert-butyl hydroperoxide, cumene hydroperoxide, and paraquat than the wild-type L. pneumophila, a phenotype that could be restored by trans-complementation. Reciprocal strategies to construct double mutants were unsuccessful. Mutant strains were not enfeebled for growth in vitro or in a U937 cell infection model. Green fluorescence protein reporter assays revealed expression to be dependent on the stage of growth, with ahpC1 appearing after the exponential phase and ahpC2 appearing during early exponential phase. Quantitative real-time PCR showed that ahpC1 mRNA levels were ~7- to 10-fold higher than ahpC2D mRNA levels. However, expression of ahpC2D was significantly increased in the ahpC1 mutant, whereas ahpC1 expression was unchanged in the ahpC2D mutant. These results indicate that AhpC1 or AhpC2D (or both) provide an essential hydrogen peroxide-scavenging function to L. pneumophila and that the compensatory activity of the ahpC2D system is most likely induced in response to oxidative stress.


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INTRODUCTION
 
In natural environments, Legionella pneumophila is an intracellular pathogen of aquatic protozoa and when transmitted in aerosols to susceptible humans is responsible for most cases of Legionnaires' disease worldwide (21-23, 41, 47). The remarkable ability of L. pneumophila to colonize a wide range of natural and mammalian host cells (36, 37) is largely attributed to a novel type IVB secretion system, encoded by the dot (defect in organelle trafficking) and icm (intracellular multiplication) genes (1, 7, 8, 10, 45, 50, 59, 64, 65, 73), which delivers effector proteins into host cells that (i) promote invasion, (ii) subvert organelle trafficking functions, and (iii) establish a replication-permissive endosome (13, 15, 16, 44, 49). In natural hosts and in certain mammalian cell lines, L. pneumophila displays a developmental cycle in which replicating bacteria differentiate into metabolically dormant cyst-like forms that enable the bacteria to survive for extended periods in a highly infectious state (24, 28). Thus, protozoan hosts not only serve as a reservoir for bacterial amplification but also prepare the pathogen for stresses that may be encountered in new hosts, including humans (9, 19, 28, 71).

Factors that enable L. pneumophila to respond to pH, nutrient starvation, osmotic shock, heat, and oxidative stress are of particular interest to fully understand how the pathogen adapts to conditions faced in natural environments or within the intracellular milieu of protozoa or macrophages. To prevent damage by reactive oxygen intermediates (ROI), microbial oxidative defenses generally include disproportionation of superoxide radicals (O2–) by superoxide dismutase (SOD) and the removal of the resulting H2O2 by catalase or alkyl hydroperoxide reductase (AhpC) (20, 38). Bacterial antioxidant enzymes usually respond to ROI such as O2– and H2O2 produced during aerobic respiration (25, 38) but have also been used effectively as survival strategies for intracellular pathogens like mycobacteria (46). Since some evidence suggests that L. pneumophila may be faced with an oxidative burst following ingestion by macrophages or amoebae (30, 39), protection against ROI may be crucial for pathogenesis. It has been proposed that the L. pneumophila KatA and KatB catalase-peroxidases might provide catalatic activity to detoxify the phagosomal milieu to promote intracellular growth (3). However, L. pneumophila has been shown to be especially susceptible to oxidizing biocides (ozone and H2O2) in vitro, indicating that the pathogen might be unusually sensitive to oxidative stress (18, 42). This later view is supported by early work demonstrating the formation of inhibitory levels of H2O2 and O2– in growth medium (35) and the growth-promoting detoxification properties of supplements, such as activated charcoal and {alpha}-ketoglutaric acid, that are present in the BCYE isolation medium (54).

The oxidative defense system of L. pneumophila has been studied in some detail and includes two bifunctional catalase-peroxidases (katA and katB) (4, 5), iron (sodB) and copper-zinc (sodC) SODs (60, 68), and AhpC (ahpC1) (58). While KatB and SodB are located in the cytoplasm, KatA and SodC are located in the periplasm and putatively protect bacteria from exogenous ROI (3-5, 60, 68). Mutational studies indicated that sodB was essential for viability (60), whereas katA, katB, and sodC mutants were enfeebled for stationary-phase survival (3-5, 68). Early work by Pine et al. (55, 56) demonstrated that L. pneumophila strains were catalase negative and peroxidase positive, whereas other species (Legionella gormanii, Legionella micdadei, and others) were catalase positive and peroxidase negative. Thus, while KatA and KatB are annotated as bifunctional catalase-peroxidases, functionally, these enzymes apparently exhibit only peroxidatic activity. Catalases exhibit a relatively low affinity for H2O2, whereas AhpC systems exhibit a much higher affinity (63). Therefore, AhpC, not catalase, is considered to be responsible for scavenging most of the peroxide generated in bacteria. AhpCs are well established detoxifiers of H2O2 and organic (lipid) peroxides (2, 11, 12, 46, 57, 63, 69, 72) and are likely candidates for peroxide-scavenging enzymes in L. pneumophila. In this regard, Rankin et al. (58) showed that AhpC1 levels increased during intracellular growth of L. pneumophila in macrophages, consistent with a putative protective role. However, the peroxide susceptibility of the ahpC1 mutant was not investigated.

We initiated this study to determine whether AhpC enzymes were primarily responsible for scavenging peroxides in the apparent absence of measurable catalase activity in L. pneumophila. Whole-genome analysis revealed a phylogenetically distinct homologue of ahpC1, designated ahpC2 that exhibited similarity with the ahpC ahpD system of Mycobacterium tuberculosis and Streptomyces coelicolor (29, 32, 67). Here we show that AhpC2 AhpD (AhpC2D) and AhpC1 protect L. pneumophila from peroxide challenge and that expression of each gene is growth phase dependent, with ahpC1 expressed during postexponential phase and ahpC2D expressed during early exponential phase. Mutational studies indicated that at least one functional AhpC is required for viability and that both genes are required for full resistance to organic peroxides and hydroperoxides. Finally, the increased expression of ahpC2D in an ahpC1 mutant background is most likely due to the increased oxidative stress caused by an absence of AhpC1 function.


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MATERIALS AND METHODS
 
Phylogenetic analysis of AhpC sequences. AhpC amino acid sequences used in phylogenetic analysis were obtained from GenBank by BLASTP search using AhpC1 of Legionella pneumophila Philadelphia-1. Phylogenetic trees were generated by multiple-sequence alignment of unedited AhpC sequences using ClustalX and were viewed with TreeView software.

Bacterial strains and growth conditions. Bacterial strains and plasmids used in this study are listed in Table 1. L. pneumophila strains were grown aerobically at 37°C on buffered charcoal yeast extract agar (BCYE) (52) or in buffered yeast extract (BYE) broth and supplemented with thymidine (100 µg/ml) and antibiotics when required. Starter cultures were prepared as previously described (35) and used to inoculate prewarmed BYE to an optical density at 620 nm (OD620) of 0.2. For in vitro growth rate determinations, samples were taken every 3 hours (in triplicate) for measures of turbidity (A620) or viability (CFU/ml). Escherichia coli strains DH5{alpha} (Clontech Laboratories, Mountain View, CA) and J1377 were grown at 37°C on Luria-Bertani (LB) agar or in LB broth supplemented with the appropriate antibiotics. E. coli J1377 cells were grown at 37°C under anaerobic conditions using the BD GasPak EZ (Becton Dickinson, Oakville, Ontario, Canada) and AnaeroGen anaerobic atmosphere generation system (Oxoid, Ltd., Nepean, Ontario, Canada). Antibiotics (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada) were added to the media at the following concentrations: streptomycin (100 µg/ml), gentamicin (10 µg/ml), kanamycin (40 µg/ml), ampicillin (100 µg/ml), metronidazole (20 µg/ml), and chloramphenicol (20 µg/ml for E. coli or 4 µg/ml for Legionella). All strains were stored at –70°C in nutrient broth containing 10% dimethyl sulfoxide.


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  		TABLE 1. Bacterial strains and plasmids

Standard genetic methods. Preparation of DNA, restriction enzyme digestions, and other molecular techniques have been described elsewhere (61). Plasmid isolation was performed with the QIAprep Spin Miniprep kit (QIAGEN Inc., Mississauga, Ontario, Canada) or by using the standard alkaline lysis method (61). Purification of DNA fragments from agarose gels for subcloning was carried out with a QIAquick gel purification kit (QIAGEN). Oligonucleotides (see supplemental material) were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). DNA sequencing was carried out by the DalGen Microbial Genetics Center (Halifax, Nova Scotia, Canada).

PCR. PCR amplifications were performed on a Perkin-Elmer Gene Amp PCR System 2400 using either Taq DNA polymerase (MBI Fermentas) or Expand High Fidelity PCR system (Roche Diagnostics, Laval, Quebec, Canada). Unless indicated otherwise, the following standard PCR conditions were used: 94°C (5 min), followed by 35 cycles of 94°C (30 s), 55°C (30 s), and 72°C (1 min per kb of amplicon), and a final extension at 72°C for 7 min.

Cloning and expression of ahpC genes in E. coli J1377. The ahpC1 and ahpC2 genes were amplified from chromosomal DNA using primer pairs FC1PTRC/RC1PTRC for ahpC1 and FC2PTRC/RC2PTRC for ahpC2 and following restriction with BamHI and HindIII were ligated into similarly restricted pTrc99A. Constructs were transformed into catalase/peroxidase-deficient E. coli strain J1377 (obtained from James A. Imlay, University of Illinois, Urbana, IL), and ampicillin-resistant transformants were confirmed by PCR analysis using the primers described above and the FBLA/RBLA primers for the ampicillin resistance cassette. The PCR-positive transformants were J1377(ptrc) (vector control), J1377(ptrcC1), and J1377(ptrcC2). All strains were grown to mid-exponential stage phase (OD620 of 0.5) and cells corresponding to 0.1 OD were suspended in 4 ml of prewarmed 0.75% LB top agar and poured onto LB ampicillin agar with or without 1 mM isopropyl-ß-D-thiogalactopyranoside (IPTG). Once the medium had solidified, 10 µl of 30% H2O2 was placed on sterile 1/4-inch antibiotic disks that were placed in the center of the plates. Blank disks with distilled water were used as controls. The plates were then incubated at 37°C overnight under anaerobic conditions, and the diameters of the zones of inhibition were measured and reported as the mean ± standard deviation (SD) for three independent experiments.

Construction of L. pneumophila ahpC1 and ahpC2D mutants. Chromosomal deletion mutants were generated by gene replacement as previously described (33). Briefly, approximately 500 bp of upstream and downstream flanking DNA sequences was amplified by PCR using primer pairs P1C1/P2C1 (upstream) and P3C1/P4C1 (downstream) for ahpC1 and P1C2D/P2C2D (upstream) and P3C2D/P4C2D (downstream) for ahpC2D. The P1/P2 amplicons containing XbaI and BamHI restriction sites and P3/P4 amplicons containing BamHI and XhoI restriction sites were ligated sequentially into pBlueScript KS (Stratagene, La Jolla, CA), creating plasmids pBS{Delta}ahpC1 and pBS{Delta}ahpC2D. BamHI restriction and subsequent ligation of the kanamycin resistance determinant of plasmid p34s-km3 (17) between the upstream and downstream regions of ahpC1 or ahpC2D resulted in the creation of pBS{Delta}ahpC1::km and pBS{Delta}ahpC2D::km. The constructs were subcloned into the NotI and XhoI sites of pBRDX (suicide vector containing sacB and rdxA counterselection markers) (26), and the resulting clones pBRDX{Delta}ahpC1::km and pBRDX{Delta}ahpC2D::km were subsequently introduced into L. pneumophila strain Lp02 by electroporation. Allelic recombinants of L. pneumophila were selected from a population of kanamycin-resistant colonies by replica plating onto medium supplemented with 5% (wt/vol) sucrose or 20 µg/ml of metronidazole (loss of plasmid) and were confirmed by testing for loss of chloramphenicol resistance. Kanamycin-resistant (Kmr), metronidazole-resistant (Mtzr), sucrose-resistant (Sacr), and chloramphenicol-sensitive (Cams) strains were screened by PCR using primers that bound outside of the cloned upstream and downstream regions, internal to the deleted coding region of ahpC1 and ahpC2D, or in combination with primers designed for the kanamycin cassette. Reverse transcriptase PCR (RT-PCR) was also used to confirm the absence of ahpC1 or ahpC2D mRNA. Both ahpC1 and ahpC2D mutants were designated ahpC1::Km and ahpC2D::Km, respectively.

Construction of double mutants. To construct ahpC1 ahpC2D double mutants (ahpC1::Km ahpC2D::Gm or ahpC1::Gm ahpC2D::Km), the kanamycin resistance cassette of pRDX{Delta}ahpC1::km or pRDX{Delta}ahpC2D::km was replaced with a gentamicin resistance cassette (Gm) from pPH1J1 (34) using primers GM1 and GM2 containing BamHI restriction sites (cassette swapping). The resulting pRDX{Delta}ahpC1::Gm and pRDX{Delta}ahpC2D::Gm were electroporated into the ahpC2D::Km and ahpC1::Km mutants, respectively. To confirm the expression of the gentamicin cassette in L. pneumophila LP02, these two constructs were also electroporated into wild-type (WT) Lp02 to generate ahpC1::Gm and ahpC2D::Gm. These two mutants were also electroporated with pRDX{Delta}ahpC2D::km and pRDX{Delta}ahpC1::km constructs, respectively, to obtain double mutants (reciprocal markers). As a control for these experiments, the pRDX{Delta}magA::Gm (nonessential gene) was electroporated into the ahpC1::Km and ahpC2::Km mutant strains. In parallel, the pRDX{Delta}magA::km construct was electroporated in the gentamicin-resistant mutants.

Complementation of mutants. Promoter and coding sequences of grlA-ahpC1 and ahpC2D were amplified using primer pairs FC1COMP/P4C1 and FC2COMP/P4C2D, respectively, and cloned into the BamHI and XbaI sites of pJB908 (kindly provided by Joseph Vogel, Washington University, St. Louis, MO). The pKB5-derived (7) plasmid pJB908 contains (i) an ampicillin resistance cassette for selection in E. coli, (ii) the td{Delta}i gene known to complement the thymidine auxotrophy of L. pneumophila Lp02, (iii) an RSF1010 origin to permit replication in L. pneumophila, and (iv) deleted origin of conjugative transfer (oriT{Delta}13) to prevent intracellular growth defects in the macrophage (66). The resulting plasmids (pC1 and pC2D) were electroporated into L. pneumophila Lp02 ahpC1::Km or ahpC2D::Km, generating ahpC1::Km(pC1) or ahpC2D::Km(pC2D) strains, respectively. Mutants capable of growth on BCYE without thymidine were screened by PCR, and expression of the target genes was confirmed by RT-PCR. Strains containing the empty vector controls were screened by PCR using the primer combination FBLA/RBLA.

Microdilution susceptibility assay. L. pneumophila strains were grown in BYE to stationary phase and diluted in 2x BYE to an OD620 of 0.2. In triplicate wells of a 96-well microtiter plate, 50 µl of bacterial suspension (giving a final OD620 of 0.01) was added to an equal volume of various concentrations of the following oxidative stressors: H2O2, tert-butyl hydroperoxide (tBOOH), cumene hydroperoxide (CHP), or methyl viologen (PQ, which is used to generate endogenous H2O2 following disproportionation of O2–) ranging from 0 to 1,000 µM (final concentration per well). Following incubation at 37°C for 24 h with gentle agitation, the lowest concentration resulting in no visible growth was deemed to be the MIC. From wells where no growth was evident, quadruplicate 10-µl samples were spotted onto BCYE agar to assess minimal bactericidal concentration (MBC).

Peroxide challenge. To eliminate the effect of the culture medium on peroxide sensitivity, L. pneumophila cells were washed with phosphate-buffered saline (PBS), normalized to an OD620 of 0.1 and were then challenged for 30 min with various concentrations (250, 500, 1,000, and 2,000 µM) of tBOOH or with 500 µM tBOOH for various periods of time (0, 30, 60, 90, and 120 min). Cells were washed twice with PBS before being serially diluted and plated on BCYE to determine the number of CFU per milliliter. Results are shown as the mean ± SD of triplicate values obtained from three independent experiments.

Infection of U937 cells. U937 cells (ATCC CRL-1593.2) were routinely grown in suspension in RPMI 1640 supplemented with 4 mM L-glutamine, an antibiotic-antimycotic solution (100 U/ml penicillin G, 100 µg/ml streptomycin, and 250 ng/ml amphotericin B), and 10% fetal bovine serum in tissue culture flasks (Falcon Plastics, Becton-Dickinson) as previously described (53). To differentiate U937 cells into nonreplicating adherent macrophage-like forms, cells were incubated in fresh medium containing 50 ng/ml of phorbol 12-myristate 13-acetate (PMA) for 48 h. PMA-activated U937 cells were washed three times and transferred to 24-well plates at approximately 1 x 106 U937 cells/well. For infection, growth from 20 h (late-log-phase) broth cultures of L. pneumophila strains were washed with PBS and resuspended in RPMI 1640 containing the proper selection agent. Bacterial suspensions were added in triplicate to differentiated U937 cell monolayers at ~106 bacteria/well followed by a brief centrifugation at low speed (500 x g, 10 min). After 1 h of incubation, the infected monolayers were washed three times with fresh RPMI 1640 and treated with 50 µg/ml gentamicin for 1 h to kill extracellular bacteria. Following three additional washes, fresh medium containing 100 µg/ml thymidine was added (without the antibiotic-antimycotic solution). U937 cell monolayers were lysed with cold deionized water (immediately for zero time point) where bacteria were harvested, serially diluted, and plated on BCYE to determine the number of CFU/ml at 24, 48, and 72 h postinfection. Since L. pneumophila does not replicate in RPMI 1640, daily determination of CFU/ml is an accurate measure of intracellular growth (66).

GFP reporter assay. Green fluorescence protein (GFP) transcriptional fusions of ahpC1 (pC1gfp) and ahpC2D (pC2gfp) were constructed using primer pairs FC1PROM/RC1PROM and FC2PROM/RC2PROM, respectively, and cloned into the EcoRI and BamHI sites of pBH6119 (gift from Michele Swanson, University of Michigan Medical School, Ann Arbor, MI). Plasmid constructs were first transformed into E. coli DH5{alpha} strains, and correct orientation was determined by PCR using the respective combination of forward primers (FC1PROM or FC2PROM) and the reverse RGFP primer. The pBH6119 plasmid or its derivatives were electroporated into wild-type L. pneumophila Lp02 or ahpC1 or ahpC2D mutant strains and confirmed by PCR using either the primers described above (or using the FBLA/RBLA primers for the empty vector controls). At 3-h intervals, bacterial samples were taken from BYE broth culture, and following determination of OD620, bacteria were collected by centrifugation and suspended in PBS. GFP levels were measured on a TD-700 fluorometer (Turner Designs Inc., Sunnyvale, CA) using 310-390-nm excitation and a 486-nm emission filter. Values are expressed as relative fluorescence units per OD620 and represent triplicate values obtained from three independent experiments.

RNA isolation and RT-PCR. Total RNA was obtained by the TRIzol method as described by the manufacturer (Invitrogen, Burlington, Ontario, Canada), and contaminating DNA was removed by DNase I treatment according to the manufacturer (Sigma). For cDNA synthesis, 2 µl of RNA was added to a 12-µl reaction mixture containing 1 µl random hexameric primers (1 µg/µl) and 1 µl deoxynucleoside triphosphate mix (10 mM each). Samples were heated to 65°C and chilled quickly on ice for 5 min. After brief centrifugation, 4 µl 5x First-Strand buffer, 2 µl 0.1 M dithiothreitol, and 1 µl RNaseOUT recombinant RNase inhibitor (Invitrogen) were added to each tube. After incubation for 2 min at 37°C, 200 units of Moloney murine leukemia virus reverse transcriptase (Invitrogen) was added. The cDNA synthesis reaction was performed for 50 min at 37°C. The enzyme was subsequently inactivated at 72°C for 15 min. Aliquots of cDNA were stored at –70°C. To determine whether grlA-ahpC1 mRNA and ahpC2D mRNA were expressed as polycistronic messages, standard PCR amplification was performed using cDNA as the template and FGRLRT/RC1RT and FC2RT/RDRT primers, respectively. To confirm the absence of DNA contamination in RNA samples, a no-RT control was included in the same PCR mixtures. Amplicons were sized on a 2% agarose gel stained with ethidium bromide and photographed using a Bio-Rad Gel Doc 2000 system equipped with a charge-coupled device camera.

qPCR. Real-time quantitative PCR (qPCR) was performed on a 36-well rotor of the RotorGene 3000 system (Corbett Research, Kirkland, Quebec, Canada). PCR amplification was performed on 2.5 µl of cDNA template in a 20-µl reaction mixture containing Taq polymerase, 104-fold dilution of SYBR green I (Molecular Probes, Invitrogen), 300 nM deoxynucleoside triphosphates, 1x reaction buffer (40), and 200 nM of the primer sets FRPLJQRT/RRPLJQRT, FC1QRT/RC1QRTR, and FC2QRT/RC2QRT for rplJ, ahpC1, and ahpC2, respectively. PCR conditions are as follows: initial denaturation (94°C, 30 s), 45 cycles (denaturation [94°C, 30 s], annealing [55°C, 30 s], and extension [72°C, 30 s]), and melting (72 to 95°C). Samples were analyzed by melting curve analysis and by electrophoresis. Sample concentrations were determined by standard curves generated under the same conditions using genomic DNA as the template. All samples were normalized to rplJ levels, and values are expressed as the change in the increase/decrease relative to the ahpC2 levels (calibrator sample = 1x) in the wild-type strain. Values represent the mean ± SD of quadruplicate samples obtained from three independent experiments.


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RESULTS
 
The genomes of L. pneumophila and Coxiella burnetii contain two loci encoding AhpC proteins with ahpC1 sharing 70% identity and 86% similarity and ahpC2 sharing 72% identity and 85% similarity. The AhpC1 class is often partnered with thioredoxin reductases (2), and upstream of ahpC is grlA, which encodes a glutaredoxin-related protein that might be involved in AhpC reduction (Fig. 1A). Upstream of this locus and in the opposite direction is the Fe-superoxide dismutase gene sodB (60). The second ahpC gene (designated ahpC2) is adjacent to ahpD, also an alkyl hydroperoxide reductase partner found in members of the genus Mycobacterium (AhpC2 shares 60% identity and 74% similarity). Downstream of ahpD and in the opposite orientation is sodC encoding the periplasmic Cu,Zn-SOD (68) (Fig. 1A). To evaluate the putative operon structure of the L. pneumophila ahpC loci, RT-PCRs were performed with primers designed to span the intergenic sequence between grlA and ahpC1 and for ahpC2 and ahpD. As seen in Fig. 1B, generation of amplicons that spanned these intergenic regions indicated that both gene sets are transcribed as single polycistronic messages.


Figure 1
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  		FIG. 1. Genetic organization of the ahpC1 and ahpC2 loci. (A) Schematic representation of the two ahpC operons. Small arrows represent primers used in RT-PCRs. Large arrows indicate putative promoter regions (PahpC1 and PahpC2). (B) RT-PCR validation of operon structure. Negative controls (lanes 2 and 4) consisted of a no-RT reaction mixture for DNase I-treated RNA or positive RT-PCRs for the grlA-ahpC1 operon (lane 3) or ahpC2-ahpD operon (lane 5). Invitrogen 100-bp DNA ladder was used as a size reference (lane 1).

Complementation of an ahpC and catalase-deficient mutant of E. coli. To determine whether both AhpC1 and AhpC2 could effectively reduce peroxides, the ahpC1 or ahpC2 genes of L. pneumophila were cloned into the pTrc99A expression vector, transformed into an ahpCF- and katG katE-defective E. coli mutant (J1377), and exposed to hydrogen peroxide (Fig. 2). In the absence of IPTG, the level of sensitivity to hydrogen peroxide of E. coli J1377 strains containing either ahpC1 or ahpC2 [J1377(ptrcC1) or J1377(ptrcC2), respectively] was similar to that of the mutant J1377 strain containing the empty vector [J1377(ptrc)]. In contrast, IPTG induction of these constructs resulted in a decrease of the zone of inhibition, indicating that both L. pneumophila AhpC1 and AhpC2 can complement the E. coli peroxidase/catalase-deficient mutant, which is consistent with the proposed role of AhpC in protecting organisms from hydroperoxides and organic peroxides.


Figure 2
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  		FIG. 2. Expression of ahpC1 and ahpC2 in E. coli J1377. Disk diffusion assays were performed using 10 µl of 30% peroxide for E. coli J1377 strains harboring the empty vector control (ptrc) or containing L. pneumophila ahpC1 (ptrcC1) or ahpC2 (ptrcC2) in the presence (+) or absence (–) of IPTG. The assay was performed in triplicate, and results are the mean diameters of clearing ± SDs (error bars).

Isolation of mutants and peroxide testing. ahpC1 and ahpC2D single deletion mutants were obtained by allelic replacement mutagenesis. Generally, the RdxA suicide vector strategy yielded >200 Kmr colonies, and of the 50 colonies randomly picked for scoring metronidazole resistance and chloramphenicol sensitivity (loss of plasmid), over 50% contained the appropriate deletion (validated by PCR techniques). To address the apparent redundancy of the two AhpC systems, we determined the MIC and MBC of H2O2, tBOOH, CHP, and PQ for each ahpC mutant. As seen in Table 2, MIC and MBC (MIC = MBC) values for WT L. pneumophila were 1,000 µM for both H2O2 and tBOOH, 500 µM for CHP, and 250 µM for PQ. PQ generates O2– internally and is converted to H2O2 by SOD (an internal measure of peroxide toxicity). In contrast, the values for the ahpC1 and ahpC2D mutants were two- to eightfold lower, indicating the mutants are more sensitive to peroxides. The ahpC1 mutant was two times more sensitive to peroxides than the ahpC2D mutant, suggesting AhpC1 may be slightly more abundant or more efficient in removal of peroxide. Complementation of these mutants by introduction of the respective plasmids expressing grlA-ahpC1 or ahpC2D restored WT resistance to H2O2, tBOOH, CHP, and PQ. In a related series of experiments, the ahpC mutants were grown to mid-log phase in BYE broth and then challenged with various concentrations of tBOOH for 30 min and plated for viability (kill curve). As seen in Fig. 3A, both ahpC mutants displayed similar kinetics of concentration-dependent killing by tBOOH compared with WT and with complemented mutants. Figure 3B shows the time-dependent kinetics of killing by 500 µM tBOOH. While WT and complemented mutants exhibited an approximately 2-log-unit decrease in viability by 2 h postchallenge, each of the mutants exhibited a 7-log-unit decrease in viability over the same period. It should also be noted that in both sets of experiments, no difference in peroxide resistance was observed when the empty vector (pJB908) was introduced into the mutant or WT strain. These results show that both ahpC genes are required for full resistance to peroxides, suggesting a synergistic rather than a redundant function.


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  		TABLE 2. Sensitivity of L. pneumophila strains to oxidative stress


Figure 3
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  		FIG. 3. Peroxide sensitivity of L. pneumophila ahpC mutants. Wild-type, ahpC1::Km, ahpC2D::Km, ahpC1::Km(pC1), and ahpC2D::Km(pC2D) strains were challenged for 30 min with various concentrations of tBOOH ranging from 250 to 2,000 µM (A) or with 500 µM tBOOH for 0, 30, 60, 90, and 120 min (B). Results are expressed as log10 of CFU per milliliter and represent values obtained from three independent experiments.

Construction of double mutants. Our attempts to construct a double mutant (ahpC1::Km ahpC2D::Gm or ahpC1::Gm ahpC2D::Km) were not successful, even under low oxygen conditions that would limit peroxide formation (7% O2, 5% CO2) or with addition of catalase to the medium. To eliminate the possibility that the gentamicin resistance cassette was not properly expressed in L. pneumophila Lp02 ahpC::Km mutants, we first created chromosomal deletions in each of the ahpC genes using the gentamicin (Gm) cassette. Once these mutants were validated, the pRDX{Delta}ahpC::km constructs were introduced into gentamicin-resistant mutants. For a control for these experiments, we were able to produce deletions in the nonessential magA gene (33) in both ahpC mutant backgrounds using both the kanamycin and gentamicin resistance markers. However, in no case were we able to isolate an ahpC1 ahpC2D double mutant, suggesting that alkyl hydroperoxide reductase function is essential for viability. Since L. pneumophila is a strict aerobe, it was not possible to isolate these mutants in the absence of molecular oxygen or under microaerobic atmospheres.

In vitro and in vivo growth rates. As seen in Fig. 4A and B, both ahpC1 and ahpC2D mutants displayed growth rates (in broth and in a U937 infection model) that were indistinguishable from those of the Lp02 parent strain. For the U937 infection model, LP02 dotB mutant was used as a negative control, since this strain had been shown to be defective in intracellular multiplication (66). While not depicted, similar results were obtained in a HeLa cell infection model.


Figure 4
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  		FIG. 4. In vitro and in vivo growth profiles of ahpC1 and ahpC2 in L. pneumophila. (A) Growth rates of wild-type and ahpC1::Km and ahpC2D::Km mutant strains of L. pneumophila in BYE broth cultures. Triplicate samples were taken, and every 3 hours, the optical density at 620 nm was reported. (B) U937 infection with wild-type and ahpC1::Km, ahpC2D::Km, and dotB mutant L. pneumophila strains. Data were reported as the mean log10 CFU/ml ± SDs (error bars) for three independent experiments.

Growth phase and compensatory expression of ahpC1 and ahpC2. To determine whether ahpC1 and ahpC2 were differentially expressed in vitro, green fluorescence protein reporter fusions were constructed for each ahpC gene. As seen in Fig. 5, ahpC1 expression decreased during lag and early exponential phases of growth in WT strain Lp02 (0 to 12 h) and was increased again in mid-log and early stationary phases (12 to 30 h). In contrast, ahpC2D levels increased during lag and early exponential phases of growth (0 to 12 h) and decreased during late log and stationary phases (16 to 36 h). The GFP reporter studies also indicated that ahpC1 is more highly expressed than ahpC2D, which is consistent with MIC/MBC findings described earlier (Table 2).


Figure 5
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  		FIG. 5. Growth phase-dependent expression profiles of ahpC1 and ahpC2 in L. pneumophila. Fluorescence was determined at 488 nm for samples taken every 3 hours of BYE-grown strains Lp02(pBH6119) ({square}), Lp02(pC1gfp) ({circ}), Lp02(pC2gfp) ({triangleup}), ahpC1::Km(pC2gfp) ({blacktriangleup}), and ahpC2D::Km(pC1gfp) (•). Relative fluorescence units (RFU) were normalized to 1.0 OD620 unit. Data are reported as the means ± SDs (error bars) for three independent experiments. The growth curves presented in Fig. 3A can be used as a reference for correlating stage of growth with expression profiles.

Since the growth rates of the ahpC mutants were similar, we considered the possibility that in each mutant, the remaining ahpC gene might compensate through increased gene expression. To determine whether the expression profiles of ahpC genes are altered in the ahpC1 and ahpC2D mutants, the pC1gfp construct was introduced into the ahpC2D mutant and the pC2gfp construct into the ahpC1 mutant. On the basis of GFP levels from the pC2gfp construct in the ahpC1 mutant, growth cycle expression was not altered; however, the relative expression increased more than twofold over levels detected in the WT strain. Furthermore, a high basal level of expression was maintained through late log and stationary phases of growth (three times higher than the WT level). However, no increase in fluorescence or change in gene expression profile was observed with pC1gfp in the ahpC2D mutant (Fig. 5). As observed in the wild-type strain, no difference in background fluorescence from the empty pBH6119 vector was observed in the mutant backgrounds (data not shown). These results indicate that AhpC2D compensates for deficiencies in AhpC1 activity.

Compensatory gene expression analysis by qPCR. To further analyze the compensatory link between the ahpC1 and ahpC2 genes, real-time quantitative PCR was used to compare the levels of ahpC1 and ahpC2 mRNA. Consistent with results depicted in Fig. 5, the qPCR analysis confirmed a significantly higher expression level of ahpC1 over ahpC2 in the WT Lp02 strain. However, ahpC2 mRNA levels increased >15-fold in the ahpC1 mutant compared to the level expressed in the wild-type strain (Fig. 6). Although significantly higher levels of ahpC1 and ahpC2 were observed in the ahpC1::Km pC1 and ahpC2D::Km pC2D (trans-complemented mutant strains), respectively, ahpC2 levels in ahpC1::Km pC1 were restored to wild-type levels. Enhanced expression in trans-complemented strains might be attributed to plasmid copy number and not to vector effects, since the empty vector control (pJB908) had no influence on ahpC1 or ahpC2 expression when introduced in wild-type or mutant strains (data no shown). It should also be noted that in all cases, grlA and ahpD levels were similar to ahpC1 and ahpC2, respectively, further confirming the operon structure described in Fig. 1 (data not shown). These results also suggest that the induction of ahpC2D in the {Delta}ahpC1 mutant is a consequence of increased oxidative stress caused by the absence of AhpC1 activity.


Figure 6
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  		FIG. 6. Compensatory expression between the ahpC genes of Legionella pneumophila. Real-time PCR (qPCR) of cDNA amplified from WT and ahpC1 and ahpC2 mutants and complemented strains. Quantification of samples was performed using standard curves generated using DNA as the template. Expression levels were normalized to the rplJ levels (internal control) and plotted as a change in the increase relative to the calibrator sample (ahpC2 levels in the wild-type strain) designated arbitrarily as 1x. The ahpC1 and ahpC2 levels in wild-type or mutant strains are shown. Results are the means ± standard deviations (error bars) of quadruplicate samples from three independently grown cultures.


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DISCUSSION
 
We have characterized two alkyl hydroperoxide reductase systems that provide essential and partially redundant peroxide-scavenging function in L. pneumophila. Phylogenetic analysis indicates that AhpC1 is similar to the NADPH-dependent thioredoxin reductase/thioredoxin-activated AhpC of Helicobacter pylori (2) and AhpC2D is similar to the pyruvate dehydrogenase-dihydrolipoamide succinyltransferase (SucB), dihydrolipoamide dehydrogenase (LpdA)-activated system of M. tuberculosis (32). Expression of these systems was growth phase dependent, with ahpC1 expressed after the exponential phase of growth and ahpC2D expressed during the early exponential phase of growth in vitro. From an efficiency perspective, exponentially growing bacteria appear to exploit catabolic (pyruvate or {alpha}-ketoglutarate oxidation) sources of reducing power to drive AhpC2D activity and then switch to a more direct NAD(P)H-driven AhpC1 system during postexponential and stationary phases when oxidative stress is increased. Alternatively, the GrlA activity might be driven by the glutathione redox system, which is absent and replaced by thioredoxin system in H. pylori (2). It is also noteworthy that the two alkyl hydroperoxide reductases of L. pneumophila are highly homologous to those found in C. burnetii, a bacterium closely related to Legionella (62, 70), suggesting that these intracellular pathogens might employ common defense strategies to detoxify their intracellular milieu.

Functional activities demonstrated in this study for the AhpC proteins of L. pneumophila included the following: (i) complementing a peroxide-sensitive E. coli mutant to WT resistance with either ahpC1 or ahpC2; (ii) determining that ahpC1 and ahpC2D deletion mutants were two- to eightfold more sensitive to H2O2, tBOOH, CHP, and PQ (MIC and MBC values and survival curves) than the WT strain and restoration to WT phenotype by trans-complementation; and (iii) showing that AhpC function is most likely essential for viability. Unlike L. pneumophila catalase mutants, ahpC mutants were not enfeebled for growth in vitro or in vivo (U937 cell model). On the basis of GFP reporter fusions and qPCR, ahpC1 was expressed at three- to sevenfold-higher levels than ahpC2 and AhpC1 levels were unchanged in an ahpC2D mutant. However, ahpC2D levels increased three- to fivefold (GFP) to compensate for a deficiency in AhpC1 production. The expression of these genes was growth phase dependent, with AhpC1 most abundant after the exponential phase and AhpC2 most abundant during early exponential phase. Both AhpC systems are required for full resistance to peroxides, and the apparent lethality of the double mutant suggests that AhpC systems provide essential catalatic activity in L. pneumophila.

Pine et al. studied the catalase, peroxidase, and superoxide dismutase enzymes in members of the genus Legionella and reported that L. pneumophila was peroxidase positive and catalase negative (56). In contrast, several other Legionella species (L. micdadei, L. jordanis, and L. bozemanii) expressed a high-molecular-weight catalase and no peroxidase (56). These observations were confirmed by direct spectrophotometric assay of catalase activity with partially purified enzymes from these various strains (55). Ironically, the initial weak catalatic activity noted in extracts from L. pneumophila strains in retrospect can be attributed to residual metabolic activities driving AhpC activity. Similarly, the weak NAD(P)H hydroperoxidatic activity reported in studies of L. pneumophila katA and katB expressed in E. coli mutant UM383 (katG and katE) is probably due to the E. coli AhpCF activity (5). The E. coli strain used in our studies contained the ahpCF deletion and katG and katE deletions (63). Several groups have shown that L. pneumophila expresses two catalase-peroxidases, with KatA located in the periplasm and KatB located in the cytoplasm (4, 5). By comparison with ahpC1 and ahpC2D mutants, katA and katB mutants and the katA katB double deletion mutants exhibit only a modest increase in susceptibility to peroxide (4, 5). While these differences might be attributable to differences in assay systems, it is also likely that KatA and KatB provide different functional roles from the degradation of H2O2 that contribute to stationary-phase survival and infectivity, since neither of these deficiencies was observed with AhpC mutants (3-5). While both KatA and KatB belong to a large family of bifunctional catalase-peroxidases, the results of studies by Pine et al. with partly purified enzymes primarily support a peroxidatic role for these enzymes (54). Since kinetic studies show that catalases exhibit low affinities for H2O2 (high Km values), even the weak reported activities for KatA and KatB would not enable them to function at low concentrations of H2O2. It is generally accepted that alkyl hydroperoxide reductases scavenge most of the H2O2 produced during microbial metabolism by virtue of their high affinities for H2O2 (low Km values) (63). These lines of evidence support the hypothesis that it is the AhpC system in L. pneumophila that is primarily responsible for the peroxide-scavenging activities that are essential for viability.

We confirmed an earlier report by Rankin et al. (58), indicating that the intracellular growth kinetics of an ahpC1 mutant in PMA-differentiated U937 cells were similar to those of the wild-type strain. We extend these findings by showing that an ahpC2D mutant is also wild type for intracellular growth in U937 cells. In contrast, Bandyopadhyay et al. proposed that KatA and KatB are crucial for pathogenesis to maintain a critically low level of H2O2 between the periplasm and cytosol to protect macromolecular targets required for invasion or survival within macrophages or to maintain a redox state necessary for metabolic changes accompanying a transition from an extracellular transmissible form to an intracellular replicative state (3). Though the catalase-peroxidases may provide some important functions necessary for intracellular survival, further investigations would be necessary to validate such a model. The sensitivity of the ahpC mutants to peroxides in vitro and absence of inhibition in vivo suggest that L. pneumophila may not be exposed to toxic concentrations of peroxides in macrophages. Our data are consistent with observations that L. pneumophila generates a unique replicative phagosome that escapes the signal transduction activities associated with phagolysosomal fusion and activation of the respiratory burst (3, 30, 39).

The absence of intracellular growth defects in L. pneumophila ahpC mutants could also be attributed to increased expression of other antioxidant enzymes that accompany loss of AhpC function. For example, catalase-deficient strains of M. tuberculosis (67) and Burkholderia pseudomallei (43) showed enhanced ahpC expression. Increased catalase levels were also observed following inactivation of ahpC in Xanthomonas campestris (14, 48) and Bacillus subtilis (12). In Pseudomonas aeruginosa, an enhanced activity of the organic hydroperoxide resistance protein (Ohr) was detected in an ahpC mutant, suggesting compensatory functions between Ohr and AhpC (51). Although no direct evidence was provided, previous studies suggested that the loss of one catalase-peroxidase (KatA or KatB) function in L. pneumophila might lead to compensation by the other to prevent the increased doubling time observed in the katA katB double mutant (4). On the basis of GFP reporter fusions and qPCR data, our results indicate that ahpC2D levels increased to compensate for a deficiency in AhpC1 production. To our knowledge, this is the first report of a compensatory link between two alkyl hydroperoxide reductases. We propose that the ahpC2D operon of L. pneumophila may act as a secondary support ("backup") system that is up-regulated with the loss of AhpC1 function, perhaps in response to increased oxidative stress, whereas high basal levels of AhpC1 would be sufficiently protective in the absence of AhpC2D.

Compensatory interactions among peroxide-detoxifying enzymes are usually mediated by thiol-reactive peroxide sensors (27), such as the peroxide-inducible transcriptional regulator OxyR (6, 14). The up-regulation of the ahpC2D operon might be induced in response to a rise in intracellular peroxides in the ahpC1 null background. Our laboratory is currently investigating whether regulation of ahpC2D is mediated by the L. pneumophila OxyR homologue. Further studies in L. pneumophila should include analysis of compensatory links between the AhpCs and other antioxidant enzymes, such as the two catalase-peroxidases. Improving our knowledge on the defense strategies used by intracellular pathogens faced with oxidative stress might help delineate the complex regulatory network involved during both environmental survival and evasion of the immune defenses during human infection.


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ACKNOWLEDGMENTS
 
We thank James Imlay, Joseph Vogel, and Michele Swanson for providing bacterial strains and plasmids and to Ralph Isberg for helpful suggestions regarding U937 cells. We thank Matthew Croxen and Fanny Ewann for critical reading of the manuscript.

This work was supported by CIHR grant MOP 14443 and start-up funds from the University of Virginia to P.S.H.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Infectious Diseases and International Health, Room 2146, MR-4 Bldg., 409 Lane Road, University of Virginia Health Systems, Charlottesville, VA 22908-1340. Phone: (434) 924-2893. Fax: (434) 924-0075. E-mail: psh2n{at}virginia.edu. Back

{dagger} Supplemental material for this article may be found at http://jb.asm.org/. Back


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Journal of Bacteriology, September 2006, p. 6235-6244, Vol. 188, No. 17
0021-9193/06/$08.00+0     doi:10.1128/JB.00635-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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