Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species

doi:10.1128/JB.00534-09

J Bacteriol. 2009 October; 191(20): 6447–6456.

Published online 2009 August 14. doi: 10.1128/JB.00534-09

PMCID: PMC2753026

Identification of Francisella tularensis Live Vaccine Strain CuZn Superoxide Dismutase as Critical for Resistance to Extracellularly Generated Reactive Oxygen Species ^†

Amanda A. Melillo,^# Manish Mahawar,^# Timothy J. Sellati, Meenakshi Malik, Dennis W. Metzger, J. Andres Melendez,^* and Chandra Shekhar Bakshi^*

Center for Immunology and Microbial Disease, Albany Medical College, Albany, New York 12208

^*Corresponding author. Mailing address: Center for Immunology and Microbial Disease, MC 151, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Phone for Chandra Shekhar Bakshi: (518) 262-6263. Fax: (518) 262-6161. E-mail: bakshis/at/mail.amc.edu. Phone for J. Andres Melendez: (518) 262-8791. Fax: (518) 262-6161. E-mail: melenda/at/mail.amc.edu

^#A.A.M. and M.M. contributed equally to this work.

Received April 21, 2009; Accepted August 3, 2009.

This article has been cited by other articles in PMC.

Abstract

Francisella tularensis is an intracellular pathogen whose survival is in part dependent on its ability to resist the microbicidal activity of host-generated reactive oxygen species (ROS) and reactive nitrogen species (RNS). In numerous bacterial pathogens, CuZn-containing superoxide dismutases (SodC) are important virulence factors, localizing to the periplasm to offer protection from host-derived superoxide radicals (O₂⁻). In the present study, mutants of F. tularensis live vaccine strain (LVS) deficient in superoxide dismutases (SODs) were used to examine their role in defense against ROS/RNS-mediated microbicidal activity of infected macrophages. An in-frame deletion F. tularensis mutant of sodC (ΔsodC) and a F. tularensis ΔsodC mutant with attenuated Fe-superoxide dismutase (sodB) gene expression (sodB ΔsodC) were constructed and evaluated for susceptibility to ROS and RNS in gamma interferon (IFN-γ)-activated macrophages and a mouse model of respiratory tularemia. The F. tularensis ΔsodC and sodB ΔsodC mutants showed attenuated intramacrophage survival in IFN-γ-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the sodC gene in the ΔsodC mutant or inhibiting the IFN-γ-dependent production of O₂⁻ or nitric oxide (NO) enhanced intramacrophage survival of the sod mutants. The ΔsodC and sodB ΔsodC mutants were also significantly attenuated for virulence in intranasally challenged C57BL/6 mice compared to the wild-type F. tularensis LVS. As observed for macrophages, the virulence of the ΔsodC mutant was restored in ifn-γ^−/−, inos⁻^/⁻, and phox⁻^/⁻ mice, indicating that SodC is required for resisting host-generated ROS. To conclude, this study demonstrates that SodB and SodC act to confer protection against host-derived oxidants and contribute to intramacrophage survival and virulence of F. tularensis in mice.

Francisella tularensis is considered a potential biological threat due to its extreme infectivity, ease of artificial dissemination via aerosols, and substantial capacity to cause illness and death. A hallmark of all F. tularensis subspecies is their ability to survive and replicate within macrophages (18) and other cell types (6, 11, 25, 28). While recent work has furthered our understanding of F. tularensis virulence mechanisms, little is known with respect to its ability to resist the microbicidal production of reactive oxygen species (ROS) or reactive nitrogen species (RNS).

Superoxide dismutases (SODs) are metalloproteins that are classified according to their coordinating active site metals. SODs catalyze the dismutation of the highly reactive superoxide (O₂⁻) anion to hydrogen peroxide (H₂O₂) and O₂ (26). The dismutation of O₂⁻ prevents accumulation of microbicidal ROS and RNS in infected macrophages. Three major categories of SODs have been identified in bacteria and include Mn-, Fe-, and CuZn-containing SODs (SodA, SodB, and SodC, respectively) and are required for aerobic survival (27). The F. tularensis genome encodes SodB (FTL_1791) and SodC (FTL_0380). In several intracellular bacterial pathogens, SodC is an important virulence factor, and its localization to the periplasmic space protects bacteria from host-derived O₂⁻ and NO radicals (8, 9, 21, 32). Moreover, many virulent bacteria possess two copies of the sodC gene (4). The evolutionary maintenance of an extra sodC gene copy suggests that it serves some essential function in survival (4). As an intracellular pathogen, F. tularensis is exposed to ROS and RNS generated by inflammatory cells during the macrophage activation process, which suggests that SODs may play an important role in its intracellular survival and pathogenesis. We have demonstrated that decreases in SodB activity render F. tularensis sensitive to ROS and attenuate virulence in mice (2). However, the contribution of F. tularensis SodC in virulence and intramacrophage survival has not been defined. In this study we have constructed a F. tularensis sodC mutant (ΔsodC) and a F. tularensis sodBC double mutant (sodB ΔsodC) and determined that SodC in conjunction with SodB primarily protects the pathogen from host-derived ROS and is required for intramacrophage survival and virulence of F. tularensis in mice.

MATERIALS AND METHODS

Bacterial strains and media.

F. tularensis subsp. holarctica live vaccine strain (LVS) (ATCC 29684; American Type Culture Collection, Rockville, MD) was used in this study. The F. tularensis ΔsodC mutant, a transcomplemented strain (ΔsodC mutant carrying psodC [ΔsodC+psodC]), and a double mutant carrying an in-frame sodC gene deletion in the sodB gene (2) (sodB ΔsodC) were constructed in the present study (Table (Table1).1). All bacterial cultures were grown on Mueller-Hinton (MH)-chocolate agar plates (BD Biosciences, San Jose, CA) supplemented with IsoVitaleX at 37°C with 5% CO₂ or in MH broth (BD Biosciences, San Jose, CA) supplemented with ferric pyrophosphate and IsoVitaleX (BD Biosciences, San Jose, CA) at 37°C with shaking (160 rpm). Active mid-log-phase bacteria grown in MH broth were harvested and stored at −80°C; 1-ml aliquots were thawed periodically for use.

TABLE 1.

Primers, plasmid vectors, and bacterial strains used and generated in the present study

Construction of F. tularensis sodC and sodB ΔsodC mutants and transcomplementation.

The plasmid constructs, bacterial strains, and the primer sequences used in this study are shown in Table Table1.1. An allelic replacement method was used to construct an in-frame sodC gene deletion mutant (ΔsodC) and sodB ΔsodC mutants of F. tularensis LVS (13). For construction of the ΔsodC mutant, the entire 557-bp coding region of the sodC gene was deleted employing an approach described earlier (23). Briefly, the regions approximately 750 and 650 bp up- and downstream of the sodC gene were PCR amplified. A previously described PCR method using splicing by overlap extension was used to join the sodC flanking regions (15). The resultant single fragment containing the flanking sequences minus the entire sodC gene was digested with XhoI/BglII and ligated into a similarly digested pDMK shuttle vector (20) to yield pDMK::ΔsodC. The deletion of the sodC gene in pDMK::ΔsodC was confirmed by PCR. The pDMK::ΔsodC plasmid was transformed in Escherichia coli S17-1 and transferred into F. tularensis LVS via conjugal transfer (13). The mutants were selected on modified chocolate agar plates (1.5% peptone, 0.1% sodium chloride, 0.4% dipotassium hydrogen phosphate, 0.1% potassium dihydrogen phosphate, 1% d-glucose, and 1.5% agar) supplemented with IsoVitaleX, l-cysteine hydrochloride, 1% hemoglobin, 10 μg/ml kanamycin, and 100 μg/ml polymyxin B (the latter component was included for counterselection of the donor E. coli). Colonies obtained on kanamycin plates following the first recombination were selected for a second recombination event by plating on medium containing 5% sucrose (13). Mutant colonies exhibiting kanamycin sensitivity and sucrose resistance following the second recombination event were confirmed for gene deletion by PCR. This mutant was referred to as the ΔsodC mutant.

We previously reported a mutant of F. tularensis LVS deficient in sodB gene expression (2). Since the sodB gene deletion mutant is not viable, a point mutation (ATG→GTG) was introduced in the initiation codon of the sodB gene to generate a sodB mutant (2). A PstI site was introduced immediately upstream of the mutated sodB gene to facilitate its differentiation from the wild-type (WT) sodB gene. We used the F. tularensis sodB mutant and deleted its sodC gene to generate a double mutant, sodB ΔsodC mutant. The sodB ΔsodC double mutation was confirmed by multiplex PCR followed by digestion of the products with the PstI restriction enzyme. The mutant cultures were stored at −80^οC.

A pKK214::gfp vector expressing green fluorescent protein under the control of the groEL promoter of F. tularensis was used for transcomplementation according to a recently reported protocol (23). The 557-bp sodC gene along with its upstream 100-bp region, and the kanamycin resistance gene of the pKK214 vector were amplified in separate PCRs using primer pairs detailed in Table Table1.1. The sodC gene and the kanamycin resistance gene were joined using an overlap extension PCR to construct a bicistron. The final PCR product containing the sodC gene and kanamycin gene was digested with PstI and ligated into similarly digested pKK214 downstream of the F. tularensis groEL promoter to yield pKK214::sodC. This approach allowed us to replace the gfp gene with the sodC gene while maintaining the kanamycin open reading frame. The pKK214::sodC plasmid was transformed in chemically competent E. coli DH5α cells and selected on LB-kanamycin plates. The plasmids were prepared from the transformants using a plasmid purification kit (Invitrogen, Carlsbad, CA), and the orientation of the sodC gene in pKK214::sodC vector was confirmed by PCR. The pKK214::sodC vector cloned in the correct orientation was electroporated in the ΔsodC mutant as described previously (3). The transformants were selected on MH-chocolate agar plates containing kanamycin (20 μg/ml). The resultant transcomplemented strain was termed the ΔsodC+psodC strain and confirmed by PCR and quantitative reverse transcription-PCR.

Susceptibility of sod mutants to ROS and RNS.

The WT F. tularensis LVS and sod mutants were tested for their susceptibilities to ROS- and RNS-producing compounds by generating growth curves and by disc diffusion and bacterial killing assays. For growth curves, single colonies of LVS and the indicated mutants were grown on MH-chocolate agar plates, then inoculated in 10 ml of MH broth, and grown overnight to an optical density at 600 nm (OD₆₀₀) of 0.20. One hundred eighty microliters of each culture was added to a 96-well microtiter plate in quadruplicate. Various concentrations of ROS- or RNS-generating drugs, including O₂⁻-generating paraquat (MP Biomedical Inc., Solon, OH), H₂O₂ (Sigma Aldrich, St. Louis, MO), the NO-generating compound (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) (Alexis Biochem, San Diego, CA), and peroxynitrite (ONOO⁻) (Calbiochem, La Jolla, CA), were added to the appropriate wells. The microtiter plate was covered to restrict evaporation and incubated at 37°C with shaking at 160 rpm for 24 h using a Biotek synergy HT plate reader (BioTek, Winoosoki, VT). OD₆₀₀ was recorded at 6-hour intervals. The OD readings were averaged and analyzed using the Tukey-Kramer multiple-comparison test. For determination of the effective 50% inhibitory dose (ED₅₀) of the redox-cycling drugs, the OD readings recorded 18 h postexposure were used and analyzed using linear regression.

The susceptibility of the sod mutants to O₂⁻-generating compounds paraquat and pyrogallol was further tested using a previously described disc diffusion assay (2). Briefly, the bacterial cultures were first spread on MH-chocolate agar plates followed by the placement of sterile filter paper discs impregnated with 5 μl of 10 mM paraquat or 1 M pyrogallol (Sigma Aldrich, St. Louis, MO). The plates were incubated at 37^οC for 48 to 72 h, and the zone of inhibition around the paper discs was measured.

Susceptibility of the sod mutants to ROS and RNS was also investigated by conventional bacterial killing assays. The LVS and sod mutants were exposed to exogenous superoxide generated by the oxidation of xanthine as described previously (29). Briefly, bacterial cultures grown on MH-chocolate agar plates were resuspended in sterile phosphate-buffered saline (PBS) at a concentration of 1 × 10⁹ CFU/ml and treated with 250 μM hypoxanthine and 0.1 U/ml of xanthine oxidase (Sigma Aldrich, St. Louis, MO). Alternatively, bovine catalase (Sigma Aldrich, St. Louis, MO) (1 U/ml) was added to hypoxanthine/xanthine oxidase to prevent H₂O₂ generation and subsequent Fenton chemistry and to ensure that killing of the sod mutants was in response to O₂⁻ radicals only. The number of viable bacteria was determined after 3 and 6 hours of incubation by plating serial dilutions on MH-chocolate agar plates. A similar protocol was adapted for examining the susceptibility of sod mutants to RNS by replacing treatment with 320 μM of pure ONOO⁻ or a mixture containing hypoxanthine/xanthine oxidase and DETA-NONOate (250 μM). Bacterial colonies were counted after 48 h and expressed as log₁₀ CFU/ml.

Macrophage invasion assay and measurements of nitrate/nitrites.

Previously described macrophage invasion assays were performed to determine the roles of SodC and SodB in intramacrophage survival (23, 24). A murine alveolar macrophage cell line, MH-S (ATTC CRL-2019), was left untreated or treated with 50 or 100 ng/ml gamma interferon (IFN-γ) (Sigma Aldrich, St. Louis, MO) for 16 h before and after infection at a multiplicity of infection (MOI) of 100 with F. tularensis LVS, sod mutants, or the transcomplemented strain. Two hours postinfection, cells were treated with gentamicin for 2 hours (100 μg/ml) to kill extracellular bacteria. Medium containing gentamicin was then replaced with IFN-γ-containing medium without antibiotics, followed by incubation at 37^οC in 5% CO₂. Samples were collected and lysed at 4, 24, and 48 h postinfection with 0.1% sodium deoxycholate. Lysates were serially diluted in sterile PBS and plated on MH-chocolate agar plates for bacterial enumeration. Parallel experiments were performed with 250 μM NADPH oxidase (PHOX) inhibitor acetovanillone (apocynin) (Arcos Organics, Morris Plains, NJ), 1 mM of the inducible nitric oxide synthase (iNOS) inhibitor, N^G-monomethyl-l-arginine (NMMLA) (Sigma Aldrich, St. Louis, MO) or its nonfunctional homolog, 2-methyl adenine (Sigma Aldrich, St. Louis, MO). The transcomplemented ΔsodC+psodC strain was also plated on MH-chocolate plates containing kanamycin (25 μg/ml) to confirm that the bacteria recovered from macrophages still carried the psodC plasmid. All statistical analyses were performed using the Tukey-Kramer multiple-comparison test. Nitrate/nitrites were also measured from the supernatants of cultured macrophages from media as treated above using gas phase chemiluminescence (NOA 280, GE, Inc., Boulder, CO) as described previously (10).

Mouse survival studies.

C57BL/6 mice, C57BL/6 inos⁻^/⁻ and phox⁻^/⁻ mice (Taconic, Germantown, NY), and C57BL/6 ifn-γ^−/− mice were obtained from Jackson Laboratories (Bar Harbor, ME). All mice were maintained in a specific-pathogen-free environment, and experiments were conducted using 6- to 8-week-old mice of both sexes. All animal procedures conformed to the institutional animal care and use committee guidelines. Prior to inoculation, mice were deeply anesthetized via intraperitoneal injection of a cocktail of ketamine (Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (Phoenix Scientific, St. Joseph, MO). Mice were challenged intranasally with 1 × 10⁴ CFU of LVS or sod mutants in a volume of 20 μl PBS (10 μl/nare). Mice were observed for a period of 21 days for morbidity and mortality. Survival results were plotted as Kaplan-Meier curves, and statistical significance was determined by the log-rank test.

RESULTS

Sequence analysis of SodC and verification of ΔsodC and sodB ΔsodC mutants of F. tularensis.

The sodC gene (FTL_0380) of F. tularensis LVS encodes a 185-amino-acid precursor protein (GenBank accession no. CAJ78820.1). The precursor protein undergoes a posttranslational modification by cleaving of the N-terminal signal peptide to yield a 148-amino-acid-long mature protein. Similar to other bacterial pathogens (22), PSORTb (http://www.psort.org/psortb/) software predicted its periplasmic localization. Amino acid sequence alignment of SodC with CuZnSOD of diverse bacteria revealed 60 to 70% homology, with high sequence conservation (~90%) in the SodC domain, including regions involved in copper and zinc binding and residues involved in dimerization (data not shown).

The genomic organization of the sodC gene is shown in Fig. Fig.1A.1A. The deletion of the entire 557-bp coding region of the sodC gene in the F. tularensis ΔsodC and sodB ΔsodC mutants was confirmed by a multiplex PCR using primer pairs flanking the sodC and sodB genes, followed by the digestion of the PCR products with PstI as described previously (2). A PCR product of ~320 bp in the ΔsodC mutant (Fig. (Fig.1B,1B, lanes 3 and 4) compared to the 880-bp product in the WT LVS (Fig. (Fig.1B,1B, lanes 1 and 2) confirmed the sodC gene deletion. Restriction digestion of the PCR product by the PstI enzyme confirmed mutation of the sodB gene (Fig. (Fig.1B,1B, lanes 2 and 4), while the WT sodB gene showed resistance to this cleavage (Fig. (Fig.1B,1B, lanes 1 and 3). Regions flanking the deleted sodC gene were also sequenced to ascertain that the gene deletions in ΔsodC and sodB ΔsodC mutants were in frame (data not shown). The transcomplementation was confirmed by PCR and quantitive reverse transcription-PCR using sodC gene-specific primers. A two- to fourfold increase in sodC transcript levels was observed for the ΔsodC+psodC strain relative to the WT F. tularensis LVS, while no sodC transcripts were observed for the ΔsodC mutant (data not shown).

FIG. 1.

Confirmation of F. tularensis ΔsodC and sodB ΔsodC mutants. (A) Genomic organization of the sodC gene of F. tularensis LVS. The small arrows indicate the locations of the primers shown in Table Table11 that were used for construction, (more ...)

F. tularensis SODs protect from ROS.

Growth curves of the F. tularensis LVS and sod mutants were generated by growing bacteria in 96-well microtiter plates and compared with those generated using large culture volumes. The microtiter plate-generated growth curves matched that of larger volume liquid cultures (data not shown). However, a rapid progression to the stationary phase was observed due to limited nutrients available in small culture volumes in the microtiter plates. The OD₆₀₀ values of microtiter plate growth curves similar to large-volume cultures were also associated with an exponential increase in the number of CFU. Although loss of SodC lead to a slight decrease in OD₆₀₀ values in the later part of the growth, no differences in the numbers of CFU for the WT LVS and ΔsodC mutant indicated that sodC gene deletion does not impair its growth (see Fig. S1 in the supplemental material).

We next evaluated the contribution of the F. tularensis SODs in protecting the pathogen from exposure to O₂⁻-generating compounds. Growth curves generated in response to paraquat revealed that both the F. tularensis ΔsodC and sodB ΔsodC mutants displayed significant increases in sensitivity to paraquat compared to the WT LVS (Fig. (Fig.2A,2A, top graphs). The effective 50% inhibitory dose was calculated using linear regression 18 h postinoculation. Both the sod mutants displayed a significantly (P < 0.01) increased sensitivity to paraquat (40 and 27 μM for the ΔsodC and sodB ΔsodC mutants, respectively) compared to the ED₅₀ for the LVS of 120 μM. A role for F. tularensis SODs in conferring resistance to H₂O₂ was also analyzed. The sodB ΔsodC mutant exhibited greater sensitivity toward 4 mM H₂O₂ (ED₅₀ of 3.28 mM), while the growth of the ΔsodC mutant or the WT LVS (ED₅₀ of 3.9 mM for both) was only slightly impaired (Fig. (Fig.2A,2A, bottom graphs). The loss of SOD increases the steady-state levels of O₂⁻, thereby elevating the intracellular pools of reduced iron (Fe²⁺) and enhancing OH radical production via Fenton chemistry (17). The F. tularensis sodB ΔsodC mutant is likely more sensitive to H₂O₂ as a result of Fenton chemistry.

FIG. 2.

Susceptibility of sod mutants to ROS. (A) Growth curves were generated as described in Materials and Methods in response to the indicated concentrations of paraquat (top graphs) and H₂O₂ (bottom graphs). OD₆₀₀ was measured every 6 h for 24 h and plotted (more ...)

The susceptibilities of F. tularensis sod mutants to O₂⁻-generating compounds paraquat and pyrogallol were further tested in a disc diffusion assay. The results corroborated those of growth curves demonstrating that ΔsodC and sodB ΔsodC mutants exhibit greater sensitivity to O₂⁻ than the WT LVS does (Fig. (Fig.2B).2B). The bacterial killing assays revealed that both the ΔsodC and sodB ΔsodC mutants were significantly more sensitive to exogenously generated O₂⁻ catalyzed by the addition of hypoxanthine and xanthine oxidase than the WT LVS was (Fig. (Fig.2C).2C). The xanthine/xanthine oxidase system generates both O₂⁻ and H₂O₂. The addition of catalase can negate the affects of H₂O₂ and would reveal the effect of O₂⁻ on bacterial killing independently of H₂O₂. The addition of catalase (1 U/ml) to the hypoxanthine/xanthine oxidase system restored growth of the sodB ΔsodC mutant to a level near that of the ΔsodC mutant 6 hours postexposure (Fig. (Fig.2D).2D). These observations suggest that the SODs act independently of one another in affording protection from extracellularly derived O₂⁻ and that catalase protects from xanthine/xanthine oxidase-generated H₂O₂.

sod mutants of F. tularensis are not sensitized to RNS.

F. tularensis sod mutants and the WT LVS were tested for sensitivity to RNS. Growth curves generated in the presence of the NO donor Deta-NONOate revealed no differences in the growth patterns of the ΔsodC or sodB ΔsodC mutant or the WT LVS. None of the strains displayed statistically significant differences in sensitivity to presynthesized ONOO⁻ in either the growth (Fig. (Fig.3)3) or bacterial killing assay (data not shown).

FIG. 3.

Susceptibility of sod mutants to RNS. (A) Growth curves were generated in response to the indicated concentrations of Deta-NONOate (top graphs) and ONOO⁻ (bottom graphs). OD₆₀₀ was measured every 6 h for 24 h and plotted against time postinoculation. (more ...)

sod mutants of F. tularensis are attenuated for growth in IFN-γ-activated macrophages.

We next evaluated the importance of F. tularensis SODs in intramacrophage survival. Infection of the MH-S alveolar macrophage cell line with the ΔsodC mutant or its trancomplement did not affect bacterial recovery 24 h postinfection compared to the WT LVS. In contrast, 5- or 10-fold-fewer bacteria were recovered from MH-S cells infected with the F. tularensis sodB or sodB ΔsodC mutant, respectively (Fig. (Fig.4A).4A). Infection of IFN-γ (50 ng/ml)-activated macrophages impeded the growth of all strains equally (10- to 15-fold) 24 h postinfection (Fig. (Fig.4A).4A). However, significantly fewer sodB, ΔsodC, and sodB ΔsodC mutants were recovered from IFN-γ-activated MH-S cells 48 h postinfection compared to infection with the WT LVS (Fig. (Fig.4B).4B). Stimulation of MH-S cells with a higher dose of IFN-γ (100 ng/ml) inhibited growth of the sodB, ΔsodC, and sodB ΔsodC mutants 24 h postinfection compared to the WT LVS and led to complete bacterial killing 48 h postinfection (Fig. 4A and B). Complementation of ΔsodC enhanced the intramacrophage survival to a level nearly similar to that of the WT LVS in IFN-γ-stimulated macrophages, demonstrating a requirement of SodC for intracellular survival. These results show that the SODs of F. tularensis play an important role in protecting the pathogen from the microbicidal activity of IFN-γ-activated macrophages and that overproduction of SodC partially suppresses this activity.

FIG. 4.

Intramacrophage survival of sod mutants of F. tularensis. MH-S cells treated with 50 or 100 ng/ml of IFN-γ for 16 h prior to infection and thereafter were infected with the WT LVS or sod mutants at an MOI of 100. Untreated cells were kept as controls. (more ...)

Intramacrophage growth defects of sod mutants of F. tularensis are rescued by inhibition of ROS or RNS production.

We next asked whether the enhanced killing of the F. tularensis sodC and sodB ΔsodC mutants by IFN-γ-activated MH-S could be blocked by PHOX or iNOS inhibition. As shown in Fig. Fig.5,5, PHOX (apocynin) or iNOS (NMMLA) inhibition partially restored the growth of the LVS in IFN-γ-activated MH-S cells. Similarly, the intracellular growth defect of the sodB, ΔsodC, and sodB ΔsodC mutants in IFN-γ-activated MH-S cells 48 h postinfection was also reversed by treatment of macrophages with both inhibitors (Fig. 5A and B). Interestingly, the survival of the sodB ΔsodC mutant in IFN-γ-activated macrophages was the most responsive to PHOX inhibition. These results support the premise that both SodB and SodC contribute to limiting the toxicity of macrophage-derived ROS. Furthermore, the survival benefits afforded by iNOS inhibition were similar in the LVS and mutant strains at both 24 and 48 h, and differences that were observed in the amplitude of the rescue when iNOS was inhibited are likely due to the enhanced susceptibility of the mutants to O₂⁻-mediated killing as observed in Fig. Fig.5A5A.

FIG. 5.

Intracellular growth of wild-type F. tularensis LVS and sod mutants in MH-S cells treated with PHOX and iNOS inhibitors. MH-S cells were treated with 50 ng/ml of IFN-γ and either PHOX inhibitor apocynin (250 μM) (A) or iNOS inhibitor NMMLA (more ...)

F. tularensis SODs are required for virulence in mice.

Recent reports have shown that SodB and KatG mutants of F. tularensis LVS are attenuated for virulence in mice (2, 20). Having observed an attenuated intramacrophage growth in IFN-γ-stimulated macrophages, we investigated the virulence of F. tularensis ΔsodC and sodB ΔsodC mutants in mice. The C57BL/6 mice were challenged intranasally with 1 × 10⁴ CFU of the ΔsodC or sodB ΔsodC mutant and the WT LVS. The mortality and morbidity of the infected mice were monitored for a period of 21 days. The mice challenged with the WT LVS succumbed to infection by day 12, while more than 50% of the mice challenged with the ΔsodC mutant and 80% of the mice challenged with the sodB ΔsodC mutant survived infection. These results suggest that although the ΔsodC mutant still retains its residual virulence, the sodB ΔsodC mutant exhibits an attenuated virulence in mice (Fig. (Fig.6A6A).

FIG. 6.

Effects of sod mutations on virulence in mice. (A) Survival of C57BL/6 mice (8 to 12 mice per group) infected intranasally with 1 × 10⁴ CFU of F. tularensis LVS or F. tularensis ΔsodC or sodB ΔsodC mutant. Comparisons between LVS (more ...)

Protection against ΔsodC mutants in mice is IFN-γ, iNOS, and PHOX dependent.

The in vitro macrophage assays suggested that ROS or RNS produced in response to IFN-γ activation plays a prominent role in restricting the intracellular survival of the F. tularensis ΔsodC mutant. It was also observed that nearly 50% of the mice infected with the ΔsodC mutant survived lethal challenge and cleared infection. It was further investigated whether IFN-γ, iNOS, and PHOX are required to provide protection against challenge with the ΔsodC mutant. C57BL/6 mice deficient for either ifn-γ^−/−, inos⁻^/⁻, or phox⁻^/⁻ and their syngeneic WT counterparts were challenged intranasally with 1 × 10⁴ CFU of LVS or the ΔsodC mutant. All of the WT C57BL/6 mice infected with LVS died by day 12 postinfection. The ifn-γ^−/−, inos⁻^/⁻, and phox⁻^/⁻ mice infected with ΔsodC mutants succumbed to infection similar to LVS-infected mice (Fig. 6B, C, and D), while 60 to 70% of the WT C57BL/6 mice infected with the ΔsodC mutant survived the infection. These results are similar to those observed in macrophages and suggest that F. tularensis SodC plays an important role in virulence in response to IFN-γ activation and that the absence of either iNOS or PHOX sensitizes mice to infection with the ΔsodC mutant.

DISCUSSION

The scavenging of O₂⁻ generated during the respiratory burst by F. tularensis SODs likely circumvents ROS-dependent killing prior to bacterial escape from phagosome to cytosol. Francisella tularensis can inhibit activation of the respiratory burst in neutrophils (25); however, this potential is limited in macrophages (1). Macrophages deficient for PHOX are less efficient at killing F. tularensis (21), suggesting a role for this enzyme in bacterial clearance. Macrophage-dependent killing of F. tularensis LVS is also associated with increased IFN-γ-dependent production of NO (12). Addition of iNOS inhibitors to IFN-γ-treated macrophages or neutralization of IFN-γ or tumor necrosis factor alpha prevents NO production and F. tularensis killing (12, 14). It has been proposed that the nearly diffusion-limited reaction product of O₂⁻ and NO, ONOO⁻, is primarily responsible for macrophage-dependent killing of F. tularensis (21). Our findings suggest that F. tularensis SODs play an important role in intramacrophage survival by primarily resisting microbicidal activity of extracellular ROS.

SODs play diverse roles in virulence and adaptation of many bacterial pathogens to their intracellular lifestyle (8, 22, 30, 34). F. tularensis encodes two putative sod genes, sodB and sodC. The constitutively expressed SodB of F. tularensis is required for survival, and its decreased activity both enhances its sensitivity to oxidative stress and attenuates bacterial virulence in mice (2). The SodB enzyme has been identified in secreted (31), soluble, and membrane fractions (our unpublished observations), while SodC is predicted to be localized to the periplasm, and its presence has been confirmed in membrane fractions (31). The transcript levels of sodB are consistently high even in the absence of an oxidative stress (our unpublished data), whereas sodC levels are induced following macrophage infection (33). The diversity of these two SODs encoded by F. tularensis prompted us to investigate whether they work cooperatively to protect from oxidative insult. Our studies indicate that F. tularensis LVS strains with single and multiple sod mutations exhibit enhanced sensitivity to ROS and RNS, reduced survival in macrophages, and an attenuated virulence in mice.

During construction of the F. tularensis LVS sodC mutant, no merodiploid reversions to the WT were observed, unlike the F. tularensis sodB mutant (2). This observation suggests that F. tularensis SodC is dispensable for survival when SodB is present. However, SodC was important for optimal growth in response to redox cycling drugs (paraquat and pyrogallol) and IFN-γ-activated macrophages. Partial restoration of intramacrophage growth of the sodC-deficient strains was observed when ROS or RNS production was impaired by PHOX or iNOS inhibition; on the other hand, the ΔsodC mutant regained its full virulence similar to WT mice in phox⁻^/⁻ or inos⁻^/⁻ mice. These findings suggest that SodC is primarily responsible for neutralizing extracellular ROS. It was also observed that the ΔsodC mutant was sensitive to paraquat similar to the sodB ΔsodC mutant. Paraquat redox cycles and generates O₂⁻ intracellularly so it was somewhat surprising to observe the loss of SodC-sensitized bacteria to paraquat while SodB was still present. Although F. tularensis SodC is predicted to reside in the periplasm, it has not been confirmed biochemically. It is possible that SodC may not be restricted to the periplasmic compartment or that periplasmic SodC may confer protection from intracellular sources of O₂⁻_. Both of these possibilities are currently being explored.

The increased sensitivity of the F. tularensis sodB ΔsodC mutant to ROS also indicates that both SODs confer combinatorial protection from oxidative stress. The sodB ΔsodC mutant was also more sensitive to H₂O₂ which is likely due to enhanced Fenton chemistry resulting from the failure to scavenge O₂⁻ (16). Both the WT LVS and the ΔsodC mutant can neutralize excess O₂⁻, restricting divalent metal reduction and subsequent OH⁻ radical production.

SOD deficiency failed to sensitize F. tularensis to the direct effects of NO when grown acellularly (Fig. (Fig.3).3). However, the growth of the SOD-deficient strains was impaired in response to long-term exposure to a low dose of IFN-γ or short-term exposure to a high dose of IFN-γ (Fig. (Fig.4)4) and was associated with higher NO production (data not shown). NO reacts with O₂⁻ to form ONOO⁻ at rates that limit diffusion (5). Furthermore, the intracellular survival of the SOD-deficient strains was partially restored by inhibiting either PHOX or iNOS activity. Further, virulence of the ΔsodC mutant was restored in both inos⁻^/⁻ and phox⁻^/⁻ mice to a level similar to that of the WT LVS, suggesting that F. tularensis SodC plays an important role in its virulence.

Our findings indicate that SODs are necessary for F. tularensis to achieve peak virulence. sod mutants of F. tularensis not only exhibit defects in their ability to survive and replicate within macrophages but are generally attenuated for virulence in the mouse model of respiratory tularemia. The virulence of the F. tularensis sodB ΔsodC mutant was significantly more attenuated than that for the ΔsodC mutant. The absence of IFN-γ, iNOS, or PHOX restored the virulence of ΔsodC mutant strains, suggesting that the CuZnSOD of F. tularensis plays a critical role in restricting the bactericidal affects of ROS and possibly RNS.

Given Francisella's need to survive in a diverse range of hosts and environments, it is not surprising it has evolved SODs that function independently. Many bacterial SODs confer redundant protection to maintain virulence (7), and on the basis of our observations, SodB appears to have an essential role in resistance to oxidative stress, and the ΔsodC mutant displays a phenotype similar to that of the attenuated F. tularensis sodB mutant (2). Given the roles of SodC in antioxidant defense, intramacrophage survival, and virulence of other bacterial pathogens (22), this enzyme may not serve a function redundant to SodB. It is also possible that SodB may cooperate with periplasmic SodC to neutralize exogenous O₂⁻ generated while the bacteria reside in phagosomes in addition to restricting endogenous production of O₂⁻. Functional redundancy and compensatory replacement for SODs have been reported in other bacterial species (7). However, the absence of one sod gene in F. tularensis does not allow for compensation by the other sod gene (our unpublished observations), suggesting that each SOD serves a unique purpose and may act in a combinatorial fashion. Our findings demonstrate that F. tularensis SODs are required for resistance to O₂⁻ generated by IFN-γ-activated macrophages and are not necessary for survival in quiescent macrophages. Overall, these studies provide evidence that F. tularensis SODs play an important role in its pathogenesis and serve to protect the pathogen from extracellular host-derived ROS.

Supplementary Material

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Acknowledgments

Technical support provided by Erin Moore and Michelle Wyland O'Brien is highly appreciated.

This work was supported by NIH grant P01 AI056320.

Footnotes

Published ahead of print on 14 August 2009.

^†Supplemental material for this article may be found at http://jb.asm.org/.

REFERENCES

1. Allen, L. A., and R. L. McCaffrey. 2007. To activate or not to activate: distinct strategies used by Helicobacter pylori and Francisella tularensis to modulate the NADPH oxidase and survive in human neutrophils. Immunol. Rev. 219:103-117. [PubMed]

2. Bakshi, C. S., M. Malik, K. Regan, J. A. Melendez, D. W. Metzger, V. M. Pavlov, and T. J. Sellati. 2006. Superoxide dismutase B gene (sodB)-deficient mutants of Francisella tularensis demonstrate hypersensitivity to oxidative stress and attenuated virulence. J. Bacteriol. 188:6443-6448. [PMC free article] [PubMed]

3. Baron, G. S., S. V. Myltseva, and F. E. Nano. 1995. Electroporation of Francisella tularensis. Methods Mol. Biol. 47:149-154. [PubMed]

4. Battistoni, A. 2003. Role of prokaryotic Cu,Zn superoxide dismutase in pathogenesis. Biochem. Soc. Trans. 31:1326-1329. [PubMed]

5. Beckman, J. S., and W. H. Koppenol. 1996. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271:C1424-C1437. [PubMed]

6. Conlan, J. W., and R. J. North. 1992. Early pathogenesis of infection in the liver with the facultative intracellular bacteria Listeria monocytogenes, Francisella tularensis, and Salmonella typhimurium involves lysis of infected hepatocytes by leukocytes. Infect. Immun. 60:5164-5171. [PMC free article] [PubMed]

7. Cybulski, R. J., Jr., P. Sanz, F. Alem, S. Stibitz, R. L. Bull, and A. D. O'Brien. 2009. Four superoxide dismutases contribute to Bacillus anthracis virulence and provide spores with redundant protection from oxidative stress. Infect. Immun. 77:274-285. [PMC free article] [PubMed]

8. De Groote, M. A., U. A. Ochsner, M. U. Shiloh, C. Nathan, J. M. McCord, M. C. Dinauer, S. J. Libby, A. Vazquez-Torres, Y. Xu, and F. C. Fang. 1997. Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase. Proc. Natl. Acad. Sci. USA 94:13997-14001. [PubMed]

9. Fang, F. C. 2004. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat. Rev. Microbiol. 2:820-832. [PubMed]

10. Feelisch, M., T. Rassaf, S. Mnaimneh, N. Singh, N. S. Bryan, D. Jourd'heuil, and M. Kelm. 2002. Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J. 16:1775-1785. [PubMed]

11. Forestal, C. A., J. L. Benach, C. Carbonara, J. K. Italo, T. J. Lisinski, and M. B. Furie. 2003. Francisella tularensis selectively induces proinflammatory changes in endothelial cells. J. Immunol. 171:2563-2570. [PubMed]

12. Fortier, A. H., T. Polsinelli, S. J. Green, and C. A. Nacy. 1992. Activation of macrophages for destruction of Francisella tularensis: identification of cytokines, effector cells, and effector molecules. Infect. Immun. 60:817-825. [PMC free article] [PubMed]

13. Golovliov, I., A. Sjostedt, A. Mokrievich, and V. Pavlov. 2003. A method for allelic replacement in Francisella tularensis. FEMS Microbiol. Lett. 222:273-280. [PubMed]

14. Green, S. J., C. A. Nacy, R. D. Schreiber, D. L. Granger, R. M. Crawford, M. S. Meltzer, and A. H. Fortier. 1993. Neutralization of gamma interferon and tumor necrosis factor alpha blocks in vivo synthesis of nitrogen oxides from l-arginine and protection against Francisella tularensis infection in Mycobacterium bovis BCG-treated mice. Infect. Immun. 61:689-698. [PMC free article] [PubMed]

15. Ho, S. N., H. D. Hunt, R. M. Horton, J. K. Pullen, and L. R. Pease. 1989. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77:51-59. [PubMed]

16. Imlay, J. A., and S. Linn. 1987. Mutagenesis and stress responses induced in Escherichia coli by hydrogen peroxide. J. Bacteriol. 169:2967-2976. [PMC free article] [PubMed]

17. Keyer, K., and J. A. Imlay. 1996. Superoxide accelerates DNA damage by elevating free-iron levels. Proc. Natl. Acad. Sci. USA 93:13635-13640. [PubMed]

18. Lauriano, C. M., J. R. Barker, S. S. Yoon, F. E. Nano, B. P. Arulanandam, D. J. Hassett, and K. E. Klose. 2004. MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc. Natl. Acad. Sci. USA 101:4246-4249. [PubMed]

19. Reference deleted.

20. Lindgren, H., H. Shen, C. Zingmark, I. Golovliov, W. Conlan, and A. Sjöstedt. 2007. Resistance of Francisella tularensis strains against reactive nitrogen and oxygen species with special reference to the role of KatG. Infect. Immun. 75:1303-1309. [PMC free article] [PubMed]

21. Lindgren, H., L. Stenman, A. Tarnvik, and A. Sjostedt. 2005. The contribution of reactive nitrogen and oxygen species to the killing of Francisella tularensis LVS by murine macrophages. Microbes Infect. 7:467-475. [PubMed]

22. Lynch, M., and H. Kuramitsu. 2000. Expression and role of superoxide dismutases (SOD) in pathogenic bacteria. Microbes Infect. 2:1245-1255. [PubMed]

23. Mahawar, M., G. S. Kirimanjeswara, D. W. Metzger, and C. S. Bakshi. 2009. Contribution of citrulline ureidase to Francisella tularensis strain Schu S4 pathogenesis. J. Bacteriol. 191:4798-4806. [PMC free article] [PubMed]

24. Malik, M., C. S. Bakshi, K. McCabe, S. V. Catlett, A. Shah, R. Singh, P. L. Jackson, A. Gaggar, D. W. Metzger, J. A. Melendez, J. E. Blalock, and T. J. Sellati. 2007. Matrix metalloproteinase 9 activity enhances host susceptibility to pulmonary infection with type A and B strains of Francisella tularensis. J. Immunol. 178:1013-1020. [PubMed]

25. McCaffrey, R. L., and L. A. Allen. 2006. Francisella tularensis LVS evades killing by human neutrophils via inhibition of the respiratory burst and phagosome escape. J. Leukoc. Biol. 80:1224-1230. [PMC free article] [PubMed]

26. McCord, J. M., and I. Fridovich. 1969. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244:6049-6055. [PubMed]

27. McCord, J. M., B. B. Keele, Jr., and I. Fridovich. 1971. An enzyme-based theory of obligate anaerobiosis: the physiological function of superoxide dismutase. Proc. Natl. Acad. Sci. USA 68:1024-1027. [PubMed]

28. Melillo, A., D. D. Sledjeski, S. Lipski, R. M. Wooten, V. Basrur, and E. R. Lafontaine. 2006. Identification of a Francisella tularensis LVS outer membrane protein that confers adherence to A549 human lung cells. FEMS Microbiol. Lett. 263:102-108. [PubMed]

29. Pacello, F., P. Ceci, S. Ammendola, P. Pasquali, E. Chiancone, and A. Battistoni. 2008. Periplasmic Cu,Zn superoxide dismutase and cytoplasmic Dps concur in protecting Salmonella enterica serovar Typhimurium from extracellular reactive oxygen species. Biochim. Biophys. Acta 1780:226-232. [PubMed]

30. Schnell, S., and H. M. Steinman. 1995. Function and stationary-phase induction of periplasmic copper-zinc superoxide dismutase and catalase/peroxidase in Caulobacter crescentus. J. Bacteriol. 177:5924-5929. [PMC free article] [PubMed]

31. Twine, S. M., N. C. Mykytczuk, M. D. Petit, H. Shen, A. Sjostedt, C. J. Wayne, and J. F. Kelly. 2006. In vivo proteomic analysis of the intracellular bacterial pathogen, Francisella tularensis, isolated from mouse spleen. Biochem. Biophys. Res. Commun. 345:1621-1633. [PubMed]

32. Vazquez-Torres, A., and F. C. Fang. 2001. Salmonella evasion of the NADPH phagocyte oxidase. Microbes Infect. 3:1313-1320. [PubMed]

33. Wehrly, T. D., A. Chong, K. Virtaneva, D. E. Sturdevant, R. Child, J. A. Edwards, D. Brouwer, V. Nair, E. R. Fischer, L. Wicke, A. J. Curda, J. J. Kupko III, C. Martens, D. D. Crane, C. M. Bosio, S. F. Porcella, and J. Celli. 2009. Intracellular biology and virulence determinants of Francisella tularensis revealed by transcriptional profiling inside macrophages. Cell. Microbiol. 11:1128-1150. [PMC free article] [PubMed]

34. Wilks, K. E., K. L. Dunn, J. L. Farrant, K. M. Reddin, A. R. Gorringe, P. R. Langford, and J. S. Kroll. 1998. Periplasmic superoxide dismutase in meningococcal pathogenicity. Infect. Immun. 66:213-217. [PMC free article] [PubMed]

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