JOURNAL OF BACTERIOLOGY, Oct. 2009, p. 6447–6456
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 191, No. 20
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
Received 21 April 2009/Accepted 3 August 2009
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
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 (?sodC) and a F. tularensis ?sodC mutant with
attenuated Fe-superoxide dismutase (sodB) gene expression (sodB ?sodC) were constructed and evaluated for
susceptibility to ROS and RNS in gamma interferon (IFN-?)-activated macrophages and a mouse model of
respiratory tularemia. The F. tularensis ?sodC and sodB ?sodC mutants showed attenuated intramacrophage
survival in IFN-?-activated macrophages compared to the wild-type F. tularensis LVS. Transcomplementing the
sodC gene in the ?sodC mutant or inhibiting the IFN-?-dependent production of O2
enhanced intramacrophage survival of the sod mutants. The ?sodC and sodB ?sodC 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 ?sodC mutant was restored in ifn-??/?,
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.
?). In the
?or nitric oxide (NO)
Francisella tularensis is considered a potential biological
threat due to its extreme infectivity, ease of artificial dissemi-
nation 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 nitro-
gen 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 superox-
The dismutation of O2
ROS and RNS in infected macrophages. Three major catego-
ries of SODs have been identified in bacteria and include Mn-,
Fe-, and CuZn-containing SODs (SodA, SodB, and SodC,
?) anion to hydrogen peroxide (H2O2) and O2(26).
?prevents accumulation of microbicidal
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
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 pro-
cess, which suggests that SODs may play an important role in
its intracellular survival and pathogenesis. We have demon-
strated 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 intra-
macrophage survival has not been defined. In this study we
have constructed a F. tularensis sodC mutant (?sodC) and a F.
tularensis sodBC double mutant (sodB ?sodC) and determined
that SodC in conjunction with SodB primarily protects the
pathogen from host-derived ROS and is required for intra-
macrophage 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 ?sodC mutant, a transcomplemented strain
(?sodC mutant carrying psodC [?sodC?psodC]), and a double mutant carrying
* 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: email@example.com.
Phone for J. Andres Melendez: (518) 262-8791. Fax: (518) 262-6161.
† Supplemental material for this article may be found at http://jb
# A.A.M. and M.M. contributed equally to this work.
?Published ahead of print on 14 August 2009.
an in-frame sodC gene deletion in the sodB gene (2) (sodB ?sodC) were con-
structed in the present study (Table 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% CO2or 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.
Construction of F. tularensis sodC and sodB ?sodC mutants and transcomple-
mentation. The plasmid constructs, bacterial strains, and the primer sequences
used in this study are shown in Table 1. An allelic replacement method was used
to construct an in-frame sodC gene deletion mutant (?sodC) and sodB ?sodC
mutants of F. tularensis LVS (13). For construction of the ?sodC 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::?sodC. The dele-
tion of the sodC gene in pDMK::?sodC was confirmed by PCR. The
pDMK::?sodC plasmid was transformed in Escherichia coli S17-1 and trans-
ferred into F. tularensis LVS via conjugal transfer (13). The mutants were se-
lected 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 hydro-
chloride, 1% hemoglobin, 10 ?g/ml kanamycin, and 100 ?g/ml polymyxin B (the
latter component was included for counterselection of the donor E. coli). Colo-
nies obtained on kanamycin plates following the first recombination were se-
lected for a second recombination event by plating on medium containing 5%
sucrose (13). Mutant colonies exhibiting kanamycin sensitivity and sucrose re-
sistance following the second recombination event were confirmed for gene
deletion by PCR. This mutant was referred to as the ?sodC 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 (ATG3GTG) 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 ?sodC mutant. The sodB ?sodC double muta-
tion was confirmed by multiplex PCR followed by digestion of the products with
the PstI restriction enzyme. The mutant cultures were stored at ?80?C.
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 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 kana-
mycin gene was digested with PstI and ligated into similarly digested pKK214
downstream of the F. tularensis groEL promoter to yield pKK214::sodC. This ap-
proach 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 DH5? 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 ?sodC mutant as described previously (3). The
transformants were selected on MH-chocolate agar plates containing kanamycin (20
?g/ml). The resultant transcomplemented strain was termed the ?sodC?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
TABLE 1. Primers, plasmid vectors, and bacterial strains used and generated in the present study
Primer,aplasmid vector, or
Primer sequence Reference
Up SodC R
Down SodC F
pKK SodC F
pKK SodC R
AGTAGG AAATAT CAACCTTAATA
E. coli strains
F. tularensis strains
sodB ?sodC double mutant
?sodC mutant carrying pKK214::sodC
aR, reverse; F, forward.
bThe primer code shows how primers are referred to in Fig. 1A.
6448MELILLO ET AL.J. BACTERIOL.
microliters of each culture was added to a 96-well microtiter plate in quadruplicate.
Various concentrations of ROS- or RNS-generating drugs, including O2?-generat-
ing 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). OD600was 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 ?l of
10 mM paraquat or 1 M pyrogallol (Sigma Aldrich, St. Louis, MO). The plates
were incubated at 37?C 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 pre-
viously (29). Briefly, bacterial cultures grown on MH-chocolate agar plates were
resuspended in sterile phosphate-buffered saline (PBS) at a concentration of 1 ?
109CFU/ml and treated with 250 ?M 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 H2O2generation 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 ?M of pure ONOO?or a mixture containing hypoxanthine/xanthine oxidase
and DETA-NONOate (250 ?M). Bacterial colonies were counted after 48 h and
expressed as log10CFU/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 mac-
rophage cell line, MH-S (ATTC CRL-2019), was left untreated or treated with
50 or 100 ng/ml gamma interferon (IFN-?) (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 postin-
fection, cells were treated with gentamicin for 2 hours (100 ?g/ml) to kill extra-
cellular bacteria. Medium containing gentamicin was then replaced with IFN-?-
containing medium without antibiotics, followed by incubation at 37?C 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 ?M NADPH oxidase (PHOX) inhibitor acetovanillone
(apocynin) (Arcos Organics, Morris Plains, NJ), 1 mM of the inducible nitric
oxide synthase (iNOS) inhibitor, NG-monomethyl-L-arginine (NMMLA) (Sigma
Aldrich, St. Louis, MO) or its nonfunctional homolog, 2-methyl adenine (Sigma
Aldrich, St. Louis, MO). The transcomplemented ?sodC?psodC strain was also
plated on MH-chocolate plates containing kanamycin (25 ?g/ml) to confirm that
the bacteria recovered from macrophages still carried the psodC plasmid. All
statistical analyses were performed using the Tukey-Kramer multiple-compari-
son 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-??/?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 ? 104CFU of LVS or sod
mutants in a volume of 20 ?l PBS (10 ?l/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.
Sequence analysis of SodC and verification of ?sodC and sodB
?sodC 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 ma-
ture 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
The genomic organization of the sodC gene is shown in Fig.
1A. The deletion of the entire 557-bp coding region of the
sodC gene in the F. tularensis ?sodC and sodB ?sodC 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 ?sodC mutant (Fig. 1B, lanes 3 and 4)
compared to the 880-bp product in the WT LVS (Fig. 1B, lanes
1 and 2) confirmed the sodC gene deletion. Restriction diges-
tion of the PCR product by the PstI enzyme confirmed muta-
tion of the sodB gene (Fig. 1B, lanes 2 and 4), while the WT
sodB gene showed resistance to this cleavage (Fig. 1B, lanes 1
FIG. 1. Confirmation of F. tularensis ?sodC and sodB ?sodC mu-
tants. (A) Genomic organization of the sodC gene of F. tularensis LVS.
The small arrows indicate the locations of the primers shown in Table
1 that were used for construction, screening, and confirmation of sodC
mutants. (B) Confirmation of sod mutations by PCR. A multiplex
colony PCR was performed to confirm the deletion/mutation of the
sod genes. Primers E and F were used for confirmation of sodC gene
deletion, while primer pairs described earlier (2) were used for con-
firmation of sodB mutation. The PCR products were digested with
PstI. The WT sodB gene is resistant to cleavage by PstI, whereas the
mutated sodB is not resistant and yields products of 600 and 650 bp.
Lanes; 1, F. tularensis LVS; 2, F. tularensis sodB mutant; 3, ?sodC
mutant; 4, sodB ?sodC mutant; 5, 1 kb plus DNA ladder.
VOL. 191, 2009SodC PROTECTS F. TULARENSIS FROM HOST-DERIVED OXIDANTS 6449
and 3). Regions flanking the deleted sodC gene were also
sequenced to ascertain that the gene deletions in ?sodC and
sodB ?sodC 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 ?sodC?psodC strain relative to the WT F. tularensis
LVS, while no sodC transcripts were observed for the ?sodC
mutant (data not shown).
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 avail-
able in small culture volumes in the microtiter plates. The
OD600values of microtiter plate growth curves similar to large-
volume cultures were also associated with an exponential in-
crease in the number of CFU. Although loss of SodC lead to a
slight decrease in OD600values in the later part of the growth,
no differences in the numbers of CFU for the WT LVS and
?sodC 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
compounds. Growth curves generated in response to paraquat
revealed that both the F. tularensis ?sodC and sodB ?sodC
mutants displayed significant increases in sensitivity to para-
quat compared to the WT LVS (Fig. 2A, top graphs). The
effective 50% inhibitory dose was calculated using linear re-
gression 18 h postinoculation. Both the sod mutants displayed
a significantly (P ? 0.01) increased sensitivity to paraquat (40
and 27 ?M for the ?sodC and sodB ?sodC mutants, respec-
tively) compared to the ED50for the LVS of 120 ?M. A role
for F. tularensis SODs in conferring resistance to H2O2was
also analyzed. The sodB ?sodC mutant exhibited greater sen-
sitivity toward 4 mM H2O2(ED50of 3.28 mM), while the
growth of the ?sodC mutant or the WT LVS (ED50of 3.9 mM
for both) was only slightly impaired (Fig. 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 ?sodC mutant is likely more sen-
sitive to H2O2as a result of Fenton chemistry.
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 ?sodC and sodB ?sodC
mutants exhibit greater sensitivity to O2
does (Fig. 2B). The bacterial killing assays revealed that both
the ?sodC and sodB ?sodC mutants were significantly more
sensitive to exogenously generated O2
tion of hypoxanthine and xanthine oxidase than the WT LVS
was (Fig. 2C). The xanthine/xanthine oxidase system generates
affects of H2O2and would reveal the effect of O2
killing independently of H2O2. The addition of catalase (1
U/ml) to the hypoxanthine/xanthine oxidase system restored
growth of the sodB ?sodC mutant to a level near that of the
?than the WT LVS
?catalyzed by the addi-
?and H2O2. The addition of catalase can negate the
?sodC mutant 6 hours postexposure (Fig. 2D). These obser-
vations suggest that the SODs act independently of one
another in affording protection from extracellularly derived
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 pat-
terns of the ?sodC or sodB ?sodC mutant or the WT LVS. None
of the strains displayed statistically significant differences in sen-
sitivity to presynthesized ONOO?in either the growth (Fig. 3) or
bacterial killing assay (data not shown).
sod mutants of F. tularensis are attenuated for growth in
IFN-?-activated macrophages. We next evaluated the impor-
tance of F. tularensis SODs in intramacrophage survival. Infec-
tion of the MH-S alveolar macrophage cell line with the ?sodC
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 in-
fected with the F. tularensis sodB or sodB ?sodC mutant, re-
spectively (Fig. 4A). Infection of IFN-? (50 ng/ml)-activated
macrophages impeded the growth of all strains equally (10- to
15-fold) 24 h postinfection (Fig. 4A). However, significantly
fewer sodB, ?sodC, and sodB ?sodC mutants were recovered
from IFN-?-activated MH-S cells 48 h postinfection compared
to infection with the WT LVS (Fig. 4B). Stimulation of MH-S
cells with a higher dose of IFN-? (100 ng/ml) inhibited growth
of the sodB, ?sodC, and sodB ?sodC mutants 24 h postinfec-
tion compared to the WT LVS and led to complete bacterial
killing 48 h postinfection (Fig. 4A and B). Complementation of
?sodC enhanced the intramacrophage survival to a level nearly
similar to that of the WT LVS in IFN-?-stimulated macro-
phages, 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 micro-
bicidal activity of IFN-?-activated macrophages and that over-
production of SodC partially suppresses this activity.
Intramacrophage growth defects of sod mutants of F. tula-
rensis are rescued by inhibition of ROS or RNS production.
We next asked whether the enhanced killing of the F. tularensis
sodC and sodB ?sodC mutants by IFN-?-activated MH-S could
be blocked by PHOX or iNOS inhibition. As shown in Fig. 5,
PHOX (apocynin) or iNOS (NMMLA) inhibition partially re-
stored the growth of the LVS in IFN-?-activated MH-S cells.
Similarly, the intracellular growth defect of the sodB,?sodC,
and sodB ?sodC mutants in IFN-?-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 ?sodC mutant in IFN-?-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. Further-
more, 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 sus-
ceptibility of the mutants to O2
in Fig. 5A.
?and that catalase protects from xanthine/xanthine oxi-
??-mediated killing as observed
6450 MELILLO ET AL.J. BACTERIOL.
F. tularensis SODs are required for virulence in mice. Re-
cent 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-?-stimulated macrophages, we investigated the virulence
of F. tularensis ?sodC and sodB ?sodC mutants in mice. The
C57BL/6 mice were challenged intranasally with 1 ? 104CFU
of the ?sodC or sodB ?sodC 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
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). OD600was measured every 6 h for 24 h and plotted against time
postinoculation. Data represent means ? standard errors (error bars) of quadruplicate samples. (B) Disc diffusion assay in response to paraquat
and pyragallol. The zones of inhibitions were measured using AlphaEase FC software (Alpha Innotech Inc., San Leandro, CA). Data represent
means plus standard errors (SE) (error bars) of the zone of inhibition determined for each strain (five plates per strain). (C) Bacterial killing assay
in response to hypoxanthine/xanthine oxidase. ND, not detected. (D) Bacterial killing assay in response to hypoxanthine/xanthine oxidase and
catalase. The numbers of viable sod mutants and F. tularensis LVS were quantified by plating bacterial dilutions on chocolate agar plates. The
results are expressed as mean plus SE. All results are representative of at least two independent experiments, statistical analysis was performed
using the Tukey-Kramer multiple-comparison test, and a P of ?0.05 was considered statistically significant.
VOL. 191, 2009SodC PROTECTS F. TULARENSIS FROM HOST-DERIVED OXIDANTS6451
succumbed to infection by day 12, while more than 50% of the
mice challenged with the ?sodC mutant and 80% of the mice
challenged with the sodB ?sodC mutant survived infection.
These results suggest that although the ?sodC mutant still
retains its residual virulence, the sodB ?sodC mutant exhibits
an attenuated virulence in mice (Fig. 6A).
Protection against ?sodC mutants in mice is IFN-?, iNOS,
and PHOX dependent. The in vitro macrophage assays sug-
gested that ROS or RNS produced in response to IFN-? acti-
vation plays a prominent role in restricting the intracellular
survival of the F. tularensis ?sodC mutant. It was also observed
that nearly 50% of the mice infected with the ?sodC mutant
survived lethal challenge and cleared infection. It was further
investigated whether IFN-?, iNOS, and PHOX are required to
provide protection against challenge with the ?sodC mutant.
C57BL/6 mice deficient for either ifn-??/?, inos?/?, or phox?/?
and their syngeneic WT counterparts were challenged intrana-
sally with 1 ? 104CFU of LVS or the ?sodC mutant. All of the
WT C57BL/6 mice infected with LVS died by day 12 postin-
fection. The ifn-??/?, inos?/?, and phox?/?mice infected with
?sodC 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 ?sodC mutant survived the infection.
These results are similar to those observed in macrophages and
suggest that F. tularensis SodC plays an important role in vir-
ulence in response to IFN-? activation and that the absence of
either iNOS or PHOX sensitizes mice to infection with the
The scavenging of O2
burst by F. tularensis SODs likely circumvents ROS-depen-
dent 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), suggest-
ing a role for this enzyme in bacterial clearance. Macroph-
age-dependent killing of F. tularensis LVS is also associated
with increased IFN-?-dependent production of NO (12).
Addition of iNOS inhibitors to IFN-?-treated macrophages
?generated during the respiratory
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). OD600was measured every 6 h for 24 h and plotted against time postinoculation.
FIG. 4. Intramacrophage survival of sod mutants of F. tularensis.
MH-S cells treated with 50 or 100 ng/ml of IFN-? for 16 h prior to
infection and thereafter were infected with the WT LVS or sod mu-
tants at an MOI of 100. Untreated cells were kept as controls. The cells
were lysed at 24 and 48 h postinfection, and 10-fold dilutions were
plated on chocolate agar plates for enumeration of bacterial CFU. The
results are cumulative of two independent experiments, each per-
formed in triplicate wells, and expressed as means plus standard errors
(error bars). The data are analyzed using the Tukey-Kramer multiple-
comparison test. Differences between the experimental groups were
considered significant if P was ?0.05. ND, not detected.
6452 MELILLO ET AL. J. BACTERIOL.
or neutralization of IFN-? 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
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 mi-
crobicidal 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 viru-
lence in mice (2). The SodB enzyme has been identified in
?and NO, ONOO?, is primarily responsible
secreted (31), soluble, and membrane fractions (our unpub-
lished observations), while SodC is predicted to be localized to
the periplasm, and its presence has been confirmed in mem-
brane fractions (31). The transcript levels of sodB are consis-
tently 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
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-? and either PHOX inhibitor apocynin (250 ?M) (A) or iNOS inhibitor NMMLA (1 mM) (B). Untreated
macrophages, macrophages treated with IFN-? alone or 2-methyladenine (2-MA) were used as controls. The cells were infected at an MOI of 100
and lysed at 24 and 48 h postinfection and plated on chocolate agar plates for enumeration of bacterial CFU. The results are representative of
two experiments performed in triplicate wells and expressed as means plus standard errors (error bars). Statistical significance was examined with
one-way analysis of variance, and P values were recorded.
VOL. 191, 2009SodC PROTECTS F. TULARENSIS FROM HOST-DERIVED OXIDANTS 6453
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-?-activated macrophages. Partial restoration of intramac-
rophage 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 ?sodC mutant re-
gained 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 ob-
served that the ?sodC mutant was sensitive to paraquat similar
to the sodB ?sodC mutant. Paraquat redox cycles and gener-
serve 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 biochem-
ically. 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
possibilities are currently being explored.
The increased sensitivity of the F. tularensis sodB ?sodC
?intracellularly so it was somewhat surprising to ob-
??.Both of these
mutant to ROS also indicates that both SODs confer combi-
natorial protection from oxidative stress. The sodB ?sodC mu-
tant was also more sensitive to H2O2which is likely due to
enhanced Fenton chemistry resulting from the failure to scav-
neutralize excess O2
subsequent OH?radical production.
SOD deficiency failed to sensitize F. tularensis to the direct
effects of NO when grown acellularly (Fig. 3). However, the
growth of the SOD-deficient strains was impaired in response
to long-term exposure to a low dose of IFN-? or short-term
exposure to a high dose of IFN-? (Fig. 4) and was associated
with higher NO production (data not shown). NO reacts with
more, the intracellular survival of the SOD-deficient strains
was partially restored by inhibiting either PHOX or iNOS
activity. Further, virulence of the ?sodC 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 im-
portant role in its virulence.
Our findings indicate that SODs are necessary for F. tula-
rensis to achieve peak virulence. sod mutants of F. tularensis
?(16). Both the WT LVS and the ?sodC mutant can
?, restricting divalent metal reduction and
?to form ONOO?at rates that limit diffusion (5). Further-
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 ? 104CFU of F. tularensis LVS or F. tularensis ?sodC or sodB ?sodC mutant. Comparisons between LVS and the sod mutants are shown.
ifn-??/?(B), inos?/?(C), and phox?/?(D) mice (six or seven mice per group) and wild-type C57BL/6 mice (six to eight mice per group) were
infected intranasally with 1 ? 104CFU of F. tularensis LVS or the ?sodC mutant. The mice were monitored for 21 days postinfection for
morbidity and mortality. The results are expressed as Kaplan-Meier survival curves, and the data were analyzed using the log-rank test. P values indicate
that the median time to death was significantly higher in WT C57BL/6 mice infected with the ?sodC mutant than in mice infected with F. tularensis LVS.
6454 MELILLO ET AL.J. BACTERIOL.
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 ?sodC mutant was significantly more
attenuated than that for the ?sodC mutant. The absence of
IFN-?, iNOS, or PHOX restored the virulence of ?sodC mu-
tant strains, suggesting that the CuZnSOD of F. tularensis plays
a critical role in restricting the bactericidal affects of ROS and
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 ?sodC
mutant displays a phenotype similar to that of the attenu-
ated F. tularensis sodB mutant (2). Given the roles of SodC
in antioxidant defense, intramacrophage survival, and viru-
lence 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
phagosomes in addition to restricting endogenous produc-
tion of O2
placement 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 combinato-
rial fashion. Our findings demonstrate that F. tularensis
SODs are required for resistance to O2
?-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 extra-
cellular host-derived ROS.
?generated while the bacteria reside in
?. Functional redundancy and compensatory re-
?generated by IFN-
Technical support provided by Erin Moore and Michelle Wyland
O’Brien is highly appreciated.
This work was supported by NIH grant P01 AI056320.
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.
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.
3. Baron, G. S., S. V. Myltseva, and F. E. Nano. 1995. Electroporation of
Francisella tularensis. Methods Mol. Biol. 47:149–154.
4. Battistoni, A. 2003. Role of prokaryotic Cu,Zn superoxide dismutase in
pathogenesis. Biochem. Soc. Trans. 31:1326–1329.
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–
6. Conlan, J. W., and R. J. North. 1992. Early pathogenesis of infection in the
liver with the facultative intracellular bacteria Listeria monocytogenes, Fran-
cisella tularensis, and Salmonella typhimurium involves lysis of infected hepa-
tocytes by leukocytes. Infect. Immun. 60:5164–5171.
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.
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.
9. Fang, F. C. 2004. Antimicrobial reactive oxygen and nitrogen species: con-
cepts and controversies. Nat. Rev. Microbiol. 2:820–832.
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 bio-
logical tissues and fluids: implications for the fate of NO in vivo. FASEB J.
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.
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–
13. Golovliov, I., A. Sjostedt, A. Mokrievich, and V. Pavlov. 2003. A method for
allelic replacement in Francisella tularensis. FEMS Microbiol. Lett. 222:273–
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.
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.
16. Imlay, J. A., and S. Linn. 1987. Mutagenesis and stress responses induced in
Escherichia coli by hydrogen peroxide. J. Bacteriol. 169:2967–2976.
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.
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 viru-
lence factors necessary for Francisella tularensis intraamoebae and intra-
macrophage survival. Proc. Natl. Acad. Sci. USA 101:4246–4249.
19. Reference deleted.
20. Lindgren, H., H. Shen, C. Zingmark, I. Golovliov, W. Conlan, and A. Sjo ¨s-
tedt. 2007. Resistance of Francisella tularensis strains against reactive nitro-
gen and oxygen species with special reference to the role of KatG. Infect.
21. Lindgren, H., L. Stenman, A. Tarnvik, and A. Sjostedt. 2005. The contribu-
tion of reactive nitrogen and oxygen species to the killing of Francisella
tularensis LVS by murine macrophages. Microbes Infect. 7:467–475.
22. Lynch, M., and H. Kuramitsu. 2000. Expression and role of superoxide
dismutases (SOD) in pathogenic bacteria. Microbes Infect. 2:1245–1255.
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.
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.
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.
26. McCord, J. M., and I. Fridovich. 1969. Superoxide dismutase. An enzymic
function for erythrocuprein (hemocuprein). J. Biol. Chem. 244:6049–
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.
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 mem-
brane protein that confers adherence to A549 human lung cells. FEMS
Microbiol. Lett. 263:102–108.
29. Pacello, F., P. Ceci, S. Ammendola, P. Pasquali, E. Chiancone, and A.
Battistoni. 2008. Periplasmic Cu,Zn superoxide dismutase and cytoplas-
mic Dps concur in protecting Salmonella enterica serovar Typhimurium
from extracellular reactive oxygen species. Biochim. Biophys. Acta 1780:
VOL. 191, 2009SodC PROTECTS F. TULARENSIS FROM HOST-DERIVED OXIDANTS6455
30. Schnell, S., and H. M. Steinman. 1995. Function and stationary-phase in- Download full-text
duction of periplasmic copper-zinc superoxide dismutase and catalase/per-
oxidase in Caulobacter crescentus. J. Bacteriol. 177:5924–5929.
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. Bio-
chem. Biophys. Res. Commun. 345:1621–1633.
32. Vazquez-Torres, A., and F. C. Fang. 2001. Salmonella evasion of the NADPH
phagocyte oxidase. Microbes Infect. 3:1313–1320.
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 tularen-
sis revealed by transcriptional profiling inside macrophages. Cell. Microbiol.
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 me-
ningococcal pathogenicity. Infect. Immun. 66:213–217.
6456 MELILLO ET AL.J. BACTERIOL.