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Infection and Immunity, October 1999, p. 5345-5351, Vol. 67, No. 10
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Neutropenia Restores Virulence to an Attenuated
Cu,Zn Superoxide Dismutase-Deficient Haemophilus ducreyi
Strain in the Swine Model of Chancroid
Lani R.
San Mateo,1
Kristen L.
Toffer,1
Paul E.
Orndorff,2 and
Thomas H.
Kawula1,*
Department of Microbiology and Immunology,
University of North Carolina School of Medicine, Chapel Hill, North
Carolina 27599,1 and Department of
Microbiology, Pathology and Parasitology, College of Veterinary
Medicine, North Carolina State University, Raleigh, North Carolina
276062
Received 22 June 1999/Accepted 21 July 1999
 |
ABSTRACT |
Haemophilus ducreyi causes chancroid, a sexually
transmitted cutaneous genital ulcer disease associated with increased
heterosexual transmission of human immunodeficiency virus. H. ducreyi expresses a periplasmic copper-zinc superoxide dismutase
(Cu,Zn SOD) that protects the bacterium from killing by exogenous
superoxide in vitro. We hypothesized that the Cu,Zn SOD would protect
H. ducreyi from immune cell killing, enhance survival, and
affect ulcer development in vivo. In order to test this hypothesis and
study the role of the Cu,Zn SOD in H. ducreyi pathogenesis,
we compared a Cu,Zn SOD-deficient H. ducreyi strain to its
isogenic wild-type parent with respect to survival and ulcer
development in immunocompetent and immunosuppressed pigs. The Cu,Zn
SOD-deficient strain was recovered from significantly fewer inoculated
sites and in significantly lower numbers than the wild-type parent
strain or a merodiploid (sodC+ sodC) strain
after infection of immunocompetent pigs. In contrast, survival of the
wild-type and Cu,Zn SOD-deficient strains was not significantly
different in pigs that were rendered neutropenic by treatment with
cyclophosphamide. Ulcer severity in pigs was not significantly
different between sites inoculated with wild type and sites inoculated
with Cu,Zn SOD-deficient H. ducreyi. Our data suggest that
the periplasmic Cu,Zn SOD is an important virulence determinant in
H. ducreyi, protecting the bacterium from host immune cell
killing and contributing to survival and persistence in the host.
| |
INTRODUCTION |
Haemophilus ducreyi is
the causative agent of chancroid, a sexually transmitted cutaneous
genital ulcer disease (13, 18). The observation that
chancroid facilitates human immunodeficiency virus transmission
(16, 19, 22-24) has provided additional impetus for the
study of the pathogenesis of H. ducreyi. Several potential
H. ducreyi virulence factors have been identified, including lipooligosaccharide (3), fine tangled pili (2), a
cytolethal distending toxin (5), and a hemolysin
(21). A recent study has established a requirement for the
hemoglobin binding protein for survival of H. ducreyi in a
temperature-dependent rabbit model of infection (29).
Histological features characteristic of chancroid include micropustules
on the skin surface consisting of neutrophils in necrotic debris and
dermal infiltrates of activated T cells and macrophages (8, 14,
26, 27). Despite the density of immune cells in chancroid
lesions, viable H. ducreyi organisms are commonly recovered
from ulcers (18, 26, 27), suggesting that H. ducreyi possesses defense mechanisms against immune cell killing.
There is evidence from in vitro studies that H. ducreyi is
resistant to the oxidative burst products of neutrophils (15, 20). Neutrophilic bactericidal activity is primarily attributed to an oxidative burst reaction that leads to the evolution of toxic
oxygen radicals, including superoxide (4, 10). In other pathogenic bacteria such as Nocardia asteroides,
Salmonella typhimurium, and Shigella flexneri,
the expression of superoxide dismutase (SOD) confers protection from
oxidative damage caused by exposure to activated neutrophils (1,
6, 7).
SODs are metalloenzymes that catalyze the conversion of superoxide
radical to oxygen and hydrogen peroxide (reviewed in references 9 and 30). Inhibition of
superoxide accumulation by SOD prevents the formation of the highly
mutagenic and cytotoxic hydroxyl radical (10). Thus,
bacterial SODs may constitute the first line of defense against
oxidative killing by host immune cells.
H. ducreyi expresses a periplasmic copper-zinc SOD (Cu,Zn
SOD) (28) that protects against superoxide killing in vitro
(25). In this study, we examined the contribution of the
periplasmic Cu,Zn SOD to H. ducreyi survival and the early
development of chancroid skin lesions in vivo. We have previously
developed a swine model of H. ducreyi infection
(12) that exploits the high degree of structural and
physiological similarity between juvenile pig skin and human skin. In
this model, ulcers histologically resembling human chancroid lesions
are produced by inoculating H. ducreyi into the skin of
young pigs by using an allergen delivery device. As in human infection,
viable H. ducreyi can be recovered from pig skin weeks after
inoculation. In addition, major features of the pig immune response are
similar to the human immune response to H. ducreyi;
neutrophils, T cells, and macrophages are predominant at the site of
infection, and protective immunity does not develop as a consequence of
infection (12).
Herein, we report the first use of the swine model for comparing
wild-type H. ducreyi to an isogenic mutant strain with an insertion at a single chromosomal locus, sodC. Since we
hypothesized that the Cu,Zn SOD enzyme protects H. ducreyi
from killing by host neutrophils, we also compared survival and lesion
formation of wild-type and sodC mutant strains in
neutrophil-depleted (neutropenic) pigs.
| |
MATERIALS AND METHODS |
Animals.
Crossbred (Yorkshire, Landrace, and Hampshire
cross) 6- to 12-week-old female pigs were used in experiments as
described previously (12). Briefly, pigs were housed at
ambient temperatures (20 to 25°C) in an American Association for
Accreditation of Laboratory Animal Care-accredited P2 containment
facility at North Carolina State University. Water and antibiotic-free
growth ration were provided ad libitum. Animals were sedated for all
procedures with 2 mg each of ketamine HCl (Fort Dodge Laboratories,
Fort Dodge, Iowa) and xylazine (Miles Laboratories, Shawnee Mission,
Kans.) per kg of body weight. Pigs were kept in individual enclosures after inoculation and biopsied on either the second or the seventh day
postinoculation. Pigs were rendered immune cell deficient by
administration of cyclophosphamide (Mead Johnson, Princeton, N.J.) at
50 mg per kg 4 days preinoculation and 20 mg per kg every other day
thereafter until the day of biopsy (17). Peripheral blood
was drawn every 48 h to monitor numbers of circulating immune cells.
Bacterial inoculum preparation.
H. ducreyi 35000 (ATCC
33922), the isogenic mutant strain 35000-sodC-cat, and the
merodiploid strain 35000-sodC+-sodC
(25) were grown from frozen stocks and passaged once on chocolate agar plates consisting of 2.5% brain heart infusion, 1.5%
Bacto agar (Difco, Detroit, Mich.), 1% hemoglobin, 1% IsoVitaleX (Becton Dickinson, Cockeysville, Md.), and 10% fetal calf serum (Life
Technologies, Gaithersburg, Md.) at 35°C in a humidified atmosphere
with 5% CO2. Chloramphenicol and vancomycin were used at 1 and 3 µg/ml, respectively.
Inocula were prepared essentially as described elsewhere
(12) except that H. ducreyi cells harvested from
plates with swabs and resuspended in phosphate-buffered saline (PBS)
were forced through 30-gauge needles to create single-cell suspensions.
Bacterial cell suspensions were quantitated by duplicate culture of
serial 10-fold dilutions. Heat-killed H. ducreyi organisms
used as controls were prepared by heating inocula for 10 min in a
boiling water bath.
Inoculation.
Each pig was inoculated with the wild-type,
mutant, and merodiploid strains or the wild-type and mutant strains and
heat-killed controls. The dorsal surface of each pig ear was cleansed
with 95% ethanol and inoculated at multiple sites with a single strain of H. ducreyi at different doses. Bacterial inocula were
vortexed vigorously prior to each inoculation. At each site, 10 µl of
bacterial suspension containing approximately 105,
106, or 107 organisms was applied to the skin
by using a multitest applicator (MTA; Lincoln Diagnostics, Decatur,
Ill.) as described elsewhere (12, 27).
Biopsy and sample preparation.
Lesion biopsy specimens were
taken with disposable 6-mm-diameter skin punches (Acuderm, Ft.
Lauderdale, Fla.). For histological study and observations of H. ducreyi recovery, biopsy specimens were cut in half. Sample halves
for histological observation were fixed in 4% paraformaldehyde in PBS
at 4°C, embedded in paraffin, sectioned, and stained with hematoxylin
and eosin (Histopathology Reference Laboratory, Richmond, Calif.). All
slides were coded and evaluated blindly by one of the authors. Sample
halves for assessment of H. ducreyi recovery were minced and
cultured on chocolate agar.
Quantitation of H. ducreyi organisms delivered into
pig skin by MTA.
Inocula were prepared as described above and
serially diluted. H. ducreyi organisms at different doses
from 103 to 107 CFU were loaded in 10-µl
volumes onto MTA pads and applied with pressure to ethanol-cleansed pig
skin for 10 s. Five minutes after application, inoculated sites
were washed with streams of 5 ml of PBS and removed with 6-mm-diameter
biopsy punches. The 6-mm-diameter punch biopsy specimens were minced
finely with scalpels, suspended in 5 ml of PBS in 50-ml polypropylene
tubes, and homogenized with a rotor-stator homogenizer (Tissue-Tearor;
Biospec Products, Bartlesville, Okla.) at 30,000 rpm for 30 s.
Dilutions of the homogenate were cultured on chocolate agar. The
undelivered H. ducreyi organisms were quantitated by
vigorously washing the MTA pads five times with 100 µl of PBS,
pooling all washes, and culturing dilutions.
Statistical procedures.
Statistical analyses were performed
with SPSS version 7.5 (SPSS, Inc., Chicago, Ill.) and Sigma Stat
version 2.0 (Jandel Scientific, San Rafael, Calif.). Data from pig
lesions were analyzed within a hierarchical linear model to accommodate
the nested nature of observing several lesions on each pig.
Calculations were performed with (i) pig and strain and (ii) strain
only as factors or covariates. Analysis of variance (ANOVA) was used
for comparing histology scores. Logistic regression was used to analyze
recovery data as a dichotomous dependent variable. The Kruskal-Wallis
ANOVA on ranks and multiple comparisons by Dunn's method were used to analyze numerical recovery data. The Mann-Whitney rank sum test was
used to determine correlation between lesion histology and presence of
live bacteria. A P value of <0.05 was considered significant.
| |
RESULTS |
Survival of the wild-type and Cu,Zn SOD-deficient H. ducreyi in the pig.
We previously created a Cu,Zn
SOD-deficient strain of H. ducreyi isogenic to H. ducreyi 35000 by inserting a chloramphenicol acetyltransferase
cassette into the open reading frame of the chromosomal sodC
gene. We showed that the Cu,Zn SOD-deficient strain had a significantly
increased susceptibility to killing by exogenous superoxide compared to
the wild-type parent or merodiploid sodC+ sodC H. ducreyi strains (25). We hypothesized that Cu,Zn SOD activity confers resistance to superoxide generated by neutrophils and
phagocytes during oxidative burst reactions in the course of the host
immune response to H. ducreyi infection. Consequently, the
Cu,Zn SOD-deficient strain would be predicted to have a decreased rate
of survival in vivo.
We inoculated pigs at multiple sites with 2.5 × 10
6
to 2.7 × 10
7 CFU (delivering an estimated dose of
10
4 to 10
5 CFU; see following section) of each
strain individually to directly
compare the ability of the wild-type,
Cu,Zn SOD-deficient, and
merodiploid
sodC+ sodC H. ducreyi strains to survive and persist in vivo. We harvested
biopsy specimens 2 and 7 days postinfection, halved the biopsy
specimens, and cultured minced halves on chocolate agar. We compared
the numbers of bacteria recovered from biopsy specimens of sites
inoculated with the three strains. We found significant differences
among the three strains with respect to the numbers of bacteria
recovered from biopsy specimens taken 2 and 7 days postinoculation
(
P = 0.049 and <0.001, respectively [Kruskal-Wallis
ANOVA]) (Fig.
1). Viable Cu,Zn
SOD-deficient
H. ducreyi organisms were recovered
from day 7 pig biopsy specimens less often and in significantly
fewer numbers than
wild-type (
P < 0.01 [Dunn's method]) and
merodiploid
sodC+ sodC (
P < 0.01 [Dunn's method])
H. ducreyi. The recovery of
merodiploid
sodC+ sodC H. ducreyi was not
significantly different from that of
the wild type, demonstrating that
the
cat insertion in the chromosome
at the
sodC
locus was not in itself responsible for the decreased
recovery of the
Cu,Zn SOD-deficient strain. Recovery of
H. ducreyi strains 2 days postinoculation followed the same trend, with Cu,Zn
SOD-deficient
H. ducreyi recovered from fewer biopsy specimens
and in
lower numbers than wild-type and merodiploid bacteria.
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FIG. 1.
Frequency distribution histogram showing number of
colonies of H. ducreyi strains recovered from biopsy
specimens 2 (A) and 7 (B) days postinoculation. Recovery numbers were
not determined for five and four sites infected with wild-type and
sodC mutant H. ducreyi, respectively. mero,
merodiploid; wt, wild type.
|
|
Using logistic regression analysis, we determined that wild-type
H. ducreyi was significantly more likely than Cu,Zn
SOD-deficient
H. ducreyi to be recovered from inoculated
sites on day 2 (97
versus 59% recovery [Table
1]; odds ratio, 15.7; 95% confidence
interval, 1.9 to 128.9;
P = 0.010) and day 7 (97 versus
64% recovery
[Table
2]; odds ratio,
17.5; 95% confidence interval, 2.1 to
143.7;
P = 0.008) postinoculation.
In contrast with the near-100% recovery of the wild-type strain, live
recovery of the
sodC mutant strain varied from pig to
pig
(Tables
1 and
2). To illustrate, 2 days postinoculation,
the
sodC mutant strain was recovered from all the inoculated
sites
on five pigs but from none of the inoculated sites on the other
four
pigs.
The recovery rate of the
sodC mutant strain 2 days
postinoculation was not significantly different from its day 7 recovery
rate. In order to test whether the Cu,Zn SOD-null
H. ducreyi
that
persisted in pig skin to days 2 and 7 still contained the
cat insertion, colonies from recovery plates were picked and
replated
on chocolate agar containing chloramphenicol. One hundred
percent
of colonies tested were chloramphenicol resistant and deficient
in Cu,Zn SOD
activity.
Our observations are consistent with the notion that the ability of
H. ducreyi to survive in normal immunocompetent pigs was
significantly impaired by the loss of periplasmic SOD
activity.
Survival of the wild-type and Cu,Zn SOD-null H. ducreyi
strains in neutropenic pigs.
We hypothesized that the Cu,Zn
SOD-deficient H. ducreyi strain survived less efficiently in
the pig because it lacks a protective mechanism against neutrophil
killing. If so, the null strain should not differ from the wild type
with respect to survival in an in vivo system without these immune
cells. To test this hypothesis, we pretreated pigs with
cyclophosphamide, a widely used immunosuppressive agent.
Cyclophosphamide treatment of pigs leads to depletion of circulating
neutrophils (data not shown) and reduction of B- and T-cell numbers
(17).
Comparing the abilities of the
H. ducreyi strains to
replicate in neutropenic pigs required quantitating the numbers of
bacteria
inoculated and recovered. The tendency of
H. ducreyi to clump
is a significant source of inaccuracy in
determining numbers of
bacteria used in in vitro and in vivo
experiments. In a series
of experiments, we loaded MTAs with
single-cell suspensions of
H. ducreyi that had been forced
through a 30-gauge needle and
applied the inocula into pig skin.
Bacteria in suspensions thus
treated were visible microscopically as
mostly single cells with
some small groups of cells (data not shown).
Five minutes after
application, we washed the inoculated pig skin
surface with streams
of sterile PBS, harvested the sites with a skin
punch, and homogenized
the biopsy specimen. Of bacteria loaded onto the
MTA pads, (0.42
± 0.22)% was delivered into the skin. This
result was corroborated
by quantitative culture of the bacteria
remaining on the pads
after application and the bacteria that were
recovered from the
PBS rinses. Thus, 2.5 × 10
6 to
2.7 × 10
7 CFU loaded onto MTA pads corresponds to
1.0 × 10
4 to 1.1 × 10
5 CFU of
H. ducreyi delivered into live pig
skin.
Using these quantitative methods to assess inocula and recovery of
H. ducreyi from pigs, we found that total numbers of both
wild-type and
sodC mutant
H. ducreyi strains
increased in neutropenic
pigs. In a representative experiment (of two
experiments performed),
wild-type recovery on day 2 was (1.2 ± 0.5) × 10
4 CFU (average of four sites) from an
inoculum of (1.1 ± 0.6) ×
10
4 CFU. Recovery of
sodC mutant
H. ducreyi was (2.7 ± 0.3) × 10
5 CFU (average of four sites) from a delivered
inoculum of (1.7
± 0.9) × 10
4 CFU. In contrast,
drastic reductions in viability of both strains,
but particularly the
Cu,Zn SOD-deficient strain, were observed
with immunocompetent pigs. In
a representative quantitative experiment
(of two), wild-type
H. ducreyi numbers decreased from (1.7 ± 0.8)
× 10
4 CFU on day of inoculation to (1.7 ± 1.0) × 10
3 CFU on day 2 (average of four sites). Numbers of
sodC mutant
H. ducreyi organisms decreased from
(7.3 ± 3.0) × 10
4 to (1.2 ± 0.9) × 10
1 CFU (average of four sites, of which two were negative
for
recovery).
In addition to the quantitative experiments above, we inoculated seven
neutropenic pigs with both
H. ducreyi strains and harvested
biopsy specimens on days 2 and 7. Biopsy specimen halves were
studied
with respect to histology and recovery of viable bacteria.
There was no
significant difference between the probability of
recovering viable
wild type and that of recovering Cu,Zn SOD-deficient
H. ducreyi on either day 2 or day 7 postinfection (logistic
regression
[Tables
3 and
4]).
Development of lesions caused by wild-type and Cu,Zn SOD-deficient
H. ducreyi.
Because heat-killed H. ducreyi
produces only mild inflammation and no ulceration in the pig model of
infection (reference 12 and data not shown), we
hypothesized that an attenuated strain with a decreased ability to
survive in vivo might show a different histological picture or time
course of ulcer development. Thus, we hypothesized that there might be
a significantly different histological manifestation in skin infected
with an H. ducreyi strain without periplasmic SOD activity.
To test this hypothesis, we compared lesions caused by wild-type and
mutant
H. ducreyi with respect to histology. Microscopic
examination of biopsy specimens revealed no gross histological
differences between lesions caused by wild type and those caused
by
sodC mutant
H. ducreyi. To allow statistical
comparisons of
relative lesion severity, we devised a numerical scoring
system
for the stages of ulcer development (Fig.
2). Skin of normal appearance
was scored
as 1; the presence of dermal perivascular and interstitial
mononuclear
and plasma cell infiltrate was assigned a score of
2; the presence of
an intraepidermal pustule consisting of neutrophils,
fibrin, and
necrotic debris was assigned a score of 3; an epidermal
pustule
accompanied by keratinocyte cytopathology and diffuse
mononuclear and
polymorphonuclear dermal infiltrate was scored
as 4; and ulceration or
epidermal necrosis and dermal erosion
accompanied by confluence of
immune cells were scored as 5. Biopsy
specimens were coded and scored
blindly. Inoculation of pig skin
with either strain most often led to
the formation of an epidermal
pustule, erosion of epidermal layers,
cytopathology of keratinocytes
around the pustule, and a diffuse
mononuclear cell infiltrate
in the dermis (Table
5 and Fig.
2D). No significant difference
in severity was found between the lesions induced by the wild
type and
those induced by Cu,Zn SOD-deficient strains on either
day 2 or day 7 (Tables
5 and
6, ANOVA). However, there
was a
significant correlation between decreased lesion severity and
the
absence of live bacteria in the same biopsy specimen (
P =
0.035 [Mann-Whitney test]).
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FIG. 2.
Hematoxylin- and eosin-stained cross sections of pig
skin inoculated with H. ducreyi, demonstrating method of
scoring lesion severity. (A) Skin of normal appearance, score = 1;
(B) Presence of dermal perivascular and interstitial mononuclear cell
infiltrate, score = 2; (C) Presence of intraepidermal pustule
consisting of neutrophils, fibrin, and necrotic debris, score = 3;
(D) Epidermal pustule accompanied by keratinocyte cytopathology and
diffuse mononuclear and polymorphonuclear dermal infiltrate, score = 4; (E) Ulceration or epidermal necrosis and dermal erosion
accompanied by confluence of immune cells, score = 5. Magnifications, ×103 for panels A to C and ×52 for panels D and E.
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|
| |
DISCUSSION |
H. ducreyi expresses a periplasmic copper- and
zinc-cofactored SOD (28) that protects this bacterium from
killing by chemically generated superoxide in vitro (25). We
hypothesized that periplasmic SOD activity protects H. ducreyi from bactericidal oxygen radicals evolved by host immune
cells during infection. In this study, we tested that hypothesis by
comparing a Cu,Zn SOD-deficient H. ducreyi strain to its
wild-type parent with respect to its survival and ability to induce
chancroid-like ulcer formation in swine.
Our results suggest that the periplasmic Cu,Zn SOD is an important
virulence determinant of H. ducreyi. Periplasmic
SOD-deficient H. ducreyi was recovered from pigs less often
and in significantly fewer numbers than wild-type or merodiploid
sodC+ sodC H. ducreyi. Whereas the recovery of
wild-type and merodiploid sodC+ sodC H. ducreyi
was consistent, the recovery of the Cu,Zn SOD-deficient H. ducreyi differed markedly between pigs. Viable Cu,Zn SOD-deficient H. ducreyi organisms were recovered from only 6 of the 10 pigs inoculated, while wild-type H. ducreyi was recovered
from all 10 pigs 2 days after inoculation. It could be that the degree of attenuation of the Cu,Zn SOD-deficient strain is such that genetic
differences among the outbred pigs, such as major histocompatibility complex alleles, or individual differences in the efficiency of nonspecific immune defenses, may have had an impact on the survival rate of the bacterium.
The differences between the recovery rates of Cu,Zn SOD-null and wild
type and those of merodiploid H. ducreyi were statistically significant, although there was variability in the actual number of
colonies recovered from different sites, even for the same strain
infecting adjacent sites on the same pig. There is evidence from
studies of naturally occurring chancroid (18), as well as
from human experimental H. ducreyi challenge studies
(26), that the outcome of infection can vary widely among
lesions on the same subject. We do not know whether the H. ducreyi organisms that persisted within skin lesions were selected
in any way from the initial infecting population.
The recovery data from cyclophosphamide-treated pigs infected with
mutant and wild-type strains contrasted with the data gathered from
immunocompetent pigs. Both wild-type and Cu,Zn SOD-deficient H. ducreyi strains survived equally well in skin of neutropenic pigs.
The difference in rates of recovery between the two strains that was
evident in immunocompetent pigs was not observed with neutropenic pigs.
These contrasting results suggest that the absence of host neutrophils
(and perhaps monocytes and lymphocytes) allowed the survival of
H. ducreyi in the absence of bacterial periplasmic SOD
activity. Conversely, the periplasmic Cu,Zn SOD protected H. ducreyi from host neutrophils in immunocompetent pigs.
The severity and gross appearance of 2- and 7-day lesions caused by the
Cu,Zn SOD-deficient strain were not significantly different from those
of lesions caused by the wild type. Pig skin inoculated with
SOD-deficient H. ducreyi showed histological changes similar
to those of skin inoculated with the wild-type strain and consistent
with chancroid ulcer development, even though roughly half of the
sodC strain-inoculated sites had no detectable live bacteria
on the day of biopsy. These observations would seem to suggest that
live bacteria were not required for chancroid ulcer formation. However,
sites inoculated with heat-killed H. ducreyi never
progressed to pustule formation and never received scores greater than
2, and statistical analysis of our data demonstrated a correlation
between decreased lesion severity and the absence of viable bacteria on
the day of biopsy. It is possible that the initial input of live
bacteria may be sufficient to induce ulcer formation (11)
and that further development of the ulcer requires the persistence of a
very small number of live H. ducreyi organisms. We predict
that the sodC mutant strain would not induce chronic ulcers
because of its decreased ability to survive in vivo.
In conclusion, we observed that lack of periplasmic Cu,Zn SOD activity
was detrimental to survival of H. ducreyi in immunocompetent pigs but not in neutropenic pigs. Our observations suggest that the
Cu,Zn SOD protects H. ducreyi from neutrophil killing and contributes to the persistence of the bacterium in chancroid lesions.
| |
ACKNOWLEDGMENTS |
This work was supported by grants NIAID AI42824 (T.H.K.) and NRSA
1 F31 AI09565-01 (L.R.S.M.).
We gratefully acknowledge our collaborator, Glen Almond, for valuable
advice on pigs; Patty Routh, John Horton, and Re Bai for technical
assistance with the pigs; Stephen Knight for help with light
micrography; Gary Gaddy and Walter Davis for advice on statistics with
hierarchical linear models; John Woosley for help with
dermatohistopathology; Janne Cannon for helpful discussions and
critical review of the manuscript; and past and present members of the
Kawula lab, particularly Marcia Hobbs, Franca Zaretzky, and Gina
Donato, for advice and practical and moral support. We are especially
grateful to Myron Cohen for his idea to use induced neutropenia to
analyze the sodC mutant.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, CB #7290, University of North Carolina
School of Medicine, Chapel Hill, NC 27599. Phone: (919) 966-9699. Fax: (919) 962-8103. E-mail: kawula{at}med.unc.edu.
Editor:
D. L. Burns
| |
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Abstract
Introduction
