Antimicrobial Effects of Interferon-Inducible CXC Chemokines against Bacillus anthracis Spores and Bacilli
Based on previous studies showing that host chemokines exert antimicrobial activities against bacteria, we sought to determine whether the interferon-inducible Glu-Leu-Arg-negative CXC chemokines CXCL9, CXCL10, and CXCL11 exhibit antimicrobial activities against Bacillus anthracis. In vitro analysis demonstrated that all three CXC chemokines exerted direct antimicrobial effects against B. anthracis spores and bacilli including marked reductions in spore and bacillus viability as determined using a fluorometric assay of bacterial viability and CFU determinations. Electron microscopy studies revealed that CXCL10-treated spores failed to undergo germination as judged by an absence of cytological changes in spore structure that occur during the process of germination. Immunogold labeling of CXCL10-treated spores demonstrated that the chemokine was located internal to the exosporium in association primarily with the spore coat and its interface with the cortex. To begin examining the potential biological relevance of chemokine-mediated antimicrobial activity, we used a murine model of inhalational anthrax. Upon spore challenge, the lungs of C57BL/6 mice (resistant to inhalational B. anthracis infection) had significantly higher levels of CXCL9, CXCL10, and CXCL11 than did the lungs of A/J mice (highly susceptible to infection). Increased CXC chemokine levels were associated with significantly reduced levels of spore germination within the lungs as determined by in vivo imaging. Taken together, our data demonstrate a novel antimicrobial role for host chemokines against B. anthracis that provides unique insight into host defense against inhalational anthrax; these data also support the notion for an innovative approach in treating B. anthracis infection as well as infections caused by other spore-forming organisms.
INFECTION AND IMMUNITY, Apr. 2009, p. 1664–1678 Vol. 77, No. 4
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Antimicrobial Effects of Interferon-Inducible CXC Chemokines against
Bacillus anthracis Spores and Bacilli
Matthew A. Crawford,
Candace S. Green,
Marie D. Burdick,
Alison D. O’Brien,
Robert M. Strieter,
and Molly A. Hughes
Department of Medicine, Division of Infectious Diseases, University of Virginia Health Sciences System, Charlottesville, Virginia
Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Virginia Health Sciences System,
; and Department of Microbiology and Immunology, Uniformed Services University of
the Health Sciences, Bethesda, Maryland
Received 29 September 2008/Returned for modiﬁcation 10 November 2008/Accepted 21 January 2009
Based on previous studies showing that host chemokines exert antimicrobial activities against bacteria, we
sought to determine whether the interferon-inducible Glu-Leu-Arg-negative CXC chemokines CXCL9,
CXCL10, and CXCL11 exhibit antimicrobial activities against Bacillus anthracis. In vitro analysis demon-
strated that all three CXC chemokines exerted direct antimicrobial effects against B. anthracis spores and
bacilli including marked reductions in spore and bacillus viability as determined using a ﬂuorometric assay of
bacterial viability and CFU determinations. Electron microscopy studies revealed that CXCL10-treated spores
failed to undergo germination as judged by an absence of cytological changes in spore structure that occur
during the process of germination. Immunogold labeling of CXCL10-treated spores demonstrated that the
chemokine was located internal to the exosporium in association primarily with the spore coat and its interface
with the cortex. To begin examining the potential biological relevance of chemokine-mediated antimicrobial
activity, we used a murine model of inhalational anthrax. Upon spore challenge, the lungs of C57BL/6 mice
(resistant to inhalational B. anthracis infection) had signiﬁcantly higher levels of CXCL9, CXCL10, and
CXCL11 than did the lungs of A/J mice (highly susceptible to infection). Increased CXC chemokine levels were
associated with signiﬁcantly reduced levels of spore germination within the lungs as determined by in vivo
imaging. Taken together, our data demonstrate a novel antimicrobial role for host chemokines against B.
anthracis that provides unique insight into host defense against inhalational anthrax; these data also support
the notion for an innovative approach in treating B. anthracis infection as well as infections caused by other
Bacillus anthracis is a gram-positive, spore-forming bacte-
rium that causes the disease anthrax. The infectious B. anthra-
cis spore is a dormant, metabolically inactive form of the or-
ganism made up of distinct, concentric layers that collectively
provide a highly structured casing capable of protecting the
spore core from high temperature, UV irradiation, lytic diges-
tion, and numerous reactive agents (31, 59). Spore germination
is initiated through receptor-mediated interactions between
soluble germinant molecules (typically nutrients such as single
amino acids, sugars, or purine nucleosides) and germinant
receptors located at the inner membrane of the dormant spore
(20, 36). Although the molecular mechanism(s) linking germi-
nant binding to the loss of dormancy is undeﬁned, germinant
receptor engagement initiates a cascade of processes, including
dipicolinic acid (DPA) release, that promote core rehydration
and result in the controlled degradation of the protective spore
structures; as germination concludes, metabolic activity re-
sumes, and vegetative outgrowth is initiated (58). Fully virulent
B. anthracis bacilli generate several virulence factors including
an antiphagocytic, poly-
D-glutamic acid capsule encoded by
pXO2 (43, 46) and two toxins, lethal toxin and edema toxin,
encoded by pXO1 and responsible for disrupting innate and
adaptive immune responses (4).
Although recent studies have demonstrated that B. anthracis
spore germination occurs primarily at initial sites of infection
along the respiratory tract (23, 24, 55) in association with macro-
phages (28, 60), dendritic cells (9, 11), and neutrophils (44, 55),
the series of events connecting spore uptake with the appearance
of extracellular bacilli remain incompletely deﬁned. It appears
that following phagocytosis, spores trafﬁc to phagolysosomes
where spore germination occurs (28, 33). While the vast majority
of germinating organisms are killed (52, 65), a small subset of
germinated spores evades cell-mediated killing mechanisms pos-
sibly through early intracellular toxin production (5, 16). Ulti-
mately, vegetative bacilli escape from host phagocytes and estab-
lish extracellular infection, leading to toxemia, septicemia, and the
subsequent death of the host (18).
Current treatment of inhalational anthrax relies on postexpo-
sure prophylaxis with standard antibiotics such as ciproﬂoxacin or
doxycycline for extended periods of time (35). Although antibi-
otics are effective against the vegetative form of B. anthracis, they
do not have activity against the dormant spore form of the or-
ganism. Thus, the use of antibiotics is limited to established in-
fections that, despite early and vigorous treatment, have high
* Corresponding author. Mailing address: Department of Medicine,
Division of Infectious Diseases, University of Virginia Health Sciences
System, P.O. Box 800513, Charlottesville, VA 22908. Phone: (434)
924-5216. Fax: (434) 982-3830. E-mail: firstname.lastname@example.org.
Published ahead of print on 29 January 2009.
morbidity and mortality (37). There are currently no therapeutic
agents available that target the spore form of B. anthracis and
thereby prevent the establishment of infection.
Chemokines are a group of structurally related, low-molecular-
mass (8- to 10-kDa) proteins originally deﬁned by their ability to
induce directed cell migration in leukocytes (40). Although some
chemokines are constitutively expressed and function in homeo-
static roles, the majority of chemokines identiﬁed thus far are
considered to be potent inﬂammatory mediators induced in re-
sponse to Toll-like receptor agonists and/or proinﬂammatory cy-
tokines (42, 51). Three members of the interferon (IFN)-induc-
ible tripeptide motif Glu-Leu-Arg-negative (ELR
chemokines, monokine-induced by IFN-␥ (CXCL9), IFN-␥-in-
ducible protein of 10 kDa (CXCL10), and IFN-inducible T-cell-
activated chemokine (CXCL11), have been shown to be strongly
induced by IFN-␥ and together comprise a family of IFN-induc-
CXC chemokines (13, 41, 69).
CXCL9, CXCL10, and CXCL11 are highly homologous pro-
teins that display several common features including a highly
positively charged C terminus, similar charge distribution, and
amphipathic character (12). CXCL9, CXCL10, and CXCL11
are produced and secreted primarily by monocytes, macro-
phages, ﬁbroblasts, and epithelial cells upon stimulation with
proinﬂammatory cytokines, especially IFN-␥ (19, 39). Each of
these chemokines signals through a common G-protein-cou-
pled receptor, CXCR3, and acts primarily in the recruitment of
T cells, NK cells, and plasmacytoid
dendritic cells to sites of inﬂammation (14, 47). In addition to
their roles in leukocyte recruitment, CXCL9, CXCL10, and
CXCL11 exert direct antimicrobial effects that are comparable
to the effects mediated by cationic antimicrobial peptides, in-
cluding defensins (12). Antimicrobial activity was subsequently
found to extend to a number of chemokines, and the organisms
targeted by speciﬁc antimicrobial chemokines include the
gram-positive bacteria Staphylococcus aureus, Streptococcus
mutans, and Listeria monocytogenes; the gram-negative bacte-
ria Pseudomonas aeruginosa and Escherichia coli; and the fungi
Candida albicans and Cryptococcus neoformans (12, 32, 61, 70).
Based on the above-described information, as well as reports
that IFN-␥ exerts protective effects against B. anthracis chal-
lenge in vitro (26) and in vivo (25), we hypothesized that the
IFN-inducible CXC chemokines CXCL9, CXCL10, and
CXCL11 exert direct antimicrobial effects against B. anthracis
and thereby promote host defense during inhalational anthrax.
CXCL9, CXCL10, and CXCL11 were found to exert direct
antimicrobial effects against both the spore and bacillus forms
of B. anthracis, establishing the ﬁrst description of direct anti-
microbial activity for host chemokines against bacterial spores.
In addition, CXC chemokine induction in the lungs of spore-
challenged C57BL/6 mice (relatively resistant to inhalational
B. anthracis infection) was signiﬁcantly higher than that in the
lungs of spore-challenged A/J mice (highly sensitive to infec-
tion). The increased level of induction of CXCL9, CXCL10,
and CXCL11 was associated with a substantial reduction in
detectable spore germination, which is suggestive of a role for
CXC chemokine-mediated antimicrobial activity in promoting
host defense during the initial stages of B. anthracis infection in
MATERIALS AND METHODS
Bacterial strains, culture conditions, and reagents. B. anthracis Sterne strain
) was used for in vitro experiments; strain 7702 spores were
kindly provided by Tod J. Merkel, U.S. Food and Drug Administration (Be-
thesda, MD). B. anthracis bacilli were prepared from the above-mentioned spore
stock by streaking diluted spore aliquots onto brain heart infusion (BHI) agar
(Remel, Lenexa, KS) plates. A single bacterial colony was subsequently inocu-
lated into 10 ml of BHI broth (BD, Franklin Lakes, NJ) and incubated overnight
at 37°C in a shaking incubator (200 rpm). The next day, the bacterial culture was
diluted 1:20 in prewarmed BHI broth, and the subculture was incubated at 37°C
with shaking until an optical density at 600 nm between 0.6 and 0.65 was
achieved. In vivo experiments were performed using B. anthracis Sterne strain
) carrying the plasmid-based sspB promoter-driven lux
germination reporter, as previously characterized (55). All work involving B.
anthracis spores and bacilli was performed using biological safety level 2 precau-
tions. Recombinant human CXCL9, CXCL10, CXCL11, CCL2, and CCL5 were
purchased from PeproTech (Rocky Hill, NJ). Chemokines were reconstituted at
1 mg/ml in sterile, distilled water stabilized with 0.3% human serum albumin
(ZLB Bioplasma AG, Berne, Switzerland) and stored at ⫺80°C.
Chemokine treatment and microscopic visualization. Before treatment, B.
anthracis spores were heat activated for 30 min in a 70°C water bath to eliminate
any germinated organisms; also, since B. anthracis bacilli grow in long chains in
vitro, bacillus cultures were vortexed before use to limit initial chain length.
Spores (0.6 ⫻ 10
to 1 ⫻ 10
total spores per sample well) or bacilli (2 ⫻ 10
4 ⫻ 10
total bacilli per sample well) were added to high-glucose Dulbecco’s
modiﬁed Eagle’s medium (Gibco-Invitrogen, Carlsbad, CA) with 10% fetal bo-
vine serum (HyClone, Logan, UT) containing 48 g/ml of individual chemokines
or an equal volume of 0.3% human serum albumin (vehicle, untreated control)
unless otherwise noted. Aliquots of 100 l were plated in triplicate into the wells
of a 96-well plate and incubated at 37°C in 5% CO
. At the same time, an aliquot
of the spore or bacillus working stock was diluted and plated onto BHI agar
plates for determinations of the initial inoculum. Samples were examined over a
6-h period for spore germination and/or vegetative outgrowth using an Olympus
IX51 inverted microscope equipped with a Q-Color3 imaging system (Olympus
America, Melville, NY). Camera control and image capture were performed
using QCapture Pro 5.1 software (QImaging, Surrey, BC, Canada); images were
processed with Adobe (San Jose, CA) Photoshop 7.0. At least eight randomly
chosen ﬁelds from each treatment group were photographed, and results were
conﬁrmed using a second spore preparation and its derived bacilli.
Alamar blue analysis and CFU determination. Active metabolism of B.
anthracis spores and bacilli with or without chemokine treatment was quantiﬁed
using the oxidation reduction indicator dye Alamar blue (AbD Serotech, Oxford,
United Kingdom), which generates ﬂuorescence in response to the chemical
reduction of the treatment medium. At the 4-h time point, Alamar blue was
added at a 1:10 dilution to each treatment well including sample blanks lacking
B. anthracis. The sample plate was then protected from light and incubated for
an additional2hat37°C in 5% CO
. The reduction of Alamar blue as an index
of bacterial cell number and proliferation was then assessed by measuring sample
well ﬂuorescence at 530-nm excitation and 590-nm emission wavelengths using a
multilabel plate reader (Perkin-Elmer, Waltham, MA).
The addition of Alamar blue at 4 h posttreatment was not found to inﬂuence the
effects of chemokine treatment or affect subsequent experimental measures.
For CFU determinations, two of the three replicate wells from each treatment
group were harvested 6 h posttreatment, and several dilutions were prepared
according to predetermined values speciﬁc for each treatment (dilution values
ranged from no dilution to 1:5,000 and typically resulted in ⬎50 colonies per
plate). Sample dilutions were plated in duplicate onto BHI agar plates and
incubated overnight at room temperature before colonies were enumerated. In
order to differentiate spores from vegetative bacilli in spore treatment groups,
sample dilutions were plated with or without heat treatment at 65°C for 30 min,
which kills vegetative bacteria.
TEM and silver-enhanced immunogold labeling. B. anthracis spores (⬃6 ⫻ 10
spores total) or bacilli (⬃8 ⫻ 10
bacilli total) were incubated in high-glucose
Dulbecco’s modiﬁed Eagle’s medium (permissive to germination) or in water
(not permissive to germination) with or without 48 g/ml CXCL10 in individual
wells of a 24-well plate (500-l ﬁnal volume). At deﬁned time points, untreated
and CXCL10-treated samples were harvested and prepared for transmission
electron microscopy (TEM) according to standard methods (27). Brieﬂy, exper-
imental samples were ﬁxed with 2.5% glutaraldehyde in 0.1 M phosphate buffer
(PB) overnight at 4°C. Samples were subsequently treated with 1% osmium
tetroxide in PB for1hatroom temperature before being dehydrated in a graded
ethanol series and embedded in epoxy resin (Epon 812; Electron Microscopy
VOL. 77, 2009 CXC CHEMOKINES AND BACILLUS ANTHRACIS 1665
Sciences, Fort Washington, PA). Ultrathin sections (80 nm) obtained with a
Diatorne (Bienne, Switzerland) diamond knife were placed onto 200-mesh nickel
grids and contrast stained with lead citrate and uranyl acetate. Sections were
examined using a Jeol 1230 transmission electron microscope operated at 80 kV;
digital images were captured using an SIA-12 16-megapixel slow-scan charge-
coupled device (Scientiﬁc Instruments and Applications, Duluth, GA).
Single immunogold labeling with silver enhancement was performed on un-
treated B. anthracis spores at 0 h and on CXCL10-treated spores at 1 h using a
preembedding protocol adapted from Aurion (Wageningen, The Netherlands)
and described in detail elsewhere (71). Ultrasmall (ⱕ1.0-nm) gold-conjugated
fragments of goat anti-murine antibody (Ab), acetylated bovine serum
albumin, cold-water ﬁsh skin gelatin, and R-Gent SE-EM electron microscopy-
grade silver enhancement mixture were purchased from Aurion and used ac-
cording to the manufacturer’s instructions. Spore samples were ﬁxed with 4%
paraformaldehyde for 30 min at 4°C followed by aldehyde inactivation using
0.1% sodium borohydride in PB. To improve reagent penetration, samples were
permeabilized with 0.1% saponin in phosphate-buffered saline (PBS) before
incubation in blocking solution (0.2% acetylated bovine serum albumin, 0.1%
cold-water ﬁsh skin gelatin, 5% normal goat serum in PB). Incubations with
primary murine anti-human CXCL10 monoclonal Ab (R&D Systems, Minneap-
olis, MN) and secondary gold-conjugated F(ab⬘)
fragments of goat anti-murine
Ab were performed overnight at 4°C in PBS supplemented with 0.2% acetylated
bovine serum albumin and 0.1% saponin. Before silver enhancement with the
R-Gent SE-EM silver enhancement mixture, postﬁxation with 2.5% glutaralde-
hyde was performed. Subsequent sample processing and visualization were per-
formed as described above. Four additional sample groups were prepared in
parallel as controls: primary Ab only, secondary Ab only, silver enhancement
only, or buffer solution only; gold particles were not visualized in the labeling
controls. All electron microscopy studies were performed at the University of
Virginia Advanced Microscopy Facility.
In vivo intranasal spore challenge and luminescence detection. In order to
analyze spore germination in vivo, intranasal B. anthracis spore challenge was
performed using B. anthracis Sterne strain 34F2 carrying a plasmid-based sspB
promoter-driven lux germination reporter (55). Brieﬂy, 6- to 8-week-old female
A/J and C57BL/6 mice (Jackson Laboratory, Bar Harbor, ME) were sedated with
isoﬂurane using the XGI-8 gas anesthesia system (Xenogen, Alameda, CA).
Thirty microliters of spore suspension (⬃1 ⫻ 10
spores total) or sterile water
was placed onto the nares of anesthetized mice, and the animals were held
upright until the inoculum was inhaled. At 1 h, 6 h, 24 h, and 48 h postchallenge,
sham-challenged mice (three to four animals per time point per strain) and B.
anthracis spore-challenged mice (eight animals per time point per strain) were
euthanized by cervical dislocation while sedated. The lungs, livers, and spleens
from sham- and spore-challenged mice were harvested and scanned for lumi-
nescence using a charge-coupled device within the Xenogen IVIS detection
chamber (Xenogen). The emission of photons from sample tissues was quantiﬁed
using the Living Image 2.5 software package (Xenogen). After luminescence was
recorded, the extracted tissues were snap-frozen in liquid nitrogen and stored at
⫺80°C; lung samples were later used for CXC chemokine protein quantiﬁcation
(described below). Animal protocols were reviewed and approved by the Insti-
tutional Animal Care and Use Committee of the University of Virginia and the
Uniformed Services University.
CXC chemokine protein quantiﬁcation. Immediately prior to endogenous
chemokine quantiﬁcation, lungs harvested from sham-challenged (three animals
per group) and spore-challenged (ﬁve animals per group) A/J and C57BL/6 mice
were homogenized in 1 ml of cold PBS containing Complete Tab protease
inhibitor cocktail (Roche, Basel, Switzerland). The lung samples were homoge-
nized using a Tissuemiser homogenizer (ThermoFisher Scientiﬁc, Waltham,
MA) and then sonicated three times for 10 s on ice. The lung homogenates were
cleared by centrifugation and ﬁltered through a 1.2-m Versapor membrane
syringe ﬁlter (Pall, East Hills, NY).
CXCL9, CXCL10, and CXCL11 protein levels in lung ﬁltrates were deter-
mined using the Luminex xMAP technology platform (Luminex, Austin, TX).
CXCL9- and CXCL10-speciﬁc beads, biotinylated Abs, and protein standards
were purchased from Biosource (Nivelles, Belgium) and used according to the
manufacturer’s instructions. Polyclonal rabbit anti-murine CXCL11 Ab was pro-
duced by the immunization of rabbits with recombinant CXCL11 (R&D Sys-
tems) as previously described (7). Bead conjugation of the CXCL11 Ab was
performed by Upstate (Lake Placid, NY) and was speciﬁc in our multiplex assays
without cross-reactivity to a panel of cytokines and other CXC and CC chemo-
kine ligands. Biotinylated anti-CXCL11 Ab and CXCL11 protein standards were
purchased from R&D Systems. CXC chemokine quantiﬁcation through multi-
plex bead array analysis was performed as previously described (64). Brieﬂy, lung
homogenate ﬁltrates were incubated in duplicate with a three-bead multiplex
suspension in individual wells of a multiscreen, 96-well ﬁlter plate (Millipore,
Bedford, MA). A mixture of biotinylated anti-CXCL9, anti-CXCL10, and anti-
CXCL11 Abs was added to each well, with the subsequent addition of strepta-
vidin-phycoerythrin (Upstate). The labeling reactions were stopped with the
addition of 0.2% paraformaldehyde to the medium, and the sample plate was
read using the Bio-Plex 200 suspension array system with Bio-Plex Manager 4.1.1
software (Bio-Rad Laboratories, Hercules, CA). CXC chemokine concentrations
were determined from a standard curve assayed at the same time with known
amounts of recombinant murine CXCL9, CXCL10, and CXCL11.
Statistical analysis. Statistical analysis and graphing were performed using
GraphPad Prism 4.0 software for Windows (GraphPad Software, San Diego,
CA). Statistically signiﬁcant differences were determined using one-way analysis
of variance with a Dunnett’s post hoc test. Logarithmic (log
) transformation of
bacterial counts (CFU/ml) was performed prior to statistical evaluation. Exper-
imental groups demonstrating statistically signiﬁcant differences were subse-
quently analyzed using an unpaired, two-tailed t test; a P value of ⬍0.05 was
considered to be signiﬁcant. Half-maximal effective concentrations (EC
determined using the sigmoidal dose-response equation of nonlinear regression
and are presented as EC
⫾ 95% conﬁdence intervals.
Direct antimicrobial effects of CXCL9, CXCL10, and
CXCL11 on B. anthracis spores. To investigate possible direct
chemokine-mediated antimicrobial effects against B. anthracis
spores, Sterne strain spores (pXO1
) were treated
with 48 g/ml recombinant human CXCL9, CXCL10, or
CXCL11 or vehicle alone (untreated control) in tissue culture
medium. The concentration chosen for these experiments was
based on previous ﬁndings that this concentration of chemo-
kine was effective at exerting antimicrobial activity against E.
coli and L. monocytogenes (12). Spore germination and subse-
quent vegetative cell outgrowth were monitored over a 6-h
period using light microscopy (Fig. 1A). Untreated spores be-
gan to germinate by 1 h, with subsequent outgrowth such that
abundant chains of bacilli were evident by 6 h. In the presence
of CXCL9 or CXCL10, spore germination and/or vegetative
cell outgrowth was markedly reduced; little to no vegetative
bacilli were detected in CXCL10-treated spore samples at 6 h,
suggesting a nearly complete absence of germination and pri-
mary outgrowth. Although less effective than CXCL9 and
CXCL10, CXCL11 also reduced spore germination and/or the
outgrowth of vegetative bacilli as determined through light
To deﬁne the direct effects of CXCL9, CXCL10, and
CXCL11 on B. anthracis spores quantitatively and determine
whether spores or germinated organisms were being affected,
two independent methods were used. First, an Alamar blue-
based ﬂuorometric assay was used to measure metabolic activ-
ity as an index of bacterial cell number and proliferation.
Second, CFU determinations were used to measure spore ger-
mination, vegetative cell outgrowth, and bacterial viability. In
these experiments, monocyte chemotactic protein 1 (CCL2),
which has not been found to exert antimicrobial effects, and
RANTES (CCL5), which was previously shown to exert anti-
microbial effects against S. aureus and C. albicans (32, 61),
were used as controls. These two CC chemokines were chosen
since their molecular masses and charges are similar to those
of the CXC chemokines tested. As shown in Fig. 1B, levels of
Alamar blue reduction were the same for untreated, CCL2-
treated, and CCL5-treated spores at 6 h. In addition, CCL2
and CCL5 had no effect on spore germination and/or out-
growth as determined by light microscopy (data not shown). In
1666 CRAWFORD ET AL. INFECT.IMMUN.
contrast, spores incubated with CXCL9 or CXCL10 displayed
little to no active metabolism (⬍10% and ⬍1% of the un-
treated control, respectively; P ⬍ 0.001), indicating that the
chemokine-treated spores consisted of a population of largely
dormant and/or dead organisms. CXCL11 treatment was less
effective against B. anthracis spores, as reﬂected by a smaller
reduction in metabolic activity (⬍60% of the untreated con-
trol; P ⬍ 0.001).
FIG. 1. Direct antimicrobial effects of CXCL9, CXCL10, and CXCL11 against B. anthracis spores. (A) B. anthracis 7702 spores were treated with
vehicle alone (untreated control) or 48 g/ml of CXCL9, CXCL10, or CXCL11. All CXC chemokines tested demonstrated antimicrobial activity as
assessed by markedly reduced numbers of vegetative bacilli 6 h posttreatment. Representative ﬁelds from ﬁve independent experiments are shown at a
⫻200 magniﬁcation. (B and C) Alamar blue reduction (B) and CFU determination (C) were determined 6 h posttreatment. In contrast to the untreated
and CC chemokine controls, CXCL9, CXCL10, and CXCL11 signiﬁcantly reduced spore germination and/or vegetative cell outgrowth. CFU determi-
nations were performed with or without heat treatment to differentiate between heat-resistant spores and heat-sensitive bacilli; the dotted line represents
the initial spore inoculum. Alamar blue data are expressed as a percentage of untreated control and represent mean values ⫾ standard errors of the
means (SEM) for three independent experiments; CFU data are expressed as CFU/ml (log
scale) ⫾ SEM, and a representative data set is shown from
three separate experiments. ⴱⴱⴱ, P value ⬍ 0.001 compared with untreated control.
OL. 77, 2009 CXC CHEMOKINES AND BACILLUS ANTHRACIS 1667
CFU determination (Fig. 1C) was performed with or without
heat treatment to differentiate between heat-resistant spores
and heat-sensitive vegetative bacilli. For experiments with
heat treatment, CFU values represent viable, heat-resistant
spores; for experiments without heat treatment, CFU values
represent both viable spores and vegetative bacilli. By 6 h,
the untreated, CCL2-treated, and CCL5-treated spores un-
derwent considerable germination and outgrowth as demon-
strated by a loss of heat-resistant CFU and a concomitant
increase in heat-sensitive CFU compared to the initial spore
inoculum. CXCL9- and CXCL10-treated spore samples dem-
onstrated an approximate 1,000-fold reduction in CFU at 6 h
compared to the untreated control. Interestingly, CFU with or
without heat treatment were nearly equal, suggesting that
there were very few germinated organisms and that the major-
ity of viable organisms were dormant spores. Furthermore, the
numbers of heat-resistant CFU (dormant spores) recovered
from CXCL9- and CXCL10-treated spore samples were sig-
niﬁcantly higher than those recovered from untreated sam-
ples, yet the CXCL9- and CXCL10-treated sample groups
contained approximately 85% fewer viable spores than were in
the initial inoculum. These results suggest that CXCL9 and
CXCL10 interfere with spore germination and may exert a
direct, sporicidal effect on B. anthracis spores. CXCL11 treat-
ment did not signiﬁcantly affect spore germination at the che-
mokine concentration tested (heat-resistant CFU not signiﬁ-
cantly greater than those found for the untreated control) but
did result in a ⬎10-fold reduction in the subsequent outgrowth
of vegetative bacilli. Washing the treatment samples before
CFU determination did not affect resultant bacterial CFU,
suggesting that chemokine-mediated reductions in viability
were irreversible by the time of plating (data not shown).
The direct effects of CXCL9, CXCL10, and CXCL11 were
dependent upon chemokine concentration as determined by
Alamar blue analysis (Fig. 2A) and CFU determinations (Fig.
2B; data not shown for CXCL9 and CXCL11). Based on
Alamar blue analysis, which correlated well with CFU data, the
values for CXCL9, CXCL10, and CXCL11 against B.
anthracis spores were 6.2 ⫾ 0.2 g/ml, 7.9 ⫾ 0.2 g/ml, and
28.2 ⫾ 1.8 g/ml, respectively. Notably, CXCL10 caused a
complete inhibition of spore germination and/or vegetative cell
outgrowth at a chemokine concentration of ⬃20 g/ml, which
was considerably lower than the concentrations of CXCL9 and
CXCL11 required to achieve the same effect (58 g/ml and 72
g/ml, respectively). The dissimilar concentration curves
among CXCL9, CXCL10, and CXCL11 are likely attributable
to differences in the cationic properties of the C-terminal re-
gion as well as overall structural characteristics as described
elsewhere (12, 70).
CXCL10 affects ungerminated spores and interferes with B.
anthracis spore germination. The data presented above indi-
cate that CXCL10 exerts direct antimicrobial effects against
either B. anthracis spores or newly forming bacilli immediately
after germination. Since we were unable to distinguish between
these two effects using light microscopy, we visualized un-
treated and CXCL10-treated spores using TEM. B. anthracis
spores were incubated in the presence or absence of CXCL10
for 6 h before being processed for TEM (Fig. 3A). The un-
treated spores underwent germination and outgrowth from the
initial spore inoculum and were visualized at6haspredomi-
nately vegetative bacilli. CXCL10-treated spores, however, re-
mained ungerminated as assessed by the lack of cytological
changes in spore structure, including cortex degradation, that
occur during the germination process in Bacillus species (54).
These data support the conclusion that CXCL10 inter-
feres with spore germination and demonstrate the ability of
CXCL10 to exert direct antimicrobial effects against B. anthra-
Since spore germination is a rapid and irreversible process,
the inhibitory effects of CXCL10 on B. anthracis spore germi-
nation likely occur during the initial chemokine interaction
with the spore. To determine the initial site(s) of interaction
between CXCL10 and B. anthracis spores, silver-enhanced im-
munogold labeling of CXCL10 was performed (Fig. 4). Con-
FIG. 2. CXCL9-, CXCL10-, and CXCL11-mediated antimicrobial
effects against B. anthracis spores are concentration dependent. (A) B.
anthracis 7702 spores were treated with increasing concentrations of
CXCL9, CXCL10, or CXCL11. Alamar blue reduction, as a measure
of active metabolism, was determined 6 h posttreatment and used to
s (6.2 ⫾ 0.2 g/ml for CXCL9, 7.9 ⫾ 0.2 g/ml for
CXCL10, and 28.8 ⫾ 1.8 g/ml for CXCL11). Alamar blue reduction
data are expressed as percentages of the control and represent mean
values ⫾ SEM for two independent experiments. For clarity, only the
lowest chemokine concentration demonstrating statistically signiﬁcant
decreases from the untreated control are labeled with asterisks.
(B) CFU determination with or without heat treatment of CXCL10-
treated B. anthracis spores was performed after6hoftreatment. The
direct effects of CXCL10 on spore germination and viability, as de-
scribed in Results, were dependent on the chemokine concentration;
the dotted line represents the initial spore inoculum. CFU data are
expressed as CFU/ml (log
scale) ⫾ SEM and are a representative
data set from two independent experiments. ⴱ, P value of ⬍0.05; ⴱⴱ, P
value of ⬍0.01; ⴱⴱⴱ, P value of ⬍0.001 (compared with the untreated
1668 CRAWFORD ET AL. I
trol spore samples without CXCL10 treatment were processed
at 0 h (prior to the initiation of germination) and prepared in
the same manner as the CXCL10-treated spores processed for
immunogold labeling after1hofchemokine treatment in
germination-permissive tissue culture medium. Control, unger-
minated spores taken from the initial inoculum demonstrated
negligible binding of primary anti-CXCL10 Ab and secondary
fragments in the presence of silver
enhancement. In the CXCL10-treated spores, silver-enhanced
gold particles were found to be associated with the exospo-
rium, spore coat, and spore coat-cortex interface by 1 h. In
addition, when CXCL10 treatment was performed in the ab-
sence of germinants (i.e., in water), CXCL10 was found to
localize to similar spore structures, supporting the conclusion
FIG. 3. CXCL10 inhibits B. anthracis spore germination prior to cortex degradation. B. anthracis 7702 spores with or without 48 g/ml CXCL10
were visualized 6 h posttreatment using TEM. Whereas untreated spores underwent germination and were visualized as vegetative bacilli,
CXCL10-treated spores failed to germinate and closely resembled spores taken from the initial inoculum. Representative ﬁelds from two
independent experiments are shown at a ⫻30,000 magniﬁcation; the scale bar equals 0.5 m.
FIG. 4. CXCL10 localizes at spore structures internal to the exosporium. Untreated B. anthracis 7702 spores (0 h of treatment) and
CXCL10-treated spores (1 h of treatment) were used for immunogold labeling with silver enhancement as described in Materials and Methods.
Untreated spores demonstrated negligible binding of labeling reagents. In spores treated with CXCL10, gold particles were found to be associated
with the exosporium (white arrow), spore coat (arrowhead), and spore coat-cortex interface (black arrow). A lower-magniﬁcation ﬁeld allowing
the visualization of multiple spores immunolabeled for CXCL10 is included. When chemokine treatment was performed in water, conditions not
permissive to germination, CXCL10 was found to localize to similar spore structures including the exosporium (white arrow), spore coat
(arrowhead), and spore coat-cortex interface (black arrow). Representative ﬁelds from 2 independent experiments are shown at a ⫻30,000 (or
⫻15,000) magniﬁcation; the scale bar equals 0.5 m.
OL. 77, 2009 CXC CHEMOKINES AND BACILLUS ANTHRACIS 1669
that CXCL10 acts directly against spores. While the limitations
of the technique used for visualization do not exclude the
possibility that CXCL10 may be located in the plane of the
exosporium, these results strongly suggest that CXCL10 is able
to penetrate the outermost spore layers and likely exerts initial
antimicrobial effects through interactions with spore compo-
nents internal to the exosporium.
Direct antimicrobial effects of CXCL9, CXCL10, and
CXCL11 on B. anthracis vegetative bacilli. To determine
whether CXCL9-, CXCL10-, and CXCL11-mediated antimi-
crobial activity extended to vegetative bacilli, we incubated B.
anthracis bacilli with 48 g/ml CXCL9, CXCL10, or CXCL11
or vehicle alone (untreated control) in tissue culture medium.
Importantly, since in vitro growth of vegetative bacilli occurs in
long chains that are difﬁcult to completely disrupt, the initial
inoculum consisted primarily of short chains (four to six bacilli)
rather than single cells. The outgrowth of vegetative bacilli was
monitored over a 6-h period using light microscopy (Fig. 5A).
Untreated bacilli underwent signiﬁcant outgrowth from the
initial inoculum by 6 h. In the presence of CXCL9 and
CXCL10, vegetative outgrowth was largely absent; although
CXCL11-treated bacilli underwent vegetative outgrowth, there
were notably fewer vegetative bacilli than the untreated con-
trol. The effects of CCL2 and CCL5 on B. anthracis bacilli were
also tested, and neither CC chemokine inhibited the outgrowth
of vegetative bacilli as determined visually (data not shown).
Interestingly, CXCL9- and CXCL10-treated bacilli demon-
strated a disruption in bacterial chain integrity in as little as 30
min, with complete chain segmentation by 3 h (Fig. 5A, ex-
panded insets). By 6 h, the majority of bacterial chains had
completely separated into individual bacilli. Chain segmenta-
tion did not occur in the untreated or CXCL11-treated sam-
ples. Bacterial chain disruption similar to that seen for
CXCL9- and CXCL10-treated bacilli was also observed when
B. anthracis bacilli were treated with penicillin G, which acts to
disrupt cell wall synthesis and integrity (data not shown). Chain
segmentation and subsequent disruption were not observed
when vegetative bacilli were treated with ciproﬂoxacin or gen-
tamicin, which inhibit DNA replication and protein synthesis,
respectively. Together, these observations indicate the CXCL9
and CXCL10 may exert antimicrobial effects on B. anthracis
bacilli by disrupting cell wall and/or cell membrane integrity.
To quantify the direct effects of CXCL9, CXCL10, and
CXCL11 on B. anthracis bacilli, Alamar blue analysis and CFU
determinations were used to measure metabolic activity and
viability, respectively. Levels of Alamar blue reduction (Fig.
5B) were similar between untreated, CCL2-treated, and
CCL5-treated bacilli at 6 h. Bacilli incubated with CXCL9 or
CXCL10, however, displayed markedly reduced levels of active
metabolism (⬍25% and ⬍1% of the untreated control, respec-
tively; P ⬍ 0.001), which is indicative of a reduction in vege-
tative cell outgrowth and viability. CXCL11-treated bacilli had
signiﬁcantly impaired outgrowth at6h(⬍70% of the untreated
control; P ⬍ 0.001), consistent with reduced numbers of veg-
etative bacilli. CFU determinations (Fig. 5C) revealed that by
6 h, the untreated, CCL2-treated, and CCL5-treated vegetative
bacilli underwent signiﬁcant outgrowth from the initial inocu-
lum. CXCL9- and CXCL11-treated bacilli underwent vegeta-
tive outgrowth but contained signiﬁcantly fewer bacilli than did
the untreated control at 6 h (CXCL9, ⬎100-fold reduction;
CXCL11, ⬎10-fold reduction). CXCL10 treatment of vegeta-
tive bacilli consistently resulted in the complete killing of the
initial inoculum, consistent with a potent bactericidal effect.
Washing the treatment samples before CFU determination did
not affect resultant CFU (data not shown).
CXCL9-, CXCL10-, and CXCL11-mediated antimicrobial
effects against B. anthracis bacilli were dependent upon che-
mokine concentrations as determined by Alamar blue analysis
(Fig. 6A) and CFU determinations (Fig. 6B; data not shown
for CXCL9 and CXCL11). Based on Alamar blue analysis,
which matched well with CFU data, the EC
s for CXCL9,
CXCL10, and CXCL11 against B. anthracis bacilli were deter-
mined to be 40.2 ⫾ 1.1 g/ml, 4.0 ⫾ 0.3 g/ml, and 72.0 ⫾ 3.4
g/ml, respectively. CXCL10, which is the most potent of the
three CXC chemokines examined, demonstrated complete
killing of the initial inoculum at concentrations of ⱖ12 g/ml.
As noted above, the dissimilar effective concentrations among
these CXC chemokines likely reﬂect molecular differences re-
lated to cationic charge distribution and structural character-
istics (12, 70).
CXCL10 disrupts the cellular integrity of B. anthracis
bacilli. The antimicrobial effects of CXCL10 against B. anthra-
cis bacilli, as described above, suggest that CXCL10 is able to
directly mediate the killing of vegetative bacilli. To determine
the extent and character of CXCL10-mediated antimicrobial
activity, we used TEM to visualize the structural integrity of
bacilli incubated for3hinthepresence or absence of CXCL10
(Fig. 7). Untreated vegetative bacilli were numerous and dis-
played intact cellular structures complete with membrane sep-
tum characteristic of vegetative growth. In contrast, CXCL10-
treated bacilli demonstrated a complete loss of cell integrity
characterized by extensive cell wall and cell membrane disrup-
tion. These observations support the conclusion that CXCL10
exerts bactericidal effects by interfering with the integrity of the
bacterial cell wall and/or cell membrane.
In vivo analysis of CXCL9, CXCL10, and CXCL11 levels in
lungs of spore-challenged mice. To examine the potential roles
of CXCL9, CXCL10, and CXCL11 in promoting host defense
against B. anthracis infection, we employed a murine model of
inhalational anthrax using intranasal spore inoculation (55).
The inbred mouse strains chosen for this study were previously
shown to be susceptible (A/J) or resistant (C57BL/6) to inha-
lational infection by the toxigenic, unencapsulated Sterne
strain of B. anthracis (34, 67). Parallel intranasal spore chal-
lenge was performed in A/J mice and C57BL/6 mice; 1 h, 6 h,
24 h, and 48 h after spore challenge, the lungs of sham- and
spore-challenged animals were harvested and used to measure
CXCL9, CXCL10, and CXCL11 protein levels. Whereas
CXCL9, CXCL10, and CXCL11 induction following spore
challenge was negligible in the lungs of A/J mice, C57BL/6
mice demonstrated considerable CXC chemokine induction
compared to sham-infected controls (Fig. 8); CXCL10 and
CXCL11 induction was seen 1 h postchallenge, and CXCL9
was markedly induced by 24 h postchallenge. Compared to
infected A/J mice, spore-challenged C57BL/6 mice had signif-
icantly higher levels of CXCL9 at 24 h and 48 h (P ⬍ 0.001)
(Fig. 8A) and signiﬁcantly higher CXCL10 levels at all time
points tested (P ⬍ 0.01 to 0.001) (Fig. 8B). Although CXCL11
levels were low in both inbred strains following spore chal-
lenge, the lungs of C57BL/6 animals demonstrated signiﬁcantly
1670 CRAWFORD ET AL. INFECT.IMMUN.
FIG. 5. Direct antimicrobial effects of CXCL9, CXCL10, and CXCL11 against B. anthracis bacilli. (A) B. anthracis 7702 bacilli were
treated with vehicle (untreated control) or 48 g/ml of CXCL9, CXCL10, or CXCL11. By 3 h (expanded inserts), CXCL9- and CXCL10-
treated bacilli had undergone substantial bacterial chain segmentation (arrowhead) resulting in nearly complete chain disruption by 6 h;
chain segmentation and subsequent disruption were not seen in untreated and CXCL11-treated bacillus samples. All three CXC chemokines
demonstrated direct antimicrobial effects against B. anthracis bacilli as assessed by reduced numbers of vegetative bacilli at 6 h posttreat-
ment. Representative ﬁelds from three independent experiments are shown at6h(3hforinsets) at a ⫻200 magniﬁcation. (B and C) Alamar
blue reduction (B) and CFU determinations (C) were determined 6 h posttreatment. Experimental measures were comparable among
untreated, CCL2-treated, and CCL5-treated control groups. CXCL11- and CXCL9-treated bacillus samples demonstrated signiﬁcantly
reduced vegetative cell numbers; CXCL10 treatment resulted in the complete killing (n.d., none detected) of the initial bacillus inoculum
(dashed line). Alamar blue data are expressed as a percentage of untreated control and represent mean values ⫾ SEM for three independent
experiments; CFU data are expressed as CFU/ml (log
scale) ⫾ SEM, and a representative data set is shown from three separate
experiments. ⴱⴱⴱ, P value of ⬍0.001 compared with untreated control.
OL. 77, 2009 CXC CHEMOKINES AND BACILLUS ANTHRACIS 1671
higher levels of CXCL11 at1hand24h(P ⬍ 0.01 and 0.05,
respectively) (Fig. 8C). These data are consistent with a role
for CXC chemokine-mediated antimicrobial activity in pro-
moting host defense against inhalational B. anthracis infection
In vivo analysis of B. anthracis spore germination in lungs of
spore-challenged mice. The B. anthracis Sterne strain spores
used in the in vivo intranasal spore challenge studies described
above contained a bioluminescent reporter plasmid in which
the expression of the lux operon signals spore germination
(55). This bioluminescent strain allowed us to determine
whether the strain-speciﬁc differences, as related to the out-
come of inhalational B. anthracis infection, between A/J and
C57BL/6 mice occur before or after spore germination. Using
In Vivo Imaging System (IVIS) technology, luminescence was
measured in the lungs of sham- and spore-challenged mice at
1 h, 6 h, 24 h, and 48 h postchallenge to determine the extent
of spore germination (summarized in Fig. 9). Lungs harvested
from spore-challenged A/J mice displayed luminescence by 1 h,
with increasing levels of luminescence through the 6-h and
24-h time points (Fig. 10A and B); by 48 h, mean luminescent
signal intensity in the lungs of spore-challenged A/J mice had
begun to decrease. In contrast, lung samples harvested from
spore-challenged C57BL/6 mice demonstrated a ⬎90% reduc-
tion in spore germination at 1 h compared to spore-challenged
A/J mice and were not found to exhibit detectable levels of
luminescence at 6 h, 24 h, or 48 h (Fig. 10A and B), indicating
that spore germination was absent. Neither inbred mouse
strain exhibited luminescence in the spleen or liver following
spore challenge, consistent with previous reports that spore
germination occurs at initial sites of infection (23, 24, 55).
These observations demonstrate that A/J and C57BL/6 mice
have differential abilities to prevent spore germination within
the lungs, which may act to disrupt the establishment of inha-
lational B. anthracis infection.
In the present study, we have established the ability of the
CXC chemokines CXCL9, CXCL10, and
CXCL11 to exert direct antimicrobial effects against B. anthra-
cis spores and bacilli in a concentration-dependent manner.
The abilities of these host chemokines to inhibit spore germi-
nation and reduce spore viability are, to our knowledge, the
ﬁrst description of direct chemokine-mediated antimicrobial
effects on bacterial spores and may represent an important
mechanism for promoting host defense during the initial stages
of inhalational anthrax. In support of this conclusion, we have
demonstrated that the induction of CXCL9, CXCL10, and
CXCL11 within the lungs of spore-challenged mice was asso-
ciated with a substantial reduction of spore germination and
subsequent disease progression. Taken together, these results
open a new avenue of research for examining host chemokines
as potential sporicidal/bactericidal agents.
The ability of the IFN-inducible ELR
CXC chemokines to
prevent spore germination and directly mediate sporicidal ef-
FIG. 6. Direct antimicrobial effects of CXCL9, CXCL10, and
CXCL11 against B. anthracis bacilli are concentration dependent.
(A) B. anthracis 7702 vegetative bacilli were treated with increasing
concentrations of CXCL9, CXCL10, or CXCL11 for 6 h before
Alamar blue reduction was measured. EC
s were determined to be
40.2 ⫾ 1.1 g/ml, 4.0 ⫾ 0.3 g/ml, and 72 ⫾ 3.4 g/ml for CXCL9,
CXCL10, and CXCL11, respectively. Alamar blue reduction data are
expressed as a percentage of control and represent mean values ⫾
SEM for one to two independent experiments. For clarity, only the
lowest chemokine concentrations demonstrating statistically signiﬁcant
decreases from the untreated control are labeled with asterisks.
(B) CFU determination of CXCL10-treated vegetative bacilli was per-
formed after6hoftreatment. CXCL10 concentrations of ⱖ12 g/ml
resulted in complete killing (n.d., none detected) of the initial inocu-
lum (dashed line). CFU data are expressed as CFU/ml (log
SEM and are a representative data set from two separate experiments.
ⴱ, P value of ⬍0.05; ⴱⴱⴱ, P value of ⬍0.001 (compared with untreated
FIG. 7. CXCL10-mediated antimicrobial activity results in the
complete loss of B. anthracis cell integrity. B. anthracis 7702 vegetative
bacilli were treated with or without 48 g/ml CXCL10 for 3 h before
being processed for TEM. Untreated bacilli were numerous and dis-
played a typical cellular structure. Conversely, CXCL10-treated bacilli
demonstrated a complete loss of cellular integrity and cell membrane/
cell wall structure. Representative ﬁelds from two independent exper-
iments are shown at a ⫻30,000 magniﬁcation; the scale bar equals
1672 CRAWFORD ET AL. INFECT.IMMUN.
fects represents novel antimicrobial roles for host chemokines.
Deﬁning spore-speciﬁc targets and the molecular mecha-
nism(s) of chemokine-mediated antimicrobial activity, how-
ever, is complicated by a lack of understanding concerning the
mechanistic details of the signaling pathways and molecular
interactions involved in spore germination (48). Whereas little
data on the inhibition of B. anthracis spore germination exist,
numerous compounds capable of inhibiting spore germination
in Bacillus species have been described and include germinant
molecule analogs (1), alkyl alcohols (62), ion channel blockers
(45), protease inhibitors (8), sulfydryl reagents (22), and a
variety of other compounds (15). While incompletely deﬁned,
the mechanisms by which these compounds inhibit spore ger-
mination appear to rely on their ability to disrupt germinant
receptor engagement and/or the subsequent signaling activities
of one or more nutrient receptors (15). Consequently, DPA
release (which is essential in triggering cortex hydrolysis during
nutrient-mediated germination) is prevented, and spore ger-
mination is blocked prior to cortex degradation (15, 58).
Notably, the abilities of the above-described compounds to inhibit
germination are largely reversible and do not affect spore via-
bility (colony-forming ability), suggesting that subsequent me-
diators of germination remain functional (15).
Although the effects of CXCL9, CXCL10, and CXCL11 on
initial germination events and the possible role of these events
FIG. 8. CXCL9, CXCL10, and CXCL11 responses in the lungs of spore-challenged C57BL/6 and A/J mice. C57BL/6 and A/J mice were challenged
intranasally with B. anthracis 34F2 spores. Lungs from sham-challenged (three animals per time point per group) and spore-challenged (ﬁve animals per
time point per group) C57BL/6 and A/J mice were harvested at 1 h, 6 h, 24 h, and 48 h postinfection. CXCL9, CXCL10, and CXCL11 protein levels were
determined using Luminex bead array analysis as described in Materials and Methods. (A) CXCL9 levels in the lungs of spore-challenged C57BL/6 mice
increased throughout the time course examined and were signiﬁcantly higher than in the lungs of spore-challenged A/J mice at 24 h and 48 h. (B) CXCL10
levels in the lungs of spore-challenged C57BL/6 mice were induced as early as 1 h and were signiﬁcantly higher than those found in spore-challenged A/J
mice at all time points tested. (C) Spore challenge induced signiﬁcantly higher CXCL11 levels in the lungs of C57BL/6 mice than in the lungs of A/J mice
at 1 h and 24 h. Luminex data are expressed as chemokine concentration (pg/ml) measured in diluted lung homogenate ﬁltrates. Data points represent
mean values ⫾ SEM. ⴱ, P value of ⬍0.05; ⴱⴱ, P value of ⬍0.01; ⴱⴱⴱ, P value of ⬍0.001.
FIG. 9. Relative spore germination in the lungs of sham- and spore-
challenged A/J and C57BL/6 mice. A/J and C57BL/6 mice were challenged
intranasally with B. anthracis 34F2 spores harboring a lux reporter construct
that is expressed during spore germination. The lungs from sham-challenged
(four animals per time point per group) and spore-challenged (eight animals
per time point per group) mice were scanned for luminescence at 1 h, 6 h,
24 h, and 48 h postchallenge using IVIS. Spore germination, as measured by
luminescence, was observed in the lungs of spore-challenged A/J mice by 1 h,
with increasing levels of germination through the 6-h and 24-h time points; by
48 h, the mean luminescent signal intensity in the lungs of spore-challenged
A/J mice had begun to decrease. Minimal luminescence (signal intensity of
⬍5) was detected in ﬁve of the eight spore-challenged C57BL/6 mice at 1 h
postchallenge; no detectable spore germination was observed in the lungs of
C57BL/6 mice at 6 h, 24 h, or 48 h as assessed by a lack of luminescent signal.
The lungs from all sham-challenged controls were nonluminescent at each
time point tested. Data points represent the maximum luminescent signals
detected in the lungs of individual mice at the time point indicated; lumines-
cence is reported as photons per second per square centimeter per steradian.
OL. 77, 2009 CXC CHEMOKINES AND BACILLUS ANTHRACIS 1673
in spore susceptibility remain to be determined, TEM visual-
ization of CXCL10-treated B. anthracis spores established that
the inhibition of spore germination and reduction in spore
viability occur prior to spore coat and cortex degradation,
demonstrating that CXCL10 directly targets spores and not
newly emerging vegetative bacilli. Since ungerminated spores
are metabolically inactive, a chemokine-mediated inhibition of
macromolecular synthesis as a mechanism for antimicrobial
activity is unlikely. These data suggest that CXCL10 exerts its
antimicrobial effects by targeting a process inherent, and likely
essential, to spore germination and the maintenance of spore
Immunoelectron microscopy demonstrated that CXCL10
associates with the outermost spore layers including the exos-
porium, spore coat, and spore coat-cortex interface by 1 h, and
this localization does not rely on the presence of germinants in
the treatment medium. The nonessential nature of the exos-
porium in spore germination and viability makes it an unlikely
target of chemokine-mediated antimicrobial activity. We can-
not exclude, however, a possible role for the exosporium, es-
pecially since it is the outermost spore layer and is rich in
negatively charged carboxylate and phosphate groups (30) that
may facilitate initial interactions with the positively charged
C-terminal regions of the ELR
CXC chemokines. Although
the roles of the spore coat and outer membrane (located at the
spore coat-cortex interface) in the process of germination are
incompletely deﬁned, studies investigating spore germination
in Bacillus subtilis and Bacillus cereus have identiﬁed several
components associated with spore germination that localize to
these structures and have orthologs in B. anthracis. For exam-
ple, cortex-lytic enzymes such as CwlJ, located in the spore
coat (3), and SleB, located at the spore coat-cortex interface
(49) and inner membrane (10), are responsible for mediating
cortex degradation during spore germination. Although the
inhibition of these enzymes is consistent with the mechanism of
action for several inhibitors of spore germination (2), a recent
study suggested that the inhibition of cortex-lytic enzymes is
likely an indirect effect and not the primary site of action for
these inhibitors (15). Studies of B. subtilis and B. cereus have
also identiﬁed a gerP operon that encodes proteins thought to
be structural components of the spore coat that are important
in inﬂuencing spore coat permeability and thereby facilitating
germinant access to the inner membrane (6). Interestingly,
mutational inactivation of the gerP locus results in germination
defects similar to those seen in CXCL10-treated B. anthracis
spores, including a block in spore germination prior to cortex
hydrolysis, a lower rate of germination, and a reduction in
colony-forming ability (6).
It is not currently known whether CXCL10 is able to interact
with spore structures in addition to the exosporium, spore coat,
and spore coat-cortex interface. Potential CXCL10 localization
at the germ cell wall and/or inner membrane may not be
identiﬁed using immunoelectron microscopy since these sites
are likely inaccessible to the relatively large labeling reagents
required for visualization. Given the proposed ability of anti-
microbial chemokines to interact with bacterial membranes
and cell wall components (described below), it is possible that
CXCL9, CXCL10, and CXCL11 exert their antimicrobial ef-
fects against B. anthracis spores by altering the structure of the
germ cell wall and/or spore membranes. As a result, the proper
FIG. 10. A/J and C57BL/6 mice differ in their abilities to prevent spore germination within the lungs. A/J and C57BL/6 mice were challenged
intranasally with B. anthracis 34F2 spores harboring a lux reporter construct expressed during spore germination. (A) Lungs, spleen, and liver from
sham-challenged (four animals per time point per group) and spore-challenged (eight animals per time point per group) A/J and C57BL/6 mice
were scanned for luminescence 6 h postchallenge. Extensive germination, as measured by luminescence signal, was detected in the lungs of
spore-challenged A/J mice; the lungs of spore-challenged C57BL/6 mice demonstrated no detectable spore germination at 6 h, and luminescence
was absent in all tissues from sham-challenged animals. (B) Tissues harvested from sham- and spore-challenged animals 24 h postchallenge were
also scanned for luminescence. Rates of germination in the lungs of spore-challenged A/J mice had increased considerably from 6 h (note that the
scale for A/J mice in B is expressed as luminescence values [10
]). Again, no detectable spore germination was observed in the lungs of C57BL/6
mice, and all sham-challenged tissues were nonluminescent. Neither inbred mouse strain exhibited luminescence in the spleen or liver following
spore challenge at any time point examined. Merged photographic and luminescence images are shown for representative animals; luminescence
is reported as photons per second per square centimeter per steradian.
1674 CRAWFORD ET AL. INFECT.IMMUN.
functioning of membrane-associated spore components may
be prevented, leading to the irreversible inhibition of spore
germination. The possibility that changes in membrane struc-
ture and/or membrane protein function can have deleterious
effects on spore germination is suggested by tetracaine- and
procaine-mediated inhibition of spore germination (45). The
mechanisms by which these germination inhibitors act are
thought to reﬂect a disruption in ion efﬂux and/or DPA release
due to increased disordering of the lipid bilayer hydrocarbon
interior (15, 73). This type of mechanism would help to explain
how chemokine treatment results in the reduction of spore
viability; however, it remains to be determined whether the
decrease in spore viability upon chemokine treatment occurs
via the same mechanism as germination inhibition or whether
these effects act through separate, independent mechanisms.
Similar to the abilities of the IFN-inducible ELR
chemokines to mediate direct antimicrobial effects against B.
anthracis spores, all three CXC chemokines were found to
exhibit direct antimicrobial activity against B. anthracis bacilli.
These data support a growing body of literature demonstrating
the ability of host chemokines to target vegetative bacteria
through a similar, as-yet-undeﬁned, mechanism. In this regard,
chemokines share several biophysical properties, including cat-
ionicity and amphipathicity, with antimicrobial peptides that
function in innate host defense against infection (72). Further-
more, the C-terminal helical region of antimicrobial chemo-
kines, which is thought to mediate the direct antimicrobial
activity of host chemokines, has a structure and amino acid
composition similar to those of classical ␣-helical antimicrobial
peptides (72). Due to such similarities, several groups have
proposed that chemokines may mediate antimicrobial activity
through a similar mechanism in which electrostatic interactions
between the cationic host molecule and the anionic microbial
surface facilitate interactions resulting in membrane permeabi-
lization and cell lysis (12, 32).
In support of this proposed mechanism of action, CXCL9-
and CXCL10-treated vegetative bacilli were found to undergo
bacterial chain segmentation with subsequent disruption, con-
sistent with the generation of defects in cell wall and/or cell
membrane integrity. These effects on the structural integrity of
bacillus chains mimic those seen when vegetative bacilli were
treated with penicillin G, which disrupts bacterial cell wall
synthesis and results in the loss of bacterial cell integrity (data
not shown). In addition, CXCL10-treated bacilli demonstrated
a complete loss of cellular integrity as determined through
TEM visualization. Although we cannot exclude the possibility
that the loss of cellular integrity is a consequence of bacteri-
cidal activity rather than the cause, our data do not contradict
the current hypothesis that chemokine-mediated antimicrobial
activity against vegetative bacteria results from a direct disrup-
tion of bacterial membranes and/or cell wall integrity.
The studies described here were performed using the toxigenic,
unencapsulated Sterne strain of B. anthracis (pXO1
Although B. anthracis strain differences are thought to be minimal
or absent with regard to spores, the presence of the poly-
tamic acid capsule may interfere with chemokine-mediated anti-
microbial activity against vegetative bacilli. In order to conﬁrm
the susceptibility of encapsulated bacilli to CXCL9, CXCL10, and
CXCL11, the ﬁndings described here will need to be further
examined using an encapsulated strain such as the nontoxigenic,
encapsulated Pasteur strain (pXO1
) or the toxigenic,
encapsulated Ames strain (pXO1
In order to begin investigating possible protective roles of
chemokine-mediated antimicrobial activity during B. anthracis
infection in vivo, we used a murine model of inhalational
anthrax. The lungs of spore-challenged A/J mice, which are
susceptible to infection by B. anthracis Sterne strain and suc-
cumb to inhalational anthrax, did not demonstrate elevated
levels of CXCL9, CXCL10, or CXCL11 compared to sham-
challenged controls, and extensive spore germination was ob-
served within the lungs at all time points examined, as deter-
mined by bioluminescent measurement. In contrast, the lungs
of spore-challenged C57BL/6 mice, which are resistant to in-
fection and survive intranasal spore challenge, were found to
have signiﬁcantly higher levels of CXCL9, CXCL10, and
CXCL11 than sham-challenged controls; increased chemokine
levels were associated with a substantial reduction (1 h) or
absence (6 h, 24 h, and 48 h) of detectable spore germination
within the lungs of C57BL/6 mice. Although these data support
a potential role for chemokine-mediated antimicrobial activity
in promoting host defense during infection, they do not differ-
entiate between direct antimicrobial effects and indirect effects
resulting from immune cell recruitment to sites of infection.
Differentiation of these activities and characterization of po-
tential direct chemokine-mediated effects in vivo will require
an infection model in which cellular inﬁltration in response to
these chemokines is prevented. While host cell recruitment to
CXCL9, CXCL10, and CXCL11 can be disrupted by using
mice deﬁcient in the chemokine receptor CXCR3, this lack of
cellular inﬁltration will likely disrupt the positive-feedback
loop whereby recruited cells produce factors (e.g., IFN-␥) that
promote the induction of these chemokines. Therefore, future
experiments will focus on the development of an in vivo model
in which indirect chemokine-mediated effects are reduced and
the overall chemokine production remains comparable to that
seen during infection. When discussing the in vivo data pre-
sented in this paper, it is also important to recognize known
differences among the mouse strains used as well as differences
between in vitro and in vivo chemokine concentrations.
Previous studies have attributed differences in susceptibility
to B. anthracis infection among inbred murine strains to a
deﬁciency in complement. Speciﬁcally, host resistance to
Sterne strain infection has been shown to be associated with
the host’s ability to produce complement component 5 (C5),
and complement depletion in normally resistant mice renders
them highly susceptible to inhalational Sterne strain infection
(29, 66). Although a direct role for C5 in controlling Sterne
strain infection has not been determined, several explanations
have been proposed, including increased phagocytic killing and
the promotion of immune cell inﬁltration to sites of infection
(66). In the present context, the latter explanation is particu-
larly interesting since a defect in C5a, a chemotactic cleavage
product of C5, would likely disrupt host cell recruitment during
inhalational B. anthracis infection and may prevent the induc-
tion of the interferon-inducible ELR
Along these lines, C5a neutralization has been shown to sig-
niﬁcantly reduce CXC and CC chemokine production by alve-
olar macrophages in vivo (17). Similarly, C5 depletion has been
found to inhibit production of IFN-␥, a potent inducer of the
CXC chemokines, and prevent the induction of proin
VOL. 77, 2009 CXC CHEMOKINES AND BACILLUS ANTHRACIS 1675
ﬂammatory cytokines and chemokines during experimental
sepsis (21, 63).
All antimicrobial chemokines characterized to date, includ-
ing those presented here, mediate direct antimicrobial effects
within a concentration range that is higher than that required
for inducing directed cell migration (12, 32, 61). In the present
study, the concentrations of CXCL9, CXCL10, and CXCL11
measured from the lungs of spore-challenged animals were
lower than the concentrations used in the in vitro studies. The
notion that direct chemokine-mediated antimicrobial activity is
likely to be relevant during infection is supported by several
published studies, including that (i) the stimulation of periph-
eral blood mononuclear cells with IFN-␥ induces the produc-
tion of CXCL9 and CXCL10 to levels calculated to be capable
of exerting direct antimicrobial effects against E. coli (12); (ii)
supernatants from IFN-␥/tumor necrosis factor alpha-stimu-
lated normal human bronchial epithelial cells demonstrate
CXC chemokine concentrations of sev
eral hundred nanograms per milliliter, with CXCL10 levels
approaching 1 g/ml (56); and (iii) markedly elevated concen-
trations of CXCL9 (⬎170 ng/ml) and CXCL10 (⬎400 ng/ml)
have been shown to be present in the plasma of patients with
melioidosis and correlate with the severity and outcome of infec-
tion (38). While the above-described studies support that the
CXC chemokines are part of the innate immune re
sponse to bacterial infections, these concentrations are lower
than the effective in vitro concentrations used in this study.
Local chemokine concentrations at sites of infection, however,
are likely higher than those measured in whole-tissue ﬁltrates
or cell culture supernatants and may reach levels sufﬁcient for
exerting antimicrobial effects through the individual or addi-
tive effects of these CXC chemokines.
Further support for a potential role of direct chemokine-
mediated antimicrobial activity during infection comes from
studies examining the effector functions of antimicrobial pep-
tides, including defensins. The ␣-, ␤-, and -defensins exhibit
antimicrobial activity in vitro at concentrations in the micro-
gram-per-milliliter range (57), much like the direct chemokine-
mediated antimicrobial activity described in the present study.
Despite relatively high effective concentrations in vitro, de-
fensins have been shown to play a critical role in innate host
defense against bacterial challenge. Several transgenic mouse
studies have provided evidence for defensin-mediated antibac-
terial effector functions in vivo and include (i) delayed clear-
ance of Haemophilus inﬂuenzae from the lungs of mice deﬁ-
cient in mouse ␤-defensin-1 (50), (ii) reductions in bacterial
burden and increased survival rates following challenge with
Salmonella enterica serovar Typhimurium in mice expressing
human defensin 5 (53), and (iii) increased virulence of E. coli
in mice deﬁcient in Paneth cell ␣-defensins (68). Taken to-
gether, these in vivo studies provide evidence for a physiolog-
ical function of defensins in promoting host defense and sug-
gest that the chemokine concentrations presented in the
current study are not so high as to preclude them from exerting
antimicrobial activity in vivo.
Given ongoing concerns about the threat posed by weap-
onized B. anthracis spores and the inability of current treat-
ment options to prevent the establishment of anthrax, novel
therapeutic strategies capable of effectively targeting the early
stages of B. anthracis infection are required. The ability of
CXCL9, CXCL10, and CXCL11 to affect both B. anthracis
spores and bacilli establishes a novel antimicrobial effect of
these chemokines. Also, their induction by the administration
of exogenous IFN-␥ may offer an effective way of augmenting
the production of protective CXCL9, CXCL10, and CXCL11
levels in the host lungs. By understanding the mechanism(s) by
which these chemokines target B. anthracis spores and vegeta-
tive bacilli and their ability to promote host defense during
infection, it is likely that innovative therapeutic strategies can
be devised for effectively treating and/or preventing inhala-
tional B. anthracis infection. In addition, these ﬁndings will
likely have a therapeutic impact on infections caused by a
range of pathogenic and potentially multidrug-resistant bacte-
ria including other spore-forming organisms such as Clostrid-
This work was supported by the Virginia Commonwealth Health
Research Board (M.A.H.) and by National Institutes of Health
National Institute of Allergy and Infectious Diseases grant U54
AI057168 and Mid-Atlantic Regional Center of Excellence in Bio-
defense and Emerging Infectious Diseases (M.A.H. and A.D.O.).
Support was also provided by National Institutes of Health grant
T32 AI055432-05, Biodefense Research Training and Career De-
We thank Erik Hewlett and the members of his laboratory for
helpful discussions and advice. We also thank Jan Redick for her
assistance with transmission electron microscopy analysis and immu-
nogold labeling procedures.
We have declared that no conﬂict of interest exists.
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