Testing nucleoside analogues as inhibitors of Bacillus anthracis spore germination in vitro and in macrophage cell culture

Article abstract of DOI:10.1128/AAC.01029-10

Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifest

Full text of DOI:10.1128/AAC.01029-10

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2010, p. 5329–5336
0066-4804/10/$12.00 doi:10.1128/AAC.01029-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 12
Testing Nucleoside Analogues as Inhibitors of Bacillus anthracis
Spore Germination In Vitro and in Macrophage Cell Cultureᰔ
Zadkiel Alvarez,1 Kyungae Lee,2 and Ernesto Abel-Santos1*
Department of Chemistry, University of Nevada, Las Vegas, Las Vegas, Nevada 89154,1 and The New England Regional Center of
Excellence in Biodefense and Emerging Infectious Diseases, Harvard Medical School, Boston, Massachusetts 021152
Received 26 July 2010/Returned for modification 6 September 2010/Accepted 26 September 2010
Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the
endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria.
In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the
respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then
produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogen-
esis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides.
Inosine and L-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that
germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG),
a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in
murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or
inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to
protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship
analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis
spores. In contrast, no structure-activity relationships were apparent for compounds that protected
macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected mac-
rophages from B. anthracis.
Anthrax is as an often-fatal, acute infectious disease
caused by the rod-shaped, Gram-positive, endospore-form-
ing bacterium Bacillus anthracis (17). The highly resistant
Bacillus anthracis spores remain dormant in soil, occasion-
ally entering mammals through skin abrasions, ingestion, or
inhalation (27). The severity of B. anthracis pathogenicity is
dependent on the uptake route. In inhalational anthrax, the
most deadly form of the disease, spores enter the host or-
ganism through the respiratory tract (27). Once inside the
lungs, spores are phagocytosed by alveolar macrophages and
germinate inside the phagosome (15, 16). The germinated
spores become metabolically active and secrete anthrax tox-
ins, the bacterium’s primary virulence factors (3, 4, 6). Veg-
etative bacilli rapidly proliferate within the lymphatic system
and disseminate throughout the bloodstream (18). The con-
tinuous secretion of toxins leads to fatal septicemia.
Although spore germination is a critical step in the estab-
lishment of anthrax infection (18), very little is known about
the signaling pathways involved in spore germination (28,
32). The first step in the germination process is most com-
monly the binding of metabolites by germination (Ger) re-
ceptors (8, 23, 38). These receptors are membrane proteins
mostly encoded by tricistronic operons. Up to seven Ger
receptors have been characterized in B. anthracis (13). Com-
binations of Ger receptors may be involved in different
interacting pathways for germination (13, 30). Usually a
purine and an amino acid are required for the efficient
germination in vitro of B. anthracis spores (2, 23, 37). Once
germination is activated, a series of degradative events
break up spore-specific structures and proteins (24, 29, 34).
Germination is followed by a period of outgrowth, during
which actively dividing cells are regenerated (19, 20, 22).
It has been observed that Bacillus and Clostridium spore
germination can be blocked by alcohols (11, 36), ion channel
blockers (26), protease inhibitors (9), sulfhydryl reagents
(14), and other miscellaneous compounds (10). Most of
these studies targeted specific germination pathways in dif-
ferent organisms and are not directly comparable. A more
recent study tested the activities of subsets of the different
types of compounds against Bacillus subtilis and Bacillus
megaterium germination (10). Research from many groups,
including ours, has shown that nucleoside and amino acid
analogues act as competitive inhibitors of B. anthracis spore
germination in vitro (2, 21, 25). Of these inhibitors, D-ala-
nine (D-Ala) and D-histidine (amino acid analogues) and
6-thioguanosine (6-TG; a nucleoside analogue) were shown
to also protect macrophages from B. anthracis-mediated ne-
crosis (2, 21, 25).
In this study, we screened 46 different nucleoside ana-
logues for their abilities to inhibit B. anthracis spore germi-
nation in vitro and in macrophage cultures. Structure-activ-
ity relationship analysis allowed identification of epitopes
necessary for nucleoside recognition by B. anthracis spores.
However, we found no correlation between in vitro germi-
nation inhibition and the ability of nucleosides to protect
macrophages from B. anthracis cytotoxicity. We also showed
that a nucleoside analogue (6-TG) and an amino acid ana-
logue (D-Ala) combined to increase macrophage protection
from B. anthracis cytotoxicity.
* Corresponding author. Mailing address: 4505 Maryland Parkway,
Campus Box 4003, Las Vegas, NV 89154. Phone: (702) 895-2608. Fax:
(702) 895-4072. E-mail: ernesto.abelsantos@unlv.edu.
ᰔ Published ahead of print on 4 October 2010.
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ANTIMICROB. AGENTS CHEMOTHER.
FIG. 1. Compounds tested as B. anthracis spore germination inhibitors in vitro and in cell culture (with the compound number shown in roman
numerals in parentheses): INO (I), 6-TG (II), 2-mercaptopyrimidine (III), 2-thiouracil (2-TU; IV), trithiocyanuric acid (TTCA; V), 2,4-diamino-
6-mercaptopyrimidine (DAMPy; VI), 2-mercaptopyridine (VII), 4-mercaptopyridine (VIII), 2-mercaptobenzimidazole (2-MBI; IX), 2-methyl-
mercaptobenzimidazole (2-MMBI; X), 6-TI (XI), ADE (XII), GUA (XIII), 6-CPR (XIV), 2-APR (XV), 6-MMPR (XVI), 6-BTI (XVII),
6-methylaminopurine riboside (6-MAPR; XVIII), 6-N-dimethyladenosine (6-DMA; XIX), 6-DEA (XX), 6-DPA (XXI), 6-DMAA (XXII), 6-BMA
(XXIII), 6-PPA (XXIV), 6-ECA (XXV), 6-PPER (XXVI), 6-OMG (XXVII), 6-NBA (XXVIII), NEB (XXIX), 2-AADE (XXX), 6-CG (XXXI),
ISO (XXXII), XAN (XXXIII), 8-AADE (XXXIV), 8-HG (XXXV), 7-MI (XXXVI), APR (XXXVII), IH (XXXVIII), TU (XXXIX), GCV (XL),
6-Tg (XLI), 6-Mp (XLII), 6-Cg (XLIII), 6-cyanopurine (6-CYp; XLIV), allopurinol (Ap, XLV), 6-BAp (XLVI).
MATERIALS AND METHODS
ethanol or DMF was heated at 60 to 90°C for 18 h and cooled to room temper-
ature. After evaporation of the solvent, the residue was dissolved in methanol (1
ml). Upon standing overnight, crystals of the desired product were formed, and
these were collected by filtration (50 to 88% yield). Compounds were charac-
terized by 1H nuclear magnetic resonance and mass spectrometry.
Cell lines, reagents, and equipment. Murine macrophage J774A.1 cells were a
generous gift from Ju¨rgen Brojatsch (Albert Einstein College of Medicine, NY).
B. anthracis Sterne 34F2 strain was a generous gift from Arturo Casadevall
(Albert Einstein College of Medicine, NY). Immunicillin H (IH; compound
XXXVIII) was a generous gift from Vern Schramm (Albert Einstein College of
Medicine, NY). Nucleoside analogues of 6-benzylthioinosine (6-BTI; compound
XVII), 6-N-diethyladenosine (6-DEA; XX), 6-N-dipropyladenosine (6-DPA;
XXI), 6-N-benzylmethyladenosine (6-BMA; XXIII), 6-N-piperidineadenosine
(6-PPA; XXIV), 6-N-((3-ethylacetyl)cyclohexyl) adenosine (6-ECA; XXV), and
ribofuranosyl-((9H-purin-6-yl)pipierazin-1-yl)ethanone (6-PPER; XXVI) were
synthesized by Kyungae Lee from NSRB. Other compounds were purchased
from Sigma-Aldrich Corporation (St. Louis, MO) or Berry & Associates Inc.
(Brea, CA) (Fig. 1). In vitro spore germination and macrophage viability were
monitored in a Tecan Infinite M200 multimode microplate reader.
Synthesis of 6-BTI (compound XXVII), 6-DEA (XX), 6-DPA (XXI), 6-BMA
(XXIII), 6-PPA (XXIV), 6-ECA (XXV), and 6-PPER (XXVI). N-6- and S-6-
subtituted purine ribosides were synthesized by substituting 6-chloropurine ri-
boside with various amines or thiols in ethanol or dimethylformamide (DMF), as
previously reported (35). In brief, a mixture of 6-chloropurine riboside (1 eq), the
corresponding amine or thiol (3 eq), and triethylamine (72 ␮l; 0.51 mmol) in
B. anthracis spore preparation. B. anthracis cells were plated in nutrient agar
(EMD Chemicals Inc.) and incubated at 37°C to yield single cell clones. Indi-
vidual B. anthracis colonies were grown in nutrient broth and replated to obtain
bacterial lawns. Plates were incubated for 5 days at 37°C. The resulting bacterial
lawns were collected by flooding with ice-cold deionized water. Spores were
pelleted by centrifugation and resuspended in fresh deionized water. After two
washing steps, spores were separated from vegetative and partially sporulated
cells by centrifugation through a 20%-to-50% HistoDenz gradient (1). Spores
were resuspended in water and washed three times before storage at 4°C. Spores
in all preparations were more than 95% pure as determined by microscopic
observation of Schaeffer-Fulton-stained aliquots. Spore viability was assessed by
heat treatment followed by serial dilution plating in nutrient agar. Spore viability
was retested every 15 to 20 days.
Activation of B. anthracis spore germination. B. anthracis spore germination
was monitored spectrophotometrically whereby the loss in light diffraction fol-
lowing addition of a germinant was reflected by a decreased optical density at 580

VOL. 54, 2010
INHIBITION OF B. ANTHRACIS SPORE GERMINATION
5331
FIG. 2. (A) B. anthracis spores were treated with different concentrations of 6-TG, and germination was monitored by decreases in the optical
density at 580 nm. The IC50 for 6-TG (1 mM) was determined based on these data. (B) Macrophages were treated with B. anthracis spores and
different concentrations of 6-TG. Macrophage killing was monitored based on increases of PI fluorescence intensity.
nm (OD580). All germination experiments were carried out in a Tecan Infinite
M200 multimode microplate reader in UV-visible mode with its monochromator
set at 580 nm. The final volume of each reaction mixture was 0.2 ml. Experiments
were carried out in triplicate on at least two different days with two different
spore preparations. Standard deviations of germination rates were calculated
from these six independent assays. Spores were heat activated at 70°C for 30 min
before resuspension in germination buffer (50 mM Tris-HCl [pH 7.5], 10 mM
NaCl) to an OD580 of 1. The spore suspension was monitored for autogermina-
tion based on the OD580 after 1 h. No autogermination was detected in any
sample used. Lack of autogermination was confirmed by phase-contrast micros-
copy on selected samples. Spore suspensions were individually supplemented
with 250 ␮M concentrations of compounds I to XLVI and 40 ␮M L-alanine to
test for compounds that activate B. anthracis spore germination. Germination
was monitored by following the decrease in OD580 for 60 min. Optical densities
were normalized by dividing each data point by the absorbance at the time of
analogue addition and were reported as the relative OD580. Germination rates
were calculated from the initial linear region of the germination curves (2).
Germination rates were set to 100% for spores treated with 250 ␮M inosine and
40 ␮M L-alanine. These inosine and L-alanine concentrations represent the
lowest concentrations that induce the maximal germination rate. Percent germi-
nation for other nucleosides was calculated as the fraction of germination rate
compared to these conditions. Germination was confirmed in selected samples
by Schaeffer-Fulton staining.
Inhibition of B. anthracis spore germination. To test for antagonists of B.
anthracis spore germination, spore aliquots were individually supplemented with
various concentrations of compounds II to XLVI and incubated for 15 min at
room temperature. Spore-analogue mixtures were then treated with 250 ␮M
inosine and 40 ␮M L-alanine to induce germination. As above, optical densities
were normalized by dividing each data point by the absorbance at the time of
inosine addition and were reported as the relative OD580. Germination rates
were calculated from the initial linear region of the germination curves, as
described above. Germination inhibition was assessed as the reduction of ger-
mination rate with increased analogue concentration. Inhibited germination
rates were plotted against inhibitor concentrations to calculate 50% inhibitory
concentration (IC50) values using SigmaPlot v.11 software.
Macrophage viability assay. Macrophage J774A.1 cells were cultured using
Dulbecco modified Eagle’s medium (DMEM) without L-glutamine (Cellgro).
The medium was supplemented with 10% fetal bovine serum (FBS; HyClone), 2
mM Glutamax (Invitrogen Corp.), and 100 U of penicillin-streptomycin. The
cells were incubated at 37°C and 5% CO2 until cultures reached approximately
80% confluence. Cells were subcultured three times before use in cell killing
assays. Near-confluent macrophages were washed twice with ice-cold phosphate-
buffered saline (PBS) and then detached by treating for 15 min with PBS sup-
plemented with 5 mM EDTA. Detached cells were resuspended in DMEM
without FBS or antibiotics and seeded at 8 ϫ 105 cells/ml on 96-well transparent
plates. After the cells had adhered, compounds I to XLVI were individually
added at different final concentrations. The multiwell plate was incubated for 30
min at 37°C. Subsequently, B. anthracis spores, suspended in Hanks balanced
saline solution (HBSS; HyClone, Logan, UT), were added to macrophages at a
multiplicity of infection of 5. To control for compound toxicity, some cells were
treated with analogues but not infected with B. anthracis spores. Infected mac-
rophages were incubated at 37°C and 5% CO2 for 75 min. To remove nonphago-
cytosed spores, all wells were washed twice with 200 ␮l HBSS and then filled with
100 ␮l of fresh medium containing identical analogue concentrations. At 3 h
postinfection, cells were treated with 20 ␮M propidium iodide (PI). Cell necrosis
was assessed by increases in PI fluorescence. Fluorescence was measured every
hour for up to 7 h postinfection by following PI fluorescence at 617 nm. Relative
fluorescence was calculated as the quotient of the difference of each data point
and the fluorescence value at the time of PI addition, divided by the fluorescence
value at the time of PI addition. Relative fluorescence intensities were plotted
against inhibitor concentrations to calculate IC50 values by using SigmaPlot v.11
software.
Testing for compounds that enhance macrophage killing. Macrophages were
individually treated with potential enhancers to a 100 ␮M final concentration. B.
anthracis spores were then added to each sample. PI fluorescence was deter-
mined 5 h postinfection. Macrophage killing enhancement was calculated by
dividing the PI fluorescence intensity observed from infected macrophages
treated with specific compounds by the PI fluorescence intensity obtained from
infected untreated macrophages. GraphPad online calculators were used to run
Student’s t test and to obtain P values.
Treatment with combinations of 6-thioguanosine and D-alanine. Macrophages
were treated with 1 mM D-Ala, 50 ␮M D-Ala, 0.1 mM 6-TG, 0.1 ␮M 6-TG, or 50
␮M D-Ala–0.1 ␮M 6-TG. B. anthracis spores were then added to each sample.
Macrophage killing was determined 5 h postinfection based on the increase of PI
fluorescence intensity, as described above. GraphPad online calculators were
used to run Student’s t test and to obtain P values.
RESULTS AND DISCUSSION
Effects of 6-TG. Our previous studies showed that purine
analogues can inhibit inosine (INO; compound I)–L-alanine–
triggered Bacillus anthracis spore germination in vitro (2). Fur-
thermore, one compound, 6-TG (compound II), was also able
to inhibit intracellular spore germination and thus protect mu-
rine RAW264.7 macrophages from B. anthracis cytotoxicity
(2). As previously shown, 6-TG is a modest inhibitor of spore
germination in vitro, with an IC50 of 1 mM (Fig. 2A). This
inhibition was not due to the reducing properties of the thiol
group, as compounds III to X, with free sulfhydryls, were
unable to inhibit spore germination. The inabilities of com-
pounds III to X to affect B. anthracis spore germination also
suggest that the purine ring is necessary for recognition by the
germination machinery. 6-TG was also able to protect J774a.1
macrophages from B. anthracis cytotoxicity, with an IC50 of 3.5
␮M (Fig. 2B). Thus, 6-TG is approximately 300-fold more

5332
ALVAREZ ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Antagonists of B. anthracis spore germination in vitroa
weak germinant in vitro. In contrast, 6-mercaptopurine ribo-
side (6-MMPR; compound XVI) and 6-BTI (XVII) are B.
anthracis spore germination inhibitors, with IC50s of 318 ␮M
and 50 ␮M, respectively. A similar correlation was observed
across other 6-substituted germination inhibitors (Table 1). A
characteristic pattern of C-6–X–R (where X is oxygen, sulfur,
or nitrogen and R is a hydrophobic group) is observed for
inhibitors of B. anthracis spore germination. Hence, while non-
substituted compounds act as germinants, addition of a hydro-
phobic side chain to the 6-exocyclic heteroatom is sufficient to
convert a germinant structure into an inhibitor (Fig. 1, com-
pounds XVI to XXVIII). However, there was no correlation
between side chain physical properties and strength of inhibi-
tion. As an example, 2-APR (compound XV), 6-MMPR
(XVI), 6-DEA (XX), 6-(␥,␥-dimethylallyl amino)purine ribo-
side (DMAA; XXII), and 6-O-methylguanosine (6-OMG;
XXVII) showed similar IC50 values (Table 1). This suggests
that binding of inhibitors in B. anthracis spores is mostly driven
by nonspecific hydrophobic interactions. A similar pattern has
been previously observed for the inhibition of Bacillus cereus
spore germination in vitro (12).
In vitro assays showed that the presence of a 2-amino exo-
cyclic group in the purine ring also contributes to spore ger-
mination inhibition. This is exemplified by 6-TI (compound
XI), a 6-TG (compound II) analogue that lacks the 2-amino
group and does not inhibit, but activates, spore germination in
vitro. Similarly, nebularine (NEB; compound XXIX), a non-
substituted purine riboside, does not affect B. anthracis spore
germination, while 2-APR (compound XV), with an exocyclic
amino group at position 2 of the purine ring, inhibits spore
germination with an IC50 of 370 ␮M (Table 1). Furthermore,
ADE (XII), with an amino group at the 6-position, is a good
germinant in vitro. Meanwhile, 2-aminoadenosine (2-AADE;
XXX), which has amino groups at both the 2- and 6-positions,
is a germination inhibitor, with an IC50 of 700 ␮M. Similarly,
6-chloroguanosine (6-CG; XXXI) is also an inhibitor, with an
IC50 of 893 ␮M. In contrast, isoguanosine (ISO; XXXII), a
guanosine positional isomer, and xanthosine (XAN; XXXIII),
with carbonyl groups at the 2-positions, are inactive in spore
germination assays.
8-AADE (compound XXIV) is a positional isomer of
2-AADE (XXX) and has similar physical and chemical prop-
erties. However, 8-AADE did not activate or inhibit spore
germination under any conditions tested. Similarly, 8-hydroxy-
guanosine (8-HG; XXXV) had no effect on B. anthracis spore
germination. The inabilities of 8-AADE and 8-HG to influence
B. anthracis spore germination could be due to steric hindrance
at the 8-position.
The inosine derivative 7-methylinosine (7-MI; compound
XXXVI) showed no effect on B. anthracis spore germination.
Similarly, inosine analogues allopurinol riboside (APR;
XXVII) and IH (XXXVIII) do not affect spore germination.
Furthermore, the adenosine analogue tubericidin (TUB;
XXXIX) is inactive in B. anthracis spore germination assays.
Hence, the presence of an unsubstituted N-7 heterocyclic ni-
trogen in the purine ring also affects the germination proper-
ties of a compound.
Antagonist
Acronym
IC50 (mM)
6-Thioguanosine
6-TG
1.00
0.89
0.05
0.32
0.35
0.70
7.19
0.19
0.38
0.05
6-Chloroguanosine
6-CG
6-Benzylthioinosine
6-Methylmercaptopurine riboside
6-O-Methylguanosine
2-Aminoadenosine
Xanthosine
6-Methylaminopurine riboside
2-Aminopurine riboside
Ribofuranosyl-((9H-purin-6-yl)
piperazin-1-yl)ethanone
6-N-Benzylmethyladenosine
6-N-Benzylaminopurine
6-BTI
6-MMPR
6-OMG
2-AADE
XAN
6-MAPR
2-APR
6-PPER
6-BMA
6-BAP
0.12
1.55
0.36
1.14
1.09
0.31
0.76
0.70
0.02
6-(␥,␥-Dimethylallyl amino)purine riboside 6-DMAA
6-N-Benzoyladenosine
6-N-Piperidineadenosine
6-N-Diethyladenosine
6-NBA
6-PPA
6-DEA
6-DPA
6-ECA
D-Ala
6-N-Dipropyladenosine
6-N-((3-Ethylacetyl)cyclohexyl) adenosine
D-Alanine
a Spore suspensions were individually supplemented with various concentra-
tion of compounds II to XLVI and then treated with 250 ␮M inosine and 40 ␮M
L-alanine. Compounds that did not inhibit germination are not shown in this
table.
effective at preventing spore germination intracellularly than in
vitro. The reason for the discrepancies in IC50 values is not
clear. In vitro spore germination assays require high spore
concentrations to obtain reliable optical density changes. As a
result, millimolar concentrations of inosine and L-alanine are
needed to activate germination. In contrast, B. anthracis spores
will encounter low inosine concentrations in the macrophage
intracellular milieu (7). Low intracellular inosine concentra-
tions might be the reason for the higher efficiency of 6-TG at
blocking germination of B. anthracis spores in macrophages. It
is conceivable that inosine is the principal germinant inside
cells and that 6-TG prevents germination by competing with
endogenous inosine.
B. anthracis spore germination in vitro. Of the 46 compounds
screened as agonists and antagonists of B. anthracis spore ger-
mination, 5 were able to act as cogerminants with L-alanine,
and 18 were able to inhibit germination of B. anthracis spores
treated with inosine and L-alanine. None of the pyrimidine
analogues (compounds III to VIII) was able to activate B.
anthracis spore germination in vitro.
Position 6 of the purine ring seems to have a significant
role in the interaction of germinants with the B. anthracis
spore germination machinery. INO, 6-thioinosine (6-TI;
compound XI), adenosine (ADE; compound XII),
guanosine (GUA; compound XIII), and 6-chloropurine ri-
boside (6-CPR; compound XIV) are germinants for B. an-
thracis spores. All five compounds have 6-exocyclic groups
with unsubstituted electronegative atoms directly bound to
C-6 of the purine ring.
Electronegative groups are also present in every inhibitor of
B. anthracis spore germination in vitro, with the exception of
2-amino riboside (2-APR; compound XV) (Table 1). However,
nucleoside inhibitors normally possess aliphatic or aromatic
chains linked to an electronegative atom. For example, 6-TI
(compound XI), with a nonsubstituted 6-thiol (-SH), was a
Similar to the purine ring, the ribosyl group seems to be
required for germination inhibition of B. anthracis spores.
Ganciclovir (GCV; compound XL), a purine riboside analogue

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INHIBITION OF B. ANTHRACIS SPORE GERMINATION
5333
FIG. 3. Kinetics of macrophage killing by B. anthracis spores. (A) Macrophages were treated with medium (E) or B. anthracis spores (F).
Macrophage necrosis was monitored over time by the increase of PI fluorescence intensity. Error bars represent standard deviations of six
independent measurements. (B) Macrophages were observed under a microscope in white light (upper panels) or fluorescence (lower panels)
modes. Macrophages showed limited cell death 3 h postinfection with B. anthracis spores (a and b). In contrast, macrophages showed extensive
necrosis at 7 h postinfection with B. anthracis spores (c and d). Macrophage-engulfed spores (e.g., black circle in panel c) and vegetative B. anthracis
cells (e.g., black arrow in panel c) were also seen under these conditions. Macrophages treated with 6-TG were protected from B. anthracis
cytotoxicity even at 7 h postinfection with B. anthracis spores (e and f). No vegetative B. anthracis cells were observed.
that lacks the 2Ј-position, fails to either trigger or hinder B.
anthracis spore germination. Similarly, purine bases lacking the
ribosyl group (compounds XLI to XLVI), with the exception of
6-benzylaminopurine (BAp; XLVI), had no effect on germina-
tion in vitro. The aromatic side chain at the C-6 position may be
responsible for the modest inhibition shown by BAp against
the germination of B. anthracis spores in vitro. Therefore, an
entire purine nucleoside frame is required for optimal germi-
nation inhibition in vitro.
compounds are intriguing, since in vitro assays showed no effect
on B. anthracis spore germination. It is worth noting that these
compounds are the corresponding bases for the protective
nucleosides 6-TG (II), 6-TI (XI), and 6-CG (XXXI), respec-
tively. Furthermore, the IC50s for nucleosides and free bases
were similar in macrophage protection assays (Table 2). Thus,
both nucleosides and their free bases are able to protect mac-
rophages from B. anthracis cytotoxicity, even though they be-
have differently in spore germination assays.
Intracellular B. anthracis germination in J774a.1 macro-
phages. Murine J774a.1 macrophages can readily phagocytose
endospores of B. anthracis. After infection, spore germination
occurs inside the phagolysosome (18). Three hours postinfec-
tion, necrosis starts to be apparent, and approximately 20%
cell death occurs. Cell death then increases rapidly, reaching
almost 100% at 5 h postinfection (Fig. 3A). The timing of cell
death is variable, but most cells have entered necrosis at 5 h
postinfection. This variability is probably due to nonuniformity
in spore intake by macrophages, which leads to heterogeneous
cell killing (33).
Due to the heterogeneity of necrosis onset, compounds were
tested for their ability to reduce B. anthracis-mediated killing
at 5 h postinfection. Of the 46 compounds screened, only 8
were able to protect macrophages from B. anthracis cytotoxic-
ity (Table 2). No compound showed toxic effects in the absence
of B. anthracis spores. The protective compounds were either
purine nucleoside or purine base derivatives. The purine nu-
cleoside derivatives 6-TG (II), 6-TI (XI), 6-BTI (XVII), 6-CG
(XXXI), and APR (XXXVII) protected macrophages, with
IC50 values ranging from 3.5 ␮M (6-TG; II) to near 1 mM
(6-BTI; XVII) (Table 2). 6-TG (II) and 6-thioguanine (6-Tg;
XLI) were the most efficient analogues and protected macro-
phages from necrosis even 6 h after infection. The remaining
nucleoside analogues started to lose effectiveness at 6 h postin-
fection. Thus, these compounds did not completely inhibit
intracellular spore germination but were able to delay the
onset of the cytotoxic effect.
Similar to in vitro germination assays, the presence of the
2-amino side group of the base favors the action of the com-
pounds in macrophage protection assay. Analogues lacking
this group showed decreased macrophage protective effects
compared to their substituted counterparts. For example,
6-TG (II) had an IC50 value of 3.5 ␮M, while the unsubstituted
analog, 6-TI (XI), had an IC50 value of 35 ␮M, a 10-fold
decrease in protection ability. Similarly, 6-CG (XXXI) had an
IC50 value of 189 ␮M, while 6-CPR (XIV) was unable to
prevent cell killing.
Comparing microscopy observations with viability assay re-
sults showed that macrophages in which B. anthracis pro-
gressed from spores to vegetative cells also showed up as PI-
positive necrotic cells. On the other hand, in cell cultures
TABLE 2. Protection of macrophages from
B. anthracis-mediated killinga
Antagonist
Acronym
IC50 (mM)
6-Thioguanosine
6-Chloroguanosine
6-Thioinosine
Allopurinol riboside
6-Benzylthioinosine
6-Thioguanine
6-Chloroguanine
6-Mercaptopurine
D-Alanine
6-TG
6-CG
6-TI
APR
6-BTI
6-Tg
6-Cg
6-Mp
D-Ala
0.004
0.19
0.04
0.65
1.04
0.002
0.20
0.40
0.02
a Macrophage cultures were individually supplemented with various concen-
trations of compounds II to XLVI and then infected with B. anthracis spores.
Compounds that did not protect macrophages from anthrax cytotoxicity are not
shown in this table.
Three purine analogues, 6-Tg (XLI), 6-mercaptopurine (6-
Mp; XLII), and 6-chloroguanine (6-Cg; XLIII), were also able
to prevent cell necrosis. The protective properties of these

5334
ALVAREZ ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 5. Macrophage protection by 6-TG and D-Ala combina-
tions. Macrophages were treated with B. anthracis spores, and kill-
ing was monitored at 5 h postinfection based on increased PI flu-
orescence intensity. Addition of 1 mM D-Ala or 0.1 mM 6-TG
significantly reduced macrophage killing. In contrast, treatment
with 50 ␮M D-Ala or 0.1 ␮M 6-TG did not prevent cell killing.
Simultaneous treatment with 50 ␮M D-Ala and 0.1 ␮M 6-TG pro-
tected macrophages from B. anthracis cytotoxicity. Error bars rep-
FIG. 4. Compounds that enhance B. anthracis cytotoxicity. BMA,
8-HG, NBA, and IH were added to macrophages to a 100 ␮M final
concentration. B. anthracis spores were then added to each sample.
Macrophage necrosis, based on increased PI fluorescence intensity,
was monitored at 5 h postinfection. Macrophage killing enhance-
ment was calculated by normalizing the PI fluorescence intensity
determined for each compound treatment by the PI fluorescence
intensity for untreated macrophages. Error bars represent standard
resent standard deviations of six independent measurements.
,
*
deviations of six independent measurements. , P Ͻ 0.05;
, P Ͻ
*
**
significantly decreased killing in the presence of both 50 ␮M D-Ala
and 0.1 ␮M 6-TG compared to samples treated with 50 ␮M D-Ala
alone (P Ͻ 0.005) or samples treated with 0.1 ␮M 6-TG alone (P Ͻ
0.0001).
0.01;
, P Ͻ 0.001 (all for comparisons to untreated samples).
***
treated with an efficient inhibitor, spores did not progress to
vegetative bacteria and the integrity of mammalian cells was
maintained (Fig. 3B). Thus, endospores per se do not show
toxin-related killing toward macrophages. This suggests that
macrophage necrosis was either stopped or slowed when B.
anthracis spores did not germinate efficiently upon treatment
with germination inhibitors.
Compounds that enhance macrophage killing. Cell viability
screening allowed the identification of compounds that coop-
erated with B. anthracis spores to increase the rate of macro-
phage killing (Fig. 4). The compounds that enhanced cell kill-
ing more efficiently were 6-BMA (XXIII), 6-benzoyladenosine
(6-NBA; XXVIII), 8-hydroxyguanosine (8-HG; XXXV), and
IH (XXXVIII). This cell killing enhancement was not due to
compound toxicity, since these compounds do not induce cell
necrosis in the absence of B. anthracis spores (data not shown).
Surprisingly, some of the compounds that enhanced cell killing
were inhibitors in vitro. Currently, the mechanism by which
these compounds are able to accelerate macrophage killing in
the presence of B. anthracis spores is not known.
Effect of D-alanine and nucleoside analogue combinations.
D-Ala is a natural antigerminant for B. anthracis spores (21).
This molecule is biosynthesized by an alanine racemase
present in the spore and can modulate germination path-
ways that require L-alanine as a germinant (25). Two re-
search groups have already shown that D-Ala protects mac-
rophages from B. anthracis toxin-mediated necrosis by
inhibiting spore germination (21, 25). We corroborated
the antigerminant ability of D-Ala both in vitro (IC50, 16.0
able to protect macrophages from B. anthracis-mediated
cytotoxicity, even at suboptimal concentrations of both in-
hibitors (Fig. 5). Similar results were obtained with D-Ala
and 6-CG (XXXI) (data no shown).
Conclusions. Spore germination is the first necessary step
in the establishment of B. anthracis infection (15). Hence,
inhibition of B. anthracis spore germination could serve as a
prophylactic approach to prevent anthrax in exposed per-
sonnel (5). Antigermination therapy will delay infection on-
set and hence allow antibiotic and/or vaccine treatments to
be more effective. We had previously tested a limited num-
ber of purine nucleosides as inhibitors of B. anthracis spore
germination (2). In this study, we tested a larger collection
of purine nucleosides, purine bases, and pyrimidine bases
for their abilities to prevent spore germination in vitro and
protect macrophages from B. anthracis-mediated killing. All
compounds (but one) that inhibited spore germination in
vitro were purine nucleoside derivatives, showing that the
sugar moiety is important for inhibitor recognition. Further-
more, hydrophobic-substituted electronegative exocyclic
groups at the 6-position and an amine at the 2-position of
the purine ring were necessary functional groups for effi-
cient germination inhibition in vitro.
Molecular interactions between the spore germination
machinery and the nucleoside inhibitors require specific
contacts. Hydrogen bonding and/or ionic interactions are
probably involved in the recognition of the amino group at
the 2-position of purine nucleoside inhibitors. Similarly, hy-
drogen bonds could also serve to recognize essential sugar
hydroxyls and the exocyclic heteroatom at position 6. Hy-
drophobic and ␲-␲ interactions would increase binding af-
finities for nucleosides with alkyl and aromatic side chains.
The efficiency of spore germination in vitro did not cor-
relate with the capacity to protect macrophages from intra-
␮M) and in the macrophage killing protection assay (IC50
22.9 ␮M).
,
Since both inosine and L-alanine are strong germinants of
B. anthracis spores, a combination of a nucleoside inhibitor
(6-TG; II) and an amino acid inhibitor (D-Ala) could in-
crease effectiveness by simultaneously blocking multiple Ger
receptors. Indeed, combinations of 6-TG and D-Ala were

VOL. 54, 2010
INHIBITION OF B. ANTHRACIS SPORE GERMINATION
5335
Support for E.A.-S. was provided by the Ellison Medical Foundation
Young Scholar Award in Global Infectious Diseases and by Public
Health Service grant 1R01A1GM053212 from the National Institutes
of Health. Support to K.L. was provided by the National Screening
Laboratory for the Regional Centers of Excellence in Biodefense and
Emerging Infectious Diseases (NIAID U54 AI057159).
cellular spore germination and cytotoxicity. Some com-
pounds were able to inhibit spore germination in vitro but
did not protect macrophages against spore-mediated killing
(e.g., 6-MMPR [compound XVI] and 6-OMG [XXVII]).
Conversely, other compounds were unable to curtail spore
germination in vitro but were able to prevent macrophage
necrosis (e.g., 6-TI [XI] and APR [XXXVII]). Yet another
compound subset was composed of efficient in vitro inhibi-
tors that enhanced macrophage killing by B. anthracis spores
(e.g., 6-BMA [XXIII] and 6-NBA [XXVIII]).
The lack of correlation between in vitro and intracellular
B. anthracis spore germination assay results was not due to
drug toxicity. These two assays measured spore germination
under different conditions. In vitro germination assays are
only dependent on the ability of compounds to cross the
spore coat and interact with their respective Ger receptor.
In contrast, intracellular germination assays are complicated
by the additional requirement for compounds to cross both
the mammalian cytoplasmic and phagosomal membranes.
Furthermore, potential germination inhibitors may have to
contend with cellular catabolism, active drug efflux pumps,
and other mammalian cellular processes.
We thank Su Chiang for helpful discussions.
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ACKNOWLEDGMENTS
This work was supported by the RING-TRUE III 0447416 award
from NSF-EPSCoR and by NIH grant no. P20 RR-016464 from the
INBRE Program of the National Center for Research Resources.

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