Innate Immunity against Granulibacter bethesdensis, an Emerging
Gram-Negative Bacterial Pathogen
Kol A. Zarember,aKimberly R. Marshall-Batty,b,cAnna R. Cruz,aJessica Chu,aMichael E. Fenster,bAdam R. Shoffner,b
Larissa S. Rogge,aAdeline R. Whitney,dMeggan Czapiga,eHelen H. Song,aPamela A. Shaw,fKunio Nagashima,gHarry L. Malech,a
Frank R. DeLeo,dSteven M. Holland,bJohn I. Gallin,aand David E. Greenbergb,e*
Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USAa; Laboratory of Clinical Infectious
Diseases, NIAID/NIH, Bethesda, Maryland, USAb; University of Texas Southwestern Medical Center, Dallas, Texas, USAc; Laboratory of Human Bacterial Pathogenesis, NIAID/
NIH, Hamilton, Montana, USAd; Research Technologies Branch, NIAID/NIH, Bethesda, Maryland, USAe; Biostatistics Research Branch, NIAID/NIH, Bethesda, Maryland, USAf;
and SAIC-Frederick, Inc., Frederick, Maryland, USAg
Acetic acid bacteria were previously considered nonpathogenic in humans. However, over the past decade, five genera of
Acetobacteraceae have been isolated from patients with inborn or iatrogenic immunodeficiencies. Here, we describe the
first studies of the interactions of the human innate immune system with a member of this bacterial family, Granulibacter
bethesdensis, an emerging pathogen in patients with chronic granulomatous disease (CGD). Efficient phagocytosis of G.
bethesdensis by normal and CGD polymorphonuclear leukocytes (CGD PMN) required heat-labile serum components
(e.g., C3), and binding of C3 and C9 to G. bethesdensis was detected by immunoblotting. However, this organism survived
in human serum concentrations of >90%, indicating a high degree of serum resistance. Consistent with the clinical host
tropism of G. bethesdensis, CGD PMN were unable to kill this organism, while normal PMN, in the presence of serum, re-
duced the number of CFU by about 50% after a 24-h coculture. This finding, together with the observations that G. bethes-
densis was sensitive to H2O2but resistant to LL-37, a human cationic antimicrobial peptide, suggests an inherent resistance
to O2-independent killing. Interestingly, 10 to 100 times greater numbers of G. bethesdensis were required to achieve the
same level of reactive oxygen species (ROS) production induced by Escherichia coli in normal PMN. In addition to the rela-
tive inability of the organism to elicit production of PMN ROS, G. bethesdensis inhibited both constitutive and FAS-
induced PMN apoptosis. These properties of reduced PMN activation and resistance to nonoxidative killing mechanisms
likely play an important role in G. bethesdensis pathogenesis.
(CGD) in 2003 (13). G. bethesdensis has since been cultured from
at least 5 patients with CGD in the United States (15, 16) and 1 in
Spain (23), justifying its classification as an emerging pathogen in
this primary immunodeficiency. Although the bacterial family to
which G. bethesdensis belongs–the Acetobacteraceae–was previ-
ously considered nonpathogenic in humans, case reports of hu-
man infections by other members of this family are being pub-
lished with increasing frequency (1, 2, 5, 6, 9, 12, 17, 22, 26, 27).
Common elements of infection by these other Acetobacteraceae
species appear to include intravenous drug administration (for
either medicinal or recreational purposes), organ transplant, or
chronic medical problems requiring dialysis. In contrast, G.
bethesdensis has been reported only in patients with CGD, a dis-
ease resulting from inadequate superoxide anion production re-
quired for normal myeloid host defenses. While G. bethesdensis is
the only member of this family for which Koch’s postulates have
been fulfilled (13), there is scant information about the mecha-
nisms of pathogenesis of infection by these organisms. Although
mice with CGD are significantly more susceptible than normal
mice to infection by G. bethesdensis, viable organisms could be
recovered from the spleens of wild-type mice more than 70 days
of long-term persistence in vivo and has a significant degree of
tical isolates from the same patient with CGD over several years
ranulibacter bethesdensis is a Gram-negative pathogen first
isolated from a patient with chronic granulomatous disease
(15). Given that G. bethesdensis in particular and the Acetobacter-
aceae in general appear to represent an emerging group of patho-
gens, we investigated the interaction of G. bethesdensis with com-
ponents of the innate immune system as a first step toward
elucidating the mechanisms of G. bethesdensis virulence.
MATERIALS AND METHODS
tained after informed consent from donors enrolled at the NIH Clinical
Center under institutional review board-approved protocols 93-I-0119,
05-I-0213, or 99-CC-0168. Human polymorphonuclear leukocytes
(PMN) were isolated from venous blood samples as previously described
(20, 31). Sera were prepared using Becton Dickenson serum separator
tubes according to manufacturer’s instructions and stored on ice for im-
mediate use or frozen at ?80°C in single-use aliquots.
Received 22 June 2011 Returned for modification 29 July 2011
Accepted 29 November 2011
Published ahead of print 19 December 2011
Editor: S. M. Payne
Address correspondence to Kol A. Zarember, firstname.lastname@example.org, or
David E. Greenberg, email@example.com.
*Present address: University of Texas Southwestern Medical Center, Dallas, Texas,
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
0019-9567/12/$12.00Infection and Immunity p. 975–981iai.asm.org
Bacterial strains and culture. G. bethesdensis strain NIH1.1 (strain
CGDNIH1T; ATCC BAA-1260) was grown to mid-log phase at 37°C in
YPG medium (5 g yeast extract, 3 g peptone, and 10 g glucose per liter).
Escherichia coli TOP10 and K1/r were grown in Luria-Bertani (LB) broth
factors (no. of CFU/optical density [OD]) were determined.
Luminol-enhanced neutrophil chemiluminescence. PMN were sus-
pended at 1.1 ? 106/ml in RPMI 1640 medium with 5.5% autologous
serum, 27.5 mM HEPES (pH 7.4), and 55 ?M luminol, and 90 ?l was
aliquoted into a 96-well polypropylene white plate. After a 30-min incu-
bation period at 37°C in a 5% CO2incubator, 10 ?l of washed bacteria
(multiplicities of infection [MOI] of 100:1, 10:1, 1:1) was suspended in
saline and added to the plate. Saline alone was added as a control to some
wells of the plate, and luminometry was performed in an Anthos Zenyth
3100 set at 37°C, with readings of 1 s per well taken every 2.4 min. Results
are expressed kinetically or converted to area under the concentration-
time curve (AUC) by the GraphPad Prism 5.0 software package.
Bacterial internalization assays. Bacteria were labeled with pHrodo
according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA)
and kept at 4°C until use. For flow cytometry experiments, bacteria and
PMN (MOI ? 10 bacteria:1 PMN) were added to a 96-well plate with
RPMI 1640 containing 25 mM HEPES (pH 7.2) and 10% autologous
serum for a final volume of 200 ?l. The plate was incubated at 37°C, and
phagocytosis was stopped at each time point by addition of 50 ?l of 10%
formaldehyde was added to the wells immediately prior to addition of
bacteria. After the final time point, the plate was stored at 4°C and read
within 24 h on a FACSCalibur flow cytometer (BD Biosciences). Cells
cellular bacteria. The pHrodo signal was measured in the FL2 channel,
and the pHrodo?threshold was set based on the 0-min time point and
applied to all samples. For microscopy studies, EtOH-washed coverslips
were placed in each well of a 24-well culture plate (Corning, NY) and
dried. Assays were performed in RPMI 1640 containing 25 mM HEPES
(pH 7.2), and the indicated numbers of PMN with or without additives
were added. After thorough mixing, plates were centrifuged at 500 ? g
and placed in a humidified incubator at 5% CO2and 37°C. At the indi-
cated times, plates were centrifuged as indicated above, and the superna-
a Leica AF 6000 LX fluorescence microscope. A minimum of 10 fields
(?100 cells) were imaged, and the numbers of PMN per field and the
durations indicated in the figure legends. After 3 washes in 1 ml ice-cold
PBS each, the bacteria were boiled in SDS-PAGE sample buffer with re-
ducing agent and then subjected to Western blotting using standard
methods. C3 and C3-deficient sera and goat anti-human C3 and C9 anti-
sera were obtained from Complement Technology, Inc. (Tyler, TX). Pri-
mary antibodies were used at 1:2,000 and detected with an anti-goat
horseradish peroxidase (HRP) conjugate (Santa Cruz) and Pierce West
Pico chemiluminescent detection reagents.
Neutrophil killing assays. All assays were performed as previously
Neutrophil apoptosis studies. Apoptosis of normal PMN or CGD
tation, or flow cytometry for active caspase-3. PMN were incubated in
RPMI 1640 buffered with 10 mM HEPES containing 10% autologous
human serum (where indicated) in the presence or absence of G. bethes-
densis and/or 1 ?g/ml anti-FAS antibody (clone CH-11; MBL) for the
indicated times. Freshly isolated PMN were also assessed by these meth-
ods and typically displayed ?5% apoptosis. For morphological analysis,
cells were subjected to cytospin, stained with the HARLECO Hemacolor
as healthy (typical multilobed nuclei) or apoptotic (condensed nuclei or
“ghost” cell membranes lacking nuclei). To assess DNA fragmentation, a
modified terminal deoxynucleotidyltransferase-mediated dUTP-biotin
nick end labeling (TUNEL) assay (APO-BRDU kit; BD Pharmingen) was
used. Briefly, cells were washed with phosphate-buffered saline (PBS),
in 70% ethanol at ?20°C. For the staining procedure, control cells and
experiment samples were washed with kit wash buffer, incubated with
DNA labeling solution for 1.5 h at 37°C with agitation every 15 min,
washed twice with kit rinse buffer, and incubated with antibody labeling
solution for 30 min at room temperature (RT) in the dark before
fluorescence-activated cell sorting (FACS) analysis (conducted within 3
caspase-3 was measured after washing cells once in 1% heat-inactivated
fetal calf serum (FCS) and 1% bovine serum albumin (BSA) in PBS and
nin in PBS), followed with incubation for 15 min on ice to complete
coerythrin (PE) antibody (BD Biosciences) for 30 min on ice in the dark,
washed with permeabilization buffer, and fixed with 2% paraformalde-
hyde before FACS analysis.
Antimicrobial activity assays. Bacteria were grown to mid-
above. Antimicrobial activity was determined in RPMI 1640 containing
10 mM HEPES (pH 7.4). A peptide corresponding to the mature human
(A) or serum treated at 56°C for 30 min (B). Results are expressed as the mean percentages of untreated control at 24 h ? standard errors of the means (SEM)
for the indicated number of experiments.
Zarember et al.
iai.asm.orgInfection and Immunity
and stored at 4°C. Hydrogen peroxide was freshly diluted from a 30%
stock solution. Growth inhibition of inocula of 106/ml was determined
after the indicated time points by plating (using sterile beads to achieve a
priate media (either YPG or LB agar) and is expressed as the number of
Pad Prism for Mac, version 5.0.
RESULTS AND DISCUSSION
G. bethesdensis is highly resistant to serum complement. The
complement system forms an important front line of innate
host defense, and microbial evasion of the complement is a
virulence determinant (32). We therefore compared the effect
of pooled human serum on the viabilities of G. bethesdensis and
E. coli K1/r, a serum-resistant encapsulated bacteremia isolate
(11). As previously reported, the polysialic acid capsular poly-
saccharide of E. coli K1/r confers serum resistance following
growth at 37°C but not at 22°C (7). While E. coli K1/r cultured
at 22°C is killed by as little as 1% (vol/vol) serum, the organism
grown at 37°C is resistant to killing by serum (up to 45% se-
rum) (Fig. 1A). In comparison, G. bethesdensis survived in 90%
serum, indicating a greater degree of serum resistance than E.
coli K1/r. Both serum-sensitive and serum-resistant bacteria
bacter oxydans, a related member of the Acetobacteraceae, was
serum sensitive following culture and testing at 25°C (it will
not grow at 37°C), indicating that serum resistance is not a
general attribute of this family (data not shown).
FIG 2 G. bethesdensis is internalized by human PMN in a time- and serum-dependent manner. (A) Internalization of pHrodo-labeled bacteria was
assessed microscopically by blinded scorers at 30 min and an MOI of 1:1. The percentage of all cells that contained at least 1 G. bethesdensis organism is
plotted as a function of the serum dose (mean ? SEM; n ? 9 healthy donors over 6 separate experiments) and the number of bacteria per PMN (mean ?
SEM; n ? 4 to 6 subjects). (B) Flow cytometry studies demonstrate internalization by normal or CGD PMN incubated with G. bethesdensis at an MOI of
10:1 in the presence of 10% autologous serum (mean ? SEM; n ? 8 healthy patients and 4 patients with CGD). (C) Internalization of G. bethesdensis was
assessed microscopically in the absence of serum or in the presence of either 10% serum or 10% heat-treated (HT; 56°C, 30 min) serum (mean ? SEM;
n ? 9). (D) Internalization of G. bethesdensis was measured microscopically in the presence of 10% C3-depleted serum alone, purified C3 at a
concentration of 10 ?g/ml, and both together (mean ? SEM of 9 healthy donors). Repletion of C3-depleted serum with C3 resulted in a significant
increase in internalization (paired t test, 2 tailed). (E) Immunodetection of C3 and C9 bound to G. bethesdensis. Lane 1, bacteria alone; lane 2, 10%
C3-deficient serum; lane 3, 10% C3-deficient serum plus 1 ?g C3/ml; lane 4, bacteria alone; lane 5, 10% serum at 0 min; lane 6, 10% serum at 5 min; lane
7, 10% serum at 10 min; lane 8, 10% serum at 30 min; lane 9, 10% serum with EGTA plus MgCl2; lane 10, 10% serum previously heat treated at 50°C for
20 min. Blots are representative of at least 3 different binding experiments.
Innate Immunity against Granulibacter bethesdensis
March 2012 Volume 80 Number 3iai.asm.org 977
Phagocytosis of G. bethesdensis by PMN. Another role of
serum is opsonization, the coating of microbes with heat-labile
components such as complement and/or heat-stable antibod-
ies that significantly enhance their phagocytosis (29). We
therefore measured the effect of serum on internalization by
PMN by labeling G. bethesdensis with pHrodo, a pH-sensitive
dye that fluoresces more intensely as the pH decreases, and
counting the number of PMN with fluorescent bacteria. Inter-
nalized bacteria were fluorescent and easily counted by micros-
copy, while extracellular bacteria were not fluorescent in this
medium and could not be counted. Internalization of the bac-
teria was confirmed by overlaying the fluorescent signal with
the differential interference contrast (DIC) image. Phagocyto-
sis of G. bethesdensis at an MOI of 1:1 was serum dependent
(e.g., in the presence of 10% autologous serum, 40% of PMN
contained bacteria by 30 min, whereas uptake was ?1% or less
for nonopsonized bacteria) (Fig. 2A), with both the number of
PMN with bacteria and the number of bacteria per cell increas-
of phagocytosis between PMN from healthy individuals and
those from patients with CGD, although there was a nonsignif-
icant trend toward increased internalization by CGD cells (Fig.
2B). Serum-dependent internalization was blocked by heat in-
activation (56°C) of serum (Fig. 2C). Since C3 is a major serum
opsonin, we obtained commercially prepared C3 and C3-
depleted human serum to test whether C3 is critical for phago-
cytosis of G. bethesdensis. As shown in Fig. 2D, C3 alone failed
to promote uptake of G. bethesdensis, whereas C3-depleted se-
rum supported some internalization (?50% of that supported
by fresh serum). However, the combination of C3 and C3-
depleted serum (C3-repleted serum) promoted internalization
significantly greater than that promoted by either component
alone (a similar significant enhancement of phagocytosis was
found using 1% serum [data not shown]).
C3 and/or C9 bound G. bethesdensis after incubation of bacte-
ria with human sera (Fig. 2E). Similar to what is seen in Fig. 2D,
was further enhanced by addition of purified C3. C3 binding, in
particular that of the opsonic C3b fragment, in 10% pooled hu-
man sera was maximal by 5 min (Fig. 2E, lane 6), whereas C9
deposition on G. bethesdensis increased for up to 30 min. Inhibi-
Mg2?-EGTA decreased binding of both C3 and C9 to bacteria
(Fig. 2E, compare lane 9 to lane 8), as did mild heat treatment
(50°C for 20 min to inhibit the alternative pathway) (Fig. 2E, lane
10), suggesting that complement activation may depend on both
pathways or that inhibition was incomplete. Consistent with its
effect of decreasing complement activation on bacteria, Mg2?-
EGTA also inhibited PMN phagocytosis of pHrodo-labeled G.
did not occur in C3- or C6-deficient sera (data not shown), indi-
nonspecific adsorption. Thus, G. bethesdensis appears to be resis-
tant to the membrane attack complex (MAC), possibly due to the
presence of a capsule (15), smooth lipopolysaccharide (LPS), or
some other mechanism.
Thus, G. bethesdensis is highly serum resistant and is internal-
ized through a serum-dependent pathway, likely involving C3 ac-
tivation through the classical or lectin pathway.
Inhibition of PMN apoptosis by G. bethesdensis. Regulation
of the PMN life span by pathogens is thought to play an important
role in microbial pathogenesis (28). To test whether G. bethesdensis
merating apoptotic nuclei and, by using two FACS-based assays,
measuring either the extent of DNA fragmentation correlating with
sis when assessed by TUNEL staining and a trend toward decreased
icantly decreased apoptosis of normal PMN and, to some extent, of
shown to increase PMN apoptosis (19), G. bethesdensis, like Ana-
(3), belongs to the relatively small list of pathogens that inhibit hu-
FIG3 Inhibition of constitutive and Fas-induced PMN apoptosis by G. bethesdensis. (A) Constitutive apoptosis of PMN was assessed by a FACS-based TUNEL
assay of normal (n ? 16) and CGD (n ? 5) PMN incubated for 24 h in the absence or presence of 10 G. bethesdensis CFU per PMN in 10% autologous serum in
of PMN from healthy subjects (N; n ? 7) or subjects with CGD (C; n ? 2). (C) Flow cytometry was used to measure activated caspase-3 in PMN treated for 6 h
with a 2-way analysis of variance (ANOVA) and is indicated with an asterisk when P is ?0.05.
Zarember et al.
iai.asm.orgInfection and Immunity
man neutrophil apoptosis. Whether such increases in the PMN life
span play a role in vivo during Granulibacter infection remains to be
bethesdensis is internalized by normal and CGD PMN in a serum-
enhanced chemiluminescence to detect superoxide production by
occurs rapidly upon exposure to bacteria. Chemiluminescence was
detected after stimulation by as few as 1 autoclaved E. coli organism
was required to achieve the same level of activation induced by a
single E. coli CFU (Fig. 4A and B). Both live and heat-killed bacteria
lently potent, live G. bethesdensis induced somewhat greater chemi-
known to directly inhibit activation of the NADPH oxidase (e.g.,
Anaplasma phagocytophilum  and Francisella tularensis ). To
test whether G. bethesdensis inhibited NADPH oxidase activation in
response to other stimuli, we incubated PMN with this organism
alone or with E. coli. As shown in Fig. 4C, G. bethesdensis did not
G. bethesdensis with different doses of phorbol ester (a potent
receptor-independent activator of the NADPH oxidase) confirmed
oxide (data not shown). Together, these results suggest that rather
Given that G. bethesdensis is a weak activator of the respiratory
burst and is a pathogen in patients with CGD, who are unable to
generate antimicrobial reactive oxygen species (ROS), including
superoxide and H2O2, we next tested the sensitivity of this organ-
ism to H2O2as a model oxidant. Ten independent experiments
ent concentrations of H2O2for 1 h. Nonlinear regression analysis
sis and E. coli are not significantly different, with 50% inhibitory
Innate Immunity against Granulibacter bethesdensis
March 2012 Volume 80 Number 3iai.asm.org 979
concentrations (IC50s) of 29.09 ?M (95% confidence interval
PMN-mediated killing of G. bethesdensis. We next tested
whether growth of G. bethesdensis could be inhibited by PMN in
the absence or presence of 10% autologous serum. As shown in
out serum grew during 24 h of incubation. However, in the pres-
ence of serum and normal PMN, G. bethesdensis was killed in a
tive with greater numbers of PMN (Fig. 5B). PMN from patients
kill about 50% of G. bethesdensis within 24 h in our bactericidal
activity assays, they killed approximately 99% of another CGD
pathogen, Burkholderia multivorans, when tested under identical
conditions in our laboratory (14). It is possible that the poor ac-
contributes to its relative resistance to killing by these cells. How-
ever, the addition of phorbol myristate acetate (PMA) at doses of
this organism (data not shown). Several microbes either fail to
activate the NADPH oxidase, actively interfere with activation or
targeting of this enzyme (e.g., Neisseria gonorrhoeae , Franci-
sella tularensis, and Helicobacter pylori ), or utilize catalase or
superoxide dismutase to inactivate ROS. Our finding that G.
bethesdensis does not inhibit PMN responses to other simultane-
rather than an inhibitor of the NADPH oxidase. Compared to E.
production of cytokines such as tumor necrosis factor alpha
Zarember and J. I. Gallin, unpublished observations). Studies are
under way to isolate and characterize the LPS of G. bethesdensis as
well as other potentially host-stimulatory compounds from this
densis CFU, we conclude that killing of G. bethesdensis by normal
to the nonoxidative antimicrobial factors of PMN. To test the
latter prediction directly, we incubated G. bethesdensis and E. coli
TOP10 with LL-37, a cationic antimicrobial cathelicidin peptide.
20 ?M peptide, G. bethesdensis survived for at least 24 h in the
presence of up to 200 ?M LL-37 (Fig. 6). Resistance to LL-37 has
been described for several pathogens and can be due to surface
properties of microbes or the production of microbial peptidases
densis genes confer resistance to LL-37 or the entire spectrum of
FIG 6 G. bethesdensis is resistant to cathelicidin peptide. Bacteria were cul-
7.4) for 1 and 24 h, and the remaining CFU/ml enumerated. Data shown
represent the mean percentages of control ? SEM for 5 or 6 independent
FIG 5 G. bethesdensis is killed by normal PMN but not CGD PMN. (A) G. bethesdensis was cultured in the absence or presence of 10% serum and in the
absence or presence of PMN at an MOI of 1:1. At the indicated time points, cultures were lysed and diluted as described in Materials and Methods, and
the CFU were enumerated and plotted as means ? SEM (n ? 6 healthy donors). (B) The number of CFU of G. bethesdensis after 24 h of culture in the
bacteriostatic toward G. bethesdensis under conditions in which normal PMN reduced the number of viable CFU by ?50% (individual data points are
shown as percent control of initial counts, and the means ? SEM are depicted (n ? 8 healthy donors and 9 donors with CGD). An unpaired t test was used
to determine significance as shown.
Zarember et al.
iai.asm.orgInfection and Immunity
nonoxidative microbicidal factors of the CGD PMN. Studies are Download full-text
under way to determine the transcriptional responses of this or-
ganism during phagocytic interaction with normal and CGD
Whether healthy humans can be infected by G. bethesdensis is
to several key innate defenses (e.g., serum and cathelicidin pep-
tide) and is a poor stimulator of the leukocyte NADPH oxidase,
microbicidal armaments of the PMN. Aside from direct antimi-
crobial effects, ROS play important roles in the production of
neutrophil extracellular traps (NETs) (10) and other antimicro-
bial host defenses. Further studies are required to determine
which cells and mechanisms are responsible for the clearance of
this organism in vivo and whether G. bethesdensis can persist in a
latent form in healthy hosts or those with CGD. It remains to be
tested whether the observed interactions between G. bethesdensis
and components of the innate immune system are unique or
whether these attributes are shared by other members of this
growing family of opportunistic pathogens.
The views expressed here are those of the authors and do not neces-
sarily reflect those of the U.S. Government.
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