Comparative Antimicrobial Activity of Granulysin against Bacterial Biothreat Agents.

Abstract

Granulysin is a cationic protein produced by human T cells and natural killer cells that can kill bacterial pathogens through disruption of microbial membrane integrity. Herein we demonstrate antimicrobial activity of the granulysin peptide derived from the active site against Bacillus anthracis, Yersinia pestis, Francisella tularensis, and Burkholderia mallei, and show pathogen-specific differences in granulysin peptide effects. The susceptibility of Y. pestis to granulysin is temperature dependent, being less susceptible when grown at the flea arthropod vector temperature (26 degrees C) than when grown at human body temperature. These studies suggest that augmentation of granulysin expression by cytotoxic lymphocytes, or therapeutic application of granulysin peptides, could constitute important strategies for protection against select agent bacterial pathogens. Investigations of the microbial surface molecules that determine susceptibility to granulysin may identify important mechanisms that contribute to pathogenesis.

Full text

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92 The Open Microbiology Journal, 2009, 3, 92-96


1874-2858/09 2009 Bentham Open
Open Access
Comparative Antimicrobial Activity of Granulysin against Bacterial
Biothreat Agents
Janice J. Endsleya,c,*, Alfredo G. Torresa,b,c, Christine M. Gonzalesa, Valeri G. Kosykhb, Vladimir L.
Motina,b,c, Johnny W. Petersona,c, D. Mark Estesa,c and Gary R. Klimpela,c
Department of Microbiology and Immunologya, Department of Pathologyb, and Sealy Center for Vaccine Developmentc,
University of Texas Medical Branch, Galveston, TX 77555-0436, USA
Abstract: Granulysin is a cationic protein produced by human T cells and natural killer cells that can kill bacterial
pathogens through disruption of microbial membrane integrity. Herein we demonstrate antimicrobial activity of the
granulysin peptide derived from the active site against Bacillus anthracis, Yersinia pestis, Francisella tularensis, and
Burkholderia mallei, and show pathogen-specific differences in granulysin peptide effects. The susceptibility of Y. pestis
to granulysin is temperature dependent, being less susceptible when grown at the flea arthropod vector temperature (26°C)
than when grown at human body temperature. These studies suggest that augmentation of granulysin expression by
cytotoxic lymphocytes, or therapeutic application of granulysin peptides, could constitute important strategies for protec-
tion against select agent bacterial pathogens. Investigations of the microbial surface molecules that determine susceptibil-
ity to granulysin may identify important mechanisms that contribute to pathogenesis.
Keywords: Granulysin, antimicrobial, B. anthracis, B. mallei, Y. pestis, F. tularensis.
INTRODUCTION

surviving phagocytosis comprise some of the most serious
microbial threats to human health. Among these are
Mycobacterium tuberculosis (Mtb) and some of the
pathogens classified by the Center for Disease Control and
Prevention as bacterial select agents, Bacillus anthracis,
Yersinia pestis, Burkholderia mallei, and Francisella
tularensis. The tropism for phagocytic cells by these patho-
gens is an important factor for their successful evasion of the
immune system and poses significant challenges for immu-
nization or therapeutic intervention. Characterizing protec-
tive immune responses to these pathogens, then, is para-
mount to development of prophylactic or early therapeutic
intervention strategies. Cytotoxic T cells (Tc) and NK cells
control intracellular pathogens through multiple killing
mechanisms, including death receptor activation and release
of cytolytic granule proteins. In human cytotoxic lympho-
cytes, granulysin is an important component of the lytic
repertoire and is stored in cytotoxic granules along with
perforin and granzymes [1-5] . Perforin and granzymes kill
the infected cell, whereas granulysin has the unique capacity
to kill the intracellular pathogen by disruption of microbial
membranes [5-7]. The lytic activity due to granulysin
has been described against several important pathogens,
including Mtb, Plasmodium falciparum, Cryptococcus
neoformans, Salmonella enterica serovar Typhimurium
(S. Typhimurium), Escherichia coli 0157:H7 and Staphylo-
coccus aureus [5-10] .
Bacteria that evade the host immune response by


*Address correspondence to this author at the University of Texas Medical
Branch, 301 University Blvd., Galveston, TX 77555-0436, USA; Tel: (409)
772-3142; Fax: (409) 747-6869; E-mail: jjendsle@utmb.edu

an important role for this molecule in the cytotoxic lympho-
cyte (Tc and NK cells) response to intracellular bacterial
pathogens that evade the immune response through residence
in macrophages. To investigate a potential role for granu-
lysin in the immune repsonse to an expanded list of intracel-
lular bacterial pathogens for which interventions are urgently
needed, we utilized a peptide derived from the active site of
human granulysin and a negative control peptide. The active
site peptide has been previously shown to reproduce the
effects of the recombinant granulysin molecule, and is
considered to have potential as an antibacterial therapeutic
[5-7, 10, 11]. In this study, we demonstrate and compare
the antibacterial activity of granulysin against B. anthracis
(Ames), Y. pestis (CO 92), F. tularensis (SHU S4 and LVS),
and B. mallei (ATCC 23344), using a peptide derived
from the active site of granulysin. These studies support
the need to further characterize the role of granulysin in
the innate and acquired CTL response to these important
pathogens.
The ability of granulysin to kill intracellular Mtb supports
MATERIALS AND METHODS

were purchased from New England Peptide LLC (Gardner,
MA, USA). A peptide corresponding to amino acid residues
34-55 of human granulysin (CRTGRSRWRDVCRNFMRR-
YQSR) was synthesized. As a negative control, a peptide
corresponding to amino acids 2-22 (RDYRTCLTIVQKL-
KKMVDKPT) of human granulysin was also synthesized.
Lyophilized peptide was stored in desiccant at -20 °C prior
to use. Peptide stocks (5 mM) were solubilized in 0.1 N
acetic acid solution, and then further diluted in sterile PBS to
1 mM, prior to use. Polypropylene tubes were used to store,
Synthetic custom peptides >95% pure by HPLC

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Granulysin Kills Select Agent Bacterial Pathogens The Open Microbiology Journal, 2009, Volume 3 93
aliquot, or perform experiments with peptides to prevent loss
of peptides due to binding to tubes. All experimentation with
select agents was performed under level 2 or 3 biosafety
conditions according to protocols approved by the University
of Texas Medical Branch Environmental Health and Safety.
S. Typhimurium, F. tularensis, B. anthracis and B. mallei
were grown in brain heart infusion broth (BHI), Muller
Hinton media with Isovitle X, Luria-Bertani (LB) media, and
LB media supplemented with 4% glucose (LBG), respec-
tively. Cultures of S. Typhimurium, F. tularensis (LVS,
SHU S4), B. anthracis (Ames strain) and B. mallei (ATCC
23344) were grown to exponential phase and diluted in
growth medium to approximately 105 colony forming units
(CFU)/ml. A 90 μl aliquot of diluted microorganism was
pre-incubated with 10 μl of PBS or 10 μl of individual
granulysin peptides serially diluted in PBS to final concen-
trations of 1, 10, and 100 μM. After 3 hours of incubation
at room temperature, 100 μl aliquots of S. Typhimurium,
F. tularensis, and B. mallei were plated on LB or LBG agar
plates, and 100 μl aliquots of peptide and B. anthracis was
plated on trypticase soy agar with 5% sheep blood. CFU’s
were counted after overnight incubation (48h for B. mallei)
at 37°C. Exponential growth phase cultures of Y. pestis
CO92 or Y. pestis CO92 caf were grown overnight at 26°C
versus 37°C in Heart-Infusion Broth (HIB, Difco). Cultures
were diluted 1:100 after overnight growth and grown at 26°C
and 37°C to log phase in HIB medium. Cultures were
harvested by centrifugation and washed once with Phosphate
buffered saline buffer (PBS, pH 7.4). Y. pestis (2 x 106 CFU/
ml) were incubated with various concentration of granulysin
as described above and plated on HIB agar plates. CFU’s
were counted after 48h incubation at 26°C. Results for
all isolates were verified by three (B. mallei, B. anthracis),
four (S. Typhimurium, F. tularensis), or five (Y. pestis)
independent experiments. A total of five independent
experiments were performed with Y. pestis to firmly
establish the temperature-dependent differences in suscepti-
bility to peptide. Data were analyzed by one-way analysis
of variance (ANOVA) followed by a Tukey’s pair wise
comparison test (GraphPad Software v4.0).
RESULTS

granulysin peptide against four aerosol-acquired select agent
bacterial pathogens, B. anthracis, Y. pestis, F. tularensis, and
B. mallei. S. Typhimurium was used as a positive control to
compare the relative antibacterial activity of the peptide
observed in earlier studies [7, 10, 12]. To normalize data
across experiments and organisms, results in Fig. (1) are
shown as percentage of control growth from each individual
experiment. Statistically significant differences due to treat-
ment, however, were determined using actual CFU relative
to peptide concentrations. A peptide derived from a region
in granulysin previously determined to lack antimicrobial
activity (helix 1) was used as a negative control [6, 7, 10].
In this study, we tested the antimicrobial activity of the

nant protein [7, 10, 12], we observed that granulysin peptide
had potent antimicrobial activity against S. Typhimurium
with a significant, dose dependent, increase in activity from
1 to 100 μM peptide concentration (Fig. 1). A significant
reduction of B. anthracis, F. tularensis, and B. mallei
was evident at 10 and 100 μM of peptide (Fig. 1), though
B. anthracis and F. tularensis were less susceptible to 100
μM levels than B. mallei or S. Typhimurium. The concentra-
tion of peptide required to reduce CFU of B. anthracis and
F. tularensis was comparable to peptide concentrations
required to reduce Mtb and M. bovis, in similar studies [5, 7,
12]. The killing activity of granulysin against F. tularensis
SHU S4 (virulent) and LVS (attenuated vaccine strain) were
similar (data not shown) indicating that susceptibility to
granulysin peptide is not a likely variable in pathogenesis.
Surprisingly, the negative control peptide derived from helix
1 had moderate activity against B. anthracis at a concentra-
tion of 100 μM (38% reduction of CFU’s as compared to
the no peptide control; data not shown). The growth of
S. Typhimurium, Y. pestis, F. tularensis, or B. mallei, was
not affected by the helix 1 peptide when compared to growth
in the absence of peptide. Due to the effects of the helix 1
peptide on B. anthracis, the no peptide control CFUs were
used to calculate the percentage of control growth shown
in Fig. (1). As shown in Fig. (2), Y. pestis growth was also
In support of previous studies with peptide and recombi-








Fig. (1). Antimicrobial activity of granulysin peptide against select agent pathogens. Reduction of S. Typhimurium, B. mallei,
F. tularensis (SHU S4), and B. anthracis (Ames) by a peptide derived from the active site of granulysin (1, 10, 100 μM) displayed as a per-
centage of control (no peptide) growth. Data shown are mean ± SEM of three to four independent experiments performed in triplicate.
*p<0.05; **p<0.01, indicate statistically significant differences between peptide treatment and negative control.
% of control growth
S. Typhimurium
B. malleiF. tularensisB. anthracis
0
20
40
60
80
100
1
10
100
*
**
*
**
*
*
*
*

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94 The Open Microbiology Journal, 2009, Volume 3 Endsley et al.
significantly reduced by the granulysin active site peptide in
a dose-dependent manner. A growth temperature-dependent
susceptibility of Y. pestis to cationic peptides has previously
been characterized [13-15]. To determine if this effect could
be observed with granulysin, Y. pestis was incubated with
the peptide after growth at ambient temperature, generally
corresponding to that in the flea arthropod vector (26°C), or
human body temperature (37°C). When grown at ambient
temperature to exponential growth phase, Y. pestis was much
more resistant to the effects of granulysin peptide. In fact,
the reduction of Y. pestis growth by granulysin peptide was
several orders of magnitude greater when grown at 37°C, as
compared to growth at 26°C (Fig. 2). As a percentage of the
negative control, however, significant reduction of Y. pestis
by 5 to 100 μM concentrations of granulysin peptide was
evident at both temperatures (Fig. 2, numbers in parenthe-
sis). Recently, surface-exposed bacterial molecules (capsular
antigen fraction 1) have been shown to modulate the suscep-
tibility of Y. pestis to antimicrobial molecules in the respira-
tory epithelium [16]. To evaluate the role of the capsule
substance in the susceptibility of Y. pestis to granulysin, a
capsule-negative mutant of Y. pestis CO 92 (Y. pestis CO 92
caf) was also grown at both temperatures prior to incubation
with peptide. The mutant contains a 1,176 bp deletion in the
caf-operon eliminating synthesis of Caf1 capsular subunit
and Caf1A usher proteins (V. Motin, unpublished). A
temperature-dependent difference in killing activity by Y.
pestis CO 92 caf was observed similar to the wild-type
strain shown in Fig. (2), while the presence or absence of the
capsule did not affect the susceptibility (data not shown).
These differences in susceptibility to the antimicrobial
effects of granulysin peptide, due to temperature, were
not caused by altered growth patterns as organisms grew to
similar density at both temperatures (Fig. 2).
DISCUSSION

protect against bacterial select agent pathogens is a critical
component in the development of control measures for these
potential biothreats. The important role of both innate and
acquired cell-mediated immune (CMI) responses to intracel-
lular pathogens strongly supports the need to characterize
CMI mechanisms that can be targeted to prevent disease or
improve clinical outcome. In this study we showed that
granulysin, a cytotoxic lymphocyte-derived antimicrobial,
may have an important role in immunity to several important
intracellular bacteria with select agent status.
The identification of immune mechanisms that can

compact ?-helical segments. Homologues of granulysin in
multiple mammalian species, fish, and birds indicates con-
servation of this antimicrobial mechanism [2, 12, 17-21].
Curiously, a granulysin homologue has not been identified in
mice, hampering studies of the effects of gene deletion. In
contrast to other antimicrobial molecules of the immune sys-
tem, granulysin expression is limited to NK cells and anti-
gen-specific T cells, the cytotoxic component of the CMI
response. Expression is constitutive in NK cells and induc-
ible in antigen specific T cells following activation with
specific antigen or cytokines[1-3]. Native granulysin has
chemoattractant capabilities [22] and has recently been
implicated in adverse drug reactions that affect epidermal
cells [23]. Normally, granulysin does not have cytotoxic
effects on non-transformed human cells, while induction of
apoptosis of tumor cells has been shown by several groups
[10, 24, 25]. Peptide mapping in three separate species has
characterized a core amino acid region including residues in
helix 2 through helix 3 as the lytic site [6, 7, 10, 12]. The
localization of the antimicrobial activity of granulysin to a
short amino acid segment indicates the potential for use as a
Granulysin is a cationic molecule that consists of five










Fig. (2). Activity of granulysin peptide against Y. pestis is growth temperature-dependent. CFU reduction of Y. pestis (CO 92) across
granulysin peptide concentration when grown at 26°C and 37°C. Reduction of CFU following 3 h incubation with peptide was determined
by overnight growth on HIB agar plates. Percentage reduction from control growth at representative peptide concentrations is shown in
parenthesis. Data shown are mean ± SEM of five independent experiments. Compared to negative control, growth was significantly (p<0.01)
reduced by 5 to 100 μM concentration of granulysin peptide at both 26 and 37°C. Effects of peptide on growth reduction were significantly
different (p<0.05) at 26°C compared to 37°C from 5 to 100 μM concentration.
1
10
101
100
102
1000
103
10000
100000
CFU/ml
1000000
10000000
0 0.55 10 2040 100
26 °C
37 °C
(2%)
(<0.01%)
(6%)
(4%)
(59%)
(0%)
104
105
106
107
peptide concentration (µ µM)

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Granulysin Kills Select Agent Bacterial Pathogens The Open Microbiology Journal, 2009, Volume 3 95
therapuetic peptide, as has been described for other small
“killer” peptides [26]. Peptides derived from the lytic site of
granulysin are able to reproduce the antimicrobial effects of
recombinant granulysin. The activity of the derived peptide
can exceed the recombinant molecule [5, 7], an effect that
may be due to the harsh denaturing conditions required to
purify the recombinant molecule or to a membrane contact
advantage of the peptide. Nonetheless, this peptide is an im-
portant screening tool to identify the antibacterial potential
of granulysin against different bacterial pathogens and may
also have utility as a bactericidal therapeutic. Granulysin-
derived peptides have been shown to neutralize LPS simul-
taneously with direct bacterial killing and may have potential
as a treatment for septic shock [11]. In the current study we
also observed antimicrobial effects of the peptide derived
from helix 1 of granulysin against B. anthracis. This result
was unexpected, as peptides corresponding to the helix 1
region of granulysin have previously been reported to have
no antimicrobial activity [6, 7, 10] Following elucidation
of the crystal structure of granulysin, however, the helix 1
region was predicted to contribute to the antimicrobial activ-
ity of granulysin due to the distribution and orientation of
positively charged amino acids in this region [27]. Further
studies with peptides and site specific amino acid substitu-
tion in recombinant protein are needed to fully characterize
the lytic sites utilized by granulysin against various classes
of microbial pathogens.

potent lytic effects on Y. pestis and B. mallei and can reduce
the growth of F. tularensis and B. anthracis at higher con-
centrations. The relative potency of granulysin to reduce
bacterial numbers in the current study varied by organism,
and in regards to Y. pestis, the effect is temperature depend-
ent. Collectively, the results from these and other studies
suggest that the surface-exposed structures on microbial
membranes that determine susceptibility to granulysin could
differ among microorganisms, or vary in response to
environmental signals. Peptides derived from granulysin
are able to bind LPS and neutralize LPS-induced secretion
of TNF? by peripheral blood mononuclear cells [11]. Differ-
ences in acylation of the LPS lipid A moiety due to
environmental temperature is a defined mechanism for bacte-
rial resistance to antimicrobial peptides [28]. Antimicrobial
peptides have a significant role in insect host defense and are
an important selective pressure for survival of bacteria that
utilize insect reservoirs. With regard to Y. pestis, growth at
environmental temperature increases the resistance of Y. pes-
tis to cationic molecules with antimicrobial activity [13-15].
These differences are frequently attributed to temperature-
induced alterations in lipid A acylation [13-15, 29], though
acyl transferase deficiency has no effect on Y. pestis suscep-
tibility to polymyxin B [30]. Very recently, bacterial capsule
polysaccharides from several species were demonstrated to
mediate resistance to both polymyxin B and alpha-defensin
from human neutrophils and were proposed to act as bacte-
rial decoys for antibacterial peptides [31]. Susceptibility of
Y. pestis to beta-defensin and cathelicidin was also recently
shown to be mediated by the capsular antigen fraction [16].
In our studies, a Y. pestis variant deficient for the capsule
substance was not more susceptible to granulysin peptide
effects. Thus, the role of capsule polysaccharides as bacterial
Our results demonstrate that granulysin may have very
decoys may be an important difference between granulysin
and other cationic antimicrobial molecules.

lysin are required to kill micro-organisms in bulk culture
compared to antibiotics or many synthetic antimicrobials [5,
7, 12]. An important difference between granulysin and other
antimicrobials is that NK cells and antigen-specific T cells
deliver granulysin to individual target cells at sites of infec-
tion. These cytotoxic immune cells are able to methodically
kill one infected target at a time using only a portion of
available granules while continually replenishing the granule
armament. Perforin functions to increase membrane perme-
ability of target cells to facilitate entry and activity of granu-
lysin and granzymes [5, 32, 33]. Granulysin molecules are
able to directly disrupt microbial membranes by electrostatic
charge disruption [7, 27, 34]. Granulysin can also elicit
extra-cellular microbicidal effects against pathogens[5, 9]. In
this regard, both virulent Francisella and LVS were shown
to have a significant extra-cellular phase in their in vivo life
cycle [35].
As previously observed, high concentrations of granu-

observed in the current study support continued investigation
of granulysin as an important cytotoxic effector molecule
contributing to the protective CMI responses to these and
other serious biothreats. Promoting activation of granulysin
expression by NK cells and T cells could represent an impor-
tant avenue for immune modulation to protect susceptible
populations from weaponized or naturally occurring bacterial
pathogens. Strategies to promote activation of NK cell an-
timicrobial activity may be very effective in the early innate
immune response to several pathogens. Promoting antimi-
crobial protein expression as part of the effector repertoire of
antigen-specific T cells may also improve vaccine efficacy.
In summary, induction of granulysin by NK cells or antigen-
specific T cells should be further characterized as a mecha-
nism to augment protective immune function against
intracellular bacterial pathogens.
The antibacterial effects of the granulysin peptide
ACKNOWLEDGEMENTS

ology and Immunology and The Sealy Center for Vaccine
Development, The University of Texas Medical Branch.
This study was supported by the Department of Microbi-
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Received: May 4, 2009

Revised: May 7, 2009 Accepted: May 8, 2009
© Endsley et al.; Licensee Bentham Open.

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