INFECTION AND IMMUNITY, Sept. 2005, p. 5319–5328
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 9
Down Regulation of Virulence Factors of Pseudomonas aeruginosa by
Salicylic Acid Attenuates Its Virulence on Arabidopsis thaliana and
B. Prithiviraj,1,2H. P. Bais,1,2T. Weir,1,2B. Suresh,1,2E. H. Najarro,1,2B. V. Dayakar,1,2
H. P. Schweizer,2,3and J. M. Vivanco1,2*
Department of Horticulture and Landscape Architecture1and Center for Rhizosphere Biology,2Colorado State University,
Fort Collins, Colorado 80523-1173, and Department of Microbiology, Immunology and Pathology,
Colorado State University, Fort Collins, Colorado 80523-16823
Received 29 November 2004/Returned for modification 13 February 2005/Accepted 9 May 2005
Salicylic acid (SA) is a phenolic metabolite produced by plants and is known to play an important role in
several physiological processes, such as the induction of plant defense responses against pathogen attack.
Here, using the Arabidopsis thaliana-Pseudomonas aeruginosa pathosystem, we provide evidence that SA acts
directly on the pathogen, down regulating fitness and virulence factor production of the bacteria. Pseudomonas
aeruginosa PA14 showed reduced attachment and biofilm formation on the roots of the Arabidopsis mutants lox2
and cpr5-2, which produce elevated amounts of SA, as well as on wild-type Arabidopsis plants primed with
exogenous SA, a treatment known to enhance endogenous SA concentration. Salicylic acid at a concentration
that did not inhibit PA14 growth was sufficient to significantly affect the ability of the bacteria to attach and
form biofilm communities on abiotic surfaces. Furthermore, SA down regulated three known virulence factors
of PA14: pyocyanin, protease, and elastase. Interestingly, P. aeruginosa produced more pyocyanin when infil-
trated into leaves of the Arabidopsis transgenic line NahG, which accumulates less SA than wild-type plants.
This finding suggests that endogenous SA plays a role in down regulating the synthesis and secretion of
pyocyanin in vivo. To further test if SA directly affects the virulence of P. aeruginosa, we used the Caenorhabiditis
elegans-P. aeruginosa infection model. The addition of SA to P. aeruginosa lawns significantly diminished the
bacterium’s ability to kill the worms, without affecting the accumulation of bacteria inside the nematodes’ guts,
suggesting that SA negatively affects factors that influence the virulence of P. aeruginosa. We employed
microarray technology to identify SA target genes. These analyses showed that SA treatment affected expres-
sion of 331 genes. It selectively repressed transcription of exoproteins and other virulence factors, while it had
no effect on expression of housekeeping genes. Our results indicate that in addition to its role as a signal
molecule in plant defense responses, SA works as an anti-infective compound by affecting the physiology of P.
aeruginosa and ultimately attenuating its virulence.
Pseudomonas aeruginosa is a prevalent opportunistic patho-
gen in humans, causing chronic lung infections in cystic fibrosis
patients, burn victims, and other immunocompromised people
(61, 62). Pathogenesis of P. aeruginosa is mediated by a suite of
cell-associated and excreted virulence factors. The cell-associ-
ated factors include flagella that aid in motility, systems that
are involved in the delivery of effector proteins into the host
cells (67), and lipopolysaccharide that suppresses host immune
responses as well as being involved in the establishment of
persistent infections (13). Secreted factors such as elastase and
protease cause the degradation of host proteins such as elastin,
collagen, and transferrins, destroying the integrity of the host
tissues (2, 34), while low-molecular-weight toxins such as pyo-
cyanin affect multiple sites of the cell machinery (38, 58).
P. aeruginosa is known for its intrinsic and acquired resis-
tance against a wide range of antimicrobial agents, which has
led to difficulty in treating infections, especially in cystic fibrosis
patients (47, 65). Antibiotic resistance in P. aeruginosa has
been partially attributed to active efflux pumps that expel an-
timicrobial compounds (1, 17, 52). Furthermore, several gram-
negative bacteria, including P. aeruginosa, form structured ag-
gregates, which under certain conditions are referred to as
biofilms (50), and biofilm formation has been found to be
partially responsible for the persistent P. aeruginosa infections
in the lungs of immunocompromised cystic fibrosis patients
(12, 23, 61, 62). Biofilms consist of a matrix of complex poly-
saccharides in which the bacteria are embedded, and the for-
mation of this structure is controlled by quorum-sensing mech-
anisms (16, 23, 43, 51, 63). Biofilm confers superior survival
capability on the bacteria by providing a physical barrier
against the entry of antimicrobial agents (12, 50); in addition,
bacteria inside the biofilm are in a quiescent state (metaboli-
cally less active) and thus relatively insensitive to antimicrobial
agents (19) and environmental stress (50). Recently, lactofer-
rin, a component of a healthy person’s innate immunity and
present in the mucosa, was found to attenuate biofilm forma-
tion in P. aeruginosa and could protect against persistent in-
fections (61). The emergence of P. aeruginosa resistance to
multiple antibiotics, including ciprofloxacin, that are com-
monly used to treat lung infections (42) highlights the need to
design more effective regimens to treat these infections. Con-
* Corresponding author. Mailing address: Department of Horticul-
ture and Landscape Architecture, Colorado State University, 217
Shepardson Building, Fort Collins, CO 80523. Phone: (970) 491-7170.
Fax: (970) 491-7745. E-mail: email@example.com.
sequently, novel strategies are being proposed, including the de-
velopment of therapeutics that have the potential to disrupt bio-
films, down regulate known virulence factors, and regulate genes
crucial for pathogenesis and quorum sensing (26, 27, 28, 29, 74).
P. aeruginosa also infects plants, eliciting soft rot symptoms
in thale cress (Arabidopsis thaliana) and lettuce (Letuca sativa)
(56, 57), and recently it has been shown to be a potent root
pathogen of Arabidopsis (71) as well as of animals such as
Caenorhabditis elegans (44, 66), Drosophila (14) and Galleria
mellonella (48). Molecular studies on the pathogenesis of P.
aeruginosa revealed that this bacterium requires similar subsets
of virulence factors for plant and animal infection (55, 56). This
finding makes Arabidopsis a convenient model to study the mo-
lecular basis of pathogenesis, which could aid in the discovery of
novel compounds for treatment of infections (53). Here, we show
evidence that the plant compound salicylic acid (SA) can atten-
Caenorhabditis elegans infectivity models without inhibiting the
growth of the bacteria, by down regulating the production of a set
of virulence factors and biofilm formation.
MATERIALS AND METHODS
Plant material, bacterial culture, Caenorhabditis elegans strains, and chemi-
cals. Arabidopsis thaliana ecotype Col-0 seeds were purchased from Lehle,
Round Rock, TX. The transgenic line NahG, carrying the salicylic acid hydrox-
ylase gene, and the mutant line npr1-1 (nonexpressor-of-pathogenesis-related
protein), were kind gifts from Xinnan Dong (Duke University, NC). The trans-
genic line 35S-LOX-2(?) (6) (ABRC accession no. CS3748) (hereafter referred
to as lox2; defective in jasmonic acid accumulation in response to stress and
hyperaccumulator of salicylic acid) and the mutant cpr5-2 (accumulates larger
amounts of salicylic acid) were obtained from the Arabidopsis Biological Resource
Center, Ohio State University, Columbus. The seeds were surface sterilized in 2%
sodium hypochlorite for 2 min, followed by three washes with sterile distilled water,
and surface-sterilized seeds were placed on static Murashige and Skoog basal me-
dium in petri dishes for germination and incubated in a growth chamber. After 2
weeks the seedlings were transferred to either 12-well plates or culture tubes con-
taining 2 to 3 ml of MS liquid medium and grown on an orbital platform shaker
(Lab-Line Instruments) set at 90 rpm with a photoperiod of 16 h light and 8 h dark
at 25 ? 2°C. Cultures of Pseudomonas aeruginosa PA14 and Caenorhabditis elegans
wild-type strain Bristol N2 were gifts from Frederick M. Ausubel (Department of
Genetics, Harvard Medical School, Boston, MA). Salicylic acid and all other chem-
icals were purchased from Sigma (St. Louis, MO) unless otherwise noted.
Effect of salicylic acid on the growth of Pseudomonas aeruginosa. P. aeruginosa
PA14 was grown overnight at 37°C in LB broth. LB broth medium (2.5 ml) was
dispensed into each well of 12-well plates (Falcon, NJ). Stock solutions (100 mM)
of salicylic acid were prepared in methanol, and filter-sterilized stock was added
to the wells of a 12-well plate to a final concentration of 0.1, 0.25, 0.5, 1, 2, 3, or
4 mM salicylic acid. PA14 inoculum was added to give a final optical density at
600 nm (OD600) of 0.04. The plates were incubated in a shaker set at 90 rpm and
37°C. The effect of SA on growth was assessed by a CFU count, using a dilution
plate technique (69).
Root inoculation. Wild-type A. thaliana Col-0, transgenic lines NahG and lox2,
and mutants npr1-1 and cpr5-2 were grown in 9-cm-diameter petri dishes con-
taining 25 ml MS basal medium for 2 weeks. The plants were transferred to glass
culture tubes containing 2 ml of liquid MS medium and placed in a rotary shaker
set at 60 rpm under a day/light cycle of 16/8 h for 5 to 7 days, after which the
plants were inoculated with appropriate aliquots of P. aeruginosa PA14 suspen-
sion added to the liquid medium to a final OD600of 0.02. The plants were
incubated at 30°C on a rotary shaker. Mortality was recorded at 5 days after
inoculation. Each experiment was conducted twice with five replicates. Heat-
killed bacteria (121°C for 5 min) were included as one of the control treatments;
the bacterial suspension was diluted to a final OD600of 0.02, and plants were
incubated as described above.
Biofilm formation on Arabidopsis thaliana roots. Wild-type Col-0, transgenic
lines (NahG and lox2), and mutants npr1-1 and cpr5-2 of A. thaliana were grown
and inoculated with PA14 in culture tubes as described above. Three days after
inoculation the roots were excised, stained with 10 ml of 75 ?g ml?1Calcofluor
(fluostain; Sigma-Aldrich) for 30 min, and observed under a fluorescence Olym-
pus BX60 microscope equipped with CoolSnap imaging software (Media-Cyber-
netics, San Diego, CA) to determine the presence of biofilm formation, as
described in the literature (4, 71).
Biofilm formation assay and biofilm quantification. The ability of PA14 to
form biofilm on abiotic surfaces was investigated according to a previously
described crystal violet (CV) assay (50), except that polypropylene tubes were
used instead of polystyrene tubes. Briefly, tubes containing 500 ?l of a 1/100
dilution of an overnight LB broth culture in BDT medium (Bushnell-Haas
mineral salts medium supplemented with 0.2% dextrose and 0.5% tryptone)
were incubated statically at 30°C for 24 h. The biofilm was qualitatively assayed
using crystal violet staining as previously described (50). Biofilm formation was
quantified using a microtiter plate assay. PA14 was grown in 96-well polyvinyl
chloride (PVC) microtiter plates (Fischer Scientific) at 37°C in biofilm growth
medium that consisted of LB medium plus 0.15 M ammonium sulfate, 100 mM
potassium phosphate (pH 7), 34 mM sodium citrate, 1 mM MgSO4, and 0.1%
(wt/vol) glucose. The inoculum for microtiter plates was obtained by growing the
cells with agitation in biofilm growth medium to mid-logarithmic phase and
diluting the cells to an OD600of 0.01 in fresh biofilm growth medium. One
hundred microliters of the diluted cells was aliquoted to each well of 96-well
PVC microtiter plates. The microtiter plates were incubated under stationary
conditions. Cells that adhered to the wells were stained with 0.1% (wt/vol) CV in
wash buffer (0.15 M ammonium sulfate, 100 mM potassium phosphate [pH 7], 34
mM sodium citrate, and 1 mM MgSO4) at room temperature for 20 min. Excess
CV was then removed, and the wells were rinsed with water. The CV that had
stained the cells was solubilized in 200 ?l of 80% (vol/vol) ethanol and 20%
(vol/vol) acetone. Biofilm formation was quantified by measuring the OD570for
each well, using an Opsys MR-Dynex plate reader (Chantilly, VA).
Effect of SA supplementation on biofilm formation on abiotic and biotic
surfaces. The effect of SA on biofilm formation by PA14 was investigated using
a crystal violet assay as described in the previous section. In the first experiment
SA was added to the BDT medium from the onset of the incubation period. The
concentrations of SA assessed were in the range of 0.1 to 5 mM. The volume of
added dimethyl sulfoxide/ethanol was adjusted so that all tubes contained the
same concentration of the solvent. Biofilm formation was quantified every 5 h
during a 50-hour incubation period. Qualitative CV staining was performed
using polypropylene tubes and photographed after 24 h of incubation with SA.
All treatments were conducted in triplicate.
Effect of salicylic acid on pyocyanin production by P. aeruginosa PA14. P.
aeruginosa PA14 was grown overnight at 37°C in LB broth. LB broth (2.5 ml) was
dispensed into each well of 12-well plates. Stock solutions (100 mM) of salicylic
acid and its derivatives acetyl salicylic acid, salicylamide, methyl salicylate, and
benzoic acid were prepared in methanol; filter-sterilized stock was added to a
12-well plate to a final concentration of 0.1, 0.5, or 1 mM. PA14 inoculum was
added to give a final OD600of 0.04. The plates were incubated in a shaker set at
90 rpm. The culture was removed at 6, 12, 18, and 24 h of incubation, and
pyocyanin was extracted as described in the literature (20). Briefly, the 2.5-ml
culture was extracted in 1.5 ml of chloroform. The chloroform was transferred to
a clean tube, and 0.8 ml of 1 N HCl was added and gently shaken to bring the
pyocyanin to the pink aqueous phase (pyocyanin extracted with chloroform turns
pink). The OD520of the aqueous solution was measured and the pyocyanin
concentration determined by multiplying this measurement by 17.07 (19). The
effect of salicylic acid on pyocyanin production by PA14 was also assessed in an
M9-glucose (0.2%, wt/vol) minimal medium following the same protocol as
described above, except that bacteria were grown in 125-ml conical flasks con-
taining 50 ml of the growth medium.
Effect of salicylic acid and its derivatives on elastase and protease production
by P. aeruginosa PA14. Plate assays for total protease and elastase activity were
performed as described by Brint and Ohman (8). To determine total protease
activity, cultures of PA14 were stab inoculated on medium containing 0.8%
nutrient broth (Luria) and 1.5% powdered skim milk amended with 1 mM or 5
mM SA and were incubated at 37°C for 12 to 24 h before visual inspection for a
zone of clearing. The same procedure was followed for elastase activity except
that 0.5% elastin (Sigma, St. Louis, MO) was incorporated into the medium in
place of 1.5% skim milk and cultures were incubated for 48 h. Protease activity
was quantified using a modification of the method described by Greene et al.
(22). Bacterial cultures were grown with and without added SA in 50 ml of 5%
peptone and 0.25% tryptic soy broth (PTSB) at 37°C for 24 h. The supernatants
were collected and filter purified using a 0.22-?m nylon filter. A 100-?l aliquot
of supernatant was then added to reaction mixtures containing 0.8% azocasein
(Sigma) in 500 ?l of 50 mM K2HPO4, pH 7. Reaction mixtures were incubated
at 25°C for 3 hours. The reaction was stopped by adding 0.5 ml of 1.5 M HCl, and
the mixture was placed on ice for 30 min and then centrifuged. After addition of
0.5 ml of 1 N NaOH, the OD440was recorded. To determine elastase activity, 100
5320PRITHIVIRAJ ET AL.INFECT. IMMUN.
?l of supernatant from the 24-hour PTSB cultures was added to tubes containing
1 ml of 10 mM Na2HPO4, pH 7, and 20 mg of elastin-Congo red (Sigma).
Reaction mixtures were incubated with agitation for 4 hours at 37°C. Tubes were
then centrifuged, and the OD495was determined.
Assessing differential pyocyanin production in the leaves of Arabidopsis with
genotypes altered in SA synthesis. Strains with three different genotypes of
Arabidopsis, i.e., the wild-type Col-0, the transgenic line NahG, and the mutant
npr1-1, were grown in solidified MS medium for 3 weeks. At this time, 1 g fully
expanded leaves was excised and vacuum infiltrated with a cell suspension
(OD600of 0.02) of PA14. The inoculated leaves were placed in moist chambers
and incubated at 30°C for 36 h. At the end of the incubation period the leaf was
extracted in 3 ml chloroform, and all further steps were similar to those described
in “Effect of salicylic acid on pyocyanin production by P. aeruginosa PA14”
above. The CFU of P. aeruginosa PA14 in the leaves of different genotypes of A.
thaliana were enumerated by weighing a 1-cm2leaf disk, and macerating the
tissue in 1 ml of sterile distilled water in a Eppendorf tube with a tissue macer-
ator (Kontes, size C). This was further serially diluted, and 20 ?l of the suspen-
sion was plated on a solid LB medium supplemented with 100 ?g ml?1rifampin.
The number of colonies was counted after overnight incubation at 37°C.
Estimation of free salicylic acid content in Arabidopsis. Free SA levels in
wild-type A. thaliana (Col-0), transgenic lines (NahG and lox2), and the mutant
npr1-1 were determined by the method of Bowling et al. (7) at 1, 2, and 3 days
following infection with P. aeruginosa PA14. Approximately 1 g of fresh tissue
was either used directly or stored at ?80°C until use. The fresh tissue was ground
in liquid nitrogen to a fine powder with a chilled pestle and mortar. Three
milliliters of 90% methanol and 250 ng o-anisic acid (internal standard) were
added to each sample. Samples were vortexed, sonicated for 20 min, and cen-
trifuged for 20 min at 1,700 ? g in a tabletop centrifuge. The supernatant was
transferred to a new tube, and the pellet was reextracted with 2 ml 90% meth-
anol. The two supernatants were combined, vacuum dried, and frozen at ?80°C;
then 2.5 ml 5% trichloroacetic acid was added, and the samples were vortexed,
sonicated for 5 min, and centrifuged at 1,700 ? g for 15 min. The supernatant was
extracted twice with 2.5 ml of a 1:1 (vol/vol) mixture of ethyl acetate and
cyclopentane. The organic phases were combined, vacuum dried, and frozen at
?80°C. Just prior to loading of samples for high-pressure liquid chromatography,
each was resuspended in 250 ?l of 20% methanol, vortexed, sonicated for 5 min,
and filtered through a 0.22-?m nylon filter. High-pressure liquid chromatography
was performed as described earlier (7).
Microarray analysis. Batch cultures of P. aeruginosa (PA14) were grown in
duplicates in LB medium supplemented with either 0 or 1.0 mM of salicylic acid
at 37°C and 200 rpm. The initial OD600was adjusted to ?0.02, and the cultures
were grown for 10 h with shaking. At the end of the incubation period, 5 ml of
the culture was retrieved and RNA was isolated using a Ribopure RNA isolation
kit (Ambion Inc., Austin, TX) according to the manufacturer’s instructions. The
concentration of the RNA was assessed using a Nanodrop system (NanoDrop,
Wilmington, DE) and the integrity estimated by 28S/18S ratio using an Agilent
2100 bioanalyzer (Agilent, Palo Alto, CA). The microarray analysis was per-
formed at Ambion Inc., Austin, TX. Two micrograms of the RNA was amplified
using a MessageAmp II-Bacteria kit (Ambion Inc., Austin, TX) according to the
manufacturer’s instructions and used in the microarray analysis. Microarray
analysis was performed by using the Affymetrix P. aeruginosa GeneChip. The
Affymetrix instrumentation consisted of a GeneChip hybridization oven 640, a
GeneChip Fluidics Station 450, and a high-resolution GeneChip scanner 3000
(GeneChip, Santa Clara, CA). The data analysis was performed using the data
analysis software GeneSpring7.2 (Silicon Genetics, Redwood City, CA) DNA-
Chip Analyzer (dChip; http://www.dchip.org). The data were normalized per
chip by dChip invariant set normalization, and each gene was normalized to the
median measurement taken for that gene across all the samples. For analysis the
average intensity of ?200 at least in one of the comparison conditions was used.
A two-sample t test using a P value cutoff of 0.05 was applied to identify genes
that were statistically differentially expressed.
Effect of salicylic acid on the efficacy of ciprofloxacin against P. aeruginosa
PA14. Pseudomonas aeruginosa PA14 was grown overnight in Mueller-Hinton
broth medium. The MIC of ciprofloxacin against PA14 was determined using a
96-well plate serial dilution method, with PA14 inoculated to a final OD600of
0.02. The 96-well plate was incubated at 37°C overnight and the growth deter-
mined by measuring the OD600using a microtiter plate reader (Dynex, VA).
Ciprofloxacin (1 ?g ml?1) significantly reduced the CFU, from 5.6 ? 109to 2.5
? 105. We used a concentration of ciprofloxacin 10 times lower than this (0.1 ?g
ml?1) to check if SA potentiates the antibacterial activity of ciprofloxacin. One
milliliter MH broth was dispensed into wells of 12-well cell culture plates, and
ciprofloxacin was added to a final concentration of 0.1 either with or without 1.0
mM SA. The PA14 suspension was added to each of the wells to a final OD600
of 0.02. The plates were incubated in a shaker set at 100 rpm at 37°C for 24 h.
At the end of the incubation period, a CFU count for each treatment was done
by serial dilution and plating on LB agar.
Nematode-killing assay with SA supplementation. PA14 was grown overnight
at 37°C in LB broth. A 1:10 dilution of the saturated culture was made in LB
broth, and 10 ?l of the diluted culture was spread on 3.5-cm-diameter plates
containing nematode growth medium (NGM). The plates were incubated at 30°C
for 24 h and allowed to equilibrate to room temperature for 30 to 60 min before
being seeded with the nematode Caenorhabditis elegans wild-type strain Bristol N2.
Nematodes were multiplied on NGM plates with Escherichia coli OP50 as the food
source. Adult populations were synchronized on similar plates by transferring two
adult nematodes to each plate and allowing them to lay eggs overnight, after which
the adult nematodes were removed and killed. The plates containing the eggs were
incubated for 4 to 5 days to get a uniform adult population. Twenty to 30 adult
nematodes were used for each assay. Similarly, to test the anti-infective property of
SA (diluted in methanol) against the bacteria, SA was administered at different
concentrations (0.1 to 2.0 mM) by dropping it on the PA14 lawn 10 to 15 min prior
to worm seeding. A separate methanol control was also tested for comparison as per
the description provided above. An additional control involved inoculating worm
plate, and each assay was carried out in triplicate. The plates were incubated at 20°C
and scored for live and dead worms at least every 24 h for 5 days. A worm was
considered dead when it failed to respond to plate tapping or a gentle touch with a
platinum wire. Worms that died as a result of getting stuck to the wall of the plate
were not included in the analysis.
Estimation of bacterial CFU within the C. elegans gut. CFU of bacteria within
the nematode gut were counted by a method described in the literature (21). For
each replication, 10 adult C. elegans worms were picked from different treatments
(PA14 and PA14 treated with different concentrations of SA), transferred into a
1.5-ml Eppendorf tube containing 500 ?l of M9 buffer supplemented with 20 ?g/ml
gentamicin, and washed with three changes of the above cocktail to remove surface
FIG. 1. P. aeruginosa attachment to and biofilm formation on A. thaliana roots. Plant roots were inoculated with PA14 cells for 5 days. Roots
were excised and stained with Calcofluor, and images were taken under a microscope and fluorescent illumination. (A) npr1-1 and (B) wild-type
Col-0 plants, which produce low levels of SA; (C) lox2 and (D) cpr5-2 plants, which produce higher levels of salicylic acid. Bars, 1 mm.
VOL. 73, 2005ANTI-INFECTIVE ROLE OF SALICYLIC ACID 5321
of M9 medium with 1% Triton X-100. The resulting slurry was serially diluted and
plated on LB agar medium containing 20 ?g/ml rifampin, and the number of CFU
RESULTS AND DISCUSSION
Salicylic acid, which is produced throughout the plant king-
dom, is known to be involved in modulating a number of
physiological processes, including thermogenesis, tolerance of
abiotic stresses (11, 31, 59), and, most importantly, defense
responses. Endogenous SA in plants modulates defense re-
sponse pathways by up regulating several pathogenesis-related
proteins (60) that are controlled through the transcriptional
regulator NPR1 (10). Several SA-dependent but NPR1-inde-
pendent defense pathways have also been reported (15, 32, 41).
Although possible direct effects of SA on an infecting pathogen
have been proposed (18, 37), this intriguing question has not
been thoroughly investigated.
Salicylic acid levels affect bacterial attachment to plant
roots. We hypothesized that SA may affect the virulence of the
pathogen without affecting its growth. To test this hypothesis, we
used Arabidopsis mutants with altered abilities to accumulate SA,
such as lox2 (5) and cpr5-2 (7), both of which accumulate elevated
concentrations of SA; npr1-1, a mutant that has normal levels of
SA but is insensitive to its activity because it lacks the transcrip-
tional regulator needed for SA-induced defense responses (10);
the transgenic line NahG (39), which constitutively expresses the
SA hydroxylase gene of Pseudomonas putida and thus has de-
pleted concentrations of SA; and the wild-type Col-0. The infec-
tivity assays were conducted by inoculating P. aeruginosa (PA14)
to the roots of plants growing under in vitro conditions (71). The
wild-type Col-0 and the mutant npr1-1 supported attachment and
the mutants lox2 and cpr5-2, which accumulate larger amounts of
SA, showed reduced attachment and/or formation of biofilm
communities on the roots (Fig. 1C and D).
Endogenous salicylic acid levels affect bacterium-induced
plant mortality. The ability of PA14 to colonize and form
biofilm communities on the roots of Arabidopsis correlated
with plant mortality (Fig. 2A). Arabidopsis wild-type Col-0, the
mutant npr1-1, and the transgenic line NahG were more sus-
ceptible to PA14 infection, resulting in almost 100% plant
mortality at 5 days postinoculation, while cpr5-2 and lox2,
which supported less colonization of PA14, were resistant as
evidenced by the lower plant mortality rates of ?50% and
?40%, respectively (Fig. 2A). P. aeruginosa PA14 inflicted
typical disease-like symptoms such as black necrotic regions
initially in the root tips, which later spread to the entire root
system, then colonized the basal leaves, and finally resulted in
the collapse of the plant (71). The enhanced susceptibility of
the transgenic line NahG and the mutant npr1-1 correlated
with higher CFU counts of the bacteria in the roots, while the
transgenic line lox2 and the pathogen-resistant mutant cpr5-2
were less susceptible to PA14 colonization and showed lower
CFU than wild-type Col-0 plants (Fig. 2B). These results
strongly suggest that SA has a role in inhibiting PA14 attach-
ment and biofilm community formation on Arabidopsis roots.
Salicylic acid affects bacterial attachment and biofilm for-
mation in vitro. To test the hypothesis that SA affects the
physiology of PA14, reducing its ability to attach to roots, we
carried out an in vitro experiment in which SA was added into the
culture medium containing salicylic acid at physiologically rele-
vant concentrations (0.1 to 1 mM) (46, 64). The ability of PA14 to
form biofilm was diminished in a concentration-dependent man-
ner when exposed to SA (Fig. 3A). It is important to note that
although SA completely inhibited the aerobic biofilms (shown as
the ring formed in the region representing the air-liquid inter-
phase of the tube), it did not decrease the formation of anaerobic
biofilm (Fig. 3A). We quantified the effect of SA on PA14’s
adherent biofilm by using a microtiter plate assay described in the
literature (25, 50). PA14 was grown in the wells of a PVC micro-
titer plate in a complex biofilm growth medium supplemented
FIG. 2. Susceptibility of A. thaliana to P. aeruginosa and bacterial
growth is dependent on endogenous salicylic acid levels. Strain PA14
was used to infect A. thaliana wild-type Col-0, the transgenic line
NahG, and the mutant npr1-1, all of which produce low levels of SA,
and the transgenic line lox2 and mutant cpr5-2, which produce higher
levels of SA. Five days after inoculation, plant mortality was recorded
(A) and bacterial numbers on roots were determined (B). Error bars
indicate standard deviations.
5322PRITHIVIRAJ ET AL.INFECT. IMMUN.
with different concentrations of SA (0.1 to 1.0 mM). Addition of
SA significantly reduced adherent cells (biofilm) over a 40-hour
period compared to the untreated control (Fig. 3B).
Salicylic acid inhibits pyocyanin formation. We also ob-
served reduced pigment accumulation in the SA-treated PA14.
P. aeruginosa produces a number of colored secondary metab-
olites commonly referred to as phenazines; one of these, the
blue pyocyanin, has been widely studied and is a potent viru-
lence factor (57, 58). We tested the effect of SA on pyocyanin
production by supplementing the growth medium with differ-
ent concentrations of SA. Addition of SA to the bacterial
growth medium significantly reduced the production of pyocy-
anin (Fig. 4A and 4B). Untreated PA14 produced ?4.5 ?g of
pyocyanin per 108cells after 24 h of incubation, while the
addition of 0.1 mM SA reduced pyocyanin production by
?50%, and 1.0 mM SA resulted in more than an 80% reduc-
tion of pyocyanin production with no apparent effect on the
growth of the bacteria. Similar reductions in the quantity of
pyocyanin were observed following the addition of SA (0.1, 0.5,
and 1.0 M) to M9-glucose (http://lamar.colostate.edu/?jvivanco
FIG. 3. Salicylic acid inhibits P. aeruginosa attachment to and bio-
film formation on an abiotic surface. PA14 cells were statically grown
for 24 h at 30°C in polypropylene tubes containing Bushnell-Haas
mineral salts medium supplemented with 0.2% dextrose and 0.5%
tryptone. Attachment and biofilm formation were then quantitated by
staining with crystal violet. (A) Biofilm formation in the presence of
increasing SA concentrations. Probable anaerobic biofilms formed in
the presence of 0.5 and 1 mM SA are circled. (B) Time course of
biofilm formation by untreated PA14 and cells treated with increasing
SA concentrations. Error bars indicate standard deviations.
FIG. 4. Salicylic acid and derivatives inhibit pyocycanin formation.
PA14 cells were grown in LB medium in the absence or presence of SA
or its derivatives acetyl salicylic acid (ASA), salicylamide (SAM),
methyl salicylic acid (MeSA), and the SA metabolic precursor benzoic
acid (BA). After 24 h, pyocyanin was extracted with chloroform and its
concentration determined spectrophotometrically. (A) Visual depic-
tion of green pyocycanin pigmentation, which is reduced in the SA-
treated cultures; (B) pyocyanin levels in untreated and SA-treated
cells; (C) pyocycanin levels in untreated cells and cells treated with the
SA derivatives ASA, SAM, and MeSA or its metabolic BA precursor.
Error bars indicate standard deviations.
VOL. 73, 2005ANTI-INFECTIVE ROLE OF SALICYLIC ACID 5323
/papers/IAI2029.pdf). We also evaluated the effect of SA deriva-
tives, including acetyl salicylic acid, salicylamide, and methyl sa-
licylate, and a precursor of SA, benzoic acid, on pyocyanin pro-
duction by PA14 (Fig. 4C). All the SA derivates led to reduced
pyocyanin production, and the levels of reduction caused by
methyl salicyate, salicylamide, and benzoic acid were comparable
to that caused by SA. Interestingly, when PA14 was infiltrated
into the leaves of Arabidopsis genotypes that accumulate different
concentrations of SA, the production of pyocyanin by PA14 was
inversely correlated with the concentration of SA found in the
plant leaves (Table 1). PA14 when infiltrated into the leaves of
the mutant npr1-1, which accumulates wild-type levels of SA but
is truncated in an upstream signaling event that leads to synthesis
of pathogenesis-related protein, accumulated 7.5 ?g pyocyanin.
Addition of 1.0 mM SA with the bacterial suspension resulted in
the reduction of pyocyanin production to 4.3 ?g per 108cells.
the leaves of Arabidopsis is sufficient to down regulate pyocyanin
production in the bacteria. Using random transposon mutagene-
sis, Rahme et al. (57) found that mutants of PA14 that were
ity in the Arabidopsis and mouse models, suggesting a main role
for pyocyanin in the virulence of P. aeruginosa, and later studies
have supported this observation (44). Recently, the molecular
mechanism by which pyocyanin exerts its cytotoxic effect has been
unraveled (58). Using Saccharomyces cerevisiae, Ran et al. (58)
have identified a number of cellular targets of pyocyanin that
encompass major cellular pathways involved in the cell cycle,
electron transport and respiration, epidermal cell growth, protein
sorting, and vesicle transport, and have determined that pyocya-
nin inactivates vacuolar ATPase, ultimately resulting in cytotox-
Salicylic acid affects exoenzyme production in P. aeruginosa
PA14. P. aeruginosa employs a repertoire of exoenzymes to
elicit disease pathology (9, 30, 44, 58). A number of studies
have found that elastases and proteases are potent virulence
factors of P. aeruginosa. Accordingly, P. aeruginosa mutants
pho34B12 and pho15, which are defective in the synthesis of
elastases and proteases, showed moderate pathogenicity on
Arabidopsis and were also attenuated in pathogenicity in the
mouse model and inflicted 56 and 62% mortality, respectively
(9, 57). We hypothesized that the reduced mortality observed
in SA-treated Arabidopsis might partly be due to down regu-
lation of virulence factors other than pyocyanin, such as elastases
and proteases. Addition of 1 mM SA to PA14 growth medium
resulted in a 50% reduction of both elastase and protease activ-
ities (Fig. 5). Acetyl salicylic acid, salicylamide, methyl salicylate,
and benzoic acid also caused similar reductions in elastase and
protease activities (Fig. 5). These results suggest that SA affects
the production of several virulence-related exoenzymes in PA14.
Global effects of salicylic acid on gene transcription. To
study the effect of SA on genome-wide changes in gene ex-
pression, we used the Affymetrix GeneChip microarray tech-
nology. The microarray data suggest that SA treatment signif-
icantly (P ? 0.05) affected the expression of 331 genes (?5%
of the total genome). A total of 3.0% of the genome of PA14
was induced while 2.7% of the genes were repressed by SA
(Fig. 6A and B; http://lamar.colostate.edu/?jvivanco/papers
/IAI2029.pdf). Several of the genes whose expression was af-
fected by SA coded for hypothetical proteins; this result was
not surprising, as almost 44% of the predicted open reading
frames of P. aeruginosa encode hypothetical proteins (73). SA
treatment, however, did not affect housekeeping genes that are
required for the growth and normal metabolism of the bacte-
ria, a finding which lends mechanistic support to our data that
SA, even at the highest concentration used in this study (1.0
mM), did not affect the growth of P. aeruginosa PA14.
Some of the genes that were down regulated by SA were
related to quorum sensing, such as rhlR and lasA. These genes
have been previously implicated in the synthesis of several
virulence factors, including pyocyanin, protease, and elastase
(29). Further, 12 genes that are involved in protein secretion
and export apparatus were significantly repressed by SA treat-
ment; for example, exoT, exsB, and exsC were repressed by SA
treatment (Fig.6B; http://lamar.colostate.edu/?jvivanco
(data not shown) were affected by 1.0 mM SA treatment, and
although the functions of many of these genes are not known,
it is plausible that these genes might affect the synthesis and
secretion of a number of virulence factors of PA14.
Salicylic acid potentiates antimicrobial activity of cipro-
floxacin. Salicylates have been shown to alter the susceptibility
FIG. 5. Exposure of P. aeruginosa to salicylic acid, its derivatives,
and a metabolic precursor inhibits protease production. PA14 cells
were grown for 24 h in peptone-tryptic soy broth medium in the
absence or presence of SA or its derivatives acetyl salicylic acid (ASA),
salicylamide (SAM), methyl salicylic acid (MeSA), and the SA meta-
bolic precursor benzoic acid (BA). Spectrophotometric assays of total
protease and elastase activities were performed using the substrates
azocasein and elastin-Congo red, respectively. The activity was mea-
sured as OD440/495/108cells. Error bars indicate standard deviations.
TABLE 1. Differential pyocyanin production by Pseudomonas
aeruginosa PA14 on the leaves of Arabidopsis thaliana with different
genotypes varying in endogenous concentrations of salicylic acid
A. thaliana genotype and SA treatment
In planta pyocyanin
concn, mean ? SD
(?g per 108cells)
Col-0 ................................................................................... 6.45 ? 0.95
NahG (no SA accumulation)........................................... 15.92 ? 0.31
npr1-1 (SA level lower than that of Col-0).................... 7.51 ? 0.14
npr1-1 ? 1mM SA ............................................................ 4.30 ? 0.45
5324 PRITHIVIRAJ ET AL.INFECT. IMMUN.
FIG. 6. Microarray analysis of P. aeruginosa transcripts from cells treated with salicylic acid. PA14 cells were grown for 10 h in the absence or
presence of 1 mM SA. Total RNA was then extracted, labeled, and hybridized to Affymetrix P. aeruginosa GeneChips. (A) Total number of genes
either affected or unaffected by treatment with SA, which significantly (P ? 0.05) affected the expression of 361 genes. (B) Functional categories
of genes that are either up or down regulated in PA14 in response to SA treatment. LPS, lipopolysaccharide; RND, resistance-modulation-cell
division; MFS, major facilitator superfamily.
VOL. 73, 2005 ANTI-INFECTIVE ROLE OF SALICYLIC ACID5325
of Helicobacter pylori to antibiotics, such as amoxicillin, clar-
ithromycin, and metronidazole, by enhancing their antibacte-
rial activity (72). Similarly, salicylate enhanced the susceptibil-
ity of E. coli to kanamycin (2). Therefore, it is reasonable to
postulate that SA treatment could alter the susceptibility of
PA14 to commercial antibiotics such as ciprofloxacin, an anti-
biotic used to treat P. aeruginosa infections in cystic fibrosis
patients (42). We found that the addition of SA in conjunction
with ciprofloxacin significantly enhanced the efficacy of the
antibiotic (Fig. 7). In the presence of 1 mM SA the antimicro-
bial activity of ciprofloxacin was potentiated about threefold,
suggesting that the production of virulence factors by PA14
may play an additional role in antibiotic resistance. Based on
these results, we propose that endogenous SA in plants may
play an important role in making pathogenic bacteria more
susceptible to preformed plant antimicrobial compounds (phy-
toanticipins) (70), as well as to those induced compounds that
are synthesized as a result of infection (phytoalexins) (24, 70).
Salicylic acid attenuates nematode killing by P. aeruginosa
PA14. Our results demonstrate that SA affects PA14 by down
regulating a number of virulence factors produced by the bac-
teria, thus affecting its virulence on Arabidopsis. However, an
intrinsic problem with using plant models to study the direct
effect of SA on the bacteria is the existence of SA-dependent
plant defense responses that are NPR1 dependent (10) or
NPR1 independent (15, 32, 41). Essentially it is very difficult to
isolate the direct effect of SA on the microbe from the effect of
SA on induction of plant defense responses. To overcome this
obstacle, we used a Caenorhabiditis elegans-P. aeruginosa infec-
tion model (44), as SA is not known to induce a defense
response in nematodes. A slow killing assay was performed to
measure the effect of SA on the ability of P. aeruginosa to infect
the worm. SA was added directly to the PA14 lawn, resulting in
attenuated nematicidal activity of PA14 (Fig. 8A), but with no
apparent reduction in bacteria accumulated in the nematode
gut as reflected by the recovered bacterial CFU (Fig. 8B).
PA14 (with no SA treatment) caused 100% mortality of adult
C. elegans after 100 h, while the addition of 0.1 mM SA to
PA14 lawns increased the time required for complete mortality
to 120 h. At higher concentrations of SA, an increasing number
of nematodes survived for over 120 h. For example, ?20% of
the animals survived at 120 h when treated with 1.0 mM SA,
while ?40% of the animals survived when treated with 2.0 mM
SA. It should be noted that neither of these SA levels affects
the growth rate of PA14 (data not shown). Antibiotics usually
target specific metabolic events and have been routinely used
to treat bacterial infections. However, the widespread emer-
gence of antibiotic-resistant strains of pathogenic bacteria has
resulted in problems in treating certain infectious diseases and
necessitates the development of alternate strategies of treat-
ment (40). SA effectively stems the infectivity of PA14 as evi-
denced by reduced C. elegans mortality after PA14 infection,
FIG. 7. Salicylic acid potentiates ciprofloxacin activity against P.
aeruginosa. PA14 cells were grown in Mueller-Hinton broth with cip-
rofloxacin (Cipro) and in the presence or absence of 1 mM SA. After
24 h, viable counts were determined in the respective cultures. Error
bars indicate standard deviations.
FIG. 8. Salicylic acid attenuates nematode killing by P. aeruginosa
but does not reduce bacterial counts in the gut. (A) Nematode growth
medium agar plates containing lawns of bacteria used for C. elegans
feeding either contained no SA (PA14) or contained increasing SA
concentrations. E. coli strain OP50 grown on NGM served as a control.
Twenty to 30 nematodes were added to each plate at time zero, and
worm survival was monitored every 20 h. (B) NGM plates containing
the indicated SA concentrations were seeded with PA14. C. elegans
adult worms were transferred to the plates, and CFU were determined
in the guts of 10 live worms removed from the plates at the indicated
time points. Error bars indicate standard deviations.
5326 PRITHIVIRAJ ET AL.INFECT. IMMUN.
possibly due to the down regulation of virulence factors. These
results suggest that in addition to its traditional role as an
analgesic, SA has the potential to be used alone or in conjunc-
tion with lower doses of antibiotics to treat bacterial infections.
Such an approach could slow the development of antibiotic-
resistant strains of bacteria.
P. aeruginosa PA14 infection affects Arabidopsis salicylic acid
levels. Since the defense-response-altered A. thaliana plants
used in this study varied in their susceptibility to PA14 infec-
tion and because some of these plants were affected in the
salicylic acid signaling pathway, we postulated that the suscep-
tibility of these plants to PA14 might be related to SA levels in
the plant. Therefore, we analyzed the endogenous accumula-
tion of SA in whole plants of wild-type A. thaliana (Col-0),
transgenic lines (NahG and lox2), and the mutant npr1-1 upon
PA14 challenge at two different time points (day 1 and day 3).
Although some of our infection assays were conducted on
roots, we choose to sample whole plants for SA analyses due to
root material limitation in some of the mutants. As expected
and reported earlier (49), both NahG and npr1-1 revealed
depleted SA levels compared to wild-type plants (http://lamar
.colostate.edu/?jvivanco/papers/IAI2029.pdf). At days 1 and 3
after inoculation with PA14, the total salicylic acid concentrations
in the tissues were essentially the same in wild-type A. thaliana
Col-0, transgenic line NahG, and the mutant npr1-1, while the
transgenic line lox-2 showed a higher concentration of SA (54)
lectively, these results show that SA may account for reduced
PA14 infectivity when higher levels of this metabolite accumulate
in the plant tissues by negatively influencing the virulence of the
Conclusions. SA is omnipresent in the plant kingdom, and
its concentration in tissue is highly regulated by abiotic and
biotic factors (59). Microbial infection causes the accumulation
of high levels of SA at the site of pathogen infection and, to a
lesser extent, also in uninfected tissues by a process known as
systemic acquired resistance (45), either by de novo synthesis
or by the release of bound SA into free SA from o-glucosides
by the action of glucosidases (45). Therefore, it is plausible that
some nonhost microbes (3, 68) may be exposed to a high
concentration of SA in infected plants, a concentration suffi-
cient to affect their virulence and pathogenicity. The results
presented in this study suggest that SA, besides triggering
defense responses, could also act on the pathogen by disrup-
tion of aggregate/biofilm formation on biotic and abiotic sur-
faces and by repression of a number of virulence factors. Con-
sistent with our results, previous studies have shown that SA
affects the ?-hemolysin secretion and fibronectin-binding ca-
pacities of Staphylococcus aureus and also that the expression
of the global regulatory genes agr and sarA is attenuated by SA
treatment (35, 36). Further, salicylic acid has been shown to
inhibit fimbria-mediated Hep-2 cell adherence of and hemag-
glutination by enteroaggregative Escherichia coli (33) and also
to inhibit attachment and colonization of contact lenses by a
number of pathogenic bacteria, including P. aeruginosa (5).
The results presented in this report largely support this hy-
pothesis and warrant an in-depth study of the direct role of SA
in plant defense, as well as its role as a potential anti-infective
compound for the treatment of human diseases.
The research presented in this paper was supported by grants from
the Colorado State University Agricultural Experiment Station (to
J.M.V) and the NIH (to H.P.S). J.M.V. is an NSF-CAREER Faculty
We thank Emily Wortman-Wunder for critical reading of the manu-
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Editor: J. B. Bliska
5328PRITHIVIRAJ ET AL.INFECT. IMMUN.