ASIAN PACIFIC JOURNAL OF ALLERGY AND IMMUNOLOGY (2008) 26: 59-70
Differential Gene Expression Profiles of
Lung Epithelial Cells Exposed to
Burkholderia pseudomallei and
Burkholderia thailandensis during the
Initial Phase of Infection
Patimaporn Wongprompitak1, Stitaya Sirisinha1,2 and Sansanee C. Chaiyaroj2
SUMMARY Burkholderia pseudomallei is the causative agent of melioidosis, and its infection usually affects pa-
tients’ lungs. The organism is a facultative intracellular Gram-negative bacillus commonly found in soil and water in
endemic tropical regions. Another closely related Burkholderia species found in soil and water is B. thailandensis.
This bacterium is a non-pathogenic environmental saprophyte. B. pseudomallei is considerably more efficient than
B. thailandensis in host cell invasion and adherence. A previous study by our group demonstrated that after suc-
cessfully invading cells, there was no difference in the ability to survive and to replicate between both Burkholderia
species in cultured A549 human lung epithelial cells. In this study, Human Affymetrix GeneChips were used to
identify the difference in gene expression profiles of A549 cells after a 2-h exposure to B. pseudomallei and B. thai-
landensis. A total of 280 of 22,283 genes were expressed at higher levels in the B. pseudomallei-infected cells than
in the B. thailandensis-infected cells, while 280 genes were expressed at lower levels in the B. pseudomallei-
infected cells. Approximately 9% of these genes were involved in immune response and apoptosis. Those genes
were further selected for gene expression analysis using reverse transcription PCR and/or real-time RT-PCR. The
results of RT-PCR and real-time RT-PCR are in accordance with data from the microarray data in that bcl2 gene
expression in the B. pseudomallei-infected cells was 2-fold higher than the level in the B. thailandensis-infected
cells even though no apoptosis was seen in the infected cells. The levels of E-selectin, ICAM-1, IL-11, IRF-1, IL-6,
IL-1? and LIF genes expression in the B. pseudomallei-infected cells were 1.5-5 times lower than in the B. thailan-
densis-infected cells. However, both species stimulated the same level of IL-8 production from the tested epithelial
cell line, and no difference in the ratio of adherent polymorphonuclear cells (PMNs) to infected A549 cells of both
species was observed. Taken together, our results suggest that B. pseudomallei manipulates host response in fa-
vor of its survival in the host cell, which may explain the more virulent characteristics of B. pseudomallei when
compared with B. thailandensis.
From the 1Department of Immunology, Faculty of Medicine Siriraj
Hospital, Bangkok, Thailand, 2Department of Microbiology, Fac-
ulty of Science, Mahidol University, Bangkok, Thailand.
Correspondence: Sansanee C. Chaiyaroj
B. pseudomallei is the causative agent of
melioidosis, a common infectious disease endemic in
Southeast Asia, especially in the northeastern part of
Thailand.1 The clinical manifestations of melioido-
sis are rather broad, ranging from fulminant septice-
mia to localized lesions and chronic disease.2-4 Al-
though every organ in the body may be involved, the
lungs are most commonly affected.5 Acute pulmo-
nary melioidosis is the most common clinical presen-
tation, and patients often have fever with cough as a
result of primary lung abscess or secondary to the
spread of septicemic.6 The disease predominantly
affects rice farmers and their family members, who
WONGPROMPITAK, ET AL.
are thought to come into contact with soil and water
in rice fields where B. pseudomallei exists as a sap-
rophyte.7 B. thailandensis is a non-pathogenic envi-
ronmental saprophyte that is genetically, biochemi-
cally, and immunologically closely related to B.
pseudomallei.8-10 In fact, a few years ago it was con-
sidered to be an avirulent biotype of B. pseudomallei,
as the two organisms are very similar to each other in
most phenotypic characteristics except for level of
virulence in man and animals.10
B. pseudomallei can adhere to and invade a
number of mammalian cells.11,12 Human lung epithe-
lial cells are particularly susceptible following expo-
sure by inhalation.12 Generally, host cell adhesion
and subsequent invasion are essential steps in the
pathogenesis of invasive bacterial infections, and in-
terfering with these processes can reduce disease in-
cidence and severity.13 An early step of host re-
sponses to bacterial invasion is the trafficking of leu-
kocytes from the vascular compartment to extravas-
cular tissues.14 Leukocyte trafficking to surrounding
tissue is often initiated by inflammatory stimuli that
induce expression of adhesion molecules on the sur-
face of the endothelial cells.14 Previous findings
showed that B. pseudomallei could enter both phago-
cytic and non-phagocytic cells and subsequently es-
cape into the cytosol.11,12
Comparative studies of B. pseudomallei and
B. thailandensis are limited. Previous studies
showed that virulent B. pseudomallei was considera-
bly more efficient than its naturally occurring aviru-
lent counterpart B. thailandensis in host cell invasion
and adherence.12 B. pseudomallei exhibited an inva-
sive capacity approximately 10-fold higher than B.
thailandensis and also exhibited a 2-fold higher ad-
herence capacity. However, after successfully invad-
ing the cells, both Burkholderia species could simi-
larly survive and replicate in the cultured A549 hu-
man lung epithelial cells.12
In the present study, the ability of B. pseu-
domallei to induce expression of adhesion molecules
and cytokine production by cultured A549 human
lung epithelial cells was assessed to gain more in-
sight into bacterial virulence and the mechanism of
pathogenicity. Gene expression profiles of A549
cells infected with B. pseudomallei and B. thailan-
densis were compared. Analysis of the data revealed
that the response of A549 cells to B. pseudomallei
infection was distinct from that of B. thailandensis
infection. The results showed that compared with B.
thailandensis, the virulent B. pseudomallei could
more readily induce expression of some anti-
apoptotic genes and at the same time suppress ex-
pression of some adhesion molecules and proin-
MATERIALS AND METHODS
Bacterial strains and growth conditions
B. pseudomallei strain 844 (arabinose-
negative strain) was originally isolated from a patient
admitted to Srinagarind Hospital, Khon Kaen, Thai-
land. The isolate was identified based on its bio-
chemical characteristics, colony morphology, reac-
tion with polyclonal antibody, and antibiotic sensi-
tivity profiles.12 B. thailandensis was isolated from
sandy loam in northeastern Thailand, and the strain
demonstrated a more than 105-fold decrease in its
virulence relative to B. pseudomallei 844 in an ani-
mal model of acute melioidosis.12 The bacteria were
cultured in trypticase soy broth at 37°C with shaking
at 150 rpm. The overnight culture was washed in
phosphate-buffered saline (PBS) and adjusted to an
appropriate concentration by measurement of the op-
tical density at 650 nm.
Lung epithelial cell and culture condition
A human lung epithelial cell line (A549,
ATCC CCL 185) was purchased from American
Type Culture Collection (ATCC), Maryland, USA.
The cells were maintained in Ham’s F-12 (GIBCO-
BRL) supplemented with 10% heat-inactivated fetal
bovine serum (FBS) (GIBCO-BRL, New York,
USA) and 1% penicillin-streptomycin antibiotic mix-
ture (Sigma, Missouri, USA) at 37°C in a humidified
atmosphere of 95% oxygen and 5% carbon dioxide.
RNA extraction and total RNA quantification
After human lung epithelial cells were in-
fected with B. pseudomallei or B. thailandensis at a
multiplicity of infection (MOI) of 10 for 2 hours,
RNA was extracted from the epithelial cells using
the RNeasy Mini kit (QIAGEN Inc., California,
USA) according to the manufacturer’s recommenda-
GENE EXPRESSION OF LUNG CELLS TO BURKHOLDERIA
sion gene (ipaB) can directly activate caspase-1,
which then induces apoptosis and inflammation.40
This form of apoptosis usually occurs early after ex-
posure of the macrophages to the pathogens, thus fa-
cilitating evasion of immune clearance. Our data are
consistent with the interpretation that B. pseu-
domallei can manipulate A549 cells for its own ad-
vantage, allowing prolonged survival inside the
harmless environmental niche of the non-phagocytic
The data presented in this study clearly show
that early phase of the host cell response to infection
by B. pseudomallei and B. thailandensis are not very
different. Both bacteria have similar ability to sur-
vive inside the host cells, to induce PMN recruit-
ment, and to inhibit host cell apoptosis. Nevertheless,
B. pseudomallei could manipulate host cell immune
response more efficiently through the suppression of
proinflammatory cytokine production. Further ex-
perimental investigation to dissect the underlying
mechanisms for such manipula
tion may explain why
infection by B. pseudomallei is more virulent than
Patimaporn Wongprompitak is a Ph.D. stu-
dent at Mahidol University. Financial support from
the Thailand Research Fund through the Royal
Golden Jubilee Ph.D. Program (Grant No. PHD/
0182/2545) to Patimaporn Wongprompitak and Prof.
Stitaya Sirisinha is acknowledged. This work is sup-
ported by Siriraj Grant for Research Development
and Medical Education a
tion in Thailan
nd Postgraduate Education
evelopment for the Development of Higher Educa-
7. Wuthiekanun V, Smith MD, Dance DA, White NJ. Isolation
of Pseudomonas pseudomallei from soil in north-eastern
Thailand. Trans R Soc Trop Med Hyg 1995; 89: 41-3.
Wuthiekanun V, Smith MD
White NJ. Biochemical characteristics of clinical and envi-
ronmental isolates of Burkholderia pseudomallei. J Med Mi-
crobiol 1996; 45: 408-12.
nose assimilation defines a nonvirulent biotype of
Burkholderia pseudomallei. Infect Immun 1997; 65: 4319-
10. Brett PJ, DeShazer D, Woods DE. Burkholderia thailanden-
sis sp. nov., a Burkholderia pseudomallei-like species. Int J
11. Jones AL, Beveridge TJ, Woods DE. Intracellular survival
of Burkholderia pseudomallei. Infect Immun 1996; 64: 782-
Kespichayawattana W, Intachote P, Utaisincharoen P, Siris-
inha S. Virulent Bu
than avirulent Burkholderia thailandensis in invasion of and
adherence to cultured human
2004; 36: 287-92.
Kaufmann SH. Immunity to intracellular bacteria. Annu Rev
Immunol 1993; 11:
14. Springer TA. Traffic signals on endothelium for lymphocyte
recirculation and leukocyte emigration. Annu Rev Physiol
1995; 57: 827-72.
Lertpatanasuwan N, Sermsri K, Petkaseam
boon S, Thamlikitkul V, Suputtamongkol Y. Arabinose-
positive Burkholderia pseudomallei infection in
case report. Clin Infect Dis 1999; 28: 927-8.
Graves DJ. Powerful tools for genetic analysis come o
Trends Biotechnol 1999; 17: 127-34.
Blohm DH, Guiseppi-Elie A. New developments in microar-
ray technology. Curr Opin B
18. Hossain H, Tchatalbachev S, Chakraborty T. Host gene ex-
pression profiling in pathogen-host interactions. Curr Opin
Immunol 2006; 18: 422-9.
19. Pruksachartvuthi S, Aswapokee N, Thankerngpol K. Sur-
vival of Pseudomonas pseudomallei in human phagocytes. J
Med Microbiol 1990; 31: 109-14.
20. Harley VS, Dance DA, Tovey G, McCrossan MV, Drasar
BS. An ultrastructural study of the phagocytosis of
Burkholderia pseudomallei. Microbios 1998; 94: 35-45.
Ekchariyawat P, Pudla S, Limposuwan K, Arjcharoen S,
Sirisinha S, Utaisincharoen P. Burkholderia pseudomallei-
induced expression of suppressor of cytokine signaling 3 and
cytokine-inducible src homolog
mouse macrophages: a possible mechanism for suppression
of the response to gamma interferon stimulation. Infect Im-
mun 2005; 73: 7332-9.
Ko J, Gen
IFN regulatory factor-1 and IFN consensus sequence binding
protein-deficient mice to brucellosis. J Immunol 2002; 168:
Utaisincharoen P, Anuntagool N, Arjcharoen S, Limposuwan
K, Chaisuriya P, Sirisinha S. Induction of iNOS expression
, Dance DA, Walsh AL, Pitt TL,
h MD, Angus BJ, Wuthiekanun V, White NJ. Arabi-
Bacteriol 1998; 48: 317-20.
rkholderia pseudomallei is more efficient
epithelial cells. Microb Pathog
iotechnol 2001; 12: 41-7.
y 2-containing protein in
dron-Fitzpatrick A, Splitter GA. Susceptibility of
and antimicrobial activity by interferon (IFN)-beta is distinct
Chaowagul W, White NJ, Dance DA, Wattanagoon Y, Nai-
gowit P, Davis TM, et al. Melioidosis: a major cause of
community-acquired septicemia in northeastern Thailand. J
Infect Dis 1989; 159: 890-9.
2. Raja NS. Localized melioidosis. J Pak Med Assoc 2003; 53:
3. Koszyca B, Currie BJ, Blumbergs PC. The neuropathology
of melioidosis: two cases and a review of the literature. Clin
Neuropathol 2004; 23: 195-203.
4. Leelarasamee A. Burkholderia pseudomallei: the unbeatable
foe? Southeast Asian J Trop Med Public Health 1998; 29:
5. Ip M, Osterberg LG, Chau PY, Raffin TA. Pulmonary
melioidosis. Chest 1995; 108: 1420-4.
White NJ. Melioidosis. Lancet 2003; 361: 1715-22.
WONGPROMPITAK, ET AL.
from IFN-gamma in Burkholderia pseudomallei-infected
mouse macrophages. Clin Exp Immunol 2004; 136: 277-83.
Santanirand P, Harley VS, Dance DA, Drasar BS, Bancroft
GJ. Obligatory role of gamma interferon for
a murine model of infection with Burkholderia pseu-
domallei. Infect Immun 1999; 67: 593-600.
Breitbach K, Klocke S, Tschernig T, van Rooijen N,
Baumann U, Steinmetz I. Role of inducible nitric oxide syn-
pseudomallei infection in mice. Infect Immun 2006; 74:
26. Kishimoto T, Akira S, Narazaki M, Taga T. Interleukin-6
family of cytokines and gp130. Blood 1995;
27. Trepicchio WL, Dorner AJ. Interleukin-11. A gp130 cyto-
kine. Ann N Y Acad Sci 1998; 856: 12-21.
28. Knight D, Bai T. Roles for Leukemia inhibitory factor in
lung biology. Drug News Perspect 1999; 12: 261-70.
Opal SM, Jhung J, Keith JC Jr, Palardy JE, Parejo N, Schaub
J. Recombinant human interleukin-11
domonas aeruginosa sepsis in immunocompromised ani-
mals. J Infect Dis 1998; 178:1205-8.
Quinton LJ, Jones MR, Robson BE, Simms BT, Whitsett JA,
Mizgerd JP. Alveolar epithelial STAT3, IL-6
kines, and host defense during Escherichia coli pneumonia.
Am J Respir Cell Mol Biol 2008; 38: 699-706.
ment elicited by bacteria in the lungs. Semin Immunol 2002;
Lasky LA. S
33. Issekutz AC, Issekutz TB. The contribution of LFA-1
(CD11a/CD18) and MAC-1 (CD11b/CD18) to the in vivo
migration of polymorphonuclear leucocytes to inflammatory
reactions in the rat. Immunology 1992; 76: 655-61.
34. Ferkol TW, Look DC. Chinks in the armor of the airway:
Pseudomonas infection in the cystic fibrosis lung. Am J
Respir Cell Mol Biol 2001; 25: 11–3.
35. Burg ND, Pillinger MH. The neutrophil: function and regula-
tion in innate and humoral immunity. Clin Immunol 2001;
36. Gibson RL, Burns JL, Ramsey BW. Pathophysiology and
management of pulmonary infections in cystic fibrosis. Am
J Respir Crit Care Med 2003; 168: 918–51.
37. Easton A, Haque A, Chu K, Lukaszewski R, Bancroft GJ. A
critical role for neutrophils in resistance to experimental in-
fection with Burkholderia pseudomallei. J Infect Dis 2007;
38. Utaisincharoen P, Anuntagool N, Arjcharoen S, Lengwe-
hasatit I, Limposuwan K, Chaisuriya P, et al. Burkholderia
pseudomallei stimulates low interleukin-8 production in the
human lung epithelial cell line A549. Clin Exp Immunol
2004; 138: 61-5.
39. Kespichayawattana W, Rattanachetkul S, Wanun T, Utaisin-
charoen P, Sirisinha S. Burkholderia pseudomallei induces
cell fusion and actin-associated membrane protrusion: a pos-
sible mechanism for cell-to-cell spreading. Infect Immun
2000; 68: 5377-84.
40. DeLeo FR. Modulation of phagocyte apoptosis by bacterial
pathogens. Apoptosis 2004; 9: 399-413.
host survival in
NADPH oxidase in early control of Burkholderia
in experimental Pseu-
Molecular mechanisms of neutrophil recruit-
electin-carbohydrate interactions and the initia-
tion of the inflammatory response. Annu Rev Biochem 1995;