Gallbladder Injury in Acute Typhoid Fever • JID 2009:200 (1 December) • 1703 MAJOR ARTICLE Salmonella Infection of Gallbladder Epithelial Cells Drives Local Inflammation and Injury in a Model of Acute Typhoid Fever Alfredo Menendez, 1,a Ellen T. Arena, 1,a Julian A. Guttman, 4 Lisa Thorson, 1 Bruce A. Vallance, 3 Wayne Vogl, 2 and B. Brett Finlay 1 1 Michael Smith Laboratories and 2 Department of Cellular and Physiological Sciences, Division of Anatomy and Cell Biology, Life Sciences Centre, University of British Columbia, and 3 Division of Gastroenterology, BC Children’s Hospital, Vancouver, and 4 Department of Biological Sciences, Simon Fraser University, Burnaby, British Columbia, Canada The gallbladder is often colonized by Salmonella during typhoid fever, yet little is known about bacterial pathogenesis in this organ. With use of a mouse model of acute typhoid fever, we demonstrate that Salmonella infect gallbladder epithelial cells in vivo. Bacteria in the gallbladder showed a unique behavior as they replicated within gallbladder epithelial cells and remained confined to those cells without translocating to the mucosa. Infected gallbladders showed histopathological damage characterized by destruction of the epithelium and massive infiltration of neutrophils, accompanied by a local increase of proinflammatory cytokines. Damage was determined by the ability of Salmonella to invade gallbladder epithelial cells and was independent of high numbers of replication-competent, although invasion-deficient, bacteria in the lumen. Our results establish gallbladder epithelial cells as a novel niche for in vivo replication of Salmonella and reveal the involvement of these cells in the pathogenesis of Salmonella in the gallbladder during the course of acute typhoid fever. Typhoid fever is a systemic disease caused by infection with the facultative, intracellular bacterium, Salmonella enterica serovar Typhi. Typhoid fever remains a serious Received 29 January 2009; accepted 20 April 2009; electronically published 23 October 2009. Potential conflicts of interest: none reported. Financial support: Canadian Institutes of Health Research (grants MOP10551 and MOP13452 to B.B.F.), Howard Hughes Medical Institute (grant 55005504 to B.B.F.) and the Foundation for the National Institutes of Health, as part of the Bill and Melinda Gates Grand Challenge program (grant BMG78589 to B.B.F.); Michael Smith Foundation for Health Research/Genome British Columbia and the Natural Sciences and Engineering Research Council of Canada (postdoctoral fellowships to A.M.); University of British Columbia (University Graduate Fellowship and Armauer Hansen Memorial Fellowship to E.T.A.). Presented in part: Banff Conference on Infectious Diseases, Banff, Alberta, Canada, 27–31 May 2008 (abstract R11); 58th Annual Conference of the Canadian Society of Microbiologists, Calgary, Alberta, Canada, 9–12 June 2008 (abstract B24); and the Gordon Research Conference on Microbial Toxins and Pathogenesis, Andover, New Hampshire, 13–18 July 2008 (abstract A23). a A.M. and E.T.A. contributed equally to this work. Reprints or correspondence: Dr B. Brett Finlay, Michael Smith Laboratories, University of British Columbia, 301–2185 E Mall, Vancouver, BC, Canada V6T 1Z4 (bfi[email protected]). The Journal of Infectious Diseases 2009; 200:1703–13 2009 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/2009/20011-0013$15.00 DOI: 10.1086/646608 public health problem in underdeveloped countries, be- cause Salmonella infection is initiated by consumption of contaminated food or water. Gallbladder infections are common in typhoid fever; Salmonella have been isolated from gallbladders from patients with acute and chronic disease [1–8]. In acute typhoid fever, coloni- zation of the gallbladder is rarely diagnosed, but it may become apparent with the onset of acalculous chole- cystitis [7, 9–11] and gallbladder perforation [7, 8, 12]. Gallbladder alterations as a result of typhoid fever are poorly characterized. When abdominal ultrasonog- raphy is performed in patients with acute typhoid fever, as many as 60% have an abnormal sonographic gall- bladder score indicative of organ damage, involving gallbladder wall thickening, pericholecystic edema and fluid collection, formation of biliary sludge, mucosal irregularity with sloughing membrane, and presence of ulcers [13, 14]. These pathological features are indic- ative of an organ’s reaction in response to local infec- tion. However, the immunopathogenesis events asso- ciated with these alterations are still undefined. Downloaded from https://academic.oup.com/jid/article-abstract/200/11/1703/832725 by guest on 23 March 2018
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Salmonella Infection of Gallbladder Epithelial CellsDrives Local Inflammation and Injury in a Modelof Acute Typhoid Fever
Alfredo Menendez,1,a Ellen T. Arena,1,a Julian A. Guttman,4 Lisa Thorson,1 Bruce A. Vallance,3 Wayne Vogl,2
and B. Brett Finlay1
1Michael Smith Laboratories and 2Department of Cellular and Physiological Sciences, Division of Anatomy and Cell Biology, Life Sciences Centre,University of British Columbia, and 3Division of Gastroenterology, BC Children’s Hospital, Vancouver, and 4Department of Biological Sciences,Simon Fraser University, Burnaby, British Columbia, Canada
The gallbladder is often colonized by Salmonella during typhoid fever, yet little is known about bacterialpathogenesis in this organ. With use of a mouse model of acute typhoid fever, we demonstrate that Salmonellainfect gallbladder epithelial cells in vivo. Bacteria in the gallbladder showed a unique behavior as they replicatedwithin gallbladder epithelial cells and remained confined to those cells without translocating to the mucosa.Infected gallbladders showed histopathological damage characterized by destruction of the epithelium andmassive infiltration of neutrophils, accompanied by a local increase of proinflammatory cytokines. Damagewas determined by the ability of Salmonella to invade gallbladder epithelial cells and was independent of highnumbers of replication-competent, although invasion-deficient, bacteria in the lumen. Our results establishgallbladder epithelial cells as a novel niche for in vivo replication of Salmonella and reveal the involvementof these cells in the pathogenesis of Salmonella in the gallbladder during the course of acute typhoid fever.
Typhoid fever is a systemic disease caused by infection
with the facultative, intracellular bacterium, Salmonella
enterica serovar Typhi. Typhoid fever remains a serious
Received 29 January 2009; accepted 20 April 2009; electronically published 23October 2009.
Potential conflicts of interest: none reported.Financial support: Canadian Institutes of Health Research (grants MOP10551
and MOP13452 to B.B.F.), Howard Hughes Medical Institute (grant 55005504 toB.B.F.) and the Foundation for the National Institutes of Health, as part of the Billand Melinda Gates Grand Challenge program (grant BMG78589 to B.B.F.); MichaelSmith Foundation for Health Research/Genome British Columbia and the NaturalSciences and Engineering Research Council of Canada (postdoctoral fellowshipsto A.M.); University of British Columbia (University Graduate Fellowship andArmauer Hansen Memorial Fellowship to E.T.A.).
Presented in part: Banff Conference on Infectious Diseases, Banff, Alberta,Canada, 27–31 May 2008 (abstract R11); 58th Annual Conference of the CanadianSociety of Microbiologists, Calgary, Alberta, Canada, 9–12 June 2008 (abstractB24); and the Gordon Research Conference on Microbial Toxins and Pathogenesis,Andover, New Hampshire, 13–18 July 2008 (abstract A23).
a A.M. and E.T.A. contributed equally to this work.Reprints or correspondence: Dr B. Brett Finlay, Michael Smith Laboratories,
University of British Columbia, 301–2185 E Mall, Vancouver, BC, Canada V6T 1Z4([email protected]).
The Journal of Infectious Diseases 2009; 200:1703–13� 2009 by the Infectious Diseases Society of America. All rights reserved.0022-1899/2009/20011-0013$15.00DOI: 10.1086/646608
public health problem in underdeveloped countries, be-
cause Salmonella infection is initiated by consumption
of contaminated food or water. Gallbladder infections
are common in typhoid fever; Salmonella have been
isolated from gallbladders from patients with acute and
chronic disease [1–8]. In acute typhoid fever, coloni-
zation of the gallbladder is rarely diagnosed, but it may
become apparent with the onset of acalculous chole-
cystitis [7, 9–11] and gallbladder perforation [7, 8, 12].
Gallbladder alterations as a result of typhoid fever
are poorly characterized. When abdominal ultrasonog-
raphy is performed in patients with acute typhoid fever,
as many as 60% have an abnormal sonographic gall-
bladder score indicative of organ damage, involving
gallbladder wall thickening, pericholecystic edema and
fluid collection, formation of biliary sludge, mucosal
irregularity with sloughing membrane, and presence of
ulcers [13, 14]. These pathological features are indic-
ative of an organ’s reaction in response to local infec-
tion. However, the immunopathogenesis events asso-
ciated with these alterations are still undefined.
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1704 • JID 2009:200 (1 December) • Menendez et al
Much of the knowledge on the pathogenesis of typhoid fever
comes from experimental animal infections with Salmonella
enterica serovar Typhimurium. Administration of this bacte-
rium to inbred mouse strains homozygous for a loss of function
in the Nramp1 allele results in a systemic, typhoid-like disease,
because macrophages from these mice have an impaired ca-
pacity to restrict the growth of intracellular pathogens [15].
Ingested Salmonella colonize the gastrointestinal tract and pen-
etrate the intestinal epithelial barrier [16]. On breaching the
epithelium, salmonellae quickly reach the underlying gut-as-
sociated lymphoid tissue, infect phagocytes that enter the lym-
phatic system and bloodstream [17], and spread systemically
to multiple organs, most notably the liver and spleen, where
the bacteria replicate primarily within macrophages [18, 19].
Experimental infections in mice have shown that, as happens
in humans with S. Typhi, S. Typhimurium can be found in the
gallbladder of acutely and chronically infected animals [4, 20].
Here, we present the first histological and immunological
account of gallbladder alterations occurring in systemic sal-
monellosis, using a well-established model of acute typhoid
fever in the susceptible mouse strain C57BL/6 (Nramp1�/�).
We show that the gallbladder is a permissive site for Salmonella
in which bacterial concentrations exceed those of the liver and
spleen. Salmonella infected the single epithelial cell layer of the
gallbladder but rarely translocated to the underlying lamina
propria; instead, they replicated and accumulated within the
gallbladder epithelial cells. Infection of the gallbladder epithe-
lium elicited a strong inflammatory response involving pro-
Figure 1. Bacteria recovered from organs of mice infected with wild-type Salmonella Typhimurium SL1344. Counts are given as colony-formingunits (CFU) per milligram of tissue. A and B, Oral infection. C and D, Intravenous infection. Error bars represent standard errors of the mean; GB,gallbladder; hpi, hours after infection.
and aliquoted. Bile samples (25 mL) were then seeded with S.
Typhimurium SB103 from an overnight, stationary-phase cul-
ture at an initial density of ∼106 bacteria/mL and incubated for
24 h at 37�C without shaking, along with control samples of
bile alone, bacteria in PBS, and bacteria in LB broth. Samples
were obtained every hour for the first 6 h and at 24 h, diluted,
and plated on LB streptomycin-kanamycin plates for colony
counts.
Statistical analyses. Data processing and statistical analyses
were performed using GraphPad Prism software, version 4.0
(GraphPad Software).
RESULTS
Salmonella invasion of gallbladder epithelial cells induces im-
munopathological damage. To study Salmonella colonization
of the gallbladder, C57BL/6 mice (Nramp1�/�) were infected
orally with 107 S. Typhimurium SL1344 and were killed at time
points ranging from 6 to 120 h after infection. Although sig-
nificant numbers of bacteria were present in the intestine and
shed in the feces throughout the infection (Figure 1A), bacteria
were detected only in the spleen and liver within the first 24
h after infection (Figure 1B). At 48 h after infection, Salmonella
were also found in the gallbladder, and by 96 h after infection,
gallbladders were colonized in 8 of 10 infected animals. By 120
h after infection, the average bacterial counts in the gallbladder
surpassed 106 cfu/mg. The concentration of bacteria in the
gallbladder at this time was slightly higher than that observed
in both the liver and spleen, showing that the gallbladder offers
a permissive environment for accumulation of Salmonella.
Infections by the intravenous route, which bypasses the initial
intestinal phase of infection, were used to determine whether
gallbladder colonization resulted from bacteria ascending di-
rectly from the intestines; such an infection route has been
proposed for various pathogens [2, 23, 24]. As shown in Figure
1C and 1D, bacteria appeared in the gallbladder but were still
undetectable in the intestine (48 h after infection). This suggests
that gallbladder colonization is not a result of Salmonella as-
cending directly from the gastrointestinal tract. Moreover, his-
tological analysis of the livers and gallbladders of infected an-
imals revealed that liver lesions appear before any pathological
alterations were apparent in the gallbladder (data not shown),
supporting the concept that bacteria are being discharged from
the liver into the gallbladder via the bile.
We investigated the location of Salmonella in infected gall-
bladders by microscopy and found bacteria in both the lumen
and tissue (Figure 2). In the lumen, Salmonella were seen as-
sociated with cells or as free, extracellular bacteria (Figure 2B).
Unexpectedly, within the tissue, Salmonella localized to epi-
thelial cells of the gallbladder and were rarely seen within the
lamina propria or the mucosa (Figure 2C and 2D). Intracellular
Salmonella normally replicate within a Salmonella-containing
vacuole (SCV) to which LAMP-1 is recruited. This finding has
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1706 • JID 2009:200 (1 December) • Menendez et al
Figure 2. Wild-type Salmonella Typhimurium SL1344 (S) in the gallbladder. A and B, Toluidine blue–stained sections from an uninfected mouse(A) and a representative orally infected mouse at 120 h after infection (B). Intracellular (arrows) and extracellular (arrowheads) bacteria are seen;scale bars indicate 10 mm. C and D, Immunostaining of gallbladder sections collected at 120 h after infection; bacteria are shown in red and cellnuclei in blue. Bacteria localize to the epithelial cells (e) but not the lamina propria (LP). Asterisks indicate luminal bacteria in association with cells;scale bars, 100 mm (C) or 50 mm (D). L, lumen.
been shown in infected epithelial cells in vitro [25, 26] and
splenic macrophages in vivo [19]. To characterize the infection
of gallbladder epithelial cells in vivo, we examined these 2 im-
portant aspects of Salmonella biology. Electron microscopy con-
firmed the intracellular location of Salmonella in epithelial cells
(Figure 3A). Bacteria were enclosed within vacuolar compart-
ments; a number of epithelial cells harbored large numbers of
Salmonella (130 bacteria) (Figure 3A), thus confirming the for-
mation of SCVs in gallbladder epithelial cells in vivo. Costaining
of infected gallbladder sections with anti-Salmonella and anti—
LAMP-1 antibodies revealed colocalization of LAMP-1 with
intracellular bacteria (Figure 3B and 3C), demonstrating an
active recruitment of LAMP-1 to the SCVs in gallbladder ep-
ithelial cells in vivo. Bacteria undergoing cell division within
SCVs were frequently observed (Figure 3D), indicating that
Salmonella replicate within the polarized gallbladder epithelium
in vivo. Salmonella microcolonies localized to a subnuclear po-
sition, as shown by immunofluorescence and electron micros-
copy (Figures 2 and 3). This positioning was often accompanied
by displacement of the nuclei toward the apex of the cells, most
likely as a result of bacterial replication and accumulation (Fig-
ure 3E and 3F).
Bacterial colonization of the gallbladder triggered a strong
inflammatory response. Local levels of the proinflammatory
age involving loss of epithelial folds, thickening of the mucosa,
exfoliation of the epithelium, and formation of luminal sludge
containing sloughed epithelial cells (indicated by positivity for
cytokeratin 19) and debris (Figure 5B). Immunostaining with
an anti–MPO-1 antibody showed a massive infiltration of neu-
trophils to the tissue and lumen of infected gallbladders (Figure
5C–5E). At the ultrastructural level, tissue alterations were evi-
denced by degeneration of the apical brush borders of the ep-
ithelial cells, loss of lateral intercellular cell processes, and ab-
sence of apical mucin granules characteristic of noninfected
gallbladder epithelial cells (Figure 3A).
Gallbladder colonization by Salmonella invasion mutants
without neutrophil infiltration and damage. Salmonella can
enter host cells by several mechanisms, including phagocytosis
and active invasion (reviewed in [27]). The invasion phenotype
of Salmonella is partly mediated by the products of genes lo-
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Figure 3. Salmonella (S) are contained in vacuoles, colocalize with host lysosome-associated membrane protein 1 (LAMP-1), replicate withingallbladder epithelial cells, and drive the cell nuclei (N) upward. A, Electron micrograph of uninfected and infected gallbladder epithelial cells. L,lumen. B and C, Immunostaining of gallbladder sections collected at 120 h after infection. Bacteria are shown in red, LAMP-1 in green, and cell nucleiin blue; scale bars indicate 10 mm. Arrows in panel B indicate apical LAMP-1 in uninfected cells; arrowhead, a Salmonella microcolony in associationwith LAMP-1. Two opposing sections of uninfected epithelium (a) separated from an infected area (b) by the lumen are shown in panel C. D, Electronmicrographs of intracellular Salmonella undergoing cell division; scale bars indicate 1 mm. E and F, Immunostaining of uninfected (E) and infected (F)gallbladder sections. Bacteria are shown in red, actin in green, and cell nuclei in blue; arrowheads show Salmonella microcolonies, and scale barsindicate 10 mm.
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1708 • JID 2009:200 (1 December) • Menendez et al
Figure 4. Cytokine levels in liver and gallbladder homogenates of orally infected mice at 72 and 120 h after infection, relative to levels in uninfected(UI) controls. (Cytokine levels were recorded as picograms of cytokine per milligram of tissue but are shown as relative levels, with control levels setat 1.) Error bars represent standard errors of the mean. GB, gallbladder; hpi, hours after infection; IFN, interferon; IL, interleukin; MCP, monocytechemoattractant protein; TNF, tumor necrosis factor.
cated in Salmonella pathogenicity island 1 (SPI-1) [28]. Prod-
ucts of SPI-1 assemble into a type 3 secretion system (T3SS)
that delivers a plethora of Salmonella proteins, termed “effec-
tors,” into the host cytosol; these effectors modify host cell
functions and promote bacterial uptake (reviewed in [28]). A
functional SPI-1 is associated with the ability of Salmonella to
infect epithelial cells in vitro and penetrate the gastrointestinal
epithelium in vivo [21, 29, 30]. Thus, we decided to determine
the role of the invasion phenotype in the colonization of the
gallbladder, the penetration of the tissue, and the resulting in-
flammatory damage. Mice were infected intravenously with the
S. Typhimurium invasion-deficient strain (invA) SB103; the
intravenous route was chosen to avoid the characteristic delayed
progression of infection observed with oral delivery of SB103
and to facilitate direct comparisons with the wild-type strain.
Colonization of systemic sites in intravenously infected mice
was very similar for the wild-type and mutant strains (Figure
6A and 6B). Bacteria were present at low levels in the gallbladder
at 72 h after infection, eventually reaching numbers comparable
to those in the liver and spleen at 120 h after infection. Sal-
monella SB103 concentrations in the gallbladder were similar
to those of SL1344 ( , .3391, and .6974 at 72, 96, andP p .2174
120 h after infection, respectively; by unpaired t test). These
results demonstrate that Salmonella do not require active in-
vasion to access the gallbladder.
In contrast to the wild-type strain, Salmonella SB103 did not
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Figure 5. Salmonella Typhimurium SL1344 infection of the gallbladder causes histopathological damage and triggers neutrophil infiltration in thetissue and the lumen (L). A, Hematoxylin-eosin–stained sections of gallbladders from an uninfected (UI) and 5 infected animals. B, Immunostainingof gallbladder sections from 3 infected animals; bacteria are shown in red, cytokeratin 19 in green, and cell nuclei in blue. C–E, Myeloperoxidase 1(MPO-1) immunostaining of gallbladder sections from an uninfected mouse (C) and 2 infected mice (D and E). Scale bars indicate 100 mm (A–D) or25 mm (E). DAPI, 4′,6-diamidino-2-phenylindole.
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1710 • JID 2009:200 (1 December) • Menendez et al
Figure 6. Invasion-deficient Salmonella Typhimurium (SB103) colonize the gallbladder but do not infect gallbladder epithelial cells. A and B, Organcolonization levels. CFU, colony-forming units; GB, gallbladder; hpi, hours after infection. C and D, Immunostaining of gallbladder sections (120 h afterinfection). Bacteria are shown in red, actin in green, and cell nuclei in blue; scale bars indicate 100 mm (C) or 50 mm (D). L, lumen. E, Electronmicrograph of a colonized gallbladder. Scale bar indicates 2 mm. S, Salmonella. F, S. Typhimurium replicate extracellularly in bile in vitro. No bacteriawere detected in unseeded bile for the duration of the experiment (data not shown). Error bars represent standard errors of the mean. LB, Luria-Bertani broth; PBS, phosphate-buffered saline.
Figure 7. Invasion-deficient Salmonella Typhimurium do not trigger tissue damage and neutrophil infiltration. A and B, Hematoxylin-eosin stainingof infected gallbladders. L, lumen. C and D, Immunostaining with an anti–myeloperoxidase 1 (MPO-1) antibody. Gallbladders are shown from 2representative animals containing 1106 cfu/mL of bile. Scale bars indicate 100 mm (A, C, and D) or 50 mm (B). DAPI, 4′,6-diamidino-2-phenylindole.
ological damage induced in the gallbladder by Salmonella is
dependent on the presence of bacteria within the epithelium
and that the presence of biliary, replication-competent bacteria
alone is unable to induce such damage.
DISCUSSION
During typhoid fever the gallbladder is often colonized by Sal-
monella, which may result in tissue damage [13, 14]. Despite
the prevalence of such infections, bacterial-host interactions in
the gallbladder have remained uncharacterized. We have shown
the presence of luminal, extracellular Salmonella in vivo and
demonstrated that bacteria grew efficiently in physiological bile
in vitro, suggesting that the gallbladder sustains luminal, ex-
tracellular replication of Salmonella in vivo. Salmonellae also
targeted the gallbladder epithelial cells, in which they replicated
and accumulated; this differs with events occurring in the
mouse intestine, where Salmonella mainly infect M cells and
translocate very rapidly to the underlying lamina propria [16].
The predominant location of Salmonella within epithelial cells
is also in stark contrast to their tropism for macrophages in
the liver and spleen [18, 19]. These differences may be partly
due to the absence of M cells and possibly to a lower number
of macrophages in the gallbladder compared with the liver and
spleen, which are phagocyte-laden organs. In summary, our
results reveal fundamental differences in the biology of sal-
monellae in the gallbladder, establish the gallbladder as a unique
replication niche for Salmonella, and provide an explanation
for the high numbers of bacteria in infected gallbladders.
Infection of gallbladder epithelial cells triggered a local in-
suggest that intracellular infection of gallbladder epithelial cells
also occurs in human typhoid fever.
Research on the intestinal, respiratory, and urogenital epi-
thelia during the past several years has changed the classic idea
that the epithelium simply acts as a physical barrier and has
introduced the alternative concept that epithelial cells are active
players in the mucosal inflammatory response to infection (re-
viewed in [40, 41]). To our knowledge, Salmonella constitutes
the first documented example of a bacterium that infects and
replicates within the gallbladder epithelium in vivo; thus, the
model we describe also provides a valuable system for detailed
studies of epithelial involvement in the pathogenesis and re-
sponse of the gallbladder bacterial infections in vivo. Our find-
ings deviate from the canonical view that Salmonella patho-
genicity is associated with intracellular replication in macro-
phages; instead, they clearly illustrate the pathogenic versatility
of this bacterium. In addition, gallbladder epithelial cells emerge
from this study as a novel cell population directly contributing
to bacterial pathogenesis and as key components of the inflam-
matory response of the gallbladder against bacterial infection.
Acknowledgments
We thank the members of the Finlay laboratory and Rick Schrieber forvaluable discussions and the critical reading of the manuscript. We aregrateful to G. Grassl for the translation of early German studies. Specialthanks to M. E. Wickham for valuable suggestions.
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