Salmonella Infection of Gallbladder Epithelial Cells Drives Local ...

Gallbladder Injury in Acute Typhoid Fever • JID 2009:200 (1 December) • 1703

M A J O R A R T I C L E

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-

inflammatory cytokine induction, massive neutrophil infiltra-

tion, and tissue injury. Salmonella invasion mutants were unable

to infect gallbladder epithelial cells, were confined to the gall-

bladder lumen, and failed to induce neutrophil influx or sig-

nificant tissue damage. Our results demonstrate that inflam-

mation and injury of the gallbladder in acute typhoid fever

result from infection of epithelial cells by invasive Salmonella.

This novel model also provides a unique opportunity to study

Salmonella interactions with epithelial cells in vivo, in regard

to both the subversion of host cell functions and the induction

of inflammatory responses at epithelial surfaces.

MATERIALS AND METHODS

Bacterial strains, mouse infections, and sample collection. S.

Typhimurium strains SL1344 (Smr) and its invasion-deficient

derivative SB103 (invA::Tn3, Smr/Kanr [21]) were used in this

study. Bacteria were grown overnight with shaking at 37�C in

Luria-Bertani (LB) broth supplemented with 100 mg/mL strep-

tomycin or 50 mg/mL kanamycin. Inocula were prepared in

sterile 100 mmol/L HEPES with 0.9% sodium chloride (pH,

8.0) or phosphate-buffered saline (PBS) for oral and intrave-

nous infections, respectively.

Infections were performed in accordance with University of

British Columbia animal protocols. Cohorts of 8-week-old fe-

male C57BL/6 mice (Nramp�/�; Jackson Laboratory) were in-

fected orally or intravenously with doses of 3– or75 � 10

bacteria, respectively. Mice were euthanized at 6, 12,25 � 10

24, 48, 72, 96, and 120 h after infection, and tissue samples

were obtained for quantification of bacteria. Intestinal tissue

samples included their respective contents; the gallbladders in-

cluded bile. Fresh stool specimens were obtained immediately

before animals were euthanized. All infection experiments were

done in duplicate using a total of 8–10 mice. Tissues were

homogenized using a mixer mill (MM 301; Retsch) at a fre-

quency of 30/s for 10 min, and dilutions were plated for colony

counts.

Microscopy. For general histological analysis, tissue sec-

tions were stained with hematoxylin-eosin (H-E). Immunoflu-

orescence microscopy was performed on frozen or paraffin-

embedded tissue sections (5 mm) using anti-Salmonella rab-

bit polyclonal (BD Biosciences 240984; 1:1000), anti-Salmo-

nella mouse monoclonal (clone 1E6; Biodesign International

C86309M; 1:1000), rat anti-mouse lysosome-associated mem-

brane protein 1 (LAMP-1; 1D4B-s; Developmental Studies Hy-

bridoma Bank, University of Iowa; 1:100), goat anti-mouse cy-

tokeratin 19 (Santa Cruz Biotechnology sc-33111; 1:20), and

rabbit anti-mouse myeloperoxidase 1 (MPO-1; NeoMarkers RB

373-AD; 1:200). Secondary antibodies conjugated to Alexa 488

or 568 were purchased from Invitrogen. Before immunostain-

ing, paraffin-embedded tissues were deparaffinized with xylene

and rehydrated by sequential immersion in 100%, 95%, and

75% ethanol and water. Antigen retrieval was performed in 10

mmol/L citric acid (pH, 6.0) at 100�C for 30 min. Immuno-

staining was performed as described elsewhere [22]. Sections

from frozen tissues were also stained for filamentous actin using

phalloidin–Alexa 488 (Invitrogen). Fluorescence was visualized

using an Olympus IX81 microscope. Electron microscopy was

performed as described elsewhere [22].

Cytokine determination. Tissue samples for cytokine as-

says were prepared by tissue homogenization in a Polytron

PT2100 homogenizer (Kinematica). Complete ethylenedia-

minetetraacetic acid–free protease inhibitor cocktail (Roche Di-

agnostics) was immediately added at the final concentration

recommended by the manufacturer. The homogenates were

centrifuged twice at 15,000 g for 20 min at 4�C to remove cell

debris, and the supernatants were aliquoted and stored at

�80�C. Cytokine levels in liver and gallbladder homogenates

were determined with the BD Cytometric Bead Array Mouse

Inflammation Kit (BD Biosciences), according to the manu-

facturer’s recommendations.

Bacterial growth in bile. Bile was collected from uninfected

8-week-old female C57BL/6 mice under sterile conditions. Typ-

ically, bile from 5–6 animals was pooled and used for single

experiments. The bile was centrifuged at 10,000 g for 10 min



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



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

mediators tumor necrosis factor (TNF)–a, interleukin (IL)–6,

and monocyte chemoattractant protein (MCP) 1 were increased

in the gallbladder �10-fold compared with uninfected controls,

whereas interferon (IFN)–g, IL-12p70, and IL-10 decreased

slightly or remained at the same levels as in uninfected controls

(Figure 4). The gallbladder cytokine response followed a pattern

clearly distinct from that of the liver; increased levels of TNF-

a, IL-6, and MCP-1 were detected in the liver—but not in the

gallbladder—as early as 72 h after infection, but at 120 h, the

fold induction was similar for both organs, corresponding to

bacterial densities at that time. Moreover, IFN-g and IL-10

levels were elevated in the liver but not the gallbladder (Figure

4). Microscopic examination of H-E–stained sections from col-

onized gallbladders (Figure 5A) showed histopathological dam-

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-


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.



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


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.



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.

infect gallbladder epithelial cells (Figure 6C–6E), demonstrating

that invasion is essential for infection of gallbladder epithelial

cells in vivo. The mutant bacteria were observed in the gall-

bladder lumen and were consistently cultured from the bile of

infected animals (data not shown). This lack of Salmonella

SB103 in gallbladder epithelial cells was not a consequence of

the infection route, because control experiments with intra-

venously administered wild-type Salmonella showed bacteria

within gallbladder epithelial cells (data not shown). Moreover,

Salmonella SB103 did replicate in physiological concentrations

of bile at the same rate as in LB broth in vitro (Figure 6F),

suggesting that these bacteria not only can survive in this en-

vironment but can also replicate extracellularly in bile in vivo.

Despite the high bacterial load, no significant histopatho-

logical damage was apparent in gallbladders of SB103-infected

animals (Figure 7A and 7B). Immunostaining for MPO-1 did

not show neutrophil infiltration (Figure 7C and 7D), and ep-

ithelial cells appeared normal by electron microscopy (Figure

6E). In addition, colonization with SB103 failed to increase

levels of proinflammatory cytokines in the gallbladder, whereas

levels in the liver were elevated in response to infection (data

not shown). These results demonstrate that the immunopath-



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-

flammatory response; proinflammatory cytokine increases co-

incided with the onset of bacterial colonization of the gall-

bladder and reached maximal levels at 120 h after infection,

when bacterial concentrations were also maximal. The source

of this cytokine response is not clear. Gallbladder epithelial cells

are able to produce proinflammatory cytokines [31, 32], so it

is likely that infected gallbladder epithelial cells are a contrib-

uting source. Invasion-deficient bacteria failed to infect the

gallbladder tissue, resulting in no significant tissue damage,

neutrophil infiltration, or proinflammatory cytokine induction

despite luminal bacterial densities comparable to wild-type lev-

els. This implies that breaching of the epithelial barrier is re-

quired to elicit a response and suggests that signals originating

from infected epithelial cells (possibly triggered by type 3 se-

cretion systems and/or their effectors) are necessary to induce

inflammation and neutrophil recruitment. The mere presence

of biliary, noninvasive bacteria in the gallbladder lumen is not



sufficient to elicit inflammatory damage. This is a surprising

finding, because primary gallbladder epithelial cells have been

found to be responsive to extracellular lipopolysaccharide (LPS)

in vitro [32]. However, our results suggest that gallbladder ep-

ithelial cells are unresponsive to biliary LPS in vivo. It has been

reported that LPS is rendered inactive in the presence of bile

[33, 34], which may account for our observation. Other factors,

such as expression levels or localization of relevant host recep-

tors for pathogen-associated molecular patterns in the polarized

epithelium, may also explain this discrepancy.

We have uncovered 2 novel aspects of Salmonella biology:

(1) intracellular replication of Salmonella occurs in gallbladder

epithelial cells in vivo, and (2) Salmonella are resistant to phys-

iological bile and can use this harsh environment for efficient

extracellular replication. This supports the notion that bacterial

bile resistance is an important trait for bacterial survival and

persistence, a concept proposed by studies using culture media

supplemented with bile extracts or purified bile salts [35–38].

Our findings suggest a scenario in which Salmonella descend

from an infected liver, reaching the gallbladder lumen and rep-

licating extracellularly in the bile; although most bacteria are

probably discharged into the intestine via the bile, some infect

gallbladder epithelial cells and replicate intracellularly, even-

tually causing the sloughing of damaged epithelial cells and

leading to loss of epithelial integrity, inflammation, and tissue

damage. The Nramp1-defective nature of the animal model that

we used does not allow for the study of chronic salmonellosis,

because most animals succumb to the infection. However, our

results help explain gallbladder pathogenesis in the context of

acute typhoid fever; the gallbladder histopathology that we ob-

served recapitulates the damage described in sonographic stud-

ies of gallbladder in acute human typhoid fever (thickened and

inflamed gallbladder wall, biliary sludge, mucosal irregularity

with sloughed membrane, and the presence of inflammatory

cell infiltrate [7, 8, 10, 11, 13, 14, 39]). Thus, our results strongly

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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