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Contents

Introduction

Intestinal inva

Dendritic cell

Macrophages

Neutrophils: r

Other potenti

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doi:10.1016/j.ijm

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International Journal of Medical Microbiology 294 (2004) 225–233

www.elsevier.de/ijmm

REVIEW

Cell tropism of Salmonella enterica

Renato L. Santosa, Andreas J. Baumlerb,�

aDepartment Clınica e Cirurgia Veterinarias, Escola de Veterinaria da Universidade Federal de Minas Gerais, Belo Horizonte,

MG, Brazil

bDepartment of Medical Microbiology and Immunology, College of Medicine, Texas A&M University Health Science Center,

407 Reynolds Medical Building, College Station, TX 77843-1114, USA

Received 3 May 2004; received in revised form 25 June 2004; accepted 28 June 2004

Abstract

Salmonella serotypes are able to actively cross the intestinal epithelium, mainly but not exclusively through M cellsin the follicle-associated epithelium of Peyer’s patches. Once reaching the basal side of the epithelium, Salmonella

serotypes are internalized by macrophages, dendritic cells, and neutrophils but are not found in fibroblasts or othermesenchymal cells. The outcome of the interaction between Salmonella serotypes and dendritic cells or neutrophils isdetrimental to the pathogen. In some host species Salmonella serotypes find a safe haven from humoral defenses andneutrophils within macrophages, and replication within this niche appears to be a prerequisite for the development of asystemic infection. In other host species, macrophages can control bacterial growth and the infection remains localizedto the intestine and mesenteric lymph nodes. This review summarizes our knowledge on the cellular tropism ofSalmonella serotypes and the bacterial and host factors relevant for these interactions.r 2004 Elsevier GmbH. All rights reserved.

Keywords: M-cells; Enterocytes; Macrophages; Dendritic cells; Neutrophils

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

sion: tropism for M cells and enterocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

s: an alternate port of entry? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

: a safe haven during systemic disease and persistence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

emoval of extracellular bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

al target cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229

e front matter r 2004 Elsevier GmbH. All rights reserved.

m.2004.06.029

ing author. Tel.: +979-862-7756, fax: +979-845-3479.

ess: abaumler@tamu.edu (A.J. Baumler).

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Fig. 1. S. Typhimurium invasion of the follicle-associated

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

R.L. Santos, A.J. Baumler / International Journal of Medical Microbiology 294 (2004) 225–233226

Introduction

Salmonella serotypes have evolved sophisticatedmolecular machineries that allow invasion, intracellularsurvival and replication within host cells. Whether theoutcome of these interactions is a localized intestinalinfection or systemic spread of the pathogen isdetermined by genetic factors of both the host speciesand the Salmonella serotype. For example, Salmonella

enterica serotype Typhi causes a systemic disease inhumans known as typhoid fever while patients infectedwith S. enterica serotype Typhimurium develop alocalized gastroenteritis and lymphadenitis resulting indiarrhea. These different disease outcomes are due togenetic differences between S. Typhi and S. Typhimu-rium that remain to be identified. Similar to humans,infection of calves with S. Typhimurium remainslocalized to the intestine. In contrast, S. Typhimuriuminfection in mice results in a systemic typhoid fever-likedisease (reviewed in Zhang et al., 2003b), althoughsusceptibility of inbred mouse strains is stronglyinfluenced by their genetic background (reviewed inLam-Yuk-Tseung and Gros, 2003). The different diseaseoutcomes in mice and calves infected with S. Typhimu-rium are due to genetic differences between these hostspecies, that likely include differences in innate immunerecognition (Zhang et al., 2003a) and the ability ofmacrophages to control bacterial replication (Barrowet al., 1994). The following review is an overview ofSalmonella-host interactions with emphasis on the celltypes S. enterica interacts with as the organism goesthrough the course of natural infection and dissemina-tion within its host. Much of the available data has beengenerated by analyzing the interaction of S. Typhimu-rium with human cell lines and two animal models, themouse and the calf.

epithelium in calves. Detection of S. Typhimurium by

immunohistochemistry (brown precipitate) in sections of the

bovine ileal mucosa collected 15min after infection of ligated

ileal loops was performed as described previously (Reis et al.,

2003) and is shown on the left. The section shows a domed

villus of a Peyer’s patch lymphoid follicle flanked by two

absorptive villi. Note the tropism of S. Typhimurium for the

follicle-associated epithelium of the domed villus while the

epithelium of the adjacent base of each absorptive villus is not

colonized. A transmission electron micrograph of the S.

Typhimurium invasion of M-cells in the bovine follicle-

associated epithelium is shown on the right. In this host

species, the follicle-associated epithelium is entirely composed

of M-cells.

Intestinal invasion: tropism for M cells and

enterocytes

Upon ingestion, Salmonella serotypes exhibit a tro-pism for intestinal lymphoid tissue, such as the Peyer’spatches in mammals (Frost et al., 1997; Jones et al.,1994; Reis et al., 2003; Santos et al., 2002; Tsoliset al., 1999) and the caecal tonsils in birds (Chadfieldet al., 2003). In mice, S. Typhimurium preferentiallyinvades the M-cells of the follicle-associated epithelium

of Peyer’s patches while it does not enter adjacententerocytes (Clark et al., 1994; Jones et al., 1994). Sincethe location of M cells is restricted to the follicle-associated epithelium, the tropism for this cell type mayin part explain why S. Typhimurium colonizes Peyer’spatches in higher numbers than adjacent areas of theileum. In calves S. Typhimurium is able to invade thefollicle-associated epithelium of the domed villi inPeyer’s patches (Fig. 1) and epithelial cells at the tipsof absorptive villi (Fig. 2). In these areas of the intestinalmucosa, bacterial invasion can be detected in allepithelial cell types, including M cells, enterocytes, andsecretory (goblet) cells (Santos et al., 2002) (Figs. 1 and2). However, attachment and invasion occurs faster atthe M cell-covered domed villi than at the tips ofabsorptive villi (Frost et al., 1997).The tropism of S. Typhimurium towards M cells may

be related to expression of adhesins. Thirteen fimbrialoperons have been identified in S. Typhimurium and atleast some of them undergo phase variation (reviewed inHumphries et al., 2001). Fimbrial adhesins play animportant role for S. Typhimurium attachment toepithelial cells in vitro (Baumler et al., 1996; Ernst etal., 1990; Thankavel et al., 1999) and their expression isinduced in bovine ligated ileal loops in vivo (Humphrieset al., 2003). Host cell surface molecules involved in

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Fig. 2. S. Typhimurium invasion of the tip of an absorptive

villus in calves. Detection of S. Typhimurium by immunohis-

tochemistry (brown precipitate) in sections of the bovine ileal

mucosa collected 1 h after infection of ligated ileal loops is

shown on the left. Transmission electron micrographs of the S.

Typhimurium invasion of a goblet cell (top) and enterocytes

(bottom) are shown on the right (From Santos et al., 2002 with

permission).

R.L. Santos, A.J. Baumler / International Journal of Medical Microbiology 294 (2004) 225–233 227

adhesion of S. Typhimurium have not yet beenidentified although a recent report indicates that themajor histocompatibility complex (MHC) class I mayfavor invasion of intestinal epithelial cells (Saarinenet al., 2002). Adhesion of S. Typhi to human cells invitro is mediated by binding of its type IVB fimbriae tothe cystic fibrosis transmembrane conductance regulator(CFTR) in the host cell membrane (Pier et al., 1998;Tsui et al., 2003).Invasion of epithelial cells is governed by the

Salmonella pathogenicity island 1 (SPI-1) encoded typeIII secretion system (T3SS-1) (reviewed in Galan, 2001).S. Typhimurium senses environmental factors such asoxygen concentration, osmolarity, and pH, that act asregulators for expression of T3SS-1 (Bajaj et al., 1996).Attachment to and invasion of the apical side of theintestinal epithelium occurs within 10–15min after S.

Typhimurium infection of bovine ligated ileal loops invivo (Frost et al., 1997; Santos et al., 2002). Themolecular basis for T3SS-1 mediated invasion has beenextensively studied in vitro (reviewed in Galan, 2001).The T3SS-1 injects several effector proteins into thecytosol of the host cell which promote changes in thecytoskeleton resulting in bacterial internalization bymacropinocytosis (Frances et al., 1993). Membraneruffles result either from direct interaction of effector

proteins (SipC and SipA) with components of thecytoskeleton of the host cell or from interference ofeffector proteins (SopE, SopE2, and SopB) with hostcell signaling pathways that indirectly induce actinrearrangements. This latter mechanism involves theactivation of Cdc42 and Rac directly by the T3SS-1effectors SopE and SopE2, and indirectly by the SopBeffector protein (reviewed in Hayward and Koronakis,2002; Zhou and Galan, 2001). In addition, T3SS-1-mediated accumulation of host cell membrane choles-terol at the site of S. Typhimurium entry is required forbacterial invasion (Garner et al., 2002).Following internalization, S. Typhimurium remains

in a membrane-bound vacuole within the epithelial cell.S. Typhimurium adapts to this new environment bychanging its pattern of gene expression, particularly byupregulating expression of a second type III secretionsystem (T3SS-2) encoded on Salmonella pathogenicityisland 2 (SPI-2) (reviewed in Waterman and Holden,2003). Induction of T3SS-2 expression correlates withincorporation of late endocytic markers to the Salmo-

nella-containing vacuole (SCV) (Brumell et al., 2001).Following intracellular bacterial replication in vitro,tubular structures projecting from the original vacuoledevelop, termed Salmonella-induced filaments (Sifs)(Garcia-del Portillo et al., 1993). Biogenesis of theSCV requires the interaction of T3SS-2 effector proteinsdirectly with the vacuole membrane (Brumell et al.,2003; Freeman et al., 2003; Knodler et al., 2003). TheT3SS-2 effector protein SseG is essential for bacterialreplication inside epithelial cells, possibly by triggering aclose association between the SCV and the Golginetwork (Salcedo and Holden, 2003). The T3SS-2effector protein SifA is required for maintaining theintegrity of the SCV membrane (Beuzon et al., 2000;Salcedo et al., 2001).One hour after infection of bovine-ligated ileal loops,

most bacteria within epithelial cells are located at thebasal side of the cells not at the apical location seen atearlier time points (Santos et al., 2002). Interestingly,very few bacteria are observed at the basal side of theepithelial layer indicating that the transit from the apicalside through the basal aspect of the epithelium into thelamina propria occurs rapidly. Detection of S. Typhi-murium by immunohistochemistry shows epithelialtransmigration takes about 1–4 h to complete in vivo(Reis et al., 2003). Bacterial factors required forexocytosis from cells during transepithelial migrationhave not yet been identified.

Dendritic cells: an alternate port of entry?

S. Typhimurium can rapidly (within 10min) accessthe blood stream from the intestinal lumen through an

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alternative, T3SS-1-independent route. This pathwayinvolves bacterial transport by CD-18-expressing pha-gocytes (macrophages and/or dendritic cells) to systemicsites of infection (Vazquez-Torres et al., 1999). Rescignoet al. (2001) demonstrated that dendritic cells are able toproject dendrites through the epithelial lining into theintestinal lumen, internalize lumenal bacteria and thentransport the organisms to the basolateral side of theepithelium. The integrity of the epithelium is maintainedthroughout this process through the expression of tightjunction proteins by dendritic cells (Rescigno et al.,2001). Specific bacterial factors may not be required toexplain the affinity of S. Typhimurium for CD18-positive cells because bacterial internalization andtransport to the spleen are thought to be part of aCD18-dependent intestinal sampling mechanism (Vaz-quez-Torres et al., 1999). However, murine dendriticcells are not highly efficient in internalizing S. Typhi-murium and complement opsonization may be requiredfor efficient uptake, since it increases the number ofintracellular bacteria after challenge of cultured den-dritic cells by 10-fold (Eriksson et al., 2003). S.

Typhimurium may exit from dendritic cells to enter itspreferred cell type, the macrophage, by inducing celldeath using its T3SS-1 (van der Velden et al., 2003).Although S. Typhimurium survives within murine

dendritic cells, this intracellular niche does not supportreplication and the numbers of intracellular bacteriatend to be stable over the course of infection (Jantschet al., 2003). Inducible nitric oxide synthase (iNOS) is ahost factor that contributes to limiting bacterial growthin dendritic cells (Eriksson et al., 2003). Neither SPI-2nor the PhoP/PhoQ system are required for intracellularsurvival in dendritic cells (Garcia-Del Portillo et al.,2000; Jantsch et al., 2003). Marked changes in dendriticcell populations are observed in the spleen of miceduring the acute phase of S. Typhimurium infection,suggesting that this cell type plays a role during thesystemic phase of infection (Kirby et al., 2001).

Macrophages: a safe haven during systemic

disease and persistence

S. Typhimurium causes a typhoid-like systemicdisease in mice in which bacteria reside intracellularlywithin macrophages and neutrophils of the liver (Nnalueet al., 1992; Richter-Dahlfors et al., 1997) and the spleen(Salcedo et al., 2001). S. Typhimurium tends to expandclonally within a given phagocyte in the liver, andindividual focal lesions harbor bacteria derived fromclonal expansion of one single bacterium (Sheppardet al., 2003). Regardless of the rate of bacterial growth invivo, the amount of bacteria in the tissue parallels thenumber of infected phagocytes with a low number of

bacteria per phagocyte (Sheppard et al., 2003). Incontrast, infection of macrophages in vitro results in amarkedly variable number of bacteria per host cell andon average much higher numbers of bacteria perphagocyte (Holden, 2002).Although genetic resistance of mice to S. Typhimu-

rium infection is the result of the expression of severalloci, the divalent cation transporter Nramp1 which islocated in the phagolysosomal membrane of macro-phages appears to be a key determinant (Gruenheidet al., 1997; Jabado et al., 2000; Vidal et al., 1993). Micewith intact Nramp1 protein are genetically resistant toS. Typhimurium infection (ItyR) while inactivation ofthe gene encoding Nramp1 by point mutation causesmice to succumb to infection within 6–10 days (ItyS). InItyR mice, S. Typhimurium persists predominantly inthe mesenteric lymph nodes, and for a shorter durationin the liver, the spleen and the cecum (Kingsley et al.,2003; Monack et al., 2004). S. Typhimurium is able topersist inside macrophages in the mesenteric lymphnodes of ItyR mice for at least 1 year in spite of highanti-Salmonella IgG titers. This chronic carrier state canrevert to acute disease by treating the mice withneutralizing anti-IFNg antibodies (Monack et al.,2004). Nramp1 is thought to confer nutritional im-munity by withholding divalent cations from bacterialocated within macrophages. Active uptake of divalentcations such as Fe++ and Mn++ by S. Typhimurium isrequired for full virulence and intracellular survival(Boyer et al., 2002).The phagosome in macrophages undergoes a well-

orchestrated maturation, that involves sequential fusionwith endosomes resulting in a progressive acidificationof the vacuole with an increasing ionizing and hydrolyticenzymatic activity (reviewed in Scott et al., 2003; Vieiraet al., 2002). S. Typhimurium manipulates maturationof the SCV using its T3SS-2 (Brumell et al., 2001; Garviset al., 2001; Kuhle and Hensel, 2002; Uchiya et al.,1999). Maturation of the SCV is delayed, but notcompletely blocked, a process modulated by the T3SS-2effector protein SifA (Brumell et al., 2001). Althoughinitially described in epithelial cells, Sifs can also bedetected in macrophage cell lines infected with S.Typhimurium (Knodler et al., 2003). Initial studiesindicated that the T3SS-2 effector protein SpiC isrequired for segregating the SCV from the late endocyticpathway (Uchiya et al., 1999). There is evidence thatPhoP-regulated genes are required for inhibition ofendosome/lysosome fusion with the SCV and that thisprocess involves bacterial proteins in addition to SpiC(Garvis et al., 2001). Lee et al. (2002) screened aleukocyte cDNA library using the yeast two-hybridsystem to find a host cell target for SpiC and found apreviously unknown gene product named TassC. TheSpiC/TassC interaction is biologically significant assuppression of TassC restores intracellular growth of

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Fig. 3. Detection of S. Typhimurium by immunohistochem-

istry (brown precipitate) in sections of the bovine ileal mucosa

collected at 10 h after infection of ligated ileal loops (on the

left). Note that most of the positive staining is localized within

inflammatory cells. On the right, transmission electron

micrograph showing phagocytosis of S. Typhimurium by a

neutrophil with some SCV.

R.L. Santos, A.J. Baumler / International Journal of Medical Microbiology 294 (2004) 225–233 229

an S. Typhimurium spiC mutant (Lee et al., 2002). Thehost cell protein Hook3 was identified as a second targetof SpiC using a GST pull-down assay. Expression ofSpiC in Vero cells results in an altered distributionof lysosomes that mimics the phenotype of cells express-ing a Hook3 dominant-negative mutant (Shotlandet al., 2003).The NADPH oxidase-dependent respiratory burst

and iNOS are partially responsible for the anti-Salmonella activity of macrophages (Mastroeni et al.,2000; Vazquez-Torres et al., 2000a). S. Typhimuriumevades macrophage NADPH oxidase by preventingrecruitment of NADPH oxidase components to thephagosome using the T3SS-2 (Gallois et al., 2001;Vazquez-Torres et al., 2000b). Similarly, a functionalT3SS-2 inhibits the co-localization of iNOS andnitrotyrosine residues with S. Typhimurium inside themacrophage (Chakravortty et al., 2002).

S. Typhimurium infection of murine macrophages invivo (Richter-Dahlfors et al., 1997) and of primarymacrophages or macrophage-like cell lines in vitroresults in induction of cell death (Chen et al., 1996;Guilloteau et al., 1996; Lindgren et al., 1996; Monack etal., 1996). Since it does not match all classical criteria forapoptosis or necrosis, the term pyroptosis has beenpostulated to describe S. Typhimurium-induced macro-phage cell death (Cookson and Brennan, 2001). S.

Typhimurium can cause pyroptosis by two independentmechanisms, an early killing that is T3SS-1 dependent(Monack et al., 1996; Chen et al., 1996; Santos et al.,2001a) and a delayed killing that is T3SS-1 independentbut requires functional spv, PhoP/Q, OmpR and T3SS-2systems (Browne et al., 2002; Detweiler et al., 2001;Kurita et al., 2003; Libby et al., 2000; Lindgren et al.,1996; Lundberg et al., 1999; Paesold et al., 2002; Santoset al., 2001a; van der Velden et al., 2000). The T3SS-1-dependent mechanism of pyroptosis depends on anSipB-mediated activation of caspase-1, resulting inactivation of IL-1 in vitro (Hersh et al., 1999). However,there is currently no direct evidence that pyroptosiscontributes to the massive neutrophil influx that is thehistopathological hallmark of S. Typhimurium infectionin the human and bovine intestine (Santos et al., 2001b).

Neutrophils: removal of extracellular bacteria

Bovine intestinal epithelial cells respond to S.

Typhimurium infection in vivo by secreting severalinflammatory mediators that trigger a massive neutro-phil influx into the intestinal mucosa (Zhang et al.,2003a). Bacteria in the lamina propria of the bovineintestine or in bovine mesenteric lymph nodes arelocated intracellularly either within mononuclear cells(dendritic cells and/or macrophages) or within neutro-

phils (Fig. 3) (Santos et al., 2002). An early studysuggests that neutrophils may represent a safe-site for S.

Typhimurium in the murine spleen (Dunlap et al., 1992).However, more recent work demonstrates that incontrast to macrophages, cultured neutrophils do notpermit intracellular replication of S. Typhimurium andkill intracellular bacteria (Papp-Szabo et al., 1994). Thefinding that neutrophils efficiently kill S. Typhimuriumin vitro is in agreement with in vivo experimentsdemonstrating that neutrophils prevent extracellularreplication of S. Typhimurium in the liver of mice(Conlan, 1996). Neutrophils also inhibit S. Typhimu-rium replication in the spleen of mice and are essential toprevent further dissemination of the infection to otherorgans such as the kidney and the lung (Conlan,1997). Neutrophils appear to be efficient killers ofS. Typhimurium in the bovine diarrhea model sinceultrastuctural analysis reveals that bacteria present inneutrophils frequently appear to lack morphologicalintegrity (Santos et al., 2002). However, active neutro-phils are not sufficient to clear S. Typhimurium fromsystemic sites of infection in mice, presumably becausebacteria find a safe haven within macrophages (Chemi-nay et al., 2004).

Other potential target cells

S. Typhimurium invades cultured cell lines represent-ing a broad variety of cell types originating from manydifferent organs and animal species. However, thesignificance of these in vitro observations for under-standing the host–pathogen interaction in vivo remainsquestionable. For example, interaction of S. Typhimu-rium with fibroblasts has been studied in vitro. S.

Typhimurium can enter into and survive within normalrat kidney fibroblasts although this cell type does not

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support rapid replication (Cano et al., 2001). Therelevance of this in vitro observation is diminished bythe finding that bacteria are not found in fibroblasts orother mesenchymal cells in bovine intestinal tissueinfected with S. Typhimurium in vivo but are locatedexclusively in epithelial cells, mononuclear cells andneutrophils (Santos et al., 2002). Similarly, S. Typhi-murium is located exclusively within macrophages andneutrophils of the murine liver in vivo (Richter-Dahlforset al., 1997).

Conclusions

While Salmonella serotypes interact with a broadspectrum of cell types in vitro, the bacteria are foundexclusively within epithelial cells, macrophages, dendri-tic cells and neutrophils in vivo. The interaction ofSalmonella serotypes with neutrophils may not beviewed as tropism since this cell type functionspredominantly in controlling infection. Dendritic cellsserve as vehicles for dissemination in the initial stages ofinfection but are not suitable as reservoirs for Salmo-

nella serotypes later on. Interactions that appear to bemost important for the tissue tropism characteristic forinfections with Salmonella serotypes are the invasion ofa selected subset of epithelial cells in the gastro-intestinaltract and the intracellular survival and replication withinmacrophages.

Acknowledgements

We would like to thank Helene Andrews-Polymenisfor helpful suggestions to improve this manuscript.Work in A. Baumler0s laboratory is supported by

USDA/NRICGP Grant #2002-35204-12247 and PublicHealth Service grants #AI40124 and #AI44170.

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