O-Antigen Delays Lipopolysaccharide Recognition and Impairs Antibacterial Host Defense in Murine Intestinal Epithelial Cells

Although Toll-like receptor (TLR) 4 signals from the cell surface of myeloid cells, it is restricted to an intracellular compartment and requires ligand internalization in intestinal epithelial cells (IECs). Yet, the functional consequence of cell-type specific receptor localization and uptake-dependent lipopolysaccharide (LPS) recognition is unknown. Here, we demonstrate a strikingly delayed activation of IECs but not macrophages by wildtype Salmonella enterica subsp. enterica sv. (S.) Typhimurium as compared to isogenic O-antigen deficient mutants. Delayed epithelial activation is associated with impaired LPS internalization and retarded TLR4-mediated immune recognition. The O-antigen-mediated evasion from early epithelial innate immune activation significantly enhances intraepithelial bacterial survival in vitro and in vivo following oral challenge. These data identify O-antigen expression as an innate immune evasion mechanism during apical intestinal epithelial invasion and illustrate the importance of early innate immune recognition for efficient host defense against invading Salmonella.


Introduction
Lipopolysaccharide (LPS) is an obligate constituent of the outer membrane of gram-negative bacteria. It is composed of three partsa conserved lipid A, a short core carbohydrate, and the O-antigen assembled by a variable number of highly polymorphic carbohydrate subunits [1]. The lipid A consists of a hexa-acylated disaccharide. It is the ligand of the innate immune receptor Tolllike receptor (TLR) 4 and represents one of the most potent immunostimulatory molecules. TLR4-mediated LPS recognition provides an important signal for activation of the antimicrobial host defense during bacterial infection [2,3]. The O-antigen confers resistance to serum complement activation during systemic infection and represents the chemical basis of bacterial serotyping [4].
Due to its amphiphilic character, LPS forms aggregates in watery solution. The serum protein LPS-binding protein (LBP) retrieves LPS from aggregates or intact bacteria and transfers it to the GPI-anchored surface-bound or soluble form of CD14. CD14 in turn presents the LPS molecule to the MD-2/TLR4 receptor complex. Alternatively, LPS bound to soluble MD-2 can bind to the TLR4 receptor facilitating efficient recognition of even minute amounts of LPS [5,6]. The structural basis of this intense interaction has recently been resolved [7]. Ligand binding induces a conformational change of the TLR4 dimer and leads to signal transduction, transcriptional activation, and the production and secretion of proinflammatory mediators. Beside professional immune cells, also other cell types such as epithelial cells express functionally active innate immune receptors [8,9]. Lack of TLR4 signaling has been associated with enhanced susceptibility to microbial challenge, increased tissue destruction during mucosal injury and cancerogenesis within the intestinal tract [10,11,12].
Strikingly, the subcellular localization of TLR4 has been demonstrated to differ between macrophages and intestinal epithelial cells (IEC) [13]. Myeloid cells harbor TLR4 on the cell surface and ligand recognition and signling occur from the plasma membrane [14]. In contrast TLR4 is restricted to an intracellular compartment in IECs [13,15]. LPS is rapidly internalized, reaches the TLR4-positive compartment and initiates signal transduction [16]. Although LPS internalization has been noted since many years [13,14,17,18] and the intracellular TLR4 localization has been confirmed in pulmonary, renal and corneal epithelial cells as well as endothelial cells [15,17,[19][20][21][22], the functional consequence of the different cellular localization of the TLR4 molecule and the functional role of ligand internalization is unknown.
Here we report a strikingly delayed recognition of wildtype Salmonella as compared to O-antigen deficient Salmonella by IECs but not macrophages. Delayed recognition of wildtype Salmonella is caused by lack of early TLR4-mediated cell activation associated with impaired LPS internalization. Importantly, lack of early epithelial activation significantly promotes intraepithelial bacterial survival and O-antigen expression is linked to enhanced numbers of intraepithelial Salmonella after oral infection in vivo. The data show that O-antigen expression contributes to bacterial virulence during apical epithelial invasion prior to contact with serum complement and illustrate the susceptibility of Salmonella to antibacterial defense activation before it reaches and establishes its protected intracellular niche.

Early activation of IECs after exposure to O-antigen deficient Salmonella
In order to evaluate a possible biological effect of LPS glycosylation on epithelial cell stimulation, differentiated and polarized intestinal epithelial m-IC cl2 cells were coincubated with wildtype Salmonella, isogenic O-antigen deficient mutants, or their respective complemented strains. The waaG (rfaG) gene encodes a UDP-glucose:(heptosyl)LPS a1,3-glucosyltransferase and mutants exhibit a rough Rd 1 LPS phenotype with only the inner core sugars attached to the lipid A molecule [23,24]. waaL (rfaL) encodes the O-antigen ligase, the last step in the LPS biosynthesis. WaaL functions within the periplasmic space at the cytoplasmic membrane to ligate the presynthesized O-antigen chain onto the lipid A core molecule [1,24]. waaL mutants therefore express the complete core sugars but completely lack the O-antigen (Ra LPS). O-antigen expression was confirmed using silver staining of LPS extracts (Fig. S1A).
Cellular activation was evaluated using (i) visualization of nuclear translocation of the NF-kB subunit p65/RelA, (ii) a stably transfected transcriptional NF-kB luciferase reporter construct, and (iii) quantification of the secreted proinflammatory chemokine MIP-2. Strikingly, a significant difference in the kinetics of cellular activation was recognized after challenge with wildtype and LPS mutant strains. Whereas no difference in the overall magnitude of epithelial cell activation was noted, waaL mutants induced a significantly earlier p65/RelA translocation (Fig. 1A, the earliest detectable p65/RelA translocation is marked with arrows) and an accelerated course of chemokine secretion and NF-kB reporter gene transcription in epithelial cells as compared to wildtype Salmonella ( Fig. 1B and C). This difference in p65/RelA translocation (Fig. 1D), chemokine secretion (Fig. 1E), and NF-kB reporter gene activation (Fig. 1F) was similarly observed using waaGand waaL-deficient mutants and reversed by the complemented strains carrying an expression plasmid encoding the waaG and waaL gene, respectively. Thus lack of O-antigen expression leads to a significantly accelerated recognition of Salmonella by IECs.
TLR4 significantly contributes to innate immune recognition of Salmonella by intestinal epithelial cells A similar delay in epithelial activation by wildtype Salmonella was also noted using heat-killed or UV-treated Salmonella suggesting structural impairment of LPS recognition by the O-antigen rather than O-antigen-mediated active inhibition of epithelial cell activation ( Fig. S1B and data not shown). To examine the contribution of TLR4-mediated epithelial cell activation and exclude indirect effects of the O-antigen on cellular activation, the role of TLR4 in Salmonella recognition during coculture with IECs over 6 hours was examined. First the stimulatory activity released in the cell culture medium at bacterial numbers corresponding to a multiplicity of infection (MOI) of 10:1 (10 6 CFU/mL) and 1:1 (10 5 CFU/mL) during one hour was completely inhibited by addition of the LPS-inhibiting agent polymyxin B ( Fig. 2A). Also, inhibition of Tlr4 (Fig. 2B) or Myd88 (Fig. 2C) expression by small interfering (si) RNA technique inhibited epithelial activation by Salmonella to a similar degree as epithelial activation by LPS. In fact, early recognition of the O-antigen deficient waaL mutant Salmonella was almost abolished in epithelial cells treated with Tlr4 siRNA (Fig. 2D). In contrast, inhibition of TLR2, TLR5, or TLR9 expression did not reduce the epithelial response to bacterial exposure (Fig. 2E). Consistently, no early epithelial stimulation was observed after apical exposure to other innate immune receptor ligands released by gram-negative bacteria such as flagellin, di-or tri-acylated lipopeptides, or CpG oligonucleotides (data not shown). The important role of LPS for the observed effect of delayed recognition of wildtype Salmonella was finally confirmed using LPS purified from O-antigen positive (smooth-type LPS, sLPS) as well as O-antigen negative (rough-type LPS, rLPS) Salmonella. Indeed, a similar pattern as compared to exposure to whole wildtype and mutant Salmonella with delayed epithelial activation in response to smooth LPS at early time points ( Fig. 2F and G) but similar levels of epithelial activation at later time points (Fig. 2H) was observed. Thus epithelial activation early during the time course of coculture is predominantly caused by TLR4mediated cell stimulation. The observed delay in the recognition of smooth Salmonella is not related to an O-antigen-mediated suppressive effect on early epithelial activation but rather caused by an inhibitory effect of the O-antigen on LPS recognition by epithelial TLR4.

IECs but not macrophages show delayed immune activation by wildtype Salmonella
Myeloid cells like macrophages carry the TLR4 receptor complex on the cell surface and signaling is initiated at the plasma membrane [14]. This is in contrast to IEC lines and isolated primary IECs that exhibit restriction of the TLR4 molecule to an intracellular compartment [13,15]. In these cells, receptor activation requires ligand internalization and signaling is initiated at the intracellular TLR4-positive compartment [17]. Using the protein delivery reagent PULSin in combination with TLR4/MD2 blocking antibodies, the different receptor localization could be

Author Summary
The mammalian host recognizes infection by the detection of particular microbial structures. Recognition of these structures leads to activation of host defense effector mechanisms that in turn combat infection. A very potent activating microbial structure is lipopolysaccharide, a cell wall component released by many bacteria such as Salmonella, one of the most frequent causative agents of foodborne infection of the gut. We previously showed that cells lining the gut surface require uptake of bacterial lipopolysaccharide for its detection. The functional consequence of lipopolysaccharide uptake, however, was unknown. Here, we demonstrate that the uptake of lipopolysaccharide released by Salmonella is impaired by its extensive sugar modification. Impaired lipopolysaccharide uptake prevents early activation of host defense mechanisms and thereby allows Salmonella to better survive and proliferate within the host's intestinal cells. Thus, this lipopolysaccharide modification represents a mechanism by which Salmonella impairs recognition by the mammalian host to more efficiently cause infection of the intestinal mucosa.
functionally demonstrated. Whereas activation of myeloid cells was readily blocked by addition of the blocking anti-TLR4 antibody MTS510 to the cell culture medium, antibody-mediated inhibition of epithelial activation was only observed in the presence of the protein delivery reagent PULSin ( Fig. 3A and B). Interestingly, both, sLPS -as well as rLPS -stimulated RAW 264.7 cells at early time points to a similar degree and with very similar kinetics ( Fig. 3C and D). Also, early p65/RelA nuclear translocation was similarly induced in macrophages by all strains, wildtype as well as DwaaL and DwaaG mutant Salmonella as well as the respective complement- cells were exposed to wildtype S. Typhimurium and to isogenic O-antigen deficient waaL mutants, both carrying a constitutive GFP plasmid. Nuclear translocation of the NF-kB subunit p65/RelA was visualized by immunostaining. The arrows mark the earliest detectable p65/RelA translocation. Bar, 5 mm. (B) m-IC cl2 cells or (C) m-IC cl2 cells stably transfected with a NF-kB-luciferase construct were exposed to S. Typhimurium wildtype, or an isogenic O-antigen deficient waaL mutant, and the secretion of MIP-2, or luciferase synthesis, respectively, was quantified after the indicated time. (D) m-IC cl2 cells were exposed to wildtype S. Typhimurium, two isogenic O-antigen deficient mutants (waaL and waaG), as well as their respective complemented controls for 1 h and the nuclear translocation of the NF-kB subunit p65/RelA was visualized by immunostaining. Bar, 5 mm. (E) m-IC cl2 cells, or (F) m-IC cl2 cells stably transfected with a NF-kB-luciferase construct were exposed to wildtype S. Typhimurium, the isogenic O-antigen deficient waaL and waaG mutants, and their respective complemented control strains, and secretion of MIP-2, or luciferase synthesis, respectively, was quantified after the indicated time points. A multiplicity of infection (MOI) of 10 was chosen for all experiments. All data presented are representative for at least three independent experiments. The asterisks indicate a significant difference between the respective rough LPS mutant (DwaaL or DwaaG) as compared to all wildtype and complemented Salmonella strains; **, p,0.01. doi:10.1371/journal.ppat.1000567.g001 ed strains ( Fig. 3E and F). Thus the delayed recognition of wildtype sLPS as compared to rLPS is restricted to IECs that are devoid of plasma membrane expression of TLR4 and rely on ligand internalization. Yet we cannot exclude that macrophage activation additionally occurs by LPS release during phagocytosis.

The kinetics of Salmonella recognition is not influenced by bacterial invasion
Genes encoded by the so called Salmonella pathogenicity island 1 (SPI-1) confer the ability to invade epithelial cells. Bacterial invasion is induced by direct translocation of effector proteins into the host cell cytoplasm which causes actin polymerization and membrane protrusions. Within one hour, this mechanism leads to bacterial internalization and localization within an endosomal compartment named Salmonella containing vacuole (SCV). Initially, the kinetics of Salmonella invasion was examined using constitutive GFP-positive wildtype Salmonella followed by immunostaining with anti-O-antigen Salmonella O4/O5 antibodies without prior cell membrane permeabilization. This technique allows the differentiation of extracellular (simultaneously green and red = orange) and intracellular (green) Salmonella. Exposure of confluent polarized m-IC cl2 cells revealed bacterial invasion starting approximately 20 minutes after challenge with significant numbers of intracellular bacteria at 2 hours after infection (Fig. 4A). Fig. 4B provides a more detailed illustration of the actin-dependent mode of Salmonella invasion at 30 minutes after infection (left panel) and the intracellular localization after 2 hours (right panel). Importantly, the O-antigen-mediated delay in LPS recognition was also observed in invasion-mutants: An isogenic pair of smooth and rough invC-mutants exhibited a similar difference in the kinetics of epithelial stimulation as compared to invasion-competent Salmonella ( Fig. 4C and D). Also, hilAand pho-24 (PhoP c ) deficient smooth Salmonella, both significantly impaired in epithelial invasion, exhibited a similar pattern of reduced activation at early time points but cellular stimulation at later time points after infection ( Fig. S2A and B). Thus the observed delay in epithelial activation by wildtype bacteria is not dependent on their ability to exhibit an epithelial cell-invasive phenotype but rather result from extracellular ligand exposure.

Delayed recognition of smooth LPS is associated with retarded ligand internalization
Viable bacteria continuously release LPS in the surrounding medium. In accordance, significant amounts of 10 2 EU/mL (approximately 10 ng/mL) LPS were found to be released from viable wildtype Salmonella into the cell culture medium within 30 minutes. The concentration increased up to 10 3 EU/mL (approximately 100 ng/mL) during the observed time period of 2 hours. No significant difference in the degree of endotoxin release between wildtype and waaLand waaG-mutants or their respective complemented Salmonella strains was noted (Fig. 5A). As expected, inhibition of CD14 and LBP expression by siRNA significantly impaired LPS and Salmonella-mediated activation of m-IC cl2 cells in accordance with the literature (Fig 5B) [25]. Strikingly, LPS internalization studies using biotinylated rLPS or sLPS preparations revealed a marked difference in the kinetics of ligand uptake. Whereas detectable amounts of rLPS were observed after 30 minutes, wildtype LPS remained undetectable until many hours after exposure (Fig 5C). Previous characterization of intestinal epithelial stimulation with rLPS identified a clathrin-and lipid raftdependent pathway of LPS internalization and receptor activation [15]. Inhibition of lipid raft formation with filipin previously linked to recognition of rLPS abolished early recognition of rLPS but left the more delayed cellular activation induced by wildtype sLPS unaffected (Fig. 5D). Also, a significant inhibitory effect of clathrin siRNA on early recognition of rLPS was noted (data not shown) and dynamin inhibition by dynasore significantly reduced activation by rough, DwaaL Salmonella consistent with this rapid internalization pathway for rLPS uptake (Fig. 5E). These data suggest that qualitative differences in the uptake and intracellular transport mechanism between sLPS and rLPS exist and account for the observed delay in the epithelial recognition of wildtype, O-antigenpositive Salmonella. Similar results obtained using viable invasive and non-invasive Salmonella, heat-killed Salmonella, or purified LPS suggest involvement of plasma membrane-to-Golgi traffic. Yet we cannot exclude that transport pathways from the SCV to the Golgi apparatus are also affected.

Early innate immune activation restricts the number of intracellular bacteria in vitro and in vivo
To examine the functional consequences of early epithelial activation, a standard Gentamicin protection-assay was performed. Strikingly, both O-antigen negative mutants but not their respective complemented strains exhibited a significantly reduced number of viably intracellular bacteria two hours after infection (Fig. 6A). This was confirmed by immunofluorescence (Fig. 6B) as well as by flow cytometry (Fig. 6C)   wildtype as compared to waaL-deficient Salmonella (Fig. 6C). Notably, this difference in the number of viable Salmonella was not due to impaired invasion of waaL-deficient Salmonella since similar numbers of intracellular bacteria were obtained 30 minutes after infection (1.460.1% versus 1.960.1%) (Fig. 6D). In fact flow cytometric quantification of intracellular bacteria after epithelial cell lysis revealed an approximately 2-fold enhanced invasion rate of the Oantigen deficient waaL mutant Salmonella as compared to wildtype bacteria ( Fig. S1C and D). The increase of the number and fluorescence intensity of wildtype Salmonella-infected epithelial cells together with the marked clusters of intracellular wildtype Salmonella 2 hours after infection suggest significant intraepithelial proliferation early after invasion. In contrast, no signs of bacterial growth were noted for the waaL-deficient Salmonella strain. In addition, reduced bacterial numbers in epithelial cells did not appear to result from general growth or viability defects of O-antigen deficient Salmonella. Wildtype, DwaaL, and DwaaG Salmonella as well as the complemented mutants exhibited comparable growth rates in LB medium, or m- IC cl2 cell lysate (Fig. S1E). Also, both wildtype and DwaaL or DwaaG Salmonella were able to induce persistent intracellular infection in m-IC cl2 cells (Fig. S1F). In accordance with the different phenotype observed in epithelial cells and macrophages ( Fig. 1 versus Fig. 3), the intracellular survival illustrated by enhanced fluorescence of infected cells with wildtype Salmonella was only found in epithelial cells. In contrast, the fluorescence of Salmonella-infected RAW 264.7 macrophages was not significantly altered during the first 2 hours of infection, irrespective of the Salmonella strain used (Fig. S2C). Of note, an enhanced internalization of the waaL-deficient Salmonella mutant was observed after macrophage infection in accordance with Ilg et al. [26] (Fig. S2C).
To confirm that the observed antibacterial effect was directly linked to early innate immune recognition and cell activation, epithelial cells were infected with wildtype bacteria in the presence or absence of rLPS. Indeed, the number of intracellular bacteria as measured by invasion assay (Fig. 6E), or immunoflu-orescence (Fig. 6F) was significantly reduced in rLPS-stimulated epithelial cells illustrating the critical importance of early cell activation to restrict intracellular bacterial growth. Wildtype Salmonella in sLPS-stimulated epithelial cells were significantly less affected (Fig. S2D). The dramatic nature of this antibacterial effect was illustrated by flow cytometric quantification of intracellular bacteria in cell lysate between 30 and 60 minutes after infection. Whereas invasion of naïve epithelial cells allowed immediate intracellular bacterial growth, rLPS-stimulated epithelial cells were able to restrict the number of Salmonella (Fig. 6G  and H). Thus early activation of intestinal epithelial cells by Oantigen-deficient Salmonella is associated with significantly reduced intraepithelial survival.
Salmonella has been shown to invade IECs in vivo after oral challenge [27]. Intestinal epithelial invasion from the luminal side occurs without prior contact with tissue macrophages or complement. To examine a possible effect of O-antigen expression on intraepithelial survival in vivo, mice were orally challenged and highly pure IECs were isolated and examined for the presence of viable Salmonella. Similar numbers of intracellular wildtype and waaL-deficient Salmonella were noted at early time points following infection (Fig. 7A). Interestingly, a significant reduction of Oantigen-deficient (waaL) Salmonella as compared to wildtype as well as the respective complemented Salmonella was detected in highly pure IECs later during the course of infection (Fig. 7B). The presence of intraepithelial wildtype Salmonella after oral challenge was also confirmed by immunohistology (Fig. 7C). Thus, lack of Oantigen expression does not influence intestinal epithelial invasion but intraepithelial survival of Salmonella in vitro and in vivo. These results identify O-antigen expression as innate immune evasion strategy to enhance intraepithelial survival. O-antigen expression might thereby promote intraepithelial proliferation and mucosal spread.

Discussion
S. Typhimurium is one of the leading causative agents of enteritis in humans. Infection is acquired by oral ingestion of contaminated food. In the intestine, Salmonella firmly attaches to the epithelial surface and induces membrane protrusions that surround the bacterium and form an endosomal vesicle called Salmonella-containing vacuole (SCV). This process has been extensively studied in vitro but also confirmed in vivo [27,28]. Intestinal epithelial invasion from the enteric lumen occurs prior to contact with serum complement or professional phagocytes such as macrophages. It plays an important role in the induction of enteritis and mucosal damage in vivo and thus represents an essential step in Salmonella pathogenesis [29,30].
Similar to professional immune cells, also intestinal epithelial cells express receptors of the innate immune system, and thus might contribute to recognition of microbial infection and antibacterial host defense during the initial phase of infection. Indeed, the LPS structure was shown to significantly influence epithelial invasion [26,31]. Also, innate immune recognition via TLR4 was reported to play a significant role in the host defense against Salmonella infection in vivo [10,[32][33][34][35]. Strikingly, the subcellular localization of TLR4 in myeloid versus epithelial cells is markedly different. Whereas the receptor molecule is situated on the cell surface of macrophages and ligand recognition and cell signaling occurs at the cell membrane, TLR4 in IECs is restricted to the intracellular compartment and ligand recognition requires uptake and intact cell traffic [13,15,16]. We could previously show that internalization of rLPS results in significant intracellular accumulation within minutes after exposure [16]. In the present study we show that qualitative differences in the uptake and intracellular transport mechanism between rLPS and sLPS might significantly contribute to immune evasion of wildtype Salmonella during the early phase of mucosal infection. Thus our results for the first time report on a biological consequence of intracellular TLR4 localization in IECs. Since apical invasion of enterocytes by Salmonella occurs prior to contact with serum complement or professional phagocytes such as tissue macrophages, the epithelial specific delay in wildtype Salmonella LPS recognition significantly contributes to bacterial virulence at the mucosal surface in addition to what has been described as serum resistance during systemic spread of the bacteria (Fig. S2E).
LPS is composed of the hydrophobic lipid A, the core polysaccharides, and the highly polymorphic and hydrophilic immunodominant O-antigen [1]. The LPS receptor TLR4 specifically interacts with and recognizes the lipid A part of the LPS molecule. Therefore, even small variations observed in the lipid A structure and their influence on TLR4-mediated recognition have extensively been studied [36]. The O-antigen is not required for the immunostimulatory activity of LPS and variations of the O-antigen and their impact on TLR4-mediated recognition have only recently attracted attention [37,38]. The Oantigen is composed of up to 100 repetitive structurally variable carbohydrate subunits and the distinction of different O-antigen subunits has been used in the serotyping of various gram-negative bacteria. It is synthesized separately from the rest of the LPS molecule on a lipid carrier by enzymes encoded by the rfb/waa locus. The O-antigen chain is subsequently transferred to the periplasmic space where ligation to the lipid A-core polysaccharide precursor takes place. Only then, the completed LPS molecule is transferred to the bacterial cell surface [39]. The O-antigen is the major determinant of complement resistance and thus represents an important virulence factor [40]. Indeed, gram-negative enteropathogenic bacteria isolated from fecal samples of diseased patients such as Yersinia enterocolitica, Salmonella enterica, Shigella dysenteriae, as well as enterohemorrhagic (EHEC) or enteropathogenic (EPEC) Escherichia coli exhibit long O-antigen chains on their respective LPS molecule. Modifications within the lipid A portion of the molecule have been described to alter the stimulatory potential of LPS [41]. The presence or absence of the O-antigen, however, has not been linked to alterations in the TLR4-mediated signaling cascade leading to MAP kinase and NF-kB activation [38].
Using X-ray diffraction of dried LPS, Kastowsky and collegues estimated the size of the lipid A molecule to measure approximately 2.4 nm in length [42]. Addition of the inner core carbohydrates (corresponding to the LPS produced by the waaG mutant Salmonella) would result in a length of approximately 3.5 nm, addition of the outer core carbohydrates (corresponding to the LPS produced by the waaL mutant Salmonella) in a length of approximately 4.4 nm. Their analysis further suggested an additional length of 1.1 to 1.6 nm per repeating carbohydrate unit of the O-antigen. A full length O-antigen with up to 100 repeating units may therefore extend the molecular length to more than 100 nm. The tertiary structure and orientation of long chain O-antigen in respect to the outer cell membrane is not fully understood [42]. Although the O-antigen might be heavily coiled and allow (and actually favor) some degree of lateral bending [42,43], addition of this long chain hydrophilic residue might dramatically enhance the spacial extension of the LPS molecule [42][43][44]. In accordance, electron microscopic images from the membrane of gram-negative bacteria suggest that the O-antigen extends from the outer cell membrane for 40-100 nm [43,44]. The length of the extending O-antigen structures are also illustrated by reports on O-antigen mediated impairment of efficient type III secretion in enteropathogenic Shigella [45]. Taking into account that the inner diameter of clathrin coated vesicles is strictly defined; one explanation for the delayed internalization of smooth LPS by epithelial cells might therefore be its physical size.
Both in vitro as well as in vivo experiments revealed comparable intestinal epithelial invasion by wildtype and O-antigen-deficient bacteria at early time points after challenge. Once inside the epithelial cell, Salmonella is able to interfere with cellular processes of endosomal maturation altering the molecular composition of its surrounding membranous compartment for its own benefit [46]. Well-established virulence determinants such as the PhoPQ regulon and the SPI-2 type III secretion system contribute to this immune evasive behavior [47]. Avoidance of epithelial activation might significantly contribute to bacterial survival since innate immune signaling has been suggested to promote maturation of endosomal compartments and to influence intracellular bacterial proliferation [48,49]. Indeed the intracellular viability of Oantigen-deficient Salmonella in intestinal epithelial cells in vitro and in vivo was significantly reduced. Previous animal studies have indicated a significant effect of Salmonella O-antigen expression after oral but not intraperitoneal or intravenous infection [50,51]. Our results provide an explanation for these findings and demonstrate that the O-antigen-modification of LPS significantly contributes to mucosal immune evasion and thus bacterial virulence in the intestine. Our data further point towards a role of intestinal epithelial infection for enteric bacterial multiplication and fecal excretion and thereby transmission of enteropathogenic bacteria like Salmonella.
In conclusion, we for the first time provide a functional consequence of internalization-dependent ligand recognition by TLR4 as compared to surface recognition in myeloid cells. We demonstrate that O-antigen modification of Salmonella LPS hinders rapid epithelial internalization and delays TLR4-mediated recognition. Evasion from early innate immune activation of IECs markedly enhances intracellular proliferation of wildtype Salmonella. This novel immune evasion mechanism might thus significantly contribute to mucosal virulence of enteropathogenic bacteria.

Ethics statement
Animals were handled in strict accordance with good animal practice as defined by the relevant local animal welfare bodies, and all animal work was approved by the appropriate committee (Landesamt für Lebensmittelsicherheit und Verbraucherschutz, Oldenburg, 07/1334).

Bacterial strains and cell culture
Salmonella enterica subsp. enterica sv. Typhimurium (S. Typhimurium) ATCC 14028 was used as wildtype strain. Isogenic mutant strains (DwaaL and DwaaG) were generated by Red recombinase mediated deletion and chromosomal insertion of a Kanamicin antibiotic resistance cassette as described elsewhere (Zenk et al., submitted). The construction of plasmids for the complementation of mutant strains is described in (Zenk et al. submitted). The LPS profiles of the various strains were analyzed using SDS-PAGE and silver staining (Fig. S1A). Bacteria were incubated at 70uC for 10 min to produce heat-killed Salmonella. The non-invasive isogenic pho-24 (PhoP constitutive) and DhilA mutants were a generous gift from Mikael Rhen (Karolinska Institute, Stockholm) and the isogenic DinvC and DinvC DwaaL double mutants were generated as described above. HilA is a central regulator of SPI-I mediated epithelial cell invasion, the PhoP/PhoQ two component system is a central regulator in Salmonella virulence, and InvC is required for type III secretion of SPI-1-encoded virulence determinants. The phenotype of the non-invasive pho-24 mutant is designated PhoP constitutive (PhoP c ). All three mutants exhibit strongly impaired epithelial invasion. Fluorescent bacteria were generated by transformation with a constitutively GFP expressing plasmid. For all experiments, bacteria were routinely grown in Luria-Bertani (LB) broth, supplemented with antibiotics if required. Murine small intestinal epithelial m-IC cl2 cells and m-IC cl2 cells stably expressing a NF-kB luciferase reporter construct were cultured as described previously [52]. RAW 264.7 macrophages were purchased from ATCC and cultured in RPMI 1640 medium (Invitrogen) supplemented with 20 mM Hepes, 2 mM Lglutamine, and 10% FCS.

Bacterial coculture and stimulation assays
For all coculture experiments, wildtype or mutant Salmonella were grown overnight at 37uC, diluted 1:10 and subcultivated with mild agitation at 37uC, until mid-logarithmic growth was reached (OD 600 : 0.5). Bacteria were adjusted by dilution, added to polarized and differentiated intestinal epithelial m-IC cl2 cells at a multiplicity of infection (MOI) of 10:1 and centrifuged at 3006g for 5 min. Following incubation for one hour, the medium was replaced with fresh medium supplemented with 50 mg/mL Gentamicin. Cell culture supernatants, as well as cell lysates, were collected after the indicated periods of time and stored at 220uC. The chemokine MIP-2 was analyzed using a commercial ELISA from Nordic Biosite (Tä by, Sweden). Luciferase in cell lysates was quantified using a luciferase reporter kit (Promega, Madison, WI). Pharmacological inhibitors were added to the cell medium 30 min prior to stimulation. NO production was determined by measurement of nitrite in cell culture supernatant using Griess reagent [53]. Stimulation with mouse recombinant TNF (R&D Systems GmbH, Wiesbaden, Germany) was performed at 100 ng/ mL. To quantify bacterial invasion coculture for one hour was followed by one hour incubation in fresh cell culture medium, supplemented with 50 mg/mL Gentamicin. After washing, cells were lysed in H 2 O/Tween 0.1% and the number of intracellular bacteria was determined by serial dilution and plating. Specific or control siRNA was transfected with INTERFERin at a final concentration of 1 or 10 nM 48 hours prior to functional analysis. TLR4 blocking experiments using the rat monoclonal anti-TLR4/ MD2 antibody MTS510 were conducted in the presence or absence of the protein delivery reagent PULSin according to the manufacturer's recommendations. R-Phycoerythrin (R-PE) is a fluorescent protein to visualize efficient protein transfection by PULSin (data not shown).
Immunostaining and flow cytometric analysis m-IC cl2 or RAW 264.7 cells were grown on 8-well chamber slides (Nunc, Rochester, NY), and infected with constitutively GFP-expressing wildtype S. Typhimurium or isogenic mutants as indicated at a MOI of 10:1, or exposed to biotinylated LPS (100 ng/mL), and incubated for the specifically mentioned period of time. Visualization of the cellular distribution of p65/RelA was performed as previously described [15]. Discrimination of extraand intracellular bacteria was achieved using Salmonella strains carrying a GFP expression construct (green) in combination with a mixture of two mouse monoclonal anti-Salmonella O-antigen (anti-O4 and anti-O5) antibodies visualized with a Texas-Redconjugated anti-mouse secondary antibody (red) in the absence of cell permeabilization. Due to impaired penetration of the anti-Salmonella antibodies into the cells, intracellular bacteria appear green, whereas extracellular bacteria exhibit an orange (green plus red) color. Biotinylated LPS was detected using Texas-Red conjugated Streptavidin (Jackson ImmunoResearch). For permeabilization of eukaryotic cell membrane, saponin was adjusted to a final concentration of 0.5%. After the indicated time periods, cells were fixed in 5% PFA and counterstained with MFP488-or MFP647-phalloidin (MoBiTec, Göttingen, Germany). Cells were subsequently mounted in DAPI containing Vectashield (Vector Laboratories) and visualized using an ApoTome-equipped Axioplan 2 microscope connected to an AxioCam M r digital Camera (Carl Zeiss MicroImaging, Inc. Jena, Germany). Flow cytometric detection of intracellular GFP-expressing bacteria in intact epithelial cells was carried out in Trypsin-EDTA 0.05% treated, fixed m-IC cl2 cells using a FACS CaliburH apparatus (BD Pharmingen). In addition, flow cytometry was used to quantify the number of GFP-expressing bacteria in cell lysates. To standardize the volume examined, a defined quantity of Cy5labelled particles was added to all samples and the data acquisition on GFP-positive bacteria (recorded in channel Fl-1) was limited until a simultaneously recorded number of 10.000 events in the far red channel (Cy5, Fl-4) was reached.

Bacterial challenge
Mice were purchased from Charles River Breeding Laboratories (Sulzfeld, Germany) and housed under specific pathogen-free conditions. 6-8-week-old female Balb/c mice were orally infected with 1610 8 CFU S. Typhimurium in 20 ml phosphate-buffered saline (PBS). 4 and 24 h post infection, mice were sacrificed and the small intestine was removed. Highly pure IECs (.98% Ecadherin + /CD45 2 ) were isolated using a recently described protocol [15], incubated in the presence of Gentamicin (50 mg/ mL) and washed. The number of viable intraepithelial bacteria was determined by serial plating.

Statistical analysis
All experiments were performed at least three times and results are given as the mean6SD of one representative experiment. Statistical analyses were performed using the Student's t test. A p value,0.05 was considered significant.