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Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK
Correspondence
Mark A. Jepson
m.a.jepson{at}bristol.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The ability of Salmonella to cross the intestinal epithelium is the critical first stage in infection and so there is a need to understand the mechanisms by which the bacteria achieve this. In common with a number of other pathogenic bacteria, Salmonella can manipulate the cytoskeleton of cultured cells through the injection of an array of bacterial effector molecules into the host cytoplasm. Numerous studies have addressed the mechanisms by which Salmonella drives its entry into cultured cells by initiating membrane ruffling, which facilitates bacterial internalization by a process thought to closely resemble macropinocytosis (Cossart & Sansonetti, 2004).
Many Salmonella virulence genes are clustered in Salmonella pathogenicity islands (SPIs). SPI-1 and SPI-2 encode type III secretion systems (TTSSs) that mediate the injection of effector proteins into the host cell cytoplasm (Galan & Wolf-Watz, 2006). Some of the effectors secreted by TTSS-1 regulate actin dynamics. For example, SipA promotes actin bundling and SopE and SopE2 activate members of the Rho GTPase family, leading to the formation of membrane ruffles (Galan, 2001). SPI-1 is primarily responsible for invasion and SPI-2 is required for intracellular survival and the systemic phase of infection, although the exclusivity of these functions has been questioned recently. For example, some SPI-1 effectors persist post-invasion and appear to contribute to modulation of the Salmonella-containing vacuole and to bacterial replication at later stages of infection (Brawn et al., 2007; Giacomodonato et al., 2007), while SPI-2 has also been found to be expressed in the intestinal lumen prior to invasion (Brown et al., 2005).
Most studies that have addressed cellular roles for SPI-1 and SPI-2 have concentrated on non-polarized cell lines, whereas the principal site of infection is the polarized epithelium lining the intestinal tract, which has features not readily modelled in conventional cell culture experiments (Buda et al., 2005). Studies of intestinal infection in intact animals have found that Salmonella invasion of villus enterocytes is accompanied by membrane ruffling that closely resembles SPI-1-dependent cellular responses seen during infection of cultured cells (Takeuchi, 1967; Frost et al., 1997). Furthermore, recent studies revealed that translocation and dissemination of Salmonella enterica serovar Dublin in calves is dependent on SPI-1 but not SPI-2 (Pullinger et al., 2007). Several studies have identified Peyer's patches of the distal ileum as preferential sites of Salmonella infection in at least some animals (Carter & Collins, 1974; Frost et al., 1997; Jepson & Clark, 2001). Here, intercalated between the enterocytes overlying lymphoid tissue, is a specialized cell type, the microfold/membranous M cell, which has an antigen-sampling function (Brayden et al., 2005), a specialization that appears to have been exploited by Salmonella and other pathogens to invade the epithelia and disseminate through the body (Clark et al., 1994; Jepson & Clark, 1998; Jones et al., 1994).
The interaction of Salmonella with M cells has mostly been investigated in vivo following oral or intragastric inoculation or infection of ligated gut loops. Some authors have reported, or assumed, that SPI-1 is necessary for M cell invasion (Jones et al., 1994; Penheiter et al., 1997; Vazquez-Torres et al., 1999). Others have suggested that SPI-1 enhances, but is by no means essential for, invasion of murine M cells by Salmonella (Clark et al., 1996, 1998; Jepson & Clark, 2001), a finding that correlates with the attenuated but significant virulence of SPI-1 mutants following oral infection (Galan & Curtiss, 1989). In vivo infection models have limited potential for elucidating molecular mechanisms underlying invasion due to inherent problems in their experimental manipulation. Recently, an in vitro M cell model has been described in which human intestinal epithelial cells (Caco-2 cells) acquire M cell-like characteristics after being co-cultured with Raji B cells (Gullberg et al., 2000; Kerneis et al., 1997). We have previously reported that Salmonella uptake and translocation across this co-culture model exceeds that observed with native Caco-2 cells (Martinez-Argudo et al., 2007). It has yet to be determined whether M cell translocation involves SPI-mediated mechanisms triggered during invasion of cultured epithelial cells.
In this study, we investigated the mechanisms involved in M cell infection by Salmonella using the in vitro co-culture model of M cells. Translocation of viable Salmonella across Caco-2 cells was dramatically increased by co-culture with Raji B cells, confirming the generation, and preferential infection, of M-like cells. Bacterial translocation was greatly reduced by heat treatment or formaldehyde fixation, was partially inhibited by the selective dynamin inhibitor dynasore, but was unaffected by the macropinocytosis inhibitor EIPA. Translocation of wild-type and mutant Salmonella strains across the M cell model indicated that neither SPI-1 nor SPI-2 effectors are essential for invasion of M cells by Salmonella.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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). The wild-type strain NCTC 12023 (equivalent to ATCC 14028s), and the ssaV mutant identified in the original signature-tagged mutagenesis screen as P2D6 were obtained from the laboratory of David Holden (Hensel et al., 1997). All strains were grown in Luria–Bertani (LB) broth and maintained on LB agar plates supplemented with appropriate antibiotics at 37 °C.
Cell culture model.
Raji B cells were grown in RPMI 1640 medium (Gibco-BRL) supplemented with 10 % fetal calf serum, 1 % Glutamax (Gibco), 0.1 mg streptomycin ml–1 and 100 units penicillin ml–1 (Sigma) at 37 °C in 5 % CO2. Caco-2 cells (3–10 passages since obtaining from ATCC) were grown in DMEM (Sigma) supplemented with 10 % fetal calf serum, 1 % non-essential amino acids, 1 % Glutamax and 12 µg gentamicin ml–1 (Sigma) at 37 °C in 5 % CO2. After trypsinization, 0.5x106 Caco-2 cells were seeded onto 1.2 cm diameter Transwell (Corning) polycarbonate inserts (3 µm pore) and incubated at 37 °C in 5 % CO2 for 14 days in order to allow full differentiation before addition of 0.5x106 Raji B cells in the basal chamber. The co-cultures were maintained for a further 6 days. Monocultures of Caco-2 cells, cultivated as above (except that Raji B cells were replaced with Raji cell medium), were used as controls.
Measurement of microsphere translocation.
Microsphere translocation was measured as described previously (Martinez-Argudo et al., 2007). Briefly, Transwells were washed with serum-free medium prior to the addition of 500 µl of 0.46 µm diameter fluorescent polystyrene (YG) microspheres (7.7x108 ml–1; Polysciences) to the apical chamber, and then incubated at 37 °C. Basolateral samples were taken after 30 min and the number of transported particles measured using a Spectramax M2 fluorescent plate reader (Molecular Devices).
Measurement of bacterial translocation.
Salmonella strains were grown overnight in LB then diluted 1 : 100 in fresh LB and incubated for 3.5 h with shaking at 37 °C. Cells were infected for 1 h by diluting 25 µl of the exponential-phase bacteria in 500 µl DMEM to give an average m.o.i. of 20–30 (approx. 107 bacteria added per layer). Serial dilutions from the inoculum were plated to calculate the actual number of bacteria inoculated. The number of translocated bacteria at 30 and 60 min was determined and expressed relative to the number of bacteria inoculated.
To kill bacteria, Salmonella cultures were incubated at 70 °C for 20 min or diluted with 2 % paraformaldehyde (1 : 1), incubated at 37 °C for 45 min and washed with fresh LB prior to infection. Cells were infected as above and the number of translocated bacteria was quantified using the LIVE/DEAD BacLight bacterial viability kit from Invitrogen.
Where indicated, inhibitors EIPA [5-(N-ethyl-N-isopropyl)amiloride; 50 µM] and dynasore (80 µM) were added 30 min prior to infection and maintained during infection.
Visualization of M cells and Salmonella.
Following Salmonella infection, Caco-2 layers cultured alone or with Raji B cells on Transwells were washed thoroughly in PBS and fixed in 2 % paraformaldehyde at 4 °C for at least 45 min. After permeabilization of cells with Triton X-100 (0.3 % in PBS for 20 min), Transwells were incubated with goat anti-Salmonella antibody (Kirkegaard & Perry Laboratories; 1 : 200 in PBS) for 45 min at room temperature and, after washing with PBS, with FITC-conjugated anti-goat antibodies (1 : 100 in PBS). F-actin was simultaneously localized by inclusion of phalloidin-TRITC (tetramethylrhodamine isothiocyanate) with the secondary antibody as previously described (Jepson et al., 1995). Transwells were then washed thoroughly in PBS and mounted in Vectashield containing DAPI (4',6-diamidino-2-phenylindole; Vector Laboratories). Distribution of F-actin and Salmonella was then examined by confocal microscopy using a Leica TCS SP2 AOBS system (Leica Microsystems) attached to a Leica DM IRE2 inverted microscope, and equipped with a 543 HeNe laser for excitation of TRITC and an argon laser for FITC excitation at 488 nm. An oil-immersion objective lens (63x, NA 1.4) was used, and imaging parameters standardized to allow direct comparison between images. Stacks of images at 0.5 µm intervals were acquired and maximum projections of these created with Leica confocal software.
Statistical analysis.
All data are expressed as means±standard error (SE). Significance of differences between mean values was assessed using two-tailed unpaired Student's t test with significance set at P
0.05.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
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; Maresca et al., 2007; Martinez-Argudo et al., 2007). The presence of cells displaying morphological and functional characteristics of M cells was confirmed by confocal microscopy following phalloidin-TRITC labelling of F-actin (Fig. 1), since M cells have reduced apical F-actin content (Martinez-Argudo et al., 2007), and by measurement of polystyrene microsphere transcytosis, as M cells exhibit enhanced transport (Gullberg et al., 2000; Martinez-Argudo et al., 2007). In agreement with previous data, translocation of fluorescent microspheres (approx. 0.5 µm) measured after 30 min was found to be increased by Raji co-culture on average 29.7±7.3-fold (n=7) (0.5–2x108 particles per co-cultured cell layer of approx. 0.5x106 cells). These findings indicate the reproducibility of the co-culture model but also reveal some variability in the extent to which the cells are phenotypically distinct from native Caco-2 cells following co-culture.
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). Furthermore, translocation of killed bacteria across the M cell model (which was below detectable levels across native Caco-2 layers) was much lower than that of viable bacteria (25±11.2 % for viable bacteria, 0.6±0.5 % for heat-killed bacteria and 3±2.7 % for paraformaldehyde-fixed bacteria per co-cultured cell layer; n=3).
The low levels of translocation of non-viable Salmonella across the M cell model confirmed the enhanced transport while indicating that translocation of Salmonella occurs via a selective process. This finding confirms previous data that the increased transport observed is not a result of loss of integrity of the cell layers, a finding we also confirmed previously by scanning electron microscopy and by measurement of transepithelial electrical resistance (Martinez-Argudo et al., 2007). Further evidence of the specificity of the M cell model is also provided by data that not all bacterial species are translocated in greater numbers across Caco-2 cells following co-culture with Raji B cells. For example, translocation of wild-type enteropathogenic Escherichia coli (EPEC), Clostridium difficile and Listeria monocytogenes was not increased in the M cell model (Daniels et al., 2000; Martinez-Argudo et al., 2007).
Salmonella translocation by M cells involves dynamin and is distinct from macropinocytosis
By analogy with growth factor-induced membrane ruffling, it has been concluded that Salmonella invades non-phagocytic cells by inducing a process akin to macropinocytosis (Cossart & Sansonetti, 2004; Francis et al., 1993). As Salmonella preferentially enter M cells that also have a propensity for transport of inert particles and since translocation of polystyrene microspheres has previously been reported to involve macropinocytosis (des Rieux et al., 2007), we sought to determine if Salmonella translocation follows a similar route. We analysed Salmonella translocation across the M cell model in the presence of two inhibitors: EIPA and dynasore. EIPA is commonly used to inhibit macropinocytosis (Amstutz et al., 2008; Fallman & Gustavsson, 2005; Wadia et al., 2004), although the selectivity of this inhibition has been questioned (Fretz et al., 2006). Dynasore is a cell-permeable specific inhibitor of the GTPase activity of dynamin (Macia et al., 2006), a molecule involved in clathrin-mediated endocytosis that may also have a role in the scission of the macropinosome from the plasma membrane (Amstutz et al., 2008; Orth et al., 2002). Initial experiments confirmed that short-term treatment with these inhibitors had no detectable effect on bacterial viability or motility (data not shown). Treatment with EIPA did not affect Salmonella translocation but when cells were preincubated with dynasore we observed a significant reduction in Salmonella translocation across both the Caco-2 monolayer and the M cell model with respect to untreated cells after 30 min (Fig. 2). The degree of inhibition of translocation associated with dynasore treatment was greater across native Caco-2 cells (64 %) than across the Caco-2/Raji co-cultured layers (27.6 %). The results obtained suggest macropinocytosis does not play a significant role in Salmonella translocation across M cells and that a minor proportion of rapid translocation is dynamin-dependent.
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) and our preliminary studies have also indicated that this inhibitor reduces translocation of 60 nm polystyrene nanoparticles across the M cell model (data not shown). The lack of effect of EIPA on Salmonella translocation thus supports our conclusion that this is mechanistically distinct from transport of inert particles. Our data are consistent with the conclusion that dynamin contributes to rapid transport across Caco-2 cells regardless of co-culture. This supports recently published data that cell invasion by some bacterial pathogens is dynamin-dependent (Veiga et al., 2007). The authors concluded that Salmonella invasion was largely, though not completely, dynamin-independent in HeLa cells, suggesting that SPI-1-mediated invasion is dynamin-independent but that an alternative SPI-1-independent invasion mechanism may also operate. Our findings in Caco-2 cells thus give additional support to the hypothesis that alternative entry mechanisms operate in some epithelial cells. At later time points (60 min) the inhibition of transport associated with dynasore treatment was no longer evident although transport across the M cell model remained higher than across mono-cultured Caco-2 cells (data not shown), further supporting the operation of alternative entry and translocation mechanisms across these cells.
SPI-1 effectors are not required for M cell translocation by Salmonella
In order to examine the role of SPI-1 effectors in M cell invasion by Salmonella, we infected the M cell model with wild-type S. Typhimurium IR715 and two strains lacking the effector genes that have been shown to contribute to Salmonella invasion of epithelial cells. It was previously reported that the two mutant strains examined, ZA20 (sopABDE2) and ZA21 (sipA sopABDE2), are essentially non-invasive in non-polarized and polarized T84 cells (Raffatellu et al., 2005).
Over a 30 min infection period, strains ZA20 and ZA21 exhibited a level of translocation across the M cell model that was not significantly different from wild-type (Fig. 3a). This suggests SPI-1 effectors that are required for Salmonella invasion of most epithelial cell lines (Clark et al., 1996; Galan & Curtiss, 1989; Raffatellu et al., 2005) are dispensable for Salmonella translocation across M cells. Furthermore, an invA mutant that lacks an intact SPI-1 TTSS was also found to translocate across the M cell model in similar numbers to its parent strain (data not shown). These data are broadly in agreement with in vivo studies, which have shown that SPI-1 mutants exhibit reduced but significant virulence after oral infection, and can also enter murine M cells, though in reduced numbers under conditions that optimize invasion (Clark et al., 1996, 1998; Galan & Curtiss, 1989). Although we cannot directly compare the present data with previous studies because here we are replicating translocation across human M cells rather than measuring intracellular bacteria in murine M cells, our findings add strong support to previous conclusions that the M cell route is an important factor in the remaining virulence of SPI-1 mutants. Of course this conclusion does not exclude the possibility that other SPI-1-independent routes of translocation exist, such as the proposed carriage by CD18-expressing phagocytes (Vazquez-Torres et al., 1999). Since M cells constitute a tiny proportion of the intestinal epithelium, it is also likely that SPI-1-dependent invasion of enterocytes is a major factor in Salmonella virulence. For example, in calf ligated gut loops, translocation of S. Dublin from lumen to mesenteric lymph nodes was recently shown to be SPI-1 dependent (Pullinger et al., 2007). However, the existence of alternative translocation routes that operate regardless of SPI-1 expression appears likely to be a significant factor in the success of Salmonella as a pathogen.
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). While these data support previous findings that SPI-1 is essential for invasion, the significant translocation of the mutants indicates that a rapid, SPI-1-independent transport route also operates in these cells. Recently it was suggested that co-culture with Raji cells amplifies an existing pool of M-like cells within the normal Caco-2 population (Blanco & DiRita, 2006). Although this idea has yet to be proven, it is consistent with the observation that a small proportion of Caco-2 cells have a poorly differentiated brush border (Blanco & DiRita, 2006), a finding we have confirmed (data not shown). The observation that rapid transport of Salmonella across Caco-2 cells is partially SPI-1-independent is also consistent with the hypothesis that a small proportion of Caco-2 cells have functional properties reminiscent of M cells. The precise nature of this cell subpopulation requires further investigation.
SPI-2 does not contribute to rapid translocation of Salmonella across the M cell model
Having shown that SPI-1 is not required for M cell translocation, we next examined whether the other TTSS encoded by SPI-2 plays a significant role in the rapid translocation of Salmonella. We tested the ability of a SPI-2-deficient mutant P2D6 carrying a transposon insertion in ssaV, which encodes an essential component of the SPI-2-encoded TTSS (Hensel et al., 1997; Shea et al., 1996), to cross the M cell model. When compared with the parent wild-type strain, the ssaV mutant showed similar levels of translocation across the M cell model and mono-cultured Caco-2 cells, suggesting SPI-2 effectors are not required (Fig. 3). To establish if there was a difference in intracellular distribution of the two strains, bacteria were localized by immunofluorescent labelling and imaged by confocal microscopy. The numbers and distribution of intracellular bacteria were similar between the two strains, with bacteria preferentially associated with cells lacking an F-actin-rich brush border (Fig. 4). It thus appears that neither of the SPI-encoded TTSSs contributes to the rapid translocation route we are modelling.
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). The finding that SPI-2 does not play a role in the rapid translocation process measured here is consistent with this TTSS being primarily involved in stages of infection that follow the initial translocation step.
Concluding remarks
Our data have confirmed that Salmonella can rapidly translocate in large numbers across intact Caco-2 monolayers, following their co-culture with Raji B cells. This rapid translocation route is dependent on bacterial viability, independent of SPI-1 and SPI-2, and appears to partially involve dynamin but not macropinocytosis. Rapid translocation of Salmonella thus appears to be a separate process from the invasion of cultured epithelial cells that has been most extensively studied.
Although the in vitro M cell model is amenable to experimental manipulation to address mechanistic questions, it is not without limitations. We saw significant variability in the extent of translocation across both co-cultured and mono-cultured Caco-2 cells, which probably reflects variation in the proportion of cell layers that functionally resemble M cells. We also found that Caco-2 cells had to be used at low passage numbers (i.e. passages 3–10) or significant phenotypic drift appeared to affect reliability of the Raji-induced differentiation (data not shown). By adopting a stringent policy of limiting passage numbers, the M cell model has proved sufficiently reproducible for us to address key questions regarding the mechanisms utilized by Salmonella to cross the intestinal epithelial barrier. Our data strongly support previous evidence that SPI-1 mutants retain significant virulence when delivered orally and that this is at least in part due to their ability to enter and translocate across M cells (Clark et al., 1996, 1998; Galan & Curtiss, 1989). We have also shown for the first time that dynamin-dependent endocytic mechanisms appear to contribute to Salmonella translocation.
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ACKNOWLEDGEMENTS |
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Edited by: D. L. Gally
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Received 4 June 2008;
revised 31 July 2008;
accepted 22 August 2008.
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  T. A. Paixao, C. M. Roux, A. B. den Hartigh, S. Sankaran-Walters, S. Dandekar, R. L. Santos, and R. M. Tsolis Establishment of Systemic Brucella melitensis Infection through the Digestive Tract Requires Urease, the Type IV Secretion System, and Lipopolysaccharide O Antigen Infect. Immun., October 1, 2009; 77(10): 4197 - 4208. [Abstract] [Full Text] [PDF]
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