1 Laboratory of Enteric and Sexually Transmitted Diseases, FDA-Center for Biologics Evaluation and Research, 29 Lincoln Drive, Bldg 29/420 HFM440, Bethesda, MD 20892, USA
2 Virulence Assessment, FDA-Center for Food Safety and Nutrition, Laurel, MD 20708, USA
Correspondence
Dennis J. Kopecko
kopecko{at}cber.fda.gov
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ABSTRACT |
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INTRODUCTION |
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Advances in our understanding of the cell biology of many infectious diseases have revealed that micro-organisms typically usurp eukaryotic cell-signalling pathways to initiate disease (e.g. to invade the mucosa, for survival within cells, for transcytosis across the mucosa, or for stimulation of diarrhoea or disease pathogenesis (Falkow et al., 1992; Galan, 1996
; Hu & Kopecko, 2000
). The divalent calcium cation (Ca2+) plays a pivotal role in host signal transduction and other cellular processes, and its cytosolic concentration is of necessity tightly regulated. Free intracellular Ca2+ has been demonstrated to link cell surface receptor stimulation, via signalling pathways, with intracellular effectors, and to modulate cytoskeletal structure, chemotaxis, membrane fluidity, chromosome segregation, cell cycle transition, enzyme activity, transmembrane ion fluxes, proteolysis and other key cell functions (Jacob, 1990
; Marks & Maxfield, 1990a
, b
; Tsien & Tsien, 1990
; Meldolesi et al., 1991
; Clapham, 1995
). The host cell has two potential sources of Ca2+: entry from the external medium and release from internal stores. Eukaryotic cells maintain an internal free calcium ion concentration ([Ca2+]i) that typically is far below that of the extracellular environment, and modulate [Ca2+]i in response to specific signalling events. Thus, eukaryotic cytosolic Ca2+ levels are mainly controlled by the action of specific molecular pumps and channels in the plasma membrane and in subcellular Ca2+-storage organelles.
Certain enteric bacterial pathogens have been found to cause a specific increase in host [Ca2+]i (Ruschkowski et al., 1992; Norris et al., 1996
; Bierne et al., 2000
; Tran Van Nhieu et al., 2004
) that may play a role in ensuing pathogenesis. For example, Pace et al. (1993)
showed that Salmonella Typhimurium strain SR11 triggers a marked [Ca2+]i increase in INT407 cells via influx from the growth medium, which reached 1 µM by 30 min post-infection. Non-invasive invA and invE SR11 mutants did not stimulate a rise in [Ca2+]i. Monolayer pretreatment with cytochalasin D blocked SR11 invasion but not the associated host-cell rise in [Ca2+]i, indicating that the bacteria are responsible for triggering this Ca2+ response. More recent studies with S. Typhimurium indicate that an increased host [Ca2+]i is involved in upregulating synthesis of the proinflammatory cytokine IL-8 (Gewirtz et al., 2000
). In contrast, Listeria monocytogenes has been reported to trigger an increase in host cytosolic Ca2+ via a release from intracellular stores (Bierne et al., 2000
).
C. jejuni invasion has been studied in some detail in only a few strains, with 81-176 arguably being the best characterized. C. jejuni 81-176 was isolated from a colitis patient and causes typical dysenteric disease when ingested by volunteers (Korlath et al., 1985; Black et al., 1988
). This C. jejuni strain triggers a microtubule-dependent internalization into intestinal epithelial cells (Hu & Kopecko, 1999
), but the invasion-associated host signalling pathway(s) or Ca2+ involvement have not been reported. The current studies were initiated to determine if C. jejuni 81-176 invasion of epithelial cells is associated with an increase in cytosolic free Ca2+ and to characterize the process(es) involved. Calcium chelators and inhibitors of calcium mobilization, as well as fluorescent indicators of cytosolic Ca2+ ions, were used to assess the timing and magnitude of the Ca2+ flux as well as the source of free Ca2+ ions. In addition, involvement of the Ca2+-activated calmodulin (CaM) or protein kinase C (PKC) was assessed with specific inhibitors.
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METHODS |
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Invasion assays in the presence of calcium chelators or inhibitors.
The assay was performed essentially as described previously (Hu & Kopecko, 1999), except that INT407 cells were attached to tissue culture plates coated with sterile aqueous 0·1 mg ml1 poly L-lysine (Sigma). To conduct these studies in Ca2+-free, serum-free MEM, 105 INT407 cells were initially grown for 24 h in complete culture medium to about 80 % confluency on 24-well plate surfaces coated with poly L-lysine, and then washed three times in calcium-free minimal essential medium (S-MEM; Sigma). Control studies using Giemsa stain showed that INT407 cells bound to poly L-lysine remained attached following inhibitor treatment, invasion, and the gentamicin-kill period totalling 4·5 h.
Calcium chelators/inhibitors included EGTA, BAPTA [1,2-bis-(2-)ethane-N,N,N',N'-tetraacetic acid], BAPTA, AM (BAPTA acetoxymethyl ester) at 0·2 mM and 50 µM dantrolene. The phospholipase C (PLC) inhibitor U73122 was used at a final concentration of 5 µM. The slow Ca2+ channel agonist (±) Bay K8644 (Calbiochem) was added to INT407 cells in complete culture media at a final concentration of 1 µM for 30 min pretreatment and during the 2 h invasion period. The CaM antagonist W7 [N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide hydrochloride] (Sigma) was used at 5 µM and the PKC inhibitor calphostin C was used at a final concentration of 0·1 µM.
Chelators or inhibitors were added to INT407 cells for a 30 min pretreatment and throughout the 2 h invasion period. Mid-exponential-phase bacteria at an m.o.i. of 20 were added and incubated with host cells for 2 h at 37 °C under 5 % CO2/95 % air to allow invasion to occur. After this invasion period, the monolayer was washed three times with PBS and incubated for another 2 h in fresh S-MEM containing 100 µg gentamicin ml1 to kill extracellular bacteria. After the gentamicin-kill period, the infected monolayers were washed as above and lysed with 0·1 % Triton X-100 in PBS for 15 min. Following serial dilution in PBS, released intracellular bacteria were enumerated by plate count on M-H agar. S. Typhi were used here at an m.o.i. of 40 as a microfilament-dependent invasion control. Trypan blue assays were employed to verify that no increased death of cultured cells occurred in the presence of inhibitors at the chosen concentrations during these studies. Also, control studies confirmed that these inhibitors had no effect on bacterial viability over the time-course of these studies. All invasion inhibition assays were conducted in two separate wells during each assay and were repeated on three separate occasions. Data are shown as the mean±SEM for all experiments. P values were calculated using Student's t test. Control studies with 2x107 C. jejuni 81-176 cells added to complete culture medium containing 100 µg gentamicin ml1 verified that all extracellular bacteria were killed within 2 h.
Measurement of intracellular free calcium by fluorescence spectrofluorimetry.
Host intracellular free calcium was measured using the fluorescent Ca2+ indicator dye Fura-2. This widely used, single-wavelength calcium-ion indicator provides a sensitive measurement capability in the range of a few nanomolar to 5 µM [Ca2+]i (Tsien, 1989). The addition of the acetoxymethyl ester (AM) moeity allowed Fura-2 to be incorporated intracellularly. The assay methodology employed here has been described elsewhere (Shevach, 2000
). In brief, INT407 cell monolayers were cultured to semiconfluence on 10x25 mm glass coverslips in 60 mm tissue-culture plates at 37 °C for 24 h. Monolayers were infected by the addition of bacteria at an m.o.i. of 100 for different time periods in MEM containing 5 % FBS and 2·0 mM glutamine. After the infection period, the monolayer was washed three times with MEM. The infected monolayer was then incubated at 30 °C for 30 min in phenol-red-free MEM containing 4 mM probenecid and 5 µM Fura-2, AM [Molecular Probes, Inc.; each 50 µg vial of Fura-2, AM was dissolved in 25 µl DMSO plus 10 % pluronic F-127, and 63 µl Hank's Balanced Salts Solution (HBSS) immediately prior to use]. Pluronic F-127 is used to help prevent compartmentalization and incomplete hydrolysis of Fura-2, AM. Probenecid improves cellular loading by minimizing leakage of organic ionic dyes and cell-to-cell variation in dye content (Vandenberghe & Ceuppens, 1990
). The treated, infected monolayer was washed three times with phenol red-free media, and incubated in phenol-red-free media containing 1 mM CaCl2 (i.e. loading buffer) for 30 min at room temperature to allow the AM derivative of Fura-2 to be cleaved by endogenous esterase to yield the free dye (Kao et al., 1989
). Anti-FITC monoclonal antibody (Sigma) was added at 0·2 µl ml1 to the monolayer 20 min before spectrofluorimetry to block fluorescence from any Fura-2 that might have leaked extracellularly. The treated coverslips were mounted into special cuvettes (kindly provided by Dr J. Lowy of the Armed Forces Radiobiology Institute, Bethesda, MD) in fresh loading buffer, maintained in the dark, and assessed within 2 h after loading with fluorescence dye. Fluorescence was measured at 37 °C in a Photon Technology International Delta Scan Spectrofluorimeter and analysed with Oscar analysis software (PTI). The excitation and emission wavelengths were 380 nm and 510 nm, respectively. The intracellular free Ca2+ was calculated according to the equation [Ca2+]i=Kd[(RRmin)/(RmaxR)]. The Kd for Fura-2Ca2+ complex is 224 nM at 37 °C in buffer simulating the mammalian cytoplasm. The maximal fluorescence ratio (Rmax) was determined by adding 0·1 % Triton X-100 to permeabilize cells, and the minimal fluorescence ratio (Rmin) was determined by the subsequent addition of 5 mM BAPTA. Addition of 5 mM BAPTA to the cuvette containing a Fura-2-loaded monolayer did not change the baseline fluorescence, indicating that no significant amount of dye leaked extracellularly during the measurement period. All studies were conducted in triplicate and on three separate days, and the data are presented as mean±SEM.
Fluorescence microscopic images of cytosolic calcium.
INT407 cells were cultured to semiconfluence on 12 mm2 circular glass coverslips. Monolayers were infected with bacteria at an m.o.i. of 100 for 10, 30 or 60 min, washed three times with HBSS and loaded with 5 µM indicator dye Fluo-3, AM, as described above for Fura-2, AM, and incubated for 30 min at room temperature. Infected monolayers were compared with uninfected INT407 cells via a Fluo-3 visualization assay (i.e. excitation and emission wavelengths of 488 and 525 nm, respectively). After washing, the coverslips were placed onto hanging-drop glass slides containing the loading buffer and viewed with a Zeiss MC100 phase-contrast/fluorescence microscope. All samples were viewed at 630x magnification, and images taken with a mounted 35 mm camera were computer-scanned and edited with Adobe Photoshop.
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RESULTS |
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Measurement of cytosolic free Ca2+
Measurement of intracellular free Ca2+ can be monitored with fluorescent probes, which provide a highly sensitive and quantitative assay. Fura-2, a ratiometric fluorescent dye, was used here to quantitate intracellular free Ca2+ levels.
The level of cytosolic free Ca2+ observed by spectrofluorimetry in control non-infected INT407 cell monolayers was typically 85 nM, similar to resting levels (70100 nM) reported for various other cell lines (Clerc et al., 1989
; Pace et al., 1993
). Infection with C. jejuni 81-176 caused a significant enhancement in Fura-2 fluorescence of INT407 cells at 10, 30 or 60 min post-infection, compared with the constant resting level in uninfected cells (P<0·10 at 10 min and P<0·05 at 30 or 60 min post-infection; Fig. 3
).
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INT407 monolayers maintained in Ca2+-free medium were also pretreated with the specific PKC inhibitor calphostin C to assess the involvement of this calcium-activated host kinase in C. jejuni invasion. As shown in Fig. 5, host-cell invasion by strain 81-176 was reduced
60 % in the presence of calphostin C (P<0·001 relative to the no-inhibitor control). In addition, the control S. Typhi strain was also markedly reduced in invasion by this inhibitor.
Together, these results suggest that both CaM and PKC are important in C. jejuni internalization. S. Typhi invasion was also strongly dependent upon the release of Ca2+ from intracellular stores, but CaM did not appear to play as prominent a role in its invasion.
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DISCUSSION |
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Previous analyses of C. jejuni 81-176 internalization into INT407 cells have revealed that this process is kinetically saturable, but gradual and dependent upon the bacteria : host cell ratio (Hu & Kopecko, 1999). In this earlier study, approximately 10, 20 and 50 % of INT407 cells were observed to be infected, respectively, at 10, 30 or 60 min post-infection, and only 67 % of all host cells were infected after 2 h at an m.o.i. of 100200. Assuming that only host cells which had been infected would be associated with increased cytosolic Ca2+, we extrapolated the intracellular Ca2+ data on a per infected cell basis, based upon the above findings (Hu & Kopecko, 1999
). The extrapolation results in a spike in free Ca2+ to 325 nM at 10 min post-infection which gradually decreased but was still 3x the resting level at 60 min (Fig. 3
), since invasion would be expected to continue for another 60 min.
In the current study, microscopic observation of Fluo-3-labelled, infected INT407 cells showed that 10 % of total host cells exhibited a marked increase (i.e. a bright fluorescence) in cytosolic Ca2+ at 60 min (Fig. 4A
). We speculate that only a fraction of the host-cell population (i.e.
10 % of cells) would be undergoing active invasion at any time-point and would exhibit the highest levels of cytosolic Ca2+. We further suggest that infected host cells lower the [Ca2+]i within 1020 min of bacterial entry. However, since continued invasion of INT407 cells by C. jejuni occurs during the 2 h invasion period, [Ca2+]i averaged over all cells is elevated above resting levels throughout this period (Fig. 3
). Further characterization of these dynamic events awaits the measurement of [Ca2+]i fluxes over time in individually infected cells versus uninfected cells.
W-7 is a widely used CaM antagonist that is both potent and selective for CaM (Miyamoto et al., 1992). W-7 can also inhibit PKC, but at a 10-fold higher concentration (about 40 µM) than that required to inhibit CaM (Tanaka et al., 1982
). Since 2 µM W7 markedly reduced the invasion ability of C. jejuni 81-176, a CaM-dependent process(es) appears to play a prominent role in host-cell invasion by C. jejuni 81-176. The marked inhibition of invasion in the presence of calphostin C also suggests a role for PKC in the C. jejuni internalization process.
Increased host intracellular free Ca2+ has been associated with pathogenicity of a number of intracellular bacterial pathogens to date. In a well-studied example, Chlamydia trachomatis elementary bodies (EB), upon internalization into vacuoles within host cells, mobilize Ca2+ and Ca2+-binding proteins such as annexins to the EB inclusions, a process which may regulate fusion events with other endosomal compartments (Majeed et al., 1994). Increased intracellular Ca2+ has also been associated with the S. Typhimurium microfilament-dependent invasion of cultured eukaryotic cells (Ruschkowski et al., 1992
; Pace et al., 1993
). Pace et al. (1993)
reported that S. Typhimurium invasion of INT407 cells occurs following Ca2+ influx from the extracellular environment through activation of the arachidonic acid pathway, leading to the production of leukotriene LTD4, a potential mediator of this Ca2+ influx. In contrast, Ruschkowski et al. (1992)
showed that S. Typhimurium infection of HeLa cells induces the inositol phosphate pathway, leading to the production of IP3, which mobilizes Ca2+ from intracellular stores. Whether these different Ca2+ mobilization pathways can function synchronously within the same host cell during S. Typhimurium infection or are dependent upon specific host cells or particular bacterial strains remains to be determined. It is interesting to note that Shigella flexneri, which triggers a type III secretion-dependent, microfilament-involved invasion pathway analogous to that of Salmonella, was found to stimulate an increase in [Ca2+]i, but it was not required to initiate invasion (Clerc et al., 1989
; Tran Van Nhieu et al., 2003
). In addition to the above reports, the invasion protein InlB of Listeria monocytogenes has recently been found to activate sequentially a p85 beta-p110 class I (A) PI-3 kinase and PLC-gamma in the human epithelial cell line HEp-2. Purified InlB was observed to stimulate the association of PLC-gamma with one or more tyrosine-phosphorylated proteins, and resulted in the release of intracellular Ca2+ via the IP3 pathway (Bierne et al., 2000
). Thus, although the data are as yet limited, increased [Ca2+]i seems to be involved in the pathogenesis of a number of different bacteria.
How is increased cytosolic Ca2+ required for Campylobacter invasion? Previous studies have shown that cycloheximide-mediated inhibition of INT407 cell protein synthesis does not reduce invasion by C. jejuni 81-176, indicating that nascent host protein synthesis is not essential for invasion during the first 2 h (Oelschlaeger et al., 1993). These data would suggest that the increased [Ca2+]i is not involved immediately in triggering invasion-essential nuclear transcription. Instead, it seems likely that Ca2+ is involved in enzyme activation, perhaps at the level of cytoskeletal rearrangements. Previous immunofluorescence antibody studies have indicated that infected INT407 cells extend a projection of the host membrane to the invading C. jejuni, which appears to interact with the tip of the host-cell extension and undergo internalization via host-membrane invagination (Hu & Kopecko, 1999
, 2000
). We speculate that interaction of C. jejuni with a membrane receptor protein in caveolae, in turn, leads to activation of a number of signal-transduction events (L. Hu and others, unpublished results) and the release of Ca2+ from intracellular stores. The general pathways of Ca2+ modulation by host cells and the steps affected by the inhibitory/stimulatory compounds employed herein are presented in Fig. 6
. Ca2+ ions are needed to activate CaM and PKC, and may be involved in aiding the necessary cytoskeletal rearrangements that occur during bacterial entry (i.e. dissociation of the cortical actin filaments resulting in localized collapse of the microvilli and the extension of peripheral microtubules to form a finger-like membrane projection). Further analyses of the host signalling pathways triggered by C. jejuni will undoubtedly lead to an improved molecular understanding of pathogenesis caused by this important food-borne bacterial pathogen.
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ACKNOWLEDGEMENTS |
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Received 4 January 2005;
revised 31 May 2005;
accepted 8 June 2005.
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