Department of Microbiology and Immunology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA1
Department of Molecular Genetics and Microbiology, PO Box 100266, University of Florida, Gainesville, FL 32610, USA2
Author for correspondence: Shouguang Jin. Tel: +1 352 392 8323. Fax: +1 352 392 3133. e-mail: sjin{at}mgm.ufl.edu
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ABSTRACT |
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Keywords: ExoS, apoptosis, type III secretion, ADP-ribosylation
Abbreviations: FCS, foetal calf serum; p.i., post infection; PI, propidium iodide; TNF, tumour necrosis factor
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INTRODUCTION |
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From a survey of various clinical isolates, Fleiszig et al. (1997) have divided isolates of P. aeruginosa into two categories, invasive and noninvasive strains, based on their abilities to invade mammalian cells. The phenotypes of these two classes are associated with the spectrum of virulence factors that are encoded within their genomes. Both types of strains harbour exoT. Typical invasive strains harbour exoS whereas noninvasive (i.e. cytolytic) strains harbour exoU. The cytolytic phenotype associated with noninvasive strains results mainly from the action of the exoU gene product (Finck-Barbancon et al., 1997
; Hauser et al., 1998
). Although the factor(s) for the invasive phenotype is not clearly understood, Cowell et al. (2000)
have recently shown that both ExoS and ExoT have an invasion-inhibitory effect on cytolytic P. aeruginosa strains. Despite extensive characterization of these factors including their abilities to cause morphological changes on various tissue culture cells (Finck-Barbancon et al., 1997
; Hauser et al., 1998
; Yahr et al., 1998
; McGuffie et al. 1999
; Vallis et al., 1999
), the mechanisms by which these virulence factors alter mammalian cell physiology remain enigmatic.
While investigating bacterial genes that are stringently induced during infection of tissue culture cells, we noted that invasive strains of P. aeruginosa produce cell-contact-dependent factors that induce an apoptosis-like morphology in cultured cells. Further characterization of this phenotype demonstrated that the ADP-ribosylating activity of ExoS is required for induction of programmed cell death by invasive P. aeruginosa.
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METHODS |
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Cells and media.
HeLa S3 cell line in suspension culture was maintained in Jokliks modified minimal essential medium supplemented with 100 µg penicillin ml-1, 100 µg streptomycin ml-1 and 7·5% horse serum (MEM+7% HS; Gibco). For the bacterial infections, HeLa monolayers were plated from suspension culture on the previous day. HeLa cell monolayers were maintained in Dulbeccos modified medium supplemented with 5% foetal calf serum (DMEM+5% FCS; Gibco). All other cell lines were maintained as monolayers in DMEM+10% FCS.
Cell infection by Pseudomonas.
Overnight bacterial cultures were pelleted and resuspended in DMEM. Cell monolayers were grown to >80% confluency in 6-well plates (5x105 cells per 35 mm well) and washed with PBS. The cell monolayer was inoculated with 1x107 bacteria resuspended in 1 ml DMEM (m.o.i.=20) and incubated for 2 h at 37 °C in a 5% CO2 incubator. The bacterial inoculum was removed, cells were washed with PBS and fresh DMEM+5% FCS supplemented with 200400 µg gentamicin ml-1 was added to the monolayer. Cells were incubated in 5% CO2 at 37 °C for an additional 324 h. As positive controls for apoptosis, HeLa cells were incubated with 10 ng tumour necrosis factor
(TNF
) ml-1 plus 20 µg cycloheximide ml-1.
Bacterial invasion tests.
Cell monolayers were infected by the bacteria for 2 h and washed once with phosphate buffered saline. Subsequently, cells were grown for an additional 2 h in fresh DMEM+5% FCS supplemented with 400 µg gentamicin ml-1 or 400 µg amikacin ml-1 to kill extracellular organisms. Cells were harvested by scraping and lysed with 0·1% Triton X-100 prior to serial dilution and plating on L-agar plates with appropriate antibiotics to obtain colony counts. Tests were performed in triplicate and the mean of three trials is reported. To inhibit invasion, the cell monolayers were incubated with the cytoskeletal poison cytochalasin D (10 µM) or the tyrosine kinase inhibitor genestein (200 µM) for 2 h prior to the addition of bacteria.
Apoptosis assays.
Caspase-3 assay.
Caspase-3 activities were measured using a commercially available kit (Caspase-3 cellular activity assay kit plus; BioMol). HeLa cells (3x107) were infected with P. aeruginosa, then cells were washed and harvested by scraping and centrifugation (1000 g, 10 min) at various times post-infection (p.i.). HeLa cells were lysed with 0·1% Tween 20 and cell lysates (supernatants) were saved following centrifugation at 10000 g for 10 min. Dilutions of the cell lysates in a 96-well plate were incubated with the DEVD-pNA substrate, or the substrate plus caspase-3 inhibitor (supplied with the assay kit). Changes in OD405 were followed for 2 h at 10 min intervals. Protein concentration was determined using the Bio-Rad Protein Assay System. The specific activity is reported as pmol substrate cleaved min-1 (µg protein)-1.
Hoechst staining of condensed chromatin.
Infected cells were recovered at 20 h p.i. by trypsinization of the cell monolayer. Cells were washed once with PBS and stained with Hoechst 33258 (1 mg ml-1) for 10 min in the dark. Chromatin condensation was examined under the fluorescence microscope using a DAPI filter.
Flow cytometry analyses of subG1 DNA populations.
Bacterial infected cells were harvested at 24 h p.i. by trypsinization of the cell monolayers and fixation overnight at 4 °C in 70% ethanol. Cells were harvested by centrifugation and stained for 30 min in 500 µl propidium iodide (PI) staining solution (0·1% BSA, 0·1% RNase, 0·5 mg PI ml-1). Cells were directly counted in batches of 10000 and shown by plotting PI fluorescence versus cell number. The percentage of apoptotic cells was calculated using the internal software system of the flow cytometer.
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RESULTS |
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Invasive strains of P. aeruginosa are more potent than the cytolytic strain PA103 at triggering apoptosis
It was noticed in the initial studies that in addition to PAK, the prototypical invasive strain, two other invasive strains (PAO1 and 388) induced consistently high levels of apoptosis. In contrast, in cells infected by the noninvasive, cytolytic strain PA103, apoptotic responses were inapparent or below the detection levels of the assays (Table 2). Although morphological changes were observed within 3 h p.i., the PA103-infected HeLa cells displayed a classic necrotic morphology, including cell swelling and subsequent lysis (Fig. 1g
i
). Furthermore, flow cytometry analysis of PI-stained PA103-infected cells for subG1 DNA levels showed only background levels of DNA fragmentation. Finally, caspase-3 activity, a definitive marker for apoptosis, was absent in PA103-infected cells (Table 2
). This suggested that apoptosis induction appeared to correlate with the invasive phenotype of the strain.
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Although bacterial invasion was not required for apoptosis induction, contact between bacteria and eukaryotic cells, and subsequent expression of the contact-dependent type III secretion system might still be required. ExsA is a global regulator of the type III secretion machinery (Hovey & Frank, 1995 ). Examination of monolayer infections with PAK or an isogenic exsA mutant revealed a profound defect in the ability of the type III mutant to provoke apoptosis. Although the invasion frequency for the exsA strain is nearly identical to that of wild-type PAK (Table 2
), infection with the exsA-deficient strain fails to induce caspase-3 activity (Fig. 2
) and produces a level of DNA fragmentation that is not significantly different from uninfected controls (Fig. 3c
). No morphological changes, specifically cell rounding and lifting, were observed in monolayers infected with the exsA mutant even after 24 h. These results demonstrate that the expression of the apoptosis-inducing factor is under the control of ExsA, suggesting a type III dependent secreted protein.
ExoS is the apoptotic inducer
Among the four type III dependent effector molecules known, only exoT is found in both invasive and non-invasive strains of P. aeruginosa, whereas exoS and exoY are specifically encoded by the invasive strains (PAK, PAO1 and 388) and exoU by the cytolytic strains (PA103) (Fleiszig et al., 1997 ; Yahr et al., 1998
). Although all four Exo products were shown to affect host-cell morphology when expressed independently (Vallis et al., 1999
), alterations in host cell morphology appear to be predominantly due to the action of ExoS and ExoT (McGuffie et al., 1999
; Olson et al., 1999
). To test the possible involvement of ExoS and ExoT in the HeLa cell apoptosis, isogenic mutants of exoS, exoT and an exoS exoT double mutant were generated in the PAK background. To confirm that these strains were ExoS and/or ExoT deficient, gene expression in these mutant strains was induced under type III secretion conditions (Yahr et al., 1997
). As expected, the mutant strains failed to secrete the corresponding ExoS and/or ExoT proteins as determined by SDS-PAGE (Fig. 4
). Apoptotic induction was assessed in cells infected with these mutants. Consistent with previous studies with similar mutants in strain 388, both PAKexoS and PAKexoT mutants failed to induce the early cytoskeletal rearrangements that manifest as cell rounding and detachment after infection. However, the PAKexoS/exoT double mutant failed to cause cytoskeletal rearrangement even after 24 h incubation. Apoptosis in exoT mutant infected cells as determined by caspase-3 activity, DNA fragmentation and chromatin condensation continued unabated at wild-type levels (Fig. 5
and data not shown). These data indicate that ExoT is not an inducer of apoptosis. In contrast, apoptosis was completely abolished in PAKexoS- and PAKexoS/exoT-infected cells. This defect was complemented back to wild-type levels by introduction of an exoS gene clone (pHW9948) but not an exoT gene clone (pHW9949), demonstrating that ExoS is the apoptotic inducer. This was further confirmed by using exoS and exoT mutations in the 388 background. Again, 388exoS::Tc was non-apoptotic while 388exoT::Tc was as potent as wild-type in induction of apoptosis (Fig. 5
). The apoptotic potential of the exoS mutant was completely complemented back to the wild-type level by introducing a plasmid containing exoS from 388, pUCPGexoS.
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These studies thus far indicate that ExoS is necessary for apoptosis induction but it may not be sufficient. Thus, to determine whether other exotoxins produced by invasive strains might also be required, further complementation studies were performed in the background of PA103. To remove the cytolytic phenotype, a PA103 strain deficient in both ExoU and ExoT production was used. The strain, PA103exoU/exoT::Tc, is both non-cytolytic as described earlier (Vallis et al., 1999
) and an apoptotic null strain (Fig. 6
). Introduction of a wild-type exoS gene from strain 388, pUCPGexoS, resulted in a strain capable of triggering apoptosis to high levels, while introduction of the mutant exoSE381A gene, pUCPGexoSE381A, failed to rescue the apoptosis-inducing activity (Fig. 6
). These confirm the requirement for the ExoS-mediated ADP-ribosylating activity in triggering apoptosis.
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DISCUSSION |
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Previous studies had suggested that apoptosis might be induced by other P. aeruginosa gene products. Introduction of purified exotoxin A or porin proteins into mammalian cells has previously been shown to cause apoptosis (Buommino et al., 1999 ; Hafkemeyer et al., 1999
; Morimoto & Bonavida, 1992
). However, the ability of these molecules to induce apoptosis within the context of bacterial infection remains unproven. In addition, it has previously been shown that exotoxin A played no role in the tissue culture and acute infection models (Apodaca et al., 1995
). This is consistent with our observation that the exoA mutant strain of PAK is unaltered in its ability to cause apoptosis of HeLa cells (data not shown) and the PA103 strain, which constitutively expresses high level of exotoxin A (Liu, 1966
), failed to cause apoptosis.
YopE of Yersinia and SptP of Salmonella are homologues of P. aeruginosa ExoS and are also secreted by type III machinery. The homology is limited to the GTPase-activating-protein (GAP) binding domain, which mediates actin disruption, and is not found in the ADP-ribosylating domain (Frithz-Lindsten et al., 1997 ; Pederson et al., 1999
). In addition, SptP has a tyrosine phosphatase domain at the C terminus while YopE has none. Neither YopE nor SptP have been reported to have the apoptotis-inducing phenotype. This is consistent with our observation that it is the ADP-ribosylating activity unique to ExoS and not the common GAP-binding domain that is responsible for the induction of apoptosis.
A number of other bacterial pathogens are known to cause host cell apoptosis by type III secreted effector molecules. For example, IpaB of Shigella spp. and SipB of Salmonella spp. trigger apoptosis by directly activating caspase-1 (Hilbi et al., 1999 ; Hersh et al., 1999
) while YopJ/P of Yersinia spp. cause apoptosis by inhibiting the NF-
B-mediated pathway (Orth et al., 1999
). Similar to these examples, our data suggest P. aeruginosa also induces apoptosis by one of its type III secreted proteins. ExoS possesses two functional domains with its N-terminal 234 amino acids capable of causing cell rounding and actin rearrangement in a Rho-dependent manner whereas the C-terminal 221 residues have a FAS-dependent ADP-ribosyltransferase activity (Pederson et al., 1999
). In addition, it has been demonstrated that exposure to ExoS-producing bacteria, but not non-producing mutants, caused cultured mammalian cells to round up and prevented entry into S phase (McGuffie et al., 1998
). Consistent with this observation, flow cytometry analysis of PI-stained infected cells indicates that P. aeruginosa preferentially induced apoptosis in G1-phase cells. It remains to be determined whether the apoptotic phenotype and the ability of ExoS to block the G1/S transition are related.
Caspase-3 activity is detected in cell lysates as early as 5 h p.i. with PAK. In vivo expression of the type III secretion system is induced and ExoS synthesis is induced only after bacteriahost cell contact. Thus, the interval during which the host cell becomes committed to apoptosis as a result of ExoS function is probably significantly shorter than the 5 h seen here. This rapidity suggests that ExoS may be acting directly to activate apoptotic pathways in the cell. In vitro, ExoS ADP-ribosylates several host proteins, including intracellular proteins such as the intermediate filament protein vimentin (Coburn et al., 1989a ), several low-molecular-mass GTP-binding proteins (Coburn et al., 1989b
) and extracellular proteins such as human immunoglobulin 3 and apolipoprotein A1 (Knight & Barbieri, 1997
). In vivo, ExoS has been shown to specifically ADP-ribosylate Ras proteins, uncoupling Ras-mediated signal-transduction pathways (Vincent et al., 1999
, McGuffie et al., 1998
; Ganesan et al., 1998
, 1999
). Among the four Ras proteins (H-Ras, N-Ras, K-RasA and K-RasB), H-Ras was found to be ADP-ribosylated most extensively. Ras is a molecular switch which controls cellular processes in response to extracellular stimuli. GTP-bound forms of Ras can activate downstream effectors (including Raf-1) which initiate MAP kinase pathways that regulate gene expression affecting cellular proliferation, differentiation and apoptosis. Of interest are also the GTPase-activating proteins (GAP), such as Ras-GAP, which accelerate the intrinsic GTPase activity of Ras resulting in inactive GDP-bound forms of Ras (McNeill & Downward, 1999
). Thus, activation of MAP kinase pathways through ADP-ribosylation of Ras is an attractive mechanistic model for the ExoS-mediated apoptosis. However, the exact pathway(s) by which ExoS triggers apoptosis remains to be elucidated and may involve other yet-to-be-identified cellular targets of the ExoS ADP-ribosyltransferase.
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ACKNOWLEDGEMENTS |
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Received 28 February 2000;
revised 17 May 2000;
accepted 25 May 2000.