Department of Pathology and Microbiology, School of Medical Sciences, University Walk, Bristol, BS8 1TD, UK1
Tel: +44 117 928 7530. Fax: +44 117 928 7896. e-mail: B.Kenny{at}bristol.ac.uk
Keywords: phosphorylation, actin, signalling, Cdc42, Map
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Overview |
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The ability to define the molecular mechanisms by which A/E pathogens induce disease arose from the discovery that infection of tissue-cultured cells could produce lesions similar to those observed in vivo, which also permitted pedestal composition to be investigated (Finlay et al., 1992 ; Knutton et al., 1987
, 1989
). This discovery also facilitated screening programmes to identify mutants defective in pedestal formation, revealing that a bacterial outer-membrane protein, intimin, was required for intimate adherence, pedestal formation and disease (Donnenberg et al., 1990
, 1993
; Jerse et al., 1990
). Another key finding came from the correlation of EPEC adherence with the tyrosine phosphorylation of a protein of
90 kDa (Hp90) within the host membrane (Rosenshine et al., 1992
). Subsequent analyses revealed that Hp90 served as a receptor for intimin, and moreover that Hp90intimin interaction was essential for pedestal formation (Kenny & Finlay, 1997
; Rosenshine et al., 1996
). A further important finding was that EPEC secreted a number of proteins (EPEC secreted proteins; Esp) whose release was linked to Hp90 phosphorylation and pedestal formation (Jarvis et al., 1995
; Kenny & Finlay, 1995
; Kenny et al., 1996
). The genes encoding these secreted proteins were mapped to the same chromosomal locus as the gene encoding intimin (eae). These genes were themselves part of a
35 kb region, named LEE for Locus of Enterocyte Effacement, that had a significantly lower GC content than the E. coli chromosome, implying that the region had been acquired via horizontal transfer (Elliott et al., 1998
; McDaniel et al., 1995
). LEE carries
41 open reading frames encoding components of a type III secretion system, secreted proteins, chaperone molecules, regulatory proteins and intimin (Elliott et al., 1998
). Cloning of this region into K-12 strains conferred an ability to induce A/E lesions, suggesting that LEE encodes all the factors required for this process, though other chromosomal encoded factors undoubtedly contribute to the appropriate regulation of gene expression (Elliott et al., 1998
; Friedberg et al., 1999
; McDaniel & Kaper, 1997
). The type III apparatus forms a supermolecular structure that spans the double membrane system of Gram-negative bacteria and in EPEC appears to be dedicated to the secretion of specific LEE-encoded proteins, including EspA, EspB and EspD (Kenny & Finlay, 1995
; Kubori et al., 1998
; Sekiya et al., 2001
). EspA is the major constituent of a filamentous structure that forms an extension of the type III apparatus to facilitate the delivery of proteins, such as EspB and EspD, directly into the host cell (Knutton et al., 1998
; Sekiya et al., 2001
; Wilson et al., 2001
). EspD appears to play a role in EspA appendage elongation and has been detected in the plasma membrane, along with EspB, where together with EspA they appear to form a pore enabling EPEC molecules to be delivered into the host cell (Daniell et al., 2001
; Ide et al., 2001
; Knutton et al., 1998
; Shaw et al., 2001
; Wachter et al., 1999
; Warawa et al., 1999
; Wolff et al., 1998
). EspB has also been detected in the cytoplasmic fraction, raising the possibility that it may have an effector function(s) (Taylor et al., 1998
, 1999
). To date, three EPEC type III secreted effector molecules have been identified, with an additional protein, EspG (Elliott et al., 2001
), reported to be translocated into host cells, though its function remains undefined.
The first EPEC effector molecule to be identified was, surprisingly, the previously characterized intimin receptor molecule Hp90, leading to its renaming to Tir for Translocated intimin receptor (Kenny et al., 1997 ). As stated above, studies with Hp90 had already revealed that this molecule becomes tyrosine-phosphorylated and inserted into the plasma membrane, where interaction with intimin mediates intimate bacteriahost cell contact and pedestal formation (Rosenshine et al., 1992
, 1996
). Tirintimin interaction can also trigger additional signalling responses such as the phosphorylation of PLC-
1 (Kenny & Finlay, 1997
), and both Tir and intimin have been shown to be essential virulence determinants in a natural animal infection model (Marches et al., 2000
). The second effector, EspF, disrupts host intestinal membrane barrier function by an unknown mechanism (McNamara et al., 2001
), while the third, Map (Mitochondrial associated proteins; formerly orf19), is targeted to host mitochondria, where it interferes with their ability to maintain their membrane potential (Kenny & Jepson, 2000
). This article will focus on recent data concerning the mechanism of action and function of the Tir and Map effector molecules within host cells, as they are the subjects of investigation within my laboratory.
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Topology of Tir within the host plasma membrane |
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Surprisingly, two secreted forms of Tir (sharing identical N-terminal sequences) have been detected migrating as 72 and
78 kDa proteins by SDS-PAGE analysis, although tir encodes 550 amino acids with a predicted molecular mass of
55 kDa (Kenny et al., 1997
). This discrepancy may be (i) a function of the amino acid composition, (ii) indicative of bacterial modification or (iii) related to Tir adopting some form of SDS-PAGE-resistant conformation/structure. Tir is predicted to possess two transmembrane regions (234259 and 363382), indicating that it may adopt a hairpin-like arrangement in the plasma membrane. This premise has been supported by topology analyses, which indicate that both termini are located inside the host cell, while the predicted central extracellular domain has been shown to mediate binding to intimin (de Grado et al., 1999
; Hartland et al., 1999
; Kenny, 1999
). This reveals that Tir is divided into roughly three similarly sized domains, with the two terminal domains presumably available for interaction with host proteins and the extracellular domain for binding intimin. It was assumed that one of these terminal domains would carry the substrate site for tyrosine phosphorylation and studies have shown that a single tyrosine residue (Y474) within the C-terminal domain of EPEC Tir is phosphorylated (Kenny, 1999
). More recently, interaction of intimin with the extracellular Tir domain has been determined at a molecular level, and is suggestive of Tir dimerization, with each molecule interacting with a lectin-like domain of intimin (Batchelor et al., 2000
; Luo et al., 2000
). It has also been suggested that a complex is formed of Tir dimers and intimin trimers to mediate the formation of symmetrically packed actin fibrils to aid in the generation of the observed pedestal-like structures beneath the adherent bacteria (Luo et al., 2000
).
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Mechanism of Tir insertion into the plasma membrane |
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Serine-phosphorylation-mediated changes to Tir structure |
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Further support for phosphorylation-mediated conformational change in Tir stems from the finding that a T0 to T'-like shift could be mediated in a phosphorylation-independent manner by specific substitutions at the PKA-modification site linked with this shift (Warawa & Kenny, 2001 ). Such substitutions, like phosphorylation, are predicted to alter the charged nature of this region, suggesting that they may serve to destabilize one Tir structure in favour of an alternative one. As unmodified Tir migrates as a
72/78 kDa protein, and not the predicted
55 kDa, it may itself have already adopted some stable structure with the sequential addition of phosphate groups triggering the adoption of alternative conformations.
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PKA modification of Tir within host cells |
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However, PKA is not the only enzyme that can modify Tir at the RRDS motif to produce a T'-like species as Yersinia-mediated delivery of Tir into cells deficient in PKA activity did not prevent its modification to the T' form, but this event was dependent on S434 (Warawa & Kenny, 2001 ). It is possible that non-PKA modification of Tir is a feature of immortalized cell lines and would not occur in polarized gut epithelial cells where more stringent control of kinase expression and their compartmentalization is expected. This may indeed be the case as preliminary data from EPEC infections of cultured polarized cell lines are suggestive of a significant reduction in Tir T' levels within the plasma membrane when Tir carried a S434A substitution. On the other hand, it is also possible that an ability to modify Tir by alternative mechanisms may provide an advantage by increasing the likelihood of Tir modification/function within new hosts, helping it to spread to new species.
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Phosphorylation on S434 is required for maximal Tir function |
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Although an important role for the T0 to T' modification process in virulence remains to be formally proven, alternative support comes from the finding that Tir mutants could be isolated that display a T'-like molecular mass in the absence of phosphorylation (Warawa & Kenny, 2001 ). Because such molecules are delivered by EPEC into host cells and modified to the T'pY actin-nucleating form, this argues that strong selection pressure has retained the phosphorylation-mediated mechanism. Indeed, the conservation of the PKA recognition motif RRXS, implicated in the T0 to T' shift, among Tir homologues further supports an important role for modification at this site in Tir function.
In contrast to the RRDS motif, the second in vitro-identified PKA modification motif (RNS463), linked with the T' to T'-like shift, is not conserved and does not appear to play a role in EPEC Tir function within host cells (Warawa & Kenny, 2001 ). At present, we believe that the in vitro PKA-mediated conformational shift simply mimics a phosphorylation event that occurs within host cells, suggesting that this second shift can, like the T0 to T' shift, be triggered by alternative mechanisms. As stated above, the current data indicate that the in vivo modification associated with the T' to T' shift is linked with Tirs correct insertion into the plasma membrane by a process dependent on bacterial, and probably host, proteins. We are currently screening Tir, which carries 98 serine/threonine residues, for the residue(s) whose modification mediates the T' to T' shift so that the role of this event in Tir membrane insertion, function and virulence can be assessed. A model representing the putative steps of EPEC Tir modification within host cells relating to its insertion into the plasma membrane is depicted in Fig. 1
.
|
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Tyrosine phosphorylation of EPEC Tir is essential for its actin nucleation function, unlike the EHEC (O157:H7 serotype) Tir molecule |
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This premise has been supported by our recent finding that EPEC Tir is functionally interchangeable for that of EHEC O157:H7, whereas the reverse is not true (Kenny, 2001 ). Furthermore, the study revealed several differences in the modification pattern of the homologues and linked the observed EHEC Tir dysfunction with an altered modification pattern. The observed difference in the EHEC Tir modification pattern following delivery into host cells by EPEC, which was linked with actin-nucleating dysfunction, was shown to be due to the presence of the EHEC C-terminal
170 residues (Kenny, 2001
). The linkage of EHEC Tir actin-nucleating activity with a specific phosphorylation-mediated banding pattern supports an important role for non-tyrosine phosphorylation in Tir function. The data also imply that EHEC expresses a factor, absent from EPEC, that facilitates the correct full modification of EHEC Tir within host cells. This conclusion has recently been verified by DeVinney and others, who also provide evidence that the putative accessory factor(s) can be delivered into host cells by EHEC to restore the function of the EPEC delivered EHEC Tir molecule (DeVinney et al., 2001
). It is possible that this putative accessory factor is encoded within LEE and has co-diverged with Tir, though the finding that EHEC LEE does not confer the A/E phenotype onto E. coli K-12 strains, unlike EPEC LEE (Elliott et al., 1999a
; McDaniel & Kaper, 1997
), raises the possibility that it lies outside LEE. Screening strategies should rapidly lead to the identification of this factor and the examination of its role in EHEC Tir modification and function.
The absence of tyrosine phosphorylation of the EHEC O157:H7 Tir molecule implies that the phosphorylation-related shifts in apparent molecular masses are due to the addition of phosphate groups, again presumably on serine and/or threonine residues. Although EPEC and EHEC Tir only share 40% identity within their C-terminal domains, both possess the putative PKA recognition motif RRXS, whose modification is linked to the EPEC Tir T0 to T' shift. Thus it is likely that this conserved site in EHEC Tir is also a substrate for modification within the host cell, though this remains to be formally tested. However, given the different modification pattern of the homologues within host cells it is also likely that EHEC Tir undergoes additional non-conserved phosphorylation events.
Recent studies have defined the importance of EPEC Tir tyrosine phosphorylation and revealed that it enables the direct binding of the Nck adaptor protein and subsequent recruitment of N-WASP and the host actin-nucleating machinery (Gruenheid et al., 2001 ; Kalman et al., 1999
). As tyrosine phosphorylation is not a feature of EHEC O157:H7 Tir, this reinforces the concept that this pathogen has evolved an alternative mechanism to trigger pedestal formation. It is possible that some of the EHEC Tir non-tyrosine modifications served to facilitate this process by, for example, triggering a conformational change to expose a docking site (perhaps explaining the divergence between the EPEC and EHEC C-terminal domains) for a host or bacterial-encoded adaptor molecule. It is intriguing to speculate that the evolution of this tyrosine-phosphorylation-independent actin-nucleating mechanism has provided EHEC O157:H7 with a selective advantage that has contributed to it being most commonly associated with EHEC disease. Studies are currently under way to identify the EHEC Tir modifications so that their role in EHEC Tir function can be determined and compared with those of EPEC Tir.
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Composition of Tirintimin pedestals |
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Identification of a second LEE-encoded effector molecule |
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Infection studies in the presence of the fluorescent dye TMRE, which is specifically taken up and retained by mitochondria as long as they maintain their membrane potential, indicate that Map disrupts mitochondrial membrane potential (Kenny & Jepson, 2000 ). This is supported by the observation that Map-associated mitochondria were poor accumulators of an alternative mitochondrial specific reagent, Mitotracker, which, like TMRE, depends on mitochondrial potential for uptake. Indeed, pre-treatment of cells with Mitotracker, which gradually becomes toxic to mitochondria, prior to Map delivery reduces the level of Map associating with mitochondria, indicating that Map also requires a membrane potential to associate with these organelles (Kenny & Jepson, 2000
). Pathogen-induced mitochondrial damage is often associated with the triggering of programmed cell death, via the release of pro-apoptotic factors and subsequent activation of proteolytic caspases (Boya et al., 2001
). Several reports suggest that EPEC can induce death of tissue-cultured cells by a mechanism displaying features of both apoptosis and necrosis, with a more recent report indicating a link with the expression of the bundle-forming pilus (Abul-Milh et al., 2001
; Barnett Foster et al., 2000
; Crane et al., 1999
). However, one must be cautious with in vitro evaluations of the apoptotic nature of pathogens because inappropriate infection conditions and/or the use of immortal cell lines may generate results that do not reflect the in vivo situation. Indeed, a recent in vivo study using a natural rabbit model infection system did not detect increases in apoptotic rates, but in contrast indicated decreases in normal cellular apoptotic rates (Heczko et al., 2001
). This raises the alternative possibility that, like other described pathogenic factors (Boya et al., 2001
), Map may have an anti-apoptotic role. Other possible roles for Mapmitochondrial interaction could be to modulate the level of mitochondrial-mediated ATP production to alter the activity of ATP-sensitive enzymes or perhaps to mediate other as yet undetermined mitochondrial controlled functions. Such possibilities are the subject of current studies, which will also address the location of Map within mitochondria, its mechanism of action and role in EPEC pathogenesis.
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Multifunctional nature of the EPEC Map and Tir effector molecules |
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So why might Tir and intimin participate in both down-regulating the Map-mediated Cdc42 activity and triggering pedestal formation? The answer may lie in the observation that the Map-mediated Cdc42-dependent signalling is inhibitory to virulence-associated pedestal formation (Kenny et al., 2002 ). This finding also argues that Map plays an important function within host cells, as otherwise this inhibitory function would have been abolished. Similarly, EPECs adaptation of Tir and intimin to down-regulate this Map-induced activity further supports an important role for this process in pathogenicity and suggests that the resulting activity must be tightly regulated. Although Map and Tir appear to be delivered into the host cells in the same time frame, Map-induced formation of filopodia can be followed for
15 min prior to down-regulation (Kenny & Jepson, 2000
; Kenny et al., 2002
). This lends itself to a mechanism whereby the duration of Cdc42-mediated Map signalling is dictated by the time taken to modify and insert Tir into the membrane for interaction with intimin, which then triggers both filopodia down-regulation and pedestal formation. An outline of the proposed steps of MapTirintimin-regulated signalling is depicted in Fig. 3
.
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Given the recent identification of a multitude of putative virulence genes within the EHEC EDL933 O157:H7 genome (Perna et al., 2001 ), it is likely that additional non-LEE, and probably LEE-encoded effector molecules will be identified. This will almost certainly lead to the discovery of additional A/E specific and conserved pathogenic mechanisms to aid the elucidation of the complex interactions between host and pathogens. Given the non-invasive lifestyle of A/E pathogens, it is likely that they evolved mechanisms to subvert pathways not targeted by invasive pathogens or manipulate such processes by an alternative mechanism or for different purposes. Thus the study of EPEC should continue to provide unique tools for understanding the pathogenic nature of bacteria and the host cellular processes that they subvert.
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ACKNOWLEDGEMENTS |
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