1 Departamento de Microbiología II, Facultad de Farmacia, Universidad Complutense de Madrid, Pza Ramón y Cajal s/n, 28040 Madrid, Spain
2 Michael Smith Laboratories, #301 - 2185 East Mall, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
Correspondence
María Molina
molmifa{at}farm.ucm.es
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ABSTRACT |
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INTRODUCTION |
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The budding yeast Saccharomyces cerevisiae has already been established as a model system to study virulence-related proteins from pathogenic bacteria (reviewed by Valdivia, 2004). Basic signalling modules and cytoskeletal components are conserved, making the yeast model a feasible approach to analyse the effects induced by bacterial virulence factors in mammalian cells. Secreted effectors from Yersinia (Pawel-Rammingen et al., 2000
; Lesser & Miller, 2001
; Skrzypek et al., 2003
; Yoon et al., 2003
; Nejedlik et al., 2004
), Salmonella (Lesser & Miller, 2001
; Rodriguez-Pachon et al., 2002
), Pseudomonas (Rabin & Hauser, 2003
; Sato et al., 2003
), Vibrio (Trosky et al., 2004
) and Legionella (Shohdy et al., 2005
) have been expressed in yeast and found to interfere with cellular functions related to their proposed targets within the host cell, such as Rho and Cdc42 small GTPases, mitogen-activated protein kinase (MAPK) cascades, membrane trafficking and the actin cytoskeleton. Here we report the systematic heterologous expression of all known LEE-encoded EPEC translocator and effector proteins in the yeast system. We show that these bacterial proteins cause differential phenotypic effects on cell growth, cytoskeletal function and signalling pathways. Furthermore, by expressing mutant versions of Map, we provide evidence that expression of bacterial TTSS effectors in yeast can be a useful tool to identify functional domains in these proteins.
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METHODS |
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YPD (1 %, w/v, yeast extract; 2 %, w/v, peptone; 2 %, w/v, glucose) broth or agar was the general nonselective medium used for growing the yeast strains. Synthetic complete medium (SC) contained 0·17 % (w/v) yeast nitrogen base without amino acids, 0·5 % (w/v) ammonium sulphate and 2 % (w/v) glucose, and was supplemented with appropriate amino acids and nucleic acid bases. SCGal and SCRaf were SD with 2 % (w/v) galactose or raffinose, respectively, instead of glucose. Galactose induction experiments in liquid media were performed by growing cells in SCRaf medium at 30 °C to exponential phase and then adding galactose to 2 % (w/v) for 68 h. Effects of the expression of EPEC genes on yeast growth were tested by spotting cells onto SC or SCGal plates lacking the corresponding auxotrophic markers to maintain plasmids. Briefly, transformants were grown overnight in SC lacking uracil, leucine or both (SCUra, SCLeu or SCUraLeu), depending on the requirements of each transformant, and adjusted to an OD600 of 0·5. Five microlitres of samples plus three serial 1/10 dilutions were spotted on the surfaces of SC or SCGal solid media lacking uracil, leucine or both. Growth was monitored after 23 days at 28 °C. Growth curves were plotted from data obtained in liquid media at 30 °C, by adjusting OD600 to 0·2 on cells growing exponentially on SCRaf and adding galactose to achieve a final concentration of 2 % (w/v). Samples were taken every 2 h and OD600 was measured.
Molecular biology techniques and plasmid construction.
Standard E. coli transformation and basic molecular biology techniques were performed. Yeast transformation was achieved by the standard lithium acetate protocol. Two series of plasmids were constructed in this study, one based on the 2µ-based pEG(KG) vector to express GST fusion proteins in yeast (Mitchell et al., 1993) and a second series based on YCpLG, a LEU2-based centromeric vector containing the GAL1 promoter, kindly provided by J. Thorner (Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA).
EPEC genes were amplified by PCR from E. coli E2348/69. Oligonucleotides for these strategies are listed in Table 1. The primers used for amplification of espA, espH and tir had BamHI (upper primer) and HindIII (lower primer) sites, the primers used for amplifications of espB, espD, espF and espG had BamHI (upper primer) and EcoRI (lower primer) sites and the primers used for map amplification had XbaI (upper primer) and HindIII (lower primer) sites in their respective non-homologous 5' tails. PCR products were cloned into the pGEM-T vector (Promega), sequenced to verify the absence of mutations and cleaved with BamHI/HindIII, BamHI/SalI (the latter site from the pGEM-T polylinker) and XbaI/HindIII respectively to be inserted into the same sites of either pEG(KG) or YCpLG. To obtain the Map-GFP fusion, YCpLG was modified by inserting the GFP sequence into BamHI/XbaI-cut vector as a PCR product obtained with the GFP1 and GFP2 oligonucleotides, thus generating YCpLG-GFP; map was amplified with the MapA and MapB oligonucleotides, which bear BglII sites, allowing the cloning of the PCR product into the BamHI site of this plasmid. To generate C-terminal truncations of Map fused to GST in yeast we cloned into XbaI/HindIII-cut pEG(KG) the PCR products resulting from combining the Map-1 upper primer with Map-2A, Map-2B and Map-3 primers respectively. Other plasmids used in this work were pLA10H to express GFP-tagged septin (Cid et al., 2001a
) and pRS315 : : SEC63-MYC (Lyman & Schekman, 1997
).
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Microscopy and immunofluorescence.
For fluorescence microscopy on live cells for the localization of GFP, cells from exponentially growing cultures were centrifuged gently, washed once with sterile water and observed. Localization of actin in yeast cells with FITC-conjugated phalloidin (Sigma) was performed as previously described (Jimenez et al., 1998). For chitin staining, cells were treated with Calcofluor white (Fluorescent Brightener 28; Sigma) as described by Pringle (1991)
. Visualization of mitochondria by in vivo DAPI staining (Williamson & Fennell, 1979
) was performed by adding DAPI (Sigma) to cells resuspended in PBS at a final concentration of 10 µg ml1 and incubating for 5 min. For statistics on cell populations, 100200 cells were counted for each experiment.
Indirect immunofluorescence on yeast cells was performed as previously described (Cid et al., 2001b). Primary antibodies in immunofluorescence experiments were used as follows: rabbit anti-GST antibodies (Santa Cruz Biotechnology) at a 1 : 500 dilution; rat anti-alpha-tubulin (YOL1/34) antibodies (Serotec) at a 1 : 500 dilution; mouse anti-V-ATPase (Molecular Probes) at 20 µg ml1; mouse anti-myc (Covance Research Products) at a 1 : 250 dilution. As secondary antibodies, Cy3-conjugated goat anti-rabbit IgG (Chemicon International) and Cy5-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch) were used at a 1 : 500 dilution; FITC-conjugated goat anti-rat IgG (Sigma) and FITC-conjugated goat anti-mouse (Sigma) were used at a 1 : 200 dilution. For phase-contrast, fluorescence microscopy and indirect immunofluorescence, cells were examined with an Eclipse TE2000U microscope (Nikon). Digital images were acquired with an Orca C4742-95-12ER charge-coupled device camera (Hamamatsu) and Aquacosmos Imaging Systems software.
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RESULTS |
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Expression of EspD, EspG and Map in yeast alters cortical actin function
Budding is a morphogenetic programme specific to yeast, but cytoskeletal structures involved in this process are conserved in mammalian cells. To determine whether the observed morphological alterations were related to interference of the EPEC effectors with the yeast actin cytoskeleton, we examined actin distribution on fixed cells stained with fluorochrome-conjugated phalloidin. During bud development, actin patches accumulate at the growing bud in control cells expressing GST. However, cells with mislocalized cortical actin were observed when expressing GST-EspD, GST-EspG or GST-Map (Fig. 4). We have previously reported that EspG disrupted cytoskeletal function in yeast, causing actin depolarization (Hardwidge et al., 2005
). A significant proportion of small-budded cells expressing GST-EspD showed loss of actin polarization to the bud (Fig. 4a
). This effect is similar to that reported for EspG, although quantitatively less severe (Fig. 4b
). The proportion of cells with random cortical actin distribution was higher for GST-Map-expressing transformants, indicating that Map strongly interferes with basic cell polarity mechanisms (Fig. 4a, b
).
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DISCUSSION |
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Expression of certain translocator proteins (EspD) and, remarkably, TTSS effectors EspF, EspG, EspH and Map led to a variety of effects in yeast, each protein causing a distinct phenotype, ranging from severe to subtle effects on growth, signalling and cytoskeletal rearrangements. We will discuss these results below, but to facilitate interpretation, a comprehensive view of the phenotypic systematic analyses carried out in this work is presented in Table 2, and a graphic overview is provided in Fig. 8
.
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EspF interferes with yeast morphogenesis and septin ring integrity
Expression of EspF caused a very peculiar effect in yeast: although actin polarity seemed normal, the accumulation of aborted tiny buds suggests that slow growth of an EspF-expressing cell population is due to an abandonment of pre-selected bud sites. Chitin distribution in the cell wall was abnormal and the MAPK pathway that monitors cell wall integrity was activated. Also, budding cells often had unusually long or unconstricted bud necks, consistent with the failure of EspF-expressing cells to assemble proper septin rings. Supporting this view, expression of EspF was more deleterious in cells with a compromised septin function, such as cdc10-11 mutants. Septin filaments regulate bud morphogenesis and cytokinesis. In the earliest stages of budding, once the bud site has been selected, a ring of septins encircles the spot in which actin starts to accumulate to direct polarized secretion for bud emergence (for reviews, see Gladfelter et al., 2001; Faty et al., 2002
). It is likely that EspF is able to sequester an important element for bud site assembly, hampering formation of the ring. EspF is a small proline-rich peptide (McNamara & Donnenberg, 1998
), so it could interact with proteins containing SH3 domains related to bud development, like Sla1 or Abp1 (Drubin et al., 1990
; Holtzman et al., 1993
), or to cytokinesis such as Hof1 (Naqvi et al., 2001
). In addition, certain mutations in the small GTPase Cdc42, which is responsible for both actin and septin assembly at presumptive bud sites, perturb septins without altering actin function (Gladfelter et al., 2002
). Therefore, it cannot be discounted that EspF interferes with Cdc42-related pathways in yeast.
During infection, EspF reportedly disrupts tight junctions in basolateral membranes of epithelial cells (McNamara et al., 2001). One of the properties of tight junctions is to block diffusion of proteins and lipids in the plane of the plasma membrane. The septin ring serves this function in yeast during cell budding, dividing the plasma membrane into two domains, the morphogenetically active daughter cell compartment and the quiescent mother cell (Barral et al., 2000
). Therefore, the modulation of septin ring assembly in yeast by EspF is consistent with its proposed role in tight junction disruption in mammalian cells. Two different groups have recently reported that EspF localized to host cell mitochondria, causing initiation of the mitochondrial death pathway (Nougayrede & Donnenberg, 2004
; Nagai et al., 2004
). Both groups also map an N-terminal mitochondrial localization signal in EspF. We did not detect such localization in yeast, probably because we expressed EspF as an N-terminal GST fusion. The fact that EspF causes cytoskeletal dysfunction in yeast when deprived of its mitochondrial localization suggests that EspF might be multifunctional. In fact, EspF has also recently been found to interact with the intermediate filament component cytokeratin 18 and adaptor 14-3-3 proteins (Batchelor et al., 2004
; Viswanathan et al., 2004
).
EspG impairs actin function in yeast and uncouples nuclear division from budding
We also expressed in S. cerevisiae two recently reported proteins translocated by EPEC into host cells, EspG and EspH. The function of EspH remains to be clarified, but it might somehow interact with the host actin cytoskeleton (Tu et al., 2003). However, its expression in yeast did not apparently affect actin function or cell growth, although it caused activation of the cell integrity MAPK pathway. EspH was produced in yeast in lower levels than the other EPEC proteins and was retained in a small compartment in the yeast cytoplasm, perhaps precluding its interaction with actin-associated targets at the plasma membrane.
EspG is homologous to the Shigella protein VirA, which interacts with mammalian tubulin and helps trigger host cytoskeletal remodelling prior to invasion (Yoshida et al., 2002). When expressed in yeast, EspG strongly depolarized actin patches without affecting septin assembly, causing an activation of the Slt2 MAPK pathway. We show here that cultures of cells expressing EspG accumulate small-budded cells, and microscopic analysis of their nuclei and microtubular apparatus revealed a loss of coordination between bud development and nuclear division, processes that are tightly synchronized in yeast (for reviews, see Cid et al., 2002
; Lew & Burke, 2003
). We have recently reported that EspG is able to interact with mammalian tubulin and affects yeast cytoplasmic microtubules (Hardwidge et al., 2005
). Another recent report presents evidence that local microtubular destabilization triggers assembly of actin stress fibres via activation of RhoA in mammalian cells (Matsuzawa et al., 2004
). The RhoA yeast homologue Rho1 is an upstream component of the Slt2 pathway. Therefore, it is temping to hypothesize that a similar mechanism occurs in yeast cells expressing EspG, leading to the activation of Slt2.
Map blocks yeast cell polarity and morphogenesis
Map, a small peptide showing slight homology to the Shigella IpgB protein, induces multiple interesting phenotypes when expressed in yeast. Map interference with the yeast actin cytoskeleton is more drastic than that of EspD and EspG. Cells expressing Map lose their normal ellipsoidal shape, turning spherical, substantially alter their cell wall composition, and completely fail to bud. Actin cortical patches are randomly distributed along the surface of Map-expressing cells, whereas septins adopt a patchy pattern, failing to assemble proper rings. These phenotypes are reminiscent of cells that have lost Cdc42 function (Johnson & Pringle, 1990), suggesting that Map might inhibit the function of this GTPase when expressed in yeast. Nevertheless, not all the effects of Map on yeast can be explained by inhibition of Cdc42. First, inability to bud in yeast Cdc42-defective cells leads to isodiametric growth, with cells reaching a large size, whereas Map-expressing cells are round and unbudded, but not oversized. Thus, some other phenomena required for growth must be affected. Second, Map-expressing cells show a very strong activation of the Slt2 MAPK, as in the case of other EPEC effectors, but also of the Kss1 MAPK. Activation of both MAPKs might reflect the existence of an altered cell wall in the Map-expressing cells, as CW staining suggests. However, Kss1 acts downstream of Cdc42 and therefore its phosphorylation could derive from a Map-mediated activation rather than an inhibition of this GTPase. This would be consistent with the proposed role of Map in the formation of filopodia in mammalian cells, a process that requires Cdc42 (Kenny et al., 2002
). However, Map is also involved in the inhibition of Tir-dependent actin recruitment for pedestal formation by an as yet unknown mechanism (Kenny et al., 2002
), and its overproduction promotes the recently described ability of EPEC to invade cultured cells (Jepson et al., 2003
). The fact that Map causes diverse effects in yeast supports the idea that Map is involved in multiple regulatory events.
Also supporting the view that Map is a multifunctional protein, we provide evidence that targeting of Map to host cell mitochondria and its toxicity can be dissected. A C-terminal GFP-tagged version of Map specifically localizes to yeast mitochondria but is not toxic, whereas N-terminal GST-tagged versions of Map do not stain mitochondria but are highly toxic. Moreover, toxicity can be eliminated by deleting the last 15 residues of Map. This deletion disrupts a C-terminal putative -helix secondary structure that is highly conserved in Map orthologues from related enteropathogenic bacteria. Further studies will help elucidate the molecular interactions in which this region is involved. This mutational analysis outlines the power of yeast genetics as a useful approach for clarifying the role of bacterial effectors in human disease.
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ACKNOWLEDGEMENTS |
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Received 28 March 2005;
revised 6 June 2005;
accepted 9 June 2005.
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