Legionella translocates an E3 ubiquitin ligase ... - Wiley Online Library

Molecular Microbiology (2008) 67(6), 1307–1319 䊏

doi:10.1111/j.1365-2958.2008.06124.x First published online 13 February 2008

Legionella translocates an E3 ubiquitin ligase that has multiple U-boxes with distinct functions Tomoko Kubori,1 Akihiro Hyakutake2 and Hiroki Nagai1* 1 The 21st Century COE Program, 2 International Research Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Yamadaoka 3–1, Suita, Osaka 565–0871, Japan.

Summary Legionella pneumophila has a Dot/Icm type IV secretion system used to translocate a number of ‘effector proteins’ which subvert host cell functions. In this study, we identified 19 novel Dot/Icm substrate proteins using a systematic screening technique. A BLAST analysis revealed that one of the substrates, which we named LubX (LegionellaU-box protein), contains two domains that have a remarkable similarity to the U-box, a domain found in eukaryotic E3 ubiquitin ligases. The expression of LubX is induced upon infection, and most of the LubX produced was translocated into the host cells. LubX has ubiquitin ligase activity in conjunction with UbcH5a or UbcH5c E2 enzymes and mediates polyubiquitination of host Clk1 (Cdc2-like kinase 1). We demonstrate that one of the U-boxes (U-box 1) is critical to the ubiquitin ligation, and the other U-box (U-box 2) mediates interaction with Clk1. Thus, the two U-boxes of LubX have distinct functions, and U-box 2 plays a non-canonical role in substrate binding. Although we demonstrate that inhibition of Clk kinase results in a marked reduction of Legionella growth within mouse macrophages, the consequence of Clk1 ubiquitination is still being elucidated. Together, these data suggest that Clk1 is the target host molecule which Legionella modulates during infection.

Introduction Legionella pneumophila is a Gram-negative bacterium ubiquitously found in freshwater environments (Fields, 1996). When inhaled by humans, Legionella enters the Accepted 12 January, 2008. *For correspondence. E-mail hnagai@ biken.osaka-u.ac.jp; Tel. (+81) 6 6879 8361; Fax (+81) 6 6879 8361.

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd

alveolar macrophages and establishes a replicative niche there, and can eventually cause a severe form of pneumonia (Fraser et al., 1977; Horwitz and Silverstein, 1980). Legionella has a Dot/Icm type IV secretion system (T4SS) which is essential for a number of virulence traits including replication within the host cells (Segal et al., 1998; Vogel et al., 1998). The T4SS is one of the secretion systems used by many important bacterial pathogens to translocate ‘effector proteins’ that interact with host factors to subvert host cellular processes. It has been shown that substrates of Legionella Dot/Icm, Agrobacterium VirB/D and Bartonella VirB/D T4SSs have carboxy-terminal translocation signals, although the features of the translocation signals of these T4SSs are distinct from each other (Nagai et al., 2005; Schulein et al., 2005; Vergunst et al., 2005). For example, we previously reported that a hydrophobic residue near the carboxy terminus (Leu372) is critical for translocation of the effector RalF by Legionella Dot/Icm T4SS (Nagai et al., 2005), whereas positively charged residues play an important role in substrate translocation of Agrobacterium VirB/D T4SS (Vergunst et al., 2000; 2005). In this study, we establish some characteristic features of the translocation signal of known Legionella Dot/Icm T4SS substrates. Using this information, we have successfully identified 19 novel Dot/Icm substrate proteins. Emerging evidence demonstrate that bacterial pathogens, as well as viruses, exploit the host ubiquitin system (Angot et al., 2007; Rytkonen and Holden, 2007). Ubiquitin, a small protein of 76 amino acids, is conjugated to substrate proteins through a cascade of reactions involving ubiquitin activating enzymes (E1), ubiquitin conjugating enzymes (E2) and ubiquitin ligases (E3). E3 ligases, in the forms of either a single peptide or a multi-subunit complex, bind to both E2 enzymes and substrates, and specifically transfer ubiquitin from the E2s to the substrates. In Legionella infections, it has been shown that anti-polyubiquitin antibodies decorate Legionellacontaining vacuoles (LCVs) using immunofluorescence microscopic analysis, suggesting that significant amount of polyubiquitinated proteins exist on LCVs (Dorer et al., 2006). The proteasome inhibitor MG132 was reported to adversely affect the intracellular growth of Legionella in mouse macrophages, illustrating the possibility that Legionella exploits the host ubiquitin-proteasome system (Dorer et al., 2006).

1308 T. Kubori, A. Hyakutake and H. Nagai 䊏

One of the novel Dot/Icm substrates we identified has a unique feature. Lpg2830 (also known as LegU2), which we named LubX (LegionellaU-box protein), contains two domains that have a striking similarity to U-boxes. A U-box is a domain closely related to a RING finger, both found among a class of eukaryotic E3 ubiquitin ligases (Koegl et al., 1999; Aravind and Koonin, 2000) and serve as a docking site for E2 ubiquitin conjugating enzymes (Zheng et al., 2000; Ohi et al., 2003). The LubX orthologue in L. pneumophila strain Paris (Lpp2887) is the first and only annotated prokaryotic protein that has domains homologous to U-boxes at the primary sequence level (Cazalet et al., 2004). Furthermore, to date no other protein, whether of eukaryotic or prokaryotic origin, is known to have multiple U-boxes. Here, we analyse the function of LubX and the roles played by the two U-boxes of LubX.

Results Screening of Dot/Icm T4SS substrate proteins We previously reported that a hydrophobic residue near the carboxy terminus of RalF (Leu372) is critical to its translocation by the Legionella Dot/Icm T4SS (Nagai et al., 2005). When we aligned the putative translocation signals of known Dot/Icm T4SS substrate proteins, we noticed several features regarding the occurrence of amino acids near the hydrophobic residues with which the sequences were aligned (Fig. S1). One of the most striking characteristics was that amino acids having tiny side-chains (alanine, glycine, serine and threonine) are frequently found at -8th to -2nd residues (from the hydrophobic residue). At the -2nd residue, the frequency of tiny amino acids (in ~80% of known substrates) is extremely high compared with that of all open reading frames (ORFs) from L. pneumophila strain Philadelphia-1 (24%). Second, polar amino acids are clearly favoured at -13th to +1st residues (in 65% of known substrates compared with 48% of all ORFs). This may reflect the solvent-exposed nature of translocation signals. Encouraged by these findings, we established a program to calculate ‘similarity score to known Dot/Icm substrates’ for any given protein from the frequency of occurrences of particular kinds of amino acids in the carboxy-terminal region spanning from -11th to +1st residues after alignment on the hydrophobic residue. The distributions of the ‘similarity scores’ of all Legionella proteins and of known effector proteins are fairly well separated but have some overlap (Fig. S2). Thus, we would expect to find Dot/Icm substrates more frequently among Legionella proteins with higher ‘similarity scores’. To evaluate the ‘similarity score’, we examined 52 proteins with the highest ‘similarity scores’ among L. pneumophila strain Philadelphia-1 proteins. We excluded: (i) four proteins (SidE, SidF, SidG and LidA) that had already been reported as Dot/Icm substrates, (ii) 16 housekeeping pro-

Fig. 1. Identification of novel Dot/Icm substrates. CHO-FcgRII cells were challenged by Legionella strains expressing Cya fusion proteins to the indicated Legionella proteins. cAMP levels in infected cells were determined as described in Experimental procedures. Black bars denote cAMP level in cells infected by wild-type Legionella strains (Lp01) expressing Cya fusions. Grey bars denote cAMP levels in cells infected by dot/icm mutant strains. Translocation of Cya-Lpg0563 and Cya-Lpg1930 from the dot/icm mutant was not tested (asterisks). RalF is included as positive control. Data are represented as mean ⫾ standard deviation.

teins that are conserved among many bacteria, (iii) six proteins that have more than two putative transmembrane domains or that are predicted to be outer membrane or periplasmic proteins and (iv) one protein that we had difficulty in cloning by polymerase chain reaction amplification. We constructed Legionella strains expressing Cya fusions to the remaining 25 proteins, and determined intracellular cAMP levels of CHO cells expressing FcgRII after infection with Legionella. Cya fusion is a remarkably sensitive reporter system to monitor protein translocation from bacteria to eukaryotic host cells, and it has now been widely used to examine protein translocation by Legionella (Sory and Cornelis, 1994; Chen et al., 2004; Bardill et al. 2005; Nagai et al., 2005). As shown in Fig. 1, most Cya fusion proteins tested were translocated in a Dot/Icm T4SS-dependent manner with two exceptions (Lpg0563, Lpg1930). Among the remaining 23 proteins that we demonstrated are Dot/Icm substrates (Fig. 1), Lpg1958 (LegL5), Lpg1890 (LegLC8), Lpg2155 (SidJ) and Lpg2508 (SdjA) have been recently reported as Dot/Icm substrates by other groups as well (Table 1) (de Felipe et al., 2005; Liu and Luo, 2007). These results illustrate that we are able to

© 2008 The Authors Journal compilation © 2008 Blackwell Publishing Ltd, Molecular Microbiology, 67, 1307–1319

Legionella E3 ubiquitin ligase 1309

Table 1. Summary of identified Dot/Icm T4SS substrates in this study. Substrates

Orthologues in L. pneumophila

Lpg No.

Alias

Philadelphia-1

Paris

2830 81 1751 365 1148 2744 2327 1689 963 1158 1273 1717 2155 634 2508 2407 1958 1890 294 2527 518 45 1588

LubX

¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥

¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥

SidJ SdjA LegL5 LegLC8

LegC6a

Homologues in Philadelphia-1 Lens ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥ ¥

¥ ¥ ¥ ¥ ¥ ¥

No. 0 0 0 1 0 0 >4 0 0 0 0 1 1 0 1 0 >4 1 0 1 0 0 3

Best E-value

Notes 2 U-boxes 1 CC

2 ¥ 10-4 1 CC, 2 TMD -27

1 ¥ 10

3 CC 2 TMD 2 TMD

3 ¥ 10-15 0

2 homologues in Coxiella

0 5 ¥ 10-45 4 ¥ 10-9

1 LRR 1 CC, 1 LRR

3 ¥ 10-15

1 CC 2 TMD

2 ¥ 10-24

3 CC

a. LegC6 was not shown to be a Dot/Icm substrate previously. CC, putative coiled-coil; TMD, transmembrane; LRR, leucine-rich repeat.

identify novel Dot/Icm substrates efficiently by the strategy utilizing the ‘similarity score’. Lpg2830 (LubX) has two U-boxes Most of the novel Dot/Icm substrates we identified have no significant homology to proteins or domains deposited in databases, although some of them have putative coiled-coil, transmembrane and/or leucine-rich repeat domains (Table 1). By contrast, Lpg2830, which we named LubX (also known as LegU2), has two domains that have a remarkable similarity to U-boxes (36% and 35% identity to pfam04564 respectively; Fig. 2A). Because bacterial cells do not have the ubiquitin system, we hypothesized that LubX functions as an E3 ubiquitin ligase in host cells rather than in bacterial cells. Furthermore, only LubX and its strain Paris orthologue Lpp2887 were found to have multiple U-box domains. Therefore, analysis of LubX could shed light on how U-boxcontaining proteins mediate cellular processes involving ubiquitins. This led us to explore more closely the expression, translocation and function of LubX. LubX is expressed and translocated in Legionella-infected cells We examined the expression and translocation of endogenously encoded LubX. The LubX antibody that we raised

and purified strongly reacted to the purified LubX protein (Fig. 2B); however, we were not able to detect LubX in lysates of Legionella grown in ACES-buffered yeast extract (AYE) liquid medium or grown on charcoal-yeast extract (CYE) solid medium. By contrast, an effector protein RalF was detected in all lysates and, as reported previously, its expression is induced upon entering stationary phase of growth (Fig. 2B) (Nagai et al., 2002). A component of the Dot/Icm T4SS, DotD, showed a similar expression profile, while the level of the bacterial chaperonin GroEL remains mostly unchanged. These data demonstrate that LubX is not expressed in AYE/CYEgrown Legionella or, if expressed, the level of expression is lower than the detection limit of the experiment. Thus, we decided to examine the LubX levels in Legionella-infected cells. CHO-FcgRII cells were challenged by Legionella strains and, at 10 h after infection, cells were extracted with a buffer containing 1% digitonin. It has been reported that 1% digitonin extraction does not liberate bacterial proteins from bacterial cells (Derre and Isberg, 2005). Consistently, bacterial DotD and GroEL proteins were detected exclusively in the digitonininsoluble fractions (Fig. 2C). Eukaryotic cytoplasmic proteins Hsp90 and b-tubulin were recovered mostly in the digitonin-soluble fractions. LubX was detected exclusively in the digitonin-soluble fraction from lysates of CHO-FcgRII cells challenged by wild-type Legionella. As expected, infection with the lubX deletion mutant did not yield the



Fig. 2. Expression and translocation of endogenously encoded LubX. A. Schematic drawing of LubX domain structure. LubX consists of two domains that have a remarkable similarity to U-boxes and its carboxy-terminal domain (CTD) contains a translocation signal. B. Protein levels of LubX, RalF, DotD and GroEL in wild-type Legionella grown in AYE liquid media for various times after inoculation or grown for 2 days on CYE solid media. C. LubX is expressed and translocated in infected CHO-FcgRII cells. CHO-FcgRII cells were challenged by Lp01 or isogenic DdotA or DlubX mutants. At 10 h after infection, cells were lysed in buffer containing 1% digitonin, and digitonin-insoluble and -soluble fractions were separated by centrifugation. LubX was pulled down by immunoprecipitation with anti-LubX antibody and immunoprecipitates were analysed by Western immunoblotting with anti-LubX antibody (IP: IB: LubX). As a control, pre-immunoprecipitation samples were analysed by Western immunoblotting with indicated antibodies (IB). GroEL and DotD are Legionella proteins. Hsp90 and b-tublin are cytoplasmic proteins of CHO-FcgRII cells. D. LubX levels in digitonin-soluble fractions prepared from CHO-FcgRII cells infected for 10 h with indicated strains were analysed as in C. E. Lub X levels in digitonin-soluble fractions prepared at indicated time points after infection from Lp01-infected CHO-FcgRII cells.

signal, and infection with the complemented strain (DlubX plubX) restored the signal (Fig. 2D). The time-course experiment demonstrated that LubX was readily detectable in digitonin-soluble fractions of infected CHO-FcgRII cells taken at 8, 10 and 12 h after infection (Fig. 2E). The DdotA strain that has defective Dot/Icm T4SS does not grow in CHO-FcgRII cells, resulting in lower levels of bacterial proteins detected (Fig. 2C). Thus, currently we are not sure whether LubX is expressed in the dot/icm mutant strain in infected cells. Importantly, LubX was detected only in the digitonin-soluble fraction, suggesting that the LubX protein is not in the bacterial cells but translocated to the host cells. LubX expression was also induced upon infection to U937 macrophages (Fig. S3A). In addition, luciferase activities of Legionella strain carrying the lubX-luciferase transcription fusion reporter were ~10fold increased at 12 h after infection to mouse macrophages or CHO-FcgRII cells (Fig. S3B). From these results, we conclude that expression of endogenously encoded LubX is induced in infected cells, and the expressed LubX is mostly translocated into the host cells. LubX is an E3 ubiquitin ligase We hypothesized that LubX is an E3 ubiquitin ligase. To

test this hypothesis, we carried out reconstitution experiments in vitro with purified ubiquitin, E1, E2 and a carboxy-terminal truncated form of LubX (LubXDC). We used LubXDC instead of full-length LubX because the purified full-length LubX forms large aggregates and the truncation resolved this problem (data not shown). The truncation removes the 25 carboxy-terminal amino acids that may carry the signal responsible for LubX translocation from bacteria, while retaining both U-boxes. Many U-box/RING finger type E3 ubiquitin ligases have autoubiquitination activity in in vitro reactions in the absence of specific substrates. We examined UbcH2, UbcH3, UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7 and UbcH10 as E2 enzymes, and found that in the presence of either UbcH5a or UbcH5c, polyubiquitin chains were formed in a LubX-dependent manner (Fig. 3A). Omission of any one component (E1, UbcH5a or LubXDC) from the reactions completely abolished the formation of the polyubiquitin chains (Fig. 3B, IB:Ubiquitin). High-molecularweight derivatives of LubXDC were detected (Fig. 3B, IB:LubX), indicating that LubXDC has autoubiquitination activity like many other U-box/RING finger E3 ligases. We thus conclude that LubX functions as a U-box/RING finger type E3 ubiquitin ligase in conjunction with an E2 enzyme, either UbcH5a or UbcH5c.



Fig. 3. LubX is an E3 ubiquitin ligase. A. In vitro reactions containing purified ubiquitin, E1, the indicated E2 and LubXDC were carried out as described in Experimental procedures. Samples were analysed by Western immunoblotting with anti-ubiquitin antibody (clone P4D1). B. Reactions were reconstituted with the indicated components, and were analysed by Western immunoblotting with anti-ubiquitin, anti-LubX (E3) and anti-UbcH5 (E2) antibodies. C. Multiple alignment of amino acid sequences of U-boxes from indicated U-box containing proteins. Residues that compose the hydrophobic patch important for E2 binding are shown in bold face. The site of the isoleucine to alanine substitutions is indicated by an asterisk. Sc Ufd2 and At PUB14 stand for Saccharomyces cerevisiae Ufd2 (swissprot accession P54860) and Arabidopsis thaliana PUB14 (swissprot accession Q8VZ40) respectively. Experimentally determined secondary structure of PUB14 is indicated above the alignment. D. LubXDC carrying the I39A mutation in U-box 1 is inactive in in vitro polyubiquitination formation. The I134A mutation in U-box 2 has no effect.

U-box 1 is critical for ubiquitin ligation Next, we asked which U-box is responsible for the formation of polyubiquitin chains. U-box/RING finger domains have a hydrophobic patch that is critical for E2 binding (shown in bold letters in Fig. 3C) (Zheng et al., 2000; Ohi et al., 2003; Andersen et al., 2004). Alanine substitutions in these hydrophobic residues typically impair E2 binding and ubiquitin ligase activity. Alignment of the U-boxes of LubX along with eukaryotic U-box containing proteins revealed that U-box 1 retains all of these hydrophobic residues intact whereas U-box 2 lacks the invariant proline in Loop 2 (Fig. 3C). This raised the possibility that U-box 1 functions as an E2 binding domain whereas U-box 2 does not. To test this possibility, we introduced alanine substitutions to isoleucines 39 and 134 in U-boxes 1 and 2 respectively (shown by the asterisk in Fig. 3C). It has been shown that alanine substitutions of the corresponding residues of RING type E3 ligases BRCA1 and CNOT4 abolish E2 binding and/or ubiquitin ligase activity (Albert et al., 2002; Brzovic et al., 2003). As shown in Fig. 3D, the ubiquitin ligase activity of LubX is completely abolished by the introduction of the I39A mutation, suggesting that U-box 1 functions as E2 binding domain. Consistent with the lack of the invariant proline in U-box 2,

the I134A mutation had no effect. These data demonstrate that U-box 1 is critical to the ubiquitin ligation by LubX.

Clk1 is a substrate of LubX ubiquitin ligase As an E3 enzyme, LubX must have a binding site for a substrate protein or an adaptor molecule that interacts with a substrate as in the case of SCF (Skp1-Cullin-F-box protein) complexes. To identify such molecules, we conducted a yeast two-hybrid screening of a mouse cDNA library using LubXDCI39A as bait. The I39A mutation was employed to suppress any possible polyubiquitination and degradation of a target protein. The screening revealed a two-hybrid interaction between LubX and Clk1(Cdc2-like kinase 1; Fig. 4A). Clk1 showed a two-hybrid interaction with wild-type LubXDC, but the b-galactosidase readout of the interaction was significantly lower than with the I39A mutant (Fig. 4A). To confirm the interaction of LubX and Clk1 in mammalian cells, we conducted coimmunoprecipitation analysis. CHO cells were co-transfected with HA-tagged Clk1, and green fluorescent protein (GFP) alone, GFP-tagged LubX or LubXDC and cell lysates were prepared at 24 h after transfection. As shown in Fig. 4B, HA-Clk1 was co-immunoprecipitated with anti-LubX anti-



Fig. 4. Interaction of LubX and Clk1. A. Summary of yeast two-hybrid analysis of interaction of LubX and Clk1. Schematic drawings of bait LubX fusions are shown in left-most panel. The prey panel indicates the appearance of yeast cells (EGY48 pSH18-34) carrying indicated bait LubX and prey Clk1 plasmids grown on Xgal plate containing galactose to induce the expression of prey fusion products. Black bars in the graph denote b-galactosidase activity of yeast expressing indicated bait LubX and prey Clk1 fusions. Grey bars denote b-galactosidase activity of yeast carrying the indicated bait and prey vector plasmids. B. Coimmunoprecipitation of mEGFP-LubX and HA-Clk1 ectopically expressed in CHO-FcgRII cells. CHO-FcgRII cells were co-transfected with expression plasmids of indicated proteins and cell lysates were subjected to immunoprecipitation with anti-HA (clone 3F10) or anti-LubX antibodies (IP). Immunoprecipitates were analysed by Western immunoblotting with indicated antibodies (IB). C. GST pull-down assay showing direct interaction of LubX U-box 2 and Clk1. Purified GST or GST-tagged LubX U-box 2 was mixed with purified Clk1mycHIS (input). The mixtures were incubated with glutathione beads, and unbound and bound proteins were analysed by Western immunoblotting using anti-myc antibody.

body from lysates of CHO cells coexpressing HA-Clk1 and GFP-LubX or GFP-LubXDC, but not from lysate of cells coexpressing HA-Clk1 and GFP. GFP-LubXDC appeared to be more potent to pull HA-Clk1 down than GFP-LubX (Fig. 4B), which raises the possibility that the carboxy-terminal domain of LubX could play a regulatory role in LubX function. Furthermore, we showed co-immunoprecipitation of HA-Clk1 with anti-myc antibody from lysates of CHO cells coexpressing HA-Clk1 and myc-LubXI39ADC (Fig. S4), suggesting that the co-immunoprecipitations were not mediated by GFP or myc tag moieties of LubX fusion proteins. The data indicate that LubX interacts with Clk1 in mammalian cells as well as in yeast cells. To further substantiate the interaction of LubX with Clk1 and, more importantly, to determine whether Clk1 is a substrate of the LubX ubiquitin ligase, we examined whether Clk1 is ubiquitinated in an in vitro ubiquitination assay. Clk1 with both carboxy-terminal myc and hexahistidine tags was expressed in Escherichia coli and purified, and included in in vitro ubiquitination reactions. Highmolecular-weight products derived from Clk1mycHIS were detected in a LubX dose-dependent manner

(Fig. 5A). Omission of any single component (ubiquitin, E1, UbcH5a or LubXDC) from the reaction totally abolished the production of the high-molecular-weight species (Fig. 5B). As expected, the I39A mutant form of LubXDC did not support the production of the high-molecularweight species (Fig. 5B). To examine whether the high-molecular-weight species contain ubiquitin, we immunoprecipitated Clk1mycHIS derivatives with antimyc antibody, and immunoprecipitates were analysed by Western immunoblotting either with anti-myc antibody or with anti-ubiquitin antibody (Fig. 5C). The data indicate that high-molecular-weight species are composed of polyubiquitinated Clk1mycHIS. We then examined whether LubX is able to ubiquitinate Clk1 in mammalian cells. HA-Clk1 was immunoprecipitated with anti-HA antibody from lysates of CHO cells coexpressing HA-Clk1 and GFP-LubXDC or GFP-LubXDCI39A. Immunoprecipitates were analysed by Western immunoblotting with either anti-HA or antiubiquitin antibodies. As shown in Fig. 5D, the amounts of polyubiquitinated HA-Clk1 were increased in lysates of cells producing HA-Clk1 and GFP-LubXDC compared with lysates of cells producing HA-Clk1 and GFP-LubXDCI39A.



Fig. 5. Clk1 is a substrate of LubX. A–C. In vitro ubiquitination assays including ubiquitin, E1, UbcH5a as E2, LubXDC as E3 and Clk1mycHIS. A. In vitro reactions contain purified Clk1mycHIS and increasing amounts of LubXDC (0–1.8 mM). Proteins in the reaction were analysed by Western immunoblotting using anti-myc antibody. High-molecular-weight species of Clk1mycHIS was generated in a LubX dose-dependent manner. B. Reactions were reconstituted with the indicated components. The data demonstrate that all components (ubiquitin, E1, E2 and LubXDC) were required for generation of high-molecular-weight species of Clk1mycHIS, and that introduction of the I39A mutation totally abolishes the generation of high-molecular-weight species of Clk1mycHIS. C. Proteins in in vitro reactions were immunoprecipitated with anti-myc antibody and analysed by Western immunoblotting using anti-myc and anti-ubiquitin antibodies. The data indicate that Clk1mycHIS is polyubiquitinated in a LubX-dependent manner. D. LubX-dependent polyubiquitination of HA-Clk1 in CHO-FcgRII cells. Immunoprecipitates with anti-HA antibody from lysates of CHO-FcgRII cells producing indicated proteins were analysed by Western immunoblotting with anti-HA or anti-ubiquitin antibodies (IP:HA IB:HA or IB:Ub). Two different exposures of the same sample (IP:HA IB:HA) were shown. Levels of HA-Clk1 and mEGFP-LubX were not affected by the I39A mutation (IP:HA IB:HA weak exposure and PreIP IB:LubX respectively).

By contrast, levels of HA-Clk1 and GFP-LubXDC derivatives were not affected by the I39A mutation. It should be noted that proteasome inhibitor was not employed in this experiment because the treatment of cells expressing HA-Clk1 with MG132 or other proteasome inhibitors we tested resulted in a rapid loss of HA-Clk1 for unknown reasons (data not shown). This would explain, at least in part, the low abundance of polyubiquitinated HA-Clk1 detected in the experiment. We do not know why HA-Clk1 level is severely reduced in response to proteasome inhibitors, but it is possible that proteasome inhibitor treatment triggers the HA-Clk1 degradation via a proteasomeindependent pathway such as a calpain protease pathway. Reduced levels of luciferase and b-galactosidase reporter proteins in response to proteasome inhibitor treatment have been reported (Deroo and Archer, 2002). We also

examined ectopically expressed HA-Clk1 levels in CHOFcgRII cells challenged with Legionella strains, and found that the HA-Clk1 levels were not significantly affected by the lubX deletion or LubX overexpression (data not shown). Thus, our data indicate that LubX is capable of catalysing polyubiquitination of Clk1 in CHO cells, although we were not able to determine whether the polyubiquitinated Clk1 is targeted to the proteasome system. Taken these results together, we conclude that Clk1 is a substrate of LubX ubiquitin ligase. Non-canonical role of U-box 2 in substrate binding LubX is a small protein consisting of two U-boxes and a small carboxy-terminal domain. As described above, U-box 1 serves as the E2 binding site, and the carboxy-



terminal domain is dispensable for interaction with polyubiquitination of Clk1. Moreover, the I39A mutation does not have an adverse effect on the interaction of LubX and Clk1. All of this is consistent with the model that U-box 2 is the binding site for Clk1. To confirm this, we constructed bait fusions containing U-box 1 alone and U-box 2 alone, and examined the two-hybrid interactions (Fig. 4A). The data clearly indicate that U-box 2, but not U-box 1, interacts with Clk1. Furthermore, we examined the direct interaction of Clk1 and LubX U-box 2 by GST pull-down assay using purified proteins (Fig. 4C). As expected, GST-U-box 2, but not GST, was able to pull down Clk1mycHIS. Together, we conclude that LubX has a non-canonical U-box domain that plays a critical role in substrate binding rather than in E2 binding. Clk kinases are required for maximum Legionella growth in mouse macrophages We were not able to detect any defect in virulence traits, including intracellular growth in mouse macrophages (Fig. 6A, open squares versus open circles), or in protozoan cells (data not shown) following LubX disruption. However, identification of Clk1 as a substrate of effector LubX raised the possibility that Clk1 plays a role in modulation of host cellular processes by Legionella infection. Clk kinases phosphorylate serine/arginine-rich proteins (SR proteins) (Colwill et al., 1996), which in turn modulate alternative splicing-site selection (Prasad et al., 1999; Hartmann et al., 2001; Schwertz et al., 2006). Mammalian cells have four Clk kinases (Clk1 to Clk4). Muraki et al. (2004) developed a highly specific and membranepermeable inhibitor of Clk kinases, TG003. TG003 inhibits kinase activities of Clk1 and its closest homologue Clk4 in vitro, whereas it is less effective on Clk2 and it does not inhibit Clk3 or various other protein kinases examined (Muraki et al., 2004). In the presence of 10 mM TG003, Legionella growth in mouse macrophages was reduced by 10 fold over 3 days after infection (Fig. 6A, filled circles). TG003 affected neither viability of macrophages (Fig. 6B) nor Legionella growth in AYE medium (Fig. 6C). These results suggest that Clk kinases play an important role in Legionella growth in macrophages.

Discussion Effector proteins play a central role in bacterial pathogenesis. Legionella has the Dot/Icm T4SS to translocate effector proteins. Mutant Legionella that have defective Dot/Icm T4SS are avirulent, and show severe defects in many virulence traits and altered host responses. Recent studies conducted by many research groups have revealed that more than 30 proteins are translocated by the Dot/Icm T4SS (Ninio and Roy, 2007).

Fig. 6. Effect of Clk kinase inhibitor TG003 on Legionella growth in mouse macrophages. A. Legionella growth in mouse bone marrow-derived macrophages. 10 mM TG003 (filled symbols) or solvent DMSO (final 1%; open symbols) were added at 30 min before infection and kept throughout the experiment. Circles, squares and triangles denote wild-type Lp01, DlubX and DdotA mutants respectively. B. TG003 did not affect macrophage viability. Macrophages were treated with 10 mM TG003 or 1% DMSO for indicated periods of time, followed by MTT assay to determine viability of macrophages. C. TG003 did not affect Legionella growth in AYE medium. Wild-type strain was grown in AYE medium at 37°C and OD600 of cultures were scored with a time-course.

However, with a few exceptions, the cellular functions of these putative effector proteins remain unknown. In this study, we identified novel Dot/Icm T4SS substrates by a systematic screening involving prefiltration of candidates by similarity in characteristics of translocation signals to known Dot/Icm substrates. Our strategy works fairly well, and we identified 19 novel Dot/Icm substrates by examining 52 candidate proteins selected by in silico pre-screening. The starting candidates contained eight previously reported substrates (SidE, SidF, SidG, LidA, LegL5, LegLC8, SidJ and SdjA) (Conover et al., 2003;



Luo and Isberg, 2004; de Felipe et al., 2005; Liu and Luo, 2007). Thus in this screening, the ratio of novel Dot/Icm substrates to previously identified substrates is roughly 2.5:1. If we simply extrapolate from this ratio and the number of previously identified Dot/Icm substrates, our prediction would be that Legionella has ~100 Dot/Icm substrates in total. Most of the novel Dot/Icm substrates we identified have no significant homology to proteins or domains deposited in databases (Table 1). However, LubX is a clear exception to this situation. LubX has two domains that show remarkable similarity to U-boxes conserved among a class of eukaryotic E3 ubiquitin ligases. Emerging evidence indicates that bacterial pathogens as well as viruses exploit the host ubiquitin system. Some bacterial effector proteins have been shown to mimic E3 ubiquitin ligase (Janjusevic et al., 2006; Zhang et al., 2006; Rohde et al., 2007), or F-box protein in the SCF type ubiquitin ligase complex (Tzfira et al., 2004; Angot et al., 2006). As the body of genomic information on bacterial pathogens grows, it is becoming clear that pathogenic bacteria including Legionella have a number of putative F-boxcontaining proteins (Angot et al., 2007). However, U-boxcontaining proteins are rarely found in the prokaryotic realm. Our attempt to find a prokaryotic U-box-containing protein, other than LubX and its orthologue, by sequence homology has failed. Recent studies demonstrated that the plant pathogen Pseudomonas syringe translocates the E3 ubiquitin ligase, AvrPtoB, to prevent programmed cell death of plant cells (Abramovitch et al., 2006; Janjusevic et al., 2006; Rosebrock et al., 2007). A carboxyterminal domain of AvrPtoB has remarkable structural homology to U-boxes, although AvrPtoB does not have significant sequence homology to U-boxes (Janjusevic et al., 2006). This illustrates the possibility that bacterial pathogens have more proteins containing a domain that structurally and functionally mimics a U-box than one can currently predict from available genomic information. Having two U-boxes is a distinctive feature of LubX and its orthologue that is not found in any other protein so far known. Our data indicate that one of the U-boxes (U-box 1) is a canonical U-box that is critical to ubiquitin ligase activity. U-box 2 interacts with Clk1 which is a substrate of LubX ubiquitin ligase. Why does the substrate binding site of LubX adopt a U-box? Some U-box/RING finger proteins have been shown to form homo- or heterodimers. Recent structural studies have demonstrated U-box-mediated dimerization of CHIP (carboxy terminus of Hsp70interacting protein) and Prp19 U-box type E3 ubiquitin ligases (Zhang et al., 2005; Vander Kooi et al., 2006). Interestingly, a homodimer of CHIP was reported to arrange in an asymmetric manner (Zhang et al., 2005). The important consequence of the asymmetric arrangement is that only one of the two U-boxes in the CHIP

homodimer is available for interaction with an E2 enzyme. Thus, the two U-boxes in a homodimer could have distinct functions in addition to serving as a dimerization interface. The two U-boxes of LubX could be arranged in the same way as U-boxes in CHIP or Prp19 homodimers, which bring substrate bound to U-box 2 in close proximity to the active site of E2 bound to U-box 1. Alternatively, substrate binding to U-box 2 could be reminiscent of protein–protein interaction between U-box/RING finger domains, although we failed to detect primary or higher-order structural similarity to U-box/RING finger in Clk1. In spite of accumulating knowledge from recent studies, the details of the ubiquitin ligation processes are not yet fully elucidated. Future structural studies of LubX will give new insight into the mechanism of ubiquitin conjugation by U-box/RING finger type E3 ubiquitin ligases. The expression of LubX is regulated differently from those of known Dot/Icm substrates. We failed to detect LubX in bacterial cells grown in laboratory media. However, we demonstrated that LubX is expressed and translocated in infected cells, suggesting that expression is induced upon exposure to a host cell environment. To date, no Dot/Icm substrates have been reported to have a similar expression profile. The reason why LubX expression is repressed in free-living bacteria is not clear, but expression on demand makes sense if LubX functions in the host cells at a later stage of infection. Consistent with this idea, the microarray analysis of Legionella gene expression demonstrated that lubX expression is upregulated by approximately threefold at 14 h post infection in amoeba host Acanthamoeba castellanii compared with at 8 h post infection (Bruggemann et al., 2006). Alternatively, the repression of LubX expression could be a strategy to avoid the formation of LubX aggregates that are likely to be transport-incompetent. However, we are currently uncertain whether (overexpressed) LubX forms aggregates in Legionella cells. We demonstrated that Clk kinases are required for maximum Legionella growth in mouse macrophages (Fig. 6A). Together with being a substrate of LubX, Clk1 seems to be a host target protein which Legionella modulates during infection. Currently, the role of Clk1 modulation in Legionella virulence traits and the role of LubX in the Clk1 modulation remain unclear. Clk1 phosphorylates SR proteins and is involved in splicing site selection. Legionella modulation of Clk1 might affect gene expression of infected host cells at the splicing level. Alternatively, Clk1 might have a distinct role in signalling that is a target of Legionella subversion. The possibility of a signalling role has been suggested by the activation of protein tyrosine phosphatase 1B by Clk1/2 (Moeslein et al., 1999). It was recently reported that the IpaH family type III effectors, Shigella IpaH9.8 and Salmonella SspH 1, are E3 ligases that target host cellular kinases involved



in MAPK and NF-kB signalling cascades respectively (Rohde et al., 2007). It would be interesting to examine if Clk1 polyubiquitination by LubX has a signalling role. One possible explanation for why we were not able to detect any defect in virulence traits due to LubX disruption could be that Legionella employs a complicated mechanism to modulate Clk1 of which LubX is only a part. For example, in silico analysis revealed that L. pneumophila strain Philadelphia-1 has five putative F-box-containing proteins (Angot et al., 2007). Some of the F-box proteins might participate in Clk1 ubiquitination as well. Although many questions remain, we believe that our results provide novel insights for understanding exploitation of the host ubiquitin system and Clk1 by bacterial pathogens.

Experimental procedures Bacterial and yeast strains, plasmids and media All Legionella strains used in this study were derivatives of L. pneumophila strain Lp01 (Berger and Isberg, 1993), and were grown on CYE plates or in AYE broth as described previously (Feeley et al., 1979). The strain defective in dotA gene was described (Zuckman et al., 1999). In-frame deletion strains of Dot/Icm substrates were constructed by allelic exchange as described previously (Zuckman et al., 1999). E. coli strain BL21 (strain B lon ompT) was used as an expression host strain for protein purification. Yeast strain EGY48 (MATa trp1 his3 ura3 leu2::6 LexAop-LEU2) was used for yeast two-hybrid analysis. Plasmids used in this study and details of plasmid construction are provided in Tables S1 and S2 respectively.

Cell culture Mouse bone marrow-derived macrophages were cultured from A/J mice as described (Celada et al., 1984). CHO-FcgRII cells were cultured at 37°C in 5% CO2 in a-MEM supplemented with 10% FBS as described (Kagan and Roy, 2002).

Antibodies Rabbit antisera against DotD, GroEL or LubX were raised by immunization of KHL-conjugated synthesized peptides CAKVEKVITPPSKDNTLT (DotD), CVVNKVAEHKDNYGFNAA (GroEL) or purified LubXDC respectively. Polyclonal antibodies against DotD, GroEL or LubX were purified from the antisera by affinity chromatography using peptide-conjugated SulfoLink resins (Pierce) or LubXDC-conjugated CNBractivated Sepharose (Sigma) respectively. The affinitypurified rabbit polyclonal antibody against RalF was described previously (Nagai et al., 2002). A horseradish peroxidaseconjugated mouse monoclonal antibody to ubiquitin (clone P4D1) was purchased from Santa Cruz Biotechnology. Antibodies against c-Myc (clone 9E10, Sigma), HA (clone 3F10, Roche), Hsp90 (BD Pharmingen) and b-tubulin (Sigma) were also purchased. Rabbit polyclonal antibody against L. pneumophila (ATCC33152) was purchased from Biodesign. Anti-

HA-agarose and mouse IgG-agarose were purchased from Sigma.

Screening of Dot/Icm substrates For in silico pre-screening, a Perl script was written to calculate the ‘similarity score’ (see Results) of all Legionella putative ORFs. The score was calculated from frequencies of tiny amino acids (alanine, glycine, serine and threonine; residues -11 to -1), polar amino acids (aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine and threonine; residues -4 to -2) and amino acids (lysine, arginine, serine, threonine, asparagine and glutamine; residue +1). Translocation of Cya-fused proteins into CHO FcgRII cells after infection with Legionella was assayed as described previously (Nagai et al., 2005) with minor modifications. Briefly, CHO-FcgRII cells replated in 48-well plates were challenged by Legionella strains expressing Cya fusions at moi 30 in the presence of opsonizing antibody (1:3000 dilution). One hour later, infected cells were lysed in 250 ml of lysis reagent 1B provided in cAMP Biotrak EIA System (GE Healthcare, RPN2251), and cAMP levels were determined as instructed by the manufacturer.

Fractionation of infected cells CHO-FcgRII cells replated in a 6-well culture dish were challenged by Legionella strains at a moi of 30 in the presence of opsonizing antibody (1:3000 dilution). At 8 h post infection, the cells were washed three times with PBS and lysed in 150 ml of PBS containing 1% (w/v) of digitonin and protease inhibitor cocktail (1:100 dilution, Sigma). The cells were scraped off and collected into microfuge tubes and centrifuged at 16 000 g for 10 min at 4°C to separate the digitoninsoluble fraction containing translocated proteins and a digitonin-insoluble fraction containing internalized bacteria. Digitonin-soluble fractions were filtrated through a 0.45 mm filter. Digitonin-insoluble pellets were dissolved in 10 mM Tris HCl (pH 8.0), 1% SDS, 1 mM EDTA by boiling for 3 min. Each fraction was diluted into 1 ml of Triton buffer [50 mM Tris HCl (pH 8.0), 2% Triton X-100, 150 mM NaCl, 0.1 mM EDTA], and the resulting mixtures were centrifuged as above to remove insoluble materials. Immunoprecipitation with anti-LubX antibody was carried out as described below. Immunoprecipitates and preimmunoprecipitation samples were analysed by SDS-PAGE followed by immunoblotting with the appropriate antibodies.

Protein purification BL21 cell pellets expressing GST-LubX (or GST-LubXDC) were suspended with 80 ml of PBS containing 1 mM EDTA, 0.4 mM PMSF (Sigma), 20 mg lysozyme (Wako Chemical) and stirred for 30 min at 4°C. The lysozyme-treated cells were lysed by sonication. Triton X-100 (to a final concentration of 1%) was added to the lysate and incubated for 30 min with gentle stirring. After centrifugation (16 000 g, 20 min) to remove unsolubilized materials, the supernatant fraction was incubated with 1 ml (bed volume) of glutathione-sepharose FF (GE Healthcare) for 30 min at room temperature. Resins



were washed three times with 10 ml of PBS containing 1 mM EDTA, 1 mM DTT, 0.1% Triton X-100, once with 10 ml of PBS containing 1 mM EDTA, 1 mM DTT, once with 10 ml of PreScission buffer [50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT]. LubX (or LubXDC) proteins were eluted from resins by incubation with GST-conjugated PreScission protease (GE Healthcare), and dialysed against PBS. GST and GST-U-box 2 were purified essentially in the same way, but were eluted with 50 mM Tris HCl (pH 8.0), 10 mM reduced glutathione. GST-Clk1mycHIS was purified essentially as GST-LubX with some modifications. BL21 cells expressing GST-Clk1mycHIS were lysed in Tris-based buffer [50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT] containing 0.4 mM PMSF, and the Tris-based buffer was used instead of PBS during purification with glutathionesepharose. The PreScission reaction was carried out in PreScission/HIS buffer [50 mM Tris (pH 7.5), 150 mM NaCl, 10 mM b-mercaptoethanol]. Fractions containing eluted proteins were pooled and incubated with 100 ml of a 50% suspension of Ni-agarose (Qiagen) for 1 h at 4°C. Resins were washed three times with PreScission/HIS buffer containing 10 mM imidazole, and the Clk1mycHIS proteins were eluted with PreScission/HIS buffer containing 250 mM imidazole. Eluted proteins were concentrated by Microcon (Millipore) and dialysed against the Tris-based buffer.

Ubiquitin ligase assays The in vitro ubiquitin polymerization assay in the substratefree system was performed as follows. Reaction mixtures (12.5 ml) containing 0.5 mg (1.8 mM) of purified LubXDC or its mutant proteins (I39A or I134A), 100 nM recombinant rabbit E1 (Boston Biochem), 400 nM recombinant human E2 enzymes (Boston Biochem), and 5 mg of recombinant human ubiquitin (Boston Biochem), 50 mM Tris-Cl (pH 7.5), 2 mM MgCl2, 120 mM NaCl, 2 mM ATP and 1 mM DTT were incubated for 2 h at 30°C. The reaction was stopped by adding 12.5 ml of 2¥ SDS sample buffer and boiling for 5 min. For the in virto ubiquitin ligation assay to the substrate Clk1, 0.3 mg (500 nM) of purified Clk1mycHIS was added to the reaction mixtures. For immunoprecipitation analysis, the ubiquitin ligation was done in a 50 ml reaction volume and disrupted by adding 0.5 ml of Triton buffer. Two microlitres of anti-myc monoclonal antibody was added to the reaction mixture and incubated for 2 h at 4°C with gentle rocking. Immunocomplexes were then recovered with Protein G sepharose FF (GE Healthcare) for 1.5 h at 4°C, and washed five times with 1 ml of the lysis buffer (12 000 g, 20 s). The samples were finally washed with 50 mM Tris-Cl (pH 7.5), and eluted by boiling in SDS sample buffer without a reducing agent. The samples were loaded on a 10% SDS-PAGE in the presence of 2-mercaptoethanol and analysed by immunoblotting using a horseradish peroxidase-conjugated mouse monoclonal antibody to ubiquitin (P4D1, Santa Cruz Biotechnology), or antibodies against LubX, UbcH5 (Boston Biochem) or myc.

with a mouse liver cDNA library based on pJG4-5 (OriGene Technologies) according to the manufacture’s instruction. b-Galactosidase activity was measured in liquid culture of the yeast strains grown in selection media to OD600 of about 5. The culture was adjusted to OD600 of 1.0 with selection media and assayed in triplicate using the Beta-Glo Assay System (Promega), and the signal was read in a luminometer.

Immunoprecipitation from cell lysates For coimmunoprecipitation analysis, CHO-FcgRII cells plated on 10 cm dish (8 ¥ 105 cells) were co-transfected with 1.5 mg of pmEGFP or its LubX derivatives and 4.5 mg of pME-HAClk1 using 36 ml of Fugene 6 reagent (Roche). Twenty-four hours later, cells were washed with cold PBS three times and lysed in 1 ml of RIPA buffer containing protease inhibitor cocktail (1:100 dilution, Sigma), 50 mM sodium fluoride, 1 mM sodium orthovanadate, 10 mM glycerol 2-phosphate and 10 mM N-ethylmaleimide. Lysates were centrifuged at 16 000 g for 10 min to remove insoluble materials. To immunoprecipitate LubX or LubX-containing complexes, cleared lysates were incubated with 40 ml of a 50% suspension of nProtein A-sepharose FF (GE Healthcare) for 1 h to pre-absorb cross-reacting proteins. After removing the resin by centrifugation, 5 mg of affinity-purified anti-LubX antibody was added to the supernatant and incubated at 4°C overnight with gentle rotation. Complexes containing antibodies were pulled down by incubation for 1 h with 40 ml of a 50% suspension of nProtein A-sepharose FF. Resins were washed three times with Triton buffer (for LubX detection in Legionella-infected cells) or RIPA buffer (for coimmunoprecipitation of LubX and Clk1), once with 50 mM Tris HCl (pH 7. 5), and immunoprecipitates were eluted with SDS-PAGE sample buffer. To immunoprecipitate HA-Clk1, cleared lysates were incubated with 40 ml of a 50% suspension of mouse IgG-agarose (Sigma) for 1 h to pre-absorb cross-reacting proteins. After removing the resin by centrifugation, 40 ml of a 50% suspension of anti-HA-agarose (Sigma) was added to the supernatant fraction and incubated at 4°C overnight with gentle rotation. Resins were washed three times with RIPA buffer, once with 50 mM Tris HCl (pH 7.5), and immunoprecipitates were eluted with SDS-PAGE sample buffer.

GST pull-down Purified GST or GST-U-box 2 (10 mg) were mixed with 1 mg of purified Clk1mycHIS in 400 ml of 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM DTT, 0.1% Triton X-100. The mixtures were mixed with 100 ml of a 20% suspension of glutathione-sepharose FF and incubated for 2 h with gentle rotation. Unbound proteins were removed by centrifugation, and resins were washed three times with the same buffer. GST and interacting proteins were eluted with 50 ml of 50 mM Tris HCl (pH 8.0), 10 mM reduced glutathione.

Yeast two-hybrid screening

Intracellular growth assay

The yeast two-hybrid screening was carried out using a DupLEX-A yeast two-hybrid system (OriGene Technologies)

Intracellular growth assays in mouse macrophages were carried out as described (Zuckman et al., 1999). 10 mM



TG003 (Sigma) or solvent 1% DMSO was added at 30 min before infection and kept throughout the experiment. Macrophage viability was monitored by a MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Sigma] assay as described (Kagan and Roy, 2002).

Acknowledgements We thank Masatoshi Hagiwara for Clk1 plasmids and discussion, May Macnab for critical reading of the manuscript. This work was supported by Grants-In-Aid (to T.K. and H.N.) and the Program of Founding Research Centers for Emerging and Reemerging Infectious Diseases (to H.N.) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Hayashi Memorial Foundation for Female Natural Scientists (to T.K.), and the Mitsubishi Foundation (to T.K.).

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