text:
Transcriptional signatures of BALB/c mouse macrophages housing multiplying Leishmania amazonensis amastigotes

Background Mammal macrophages (MΦ) display a wide range of functions which contribute to surveying and maintaining tissue integrity. One such function is phagocytosis, a process known to be subverted by parasites like Leishmania (L). Indeed, the intracellular development of L. amazonensis amastigote relies on the biogenesis and dynamic remodelling of a phagolysosome, termed the parasitophorous vacuole, primarily within dermal MΦ. Results Using BALB/c mouse bone marrow-derived MΦ loaded or not with amastigotes, we analyzed the transcriptional signatures of MΦ 24 h later, when the amastigote population was growing. Total RNA from MΦ cultures were processed and hybridized onto Affymetrix Mouse430_2 GeneChips®, and some transcripts were also analyzed by Real-Time quantitative PCR (RTQPCR). A total of 1,248 probe-sets showed significant differential expression. Comparable fold-change values were obtained between the Affymetrix technology and the RTQPCR method. Ingenuity Pathway Analysis software® pinpointed the up-regulation of the sterol biosynthesis pathway (p-value = 1.31e-02) involving several genes (1.95 to 4.30 fold change values), and the modulation of various genes involved in polyamine synthesis and in pro/counter-inflammatory signalling. Conclusion Our findings suggest that the amastigote growth relies on early coordinated gene expression of the MΦ lipid and polyamine pathways. Moreover, these MΦ hosting multiplying L. amazonensis amastigotes display a transcriptional profile biased towards parasite-and host tissue-protective processes.

Background
L. amazonensis are protozoan parasites belonging to the trypanosomatidae family. In natural settings, the L. amazonensis perpetuation relies on blood-feeding sand fly and rodent hosts. The development of promastigotes proceeds within the gut lumen of the sand fly hosts and ends with metacyclic promastigotes. The latter, once delivered into the mammal dermis, differentiate as amastigotes mainly within the resident dermal macrophage (MΦ) acting as bona fide host cells. Following the parasite inoculation and before the development of the more or less transient skin damages that characterize cutaneous leishmaniasis there is an asymptomatic phase lasting for several days or weeks during which the intracellular amastigote progeny expands. This expansion takes place within a compartment named parasitophorous vacuole (PV) that displays properties similar to late endosomes/lysosomes and the size of which grows significantly for Leishmania belonging to the mexicana complex [1,2]. In this study we sought to analyze the transcriptional signatures of a homogeneous population of MΦ derived in vitro from BALB/c mouse bone marrow CSF-1 dependent progenitors and hosting amastigotes that are actively multiplying. The Affymetrix GeneChip technology was used to compare the gene expression profiles of L. amazonensis amastigotes-hosting bone marrow-derived MΦ and parasite-free ones. This in vitro transcriptomics approach was combined with the Ingenuity biological network analysis to highlight the mouse MΦ biological processes the multiplying L. amazonensis amastigotes rely on within their giant communal PV. Our findings suggest that MΦ hosting multiplying amastigotes contribute to carve a parasite-as well as a host tissue-protective environment.

Results and Discussion
L. amazonensis amastigotes subvert MΦ as host cells where they enter a cell-cycling phase lasting several days (Fig.  1A). We compared the transcriptomes of amastigote-free MΦ and amastigote-harbouring MΦ 24 h after the uptake of amastigotes carefully purified from nude mouse lesions. At this time-point amastigotes were multiplying within a huge PV (Fig. 1B) and their population size had almost doubled (Fig. 1A). Among the 45,101 probe-sets of the Mouse430_2 GeneChip, 1,248 (2.77%) were displaying features of differential expression at the 5% significance level (Fig. 2, see Additional file 1). Of these, 1,206 matched Ingenuity Pathway Analysis database version 5.5.1 which represented 898 genes with a known function. About 80% of these genes were incorporated into either Ingenuity's canonical pathway or biological network (i.e. their products interact with other molecules in Ingenuity's knowledge base). The symbols of the modulated genes are specified in the text (fold change [FC] values between brackets), while their full names are given in Additional file 1. Furthermore, comparable FC values were obtained between the Affymetrix technology and the Real Time quantitative Polymerase Chain Reaction (RTQPCR) method (Table 1) [3].
Though transcriptional changes due to the phagocytic uptake process per se -known to occur mostly within the first 2 hours post particle addition -cannot be completely excluded, the MΦ transcript modulation -detected at 24 h post the amastigote addition -very likely reflects MΦ reprogramming due to the presence of cell cycling amas-tigotes within giant PV. Indeed, in our experimental conditions, no extracellular amastigotes could be evidenced in the MΦ culture (a) after a brief centrifugation step and (b) one hour contact with adherent MΦ indicating that the phagocytic uptake of L. amazonensis amastigotes is a rapid and efficient process. Furthermore, it is worth mentioning that the size of the amastigote population hosted within the MΦ PV rapidly expands within the first 24 h (Fig. 1A) [4]. Using also mouse bone marrow-derived MΦ as host cells for Leishmania, Gregory and coworkers demonstrated that the gene expression profiles of control MΦ and MΦ that have phagocytosed latex beads 24 h before were very similar. They evidenced a statistically significant difference for only 15 probe sets. None of the 29 corresponding probe sets in the mouse 430 DNA Affymetrix gene chip was present in the list of 1248 modulated probe sets observed in presence of L. amazonensis amastigotes. Thus, these data strongly support our conclusion that the gene expression profile observed 24 h after the phagocytosis of L. amazonensis amastigotes was due to the presence of intracellular cell-cycling parasites.

L. amazonensis amastigotes set up an optimal sub cellular niche Modulation of MΦ genes encoding vacuolar proton ATPase sub-units
Within their host cells, L. amazonensis amastigotes are known to multiply efficiently in the acidic environment of the MΦ PV [1]. In presence of amastigotes, we observed an up-regulation of the gene expression of eight isoforms of the V0 and V1 sub-units of the MΦ vacuolar proton ATPase (atp6V0a1, atp6V0c, atp6V0d2, atp6V1a, atp6V1c1, atp6V1d, atp6V1g1 and atp6V1h: +1.27 < FC < +2.32) [5]. This could contribute to the sustained acidification of the PV lumen which has been shown to be important at least for the optimal amastigote nutrient acquisition [6,7].

Coordinated modulation of MΦ lipid metabolism
The most relevant biological networks fitting our dataset were strongly associated to the function "lipid metabolism", the most significant canonical metabolic pathway being "biosynthesis of steroids" (p-value = 1.31e-02). Indeed, several up-regulated genes (Fig. 3, Table 1) were involved i) in cholesterol uptake (ldlr: + 4.68), ii) in cholesterol transport (fabp4: + 6.42 and stard4: + 2.31) and iii) in sterol biosynthesis (hmgcs1, hmgcr, mvd, idi1, fdps, fdft1, sqle, lss, cyp51, sc4mol, hsd17b7, sc5d and dhcr24: +1.95 < FC < +4.30). Worth is mentioning the most up-regulated gene encoding type II deiodinase (dio2, + 25.92), an enzyme converting intracellular thyroxin (T4) to triiodothyronine (T3), the more active form of thyroid hormone. It has previously been demonstrated in mouse hepatocytes that the molecular basis for the connection of T3 and cholesterol metabolism involves the master transcriptional activator of the aforementioned genes, namely srebf2 (+ 1.84) the promoter of which contains a thyroid  hormone response element [8]. Furthermore, thyroid hormone receptors can activate transcription of target genes upon T3 binding and this could be facilitated by co-activators ncoa4 (+ 1.65) and brd8 (+ 1.08). Interestingly, opposite to dio2, the most down-regulated gene was cholesterol-25-hydrolase (ch25h: -6.57), an enzyme acting downstream this pathway by breaking down cholesterol and by synthesizing a co-repressor of srebf2 transcriptional activation [9]. Upstream this pathway, several up-regulated genes involved in glycolysis could also contribute to increase the supply of acetate (acsl3, adhfe1, akr1a1, aldoa, aldoc, eno2, hk2, hk3, ldha, pfkl, pkg1 and pkm2: +1.13 < FC < +2.61). Of note was the down-regulation of genes encoding enzymes competing i) with hmgcs1 for acetate (acaca: -1.32) and ii) with aldoa and aldoc for fructose, 1-6, biphosphate, which is needed to produce glyceraldehyde-3-phosphate upstream the sterol biosynthesis path-way (fbp1: -2.16). In addition, the up-regulation of the transcription factor encoded by atf3 (+ 1.77) was consistent with the down-modulation of fbp1. These data suggest that the available intracellular pool of sterol-synthesis molecular intermediates was maintained by a gene expression program relying on a coordinated regulation at both the transcriptional level by srebf2, atf1 (+ 1.84) and atf3, and also most likely at the post-transcriptional level by insig1 (+ 2.62) encoding a sterol-sensing protein that regulates the intracellular cholesterol level [10].
The expression of several genes involved in the fatty acid biosynthesis pathway was also up-regulated with the modulation of ppap2b (+ 8.53), scd1 (+ 2.68), scd2 (+ 2.45) and acsl3 (+ 2.09). Moreover, genes encoding fatty acid binding proteins that play a role in fatty acid uptake and transport were up-regulated (fabp3: + 2.29, fabp4: + Time course of intracellular amastigote population size increase and MΦ culture imaging Fatty acid transport to peroxisome was diminished with abcd2 down-modulation (-2.11). Since this was not described neither for L. major nor L. donovani [11], this could be unique for the L. mexicana complex, all sub-species of which multiply within giant communal PV [1]. Indeed, previous experimental work performed with L. mexicana [12,13], which is very close to L. amazonensis (both share the same distinctive feature to multiply within a communal PV), has shown that amastigotes could take advantage of the MΦ sterol biosynthesis pathway to produce ergosterol.
These data were in agreement with the sterol biosynthesis machinery of the MΦ host cell being exploited by the cell-cycling amastigotes for both their own cell membrane sterols, in particular ergosterol and the PV membrane sterol-dependent remodelling. Indeed, cholesterol availability might play a role in the formation of the PV lipid rafts [14] that could be involved in the control of fusion events leading to the sustained remodelling of the huge communal PV membrane where the aforementioned proton pump components are regularly delivered.

Modulation of MΦ polyamine metabolism
Polyamines (e.g. putrescine) derived from arginine catabolism are essential compounds for amastigote growth [15]. Using the Affymetrix technology we failed to detect, at the 5% significance threshold, arginase-2 (arg2) and ornithine decarboxylase-1 (odc1), two enzymes leading to the formation of polyamines through arginine catabolism. Indeed, while for arg2 the raw fluorescence intensity values were below or close to the background level, for odc1 the raw fluorescence intensities before data processing displayed only a slight increase (+ 1.21) in presence of amastigotes (see Additional file 1). However, the up-regulation of SLC7A2 (+ 4.14) in MΦ hosting amastigotes was a strong incentive for monitoring the abundance of arg2 and odc1 transcripts with a validated RTQPCR method. Using this method we did detect a slight variation of the expression of arg2 (+ 1.91) and odc1 (+ 1.18) (Table 1). Therefore, in presence of amastigotes, arg2 could favour arginine transformation into ornithine, the latter being catalyzed in turn by odc1 to generate putrescine (Fig. 4).

Affymetrix outcome
ODC1-antizyme plays a role in the regulation of polyamine synthesis by binding to and inhibiting ODC1. The transcript abundance of azin1 encoding ODC1antizyme inhibitor-1 was higher (+ 1.96) when amastigotes were present, so that this inhibitor might prevent antizyme-mediated ODC1 degradation. Of note, ornithine could also be generated from proline by p4ha2 (+ 2.27), and putrescine from spermine and spermidine by the successive action of sat1 (+ 1.47) and maoa (+ 2.56).
Spermidine synthase (srm) and spermine synthase (sms), two enzymes catalyzing the reverse reactions leading to the formation of spermine from putrescine, were not detected with Affymetrix (5% threshold), although their transcript abundance decreased in presence of amastigotes (-1.22 and -1.38, respectively; see Additional file 1). No gene expression modulation was detected with Affymetrix for nos2 (5% threshold) that encodes a competing enzyme for arginine substrate leading to the production of microbe-targeting nitric oxide derivatives (fluorescence intensity was below the background level, see Additional file 1), and only a slight up-regulation was detected with RTQPCR (+ 1.28) ( Table 1). The present data further extend former observations [15,16], and highlight a coordinated gene expression modulation that sustains a metabolic flux leading to the biosynthesis of putrescine from arginine and proline via ornithine, and from spermine and spermidine. 1.35) as well as some genes of the Toll-like receptor signalling pathway (tlr2, tlr7, tlr8, cd14, mapk14, c-fos and nfkbia: -3.11 < FC < -1.61. Furthermore, the negative regulator tollip also was up-regulated (+ 1.69). These pathways are known to contribute to the entry of micro-organisms and the sensing/processing of microbial molecules. In presence of the intracellular cell-cycling amastigotes these biological processes would be restricted, if not prevented. Indeed, it is conceivable that non-Leishmania microorganisms or microbial molecules might trigger a different MΦ transcriptional program that could interfere with the one already set up by L. amazonensis amastigotes for their multiplication. Nevertheless, it has recently been demonstrated that the other L. amazonensis developmental stage, the promastigote, was still able to enter MΦ already hosting amastigotes, to transform into amastigote and to multiply efficiently within the PV [17].

L. amazonensis amastigotes set up an optimal dermis niche Decreased expression of genes involved in the entry of non leishmanial micro-organisms as well as in the sensing and processing of microbial molecules
The above data suggested that L. amazonensis amastigotes were able to control MΦ expression of the early complement components, the proteolytic products of which are known to be pro-inflammatory. This complement component pathway down-modulation was also recently described for human MΦ housing L. major parasites [18]. The down-modulation of the Toll-like receptor pathway also suggested prevention of the inflammatory process signalling. At this stage, although some anti-inflammatory genes were not up-modulated (il10: -2.97 and il10ra: -2.16) the gene expression modulation for the majority of the listed genes involved in inflammatory processes showed that the presence of cell-cycling amastigotes imposed an immune unbalance favouring the shaping of a counter-inflammatory and safe dermis niche for these parasites (il1rn, il1b, il11ra1, il17rb, il18, socs6, cd200, nfkbia, relB, c-fos and anxA1, an inhibitor of phospholipase A2 mediated-inflammation: 1.41 < | FC | < 4.19).

Decreased expression of genes involved in the chemokine-dependent MΦ traffic
The down-modulation of the expression of genes encoding chemokine receptors (ccr2, ccr3, cx3cr1 and cmklr1: -2.65 < FC < -1.83) suggested that amastigote-harbouring MΦ were less responsive to chemo-attractant gradients and thus less amenable to enter into the afferent lymphatics. This is consistent with the dominant residence of L. amazonensis-hosting MΦ in the skin. In favour of this possible reduced emigration of MΦ from the dermis niche was the down-regulation of itga4 (-2.06) encoding an integrin shown to contribute to the lymphatic adhesion/ transmigration [19]. It is beyond the scope of this article to discuss about more than a dozen of chemokine receptor ligands the gene expression of which was modulated (see Additional file 1). Indeed, the interpretation is not that straightforward because of the complexity of their partial overlapping functions and/or common receptors.

Decreased expression of genes involved in the cellular communication with leukocytes prone to display parasite-damaging functions
The modulation of several transcripts indicated a prevention of MΦ communication with leukocytes that could be rapidly recruited such as NK lymphocytes, and T-lymphocytes. For instance, H60 is one of the ligand able to efficiently activate NK-lymphocytes by binding to the NKG2D receptor (encoded by klrk1). In presence of amastigotes, the h60 MΦ expression was down-modulated (-2.07), suggesting the prevention of this "immune synapse" by which parasitized MΦ and NK lymphocytes can communicate. Interestingly, NKG2D receptor engagement by H60 ligand in MΦ, that normally leads to the production of MΦ leishmanicidal molecules such as NO and TNF-α [20], could be impaired in MΦ hosting amastigotes since the expression of klrk1 gene was also downmodulated (-1.72). Besides, the gene expression of the costimulatory molecule CD86 was reduced (-1.83), while that of the inhibitory receptor CD274 (also referred to as B7-H1) was increased (+ 1.93). In addition, the transcript abundance of the co-stimulatory molecules ICAM1 (-1.75), ICAM2 (-1.85) and LFA-1 (or integrin-alpha L, -2.0) was also reduced. The down-modulation of several genes involved in antigen presentation by MHC class II molecules was recently discussed for human MΦ housing L. major parasites [18]. This data suggested plausible reduced effectiveness of this other "immune synapse" involving TCR-dependent signalling by which MΦ and Tlymphocytes can communicate. Consistent with this was the reduced transcription level in MΦ hosting L. amazonensis amastigotes of h-2ma (-1.88) and of ifngr1 (-1.83 FC) that encodes the receptor for IFNγ, a cytokine secreted by both activated NK-and T-lymphocytes and involved upstream the MHC class II gene up-regulation.

Conclusion
The Affymetrix GeneChip technology has allowed -for many cell lineages -the global analysis of several thousand transcripts simultaneously to be carried out in a robust fashion [21]. The remarkable coordination of gene expression as well as coherent biological interaction networks displayed by MΦ subverted as host cells by the multiplying L. amazonensis amastigotes allow highlighting the power of this technology at two different levels: (a) the amastigote-hosting MΦ transcriptional features per se and (b) the features of MΦ hosting cell-cycling amastigotes which would have been captured within the dermal environment. Further in vivo quantitative analysis will have to be set up for validating or not the present transcriptional profile at early stage after the first wave of amastigote multiplication in the ear dermis of naïve BALB/c mice. Overall, the gene expression profile of MΦ hosting amastigotes did not strictly fall into either of the MΦ "activation" profiles, as it was also the case for L. chagasi [22]. Neverthe-less, consistent with the multiplication of the amastigote developmental stage, some overlap with features of the alternative MΦ activation could be observed, such as the up-regulation of arg2 and il1rn, and the down-regulation of cd14 (-1.73 FC).
In addition to the conversion of the MΦ arginine metabolism from a parasite-damaging pathway to a parasitesupportive one, the most clear-cut and novel output of the present analysis was the up-regulation of the MΦ fatty acid biosynthesis pathway. Coupled to the polyamine biosynthesis the MΦ lipids could not only be a source of nutrients for the amastigotes but could also contribute to the PV unique membrane features [2,23]. Lipids could not only influence the PV membrane curvature but also coordinate the recruitment and retention of key protein export to the PV where multiplying amastigotes are known to be attached [2]. This makes it conceivable that the multiplying amastigotes could take up trophic resources and sense non-trophic signals.
We have highlighted a promising set of transcripts accounting for the BALB/c mouse macrophage reprogrammed as cell-cycling amastigote hosting cells. We do not ignore that transcript modulation changes revealed by microarray analysis could be uncoupled to changes revealed by proteomic and phosphoproteomic analysis. We did not explore how these mRNA changes manifest at the level of the proteome but the present genomewide data will provide a unique resource (a) against which to compare any proteomic/phosphoproteomic data (b) to allow identifying novel small compounds displaying static or cidal activity towards cell-cycling amastigotes hosted within the macrophage PV. Indeed the readout assay we designed allows high content imaging in real time of (a) the amastigotes (b) the amastigotes-hosting PV as well as the macrophages per se [24] and can be upscaled for high throughput screening of small compound libraries.

Mice, MΦ and amastigotes
Swiss nu/nu and BALB/c mice were used (following National Scientific Ethics Committee guidelines) for L. amazonensis (LV79, MPRO/BR/1972/M1841) amastigote propagation and bone marrow-derived MΦ preparation, respectively. Four amastigotes per MΦ were added. Parasite-harbouring MΦ (>98%) and parasite-free ones were cultured at 34°C either for 24 h for transcriptomic studies or for different time periods for microscopy analyses [25].

Kinetic study of the intracellular amastigote population size
At different time points post amastigote addition, MΦ cultures were processed for immunofluorescence and phase contrast microscopy. Briefly, MΦ cultures on coverslips were fixed, permeabilized, incubated with the amastigotespecific mAb 2A3. 26 and Texas Red-labelled conjugate, stained with Hoechst 33342 and mounted in Mowiol for observation under an inverted microscope as previously described [25]. Ratios of amastigotes per MΦ (between 200 and 700 MΦ nuclei being counted) were calculated and expressed as mean numbers of amastigotes per MΦ at each time point.

GeneChip hybridization and data analysis
Total RNA were extracted from MΦ (RNeasy+ Mini-Kit, Qiagen), their quality control (QC) and concentration were determined using NanoDrop ND-1000 micro-spectrophotometer and their integrity was assessed [26] using Agilent-2100 Bioanalyzer (RNA Integrity Numbers ≥ 9). Hybridizations were performed following the Affymetrix protocol http://www.affymetrix.com/support/down loads/manuals/ expression_analysis_technical_manual.pdf. MIAME-compliant data are available through ArrayExpress and GEO databases http://www.ebi.ac.uk/microarray-as/ae/, accession: E-MEXP-1595; http://www.ncbi.nlm.nih.gov/ projects/geo/, accession: GSE11497). Based on AffyGCQC program QC assessment [27], hybridizations of biological duplicates were retained for downstream analysis. Raw data were pre-processed to obtain expression values using GC-RMA algorithm [28]. Unreliable probe-sets called "absent" by Affymetrix GCOS software http:// www.affymetrix.com/support/downloads/manuals/ data_analysis_fundamentals_manual.pdf for at least 3 GeneChips out of 4 were discarded, as well as probe-sets called "absent" once within samples plus once within controls. LPE tests [29] were performed to identify significant differences in gene expression between parasite-free and parasite-harbouring MΦ. Benjamini-Hochberg (BH) multiple-test correction [30] was applied to control for the number of false positives with an adjusted 5% statistical significance threshold. A total of 1,248 probe-sets showing significant differential expression were input into Ingenuity Pathway Analysis software v5.5.1 http:// www.ingenuity.com to perform a biological interaction network analysis. Although a cross-hybridization study was performed by Gregrogy and coworkers (11) on a mouse U74av2 DNA Affymetrix gene chip (12,488 transcripts) with RNA from Leishmania donovani, it was important to also assess the absence of significant crosshybridization in our experimental conditions. To this end, we compared the gene chip data obtained with MΦ RNA alone with those obtained with the same RNA preparation spiked with different amount of L. amazonensis RNA. Our data showed that L. amazonensis RNA did not interfere with mouse RNA hybridization onto GeneChips (data not shown). Indeed, fold-change values for a technical replicate of mouse RNA were not significantly different from those observed for mouse RNA spiked with up to 10% of L. amazonensis RNA taking the non-spiked mouse RNA as reference (one-sample one-sided Student's t-test P-values < 5% for all 45,101 probe-sets, the 1,248 significantly modulated probe-sets, the probe-sets of the 107 genes in Table 1 and the probe-sets of the 13 genes in Figure 3). Therefore, the observed over-expressions were not due to cross-hybridization between the mouse and the amastigote transcripts, thus providing valid information about the reprogramming of MΦ hosting cell-cycling amastigotes.

Real-time quantitative PCR
RTQPCR were performed on cDNA from various biological samples including those used for the hybridizations using a LightCycler ® 480 (Roche Diagnostics). Primer sequences are available upon request. Gene expression analysis using qBase [31] allowed determining the normalized relative quantities between parasite-free and parasite-harbouring MΦ.


extfieldsofstudy: Biology Medicine

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Inter‐conversion of catalytic abilities in a bifunctional carboxyl/feruloyl‐esterase from earthworm gut metagenome

Summary Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p‐nitrophenyl and cinnamate esters, respectively, with a [(kcat/Km)]CE/[(kcat/Km)]FAE factor of 17. Modelling‐guided saturation mutagenesis at specific hotspots (Lys281, Asp282, Asn316 and Lys317) situated close to the catalytic core (Ser143/Asp273/His305) and a deletion of a 34‐AA–long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180‐fold in kcat/Km values) and enzymes with inverted specificity ((kcat/Km)CE/(kcat/Km)FAE ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error‐prone PCR‐generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to −5.6 J mol−1), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to −13.7 J mol−1) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of ‘hot spot’ mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.

Introduction
Carboxyl-esterases (CEs; EC 3.1.1.X) are ubiquitous a/bhydrolases with representatives in all three domains of life, Eukarya, Bacteria and Archaea (Bornscheuer, 2002). These proteins catalyse the cleavage and formation of ester bonds whose components differ in chain nature and length. Within the a/b-hydrolase superfamily, CEs belong to the best structurally and functionally characterized, with more than 6016 members divided in 90 subfamilies after a comprehensive non-redundant search in the nucleotide collections, reference genomic sequences, whole genome shotgun reads and environmental samples databases. Site-directed mutagenesis (Manco et al., 2001;Reyes-Duarte et al., 2005), saturation mutagenesis (Mee-Hie Cho et al., 2006;Wang et al., 2006;Nakagawa et al., 2007) and a number of directed evolution methods (Funke et al., 2005;Ivancic et al., 2007) have successfully been applied to identify the molecular determinants of substrate specificity and enantio-selectivity and to gain some knowledge on molecular evolution of this super-family of proteins.
We focused in this study on esterases able to attack the ester bond between hydroxyl-cinnamic acids, such as ferulic, p-coumaric and sinnapic acid, and sugars present in intricate structure of the plant cell wall (Fazary and Ju, 2007). The hydrolysis of these compounds is catalysed by feruloyl esterases (FAEs; EC 3.1.1.73). About 30 FAEs have been described and based on structural and functional factors a division into four types (A-D) has been established (Crepin et al., 2004;Wong, 2006;Benoit et al., 2008). They have been purified and characterized from bacteria (Wang et al., 2004;Aurilia et al., 2007; and fungi (Shin and Chen, 2007;Kanauchi et al., 2008;Moukouli et al., 2008;Koseki et al., 2009), and showed significant variations in activities. Despite all of them having the typical a/b fold and the common CE-like catalytic triad, Ser-Asp/Glu-His (Fazary and Ju, 2007), little is known about the factors determining substrate specificity of these enzymes compared with 'common' CEs, which has inter alia motivated present work. Only few studies have demonstrated that few mutations at the ligand-binding cavity (Tarbouriech et al., 2005) or in insertions such as lids (Hermoso et al., 2004;Faulds et al., 2005;Koseki et al., 2005) have important effects in controlling the binding and hydrolysis of esters with m-methoxy groups. Following on from this, Levasseur's group studied the evolutionary relationships between fungal lipases and FAEs through the study of their phylogenies (Levasseur et al., 2006). Authors concluded that lipases appear to be primitive functions from which FAE functionality could have been derived bought duplication events probably induced by environmental stresses, i.e. those caused by new substrates. Although, authors suggest that modifications within the active site might thus be positively selected, causing the shift in functionality at the organismal level (Hermoso et al., 2004;Levasseur et al., 2006), no experimental evidence for such hypothesis has been provided.
In this work, the first systematic study was performed to investigate the structural and energetic determinants for the transition between CEs and FAEs. Here, we used a novel bi-functional CE/FAE protein (3A6) that catalyses hydrolytic cleavage of 'common' and 'cinnamate' esters, isolated from the microbial community of the gut of the earthworm Aporrectodea caliginosa. Invertebrate guts are certainly one of the most diverse environments yet studied and, in particular, the earthworm gut associated-organisms require an efficient enzymatic machinery to cope with a continuous flux of various polymeric materials (including cinnamate esters) from the soil and from plant litter passing through intestinal tract, which make it attractive for isolating hydrolytic activities. We show here by examining saturation mutagenesis and error-prone PCR libraries, that a reduced number of 'hot-spot' mutations could alter the overall substrate hydrolysis and, more importantly, evidence is presented for the first time that suggests that the acquisition of FAE phenotype, from an ancestor CE activity, is energetically favourable. Moreover, to the best of our knowledge, this work is the first example for a successful conversion of an FAE into a common CE.

General characteristics of the 3A6 protein
The fosmid carrying the 3A6 gene was isolated by screening a metagenomic library from the cellulose-enriched microbial community from the gut of the earthworm A. caliginosa, based on its ability to hydrolyse methyl ferulate (MF). The analysis of 3081-bp-long subcloned DNA fragment (GC-content 42.2%; Fig. S1) revealed the presence of two oppositely oriented open reading frames (ORFs), the first of which (1026 bp, positions 1144 to 119) encodes a putative 341-amino-acid protein with a predicted molecular mass of 37 502 Da that exhibited high homology (54% identity and 71% of similarity) to proteins of the esterase/lipase superfamily (pfam00135) ( Table S1). The 3A6 gene was subcloned into a plasmid vector and after that, inserted into the pET41 Ek/LIC vector for expression in Escherichia coli. The protein, purified to homogeneity as a single monomer of 38 000 Ϯ 3200 Da, was tested for its catalytic ability to hydrolyse a series of commercially available substrates (Table 1). We show that besides   hydrolysing p-nitrophenyl (pNP) esters, 3A6 efficiently catalysed the hydrolysis of cinnamates with a (kcat/Km) factor of~17:1 with pNP acetate (pNPC2) and MF as substrates. Overall, the enzyme was able to cleavage most efficiently methyl sinapinate, followed by MF (2.5-fold), and methyl-p-coumarate (16-fold), but not methyl caffeate. As judged by the kcat/Km values, the enzyme functions with short chain substrates from 2-to 1500-fold better than the longer substrates. The enzyme was also able to hydrolyse, to some extent, p-nitrophenyl 5-O-trans-feruloyl-a-Larabinofuranoside (kcat of 1 s -1 ) and (5-O-(trans-feruloyl)a-L-arabinofuranosyl)-(1,3)-b-D-xylopyranosyl-(1,4)-Dxylopyranose (kcat of 10 s -1 ). According to its substrate specificity, the enzyme was classified as an FAE type A (Crepin et al., 2004). The protein showed maximal activity at 45-50°C and pH 7.5-8.5 (Fig. S2). The substrate specificity showed strikingly different results with those reported in literature: nearly all characterized a/b hydrolases that share sequence similarity with 3A6 cleaved p-nitrophenyl esters but not cinnamates (Table S1). Following on from this, one of the main objectives of this study was to complement the kinetic data with mechanistic and engineering protocols so as to facilitate examination the essential residues that might hinder the recognition of cinnamate esters by steric or catalytic effects in the 3A6 protein.

Saturation mutagenesis strategy for altering the enzyme's specificity
A model of the enzyme was obtained by homology using the crystal structure of carboxyl-esterase Este1 of metagenomic origin (PDB 2C7B) as a template (Fig. S3), given the high level of identity (38% of the overall sequence, close to 70% in the active-site region). The quality of the obtained model was judged to be adequate for our purposes as judged by the Ramachandran plots (not shown), and a putative active-site pocket was identified (Ser 143 /Asp 273 /His 305 ) and further confirmed by mutagenic analysis: mutations of those residues by Gly, Gln and Asn, in the same order, results in a dramatic effect on the kcat/Km for pNPC2 and MF cleavage (> 10 000-fold). To further identify key residues that might define additional stabilization, subtraction or orientation effects on substrate preference, a detailed multiple (structural) alignment of 3A6 with homologous proteins was performed (Figs S4 and S5). The attention was focused on identifying regions that might have a fundamental role in substrate specificity and preference and that are not found in homologue proteins. The regions that were selected for mutagenesis were: Asn 109 -Leu 111 , Lys 281 -Asp 282 and Tyr 315 -Lys 317 (Fig. S3).
Saturation mutagenesis libraries (Asn 109 X, Glu 110 X, Leu 111 X, Lys 281 X, Asp 282 X, Tyr 315 X, Asn 316 X and Lys 317 X) were initially constructed, and crude cell extracts from 100 colonies (found to be sufficient to ensure that the 20 AA were represented) from each library were screened for activity with both pNPC 2 and MF. The results corrected for cell growth level, are shown in Fig. 1 (mutations at positions 109-111 did not affect hydrolytic proficiency and are not shown). In each case, a minor proportion of the library members were inactive with both substrates, showing that only few amino acid substitutions were not tolerated for activity. For each library some of the variants showed altered improved activity but no change in substrate preference. However, a number of particularly interesting variants were identified in the K 281 X, D 282 X, N 316 X and K 317 X libraries, suggesting that they may play important roles in controlling substrate recognition. It is particularly interesting to note that some of the subpopulations of variants in the libraries appeared to have a significant preference for the pNP substrate, with some of N 316X and K 317 X variants having no measurable activity against MF, whereas some (i.e. K 281 X and D 282 X) showed higher overall activity for the MF screening substrate. Interestingly, pNPC2 inactive variants were not detected, suggesting that CE activity is more robust to amino acid substitutions. Detailed kinetic characterization of fourteen purified enzymes selected on the basis of their substrate preferences was carried out in order to accurately determine the importance of each of the identified amino acid substitutions in the discrimination between the pNPC2 and MF screening substrates ( Table 2).

Construction of a CE-selective enzyme.
Screening of the saturation mutagenesis libraries ( Fig. 1) identified three substitutions (populations B, D and E) that yield pNPC2 selective catalysts. In the initial screen, members of these populations showed similar or higher activity with the pNPC2 substrate than the wild-type enzyme, but most importantly for our selection criteria, those appeared to have little or no measurable activity with MF. Sequence analysis revealed that three simple amino acid substitutions (D 282 L, N 316 STOP and K 317 H) are responsible for the excellent substrate discrimination: from (kcat/Km)pNPC2/(kcat/Km)MF of~17 for the wild-type to 69 (for D 282 L) to 3735 (for N 316 STOP) and 2 ¥ 10 6 (for K 317 H). This result can be explained by the up to 10-fold greater Km values coupled with a significant (from 1.5-to 29 000-fold) reduction in the kcat values for MF. This result is entirely consistent with the apparent~3 to > 700-fold discrimination displayed by populations B, D and E in the initial screen ( Fig. 1). Our data suggest that neither of the Y 315 X variants were individually responsible for discrimination of esterase and feruloyl esterase activities, although mutations at this residue equally increased both activities.   Construction of FAE-selective enzymes. As shown in Fig. 1, populations A, C and F were identified as leading to the most MF-selective variants. Sequencing identified 10 new mutations. The K 281 I mutant displayed the highest (34-fold increase) hydrolytic efficiency for MF, mainly due to a 14-fold reduction in binding capacity (Table 2). Overall, K 281 I substitution produced the only enzyme variant more selective towards MF with a (kcat/Km)pNPC2/(kcat/Km)MF ratio of 0.7 (24 times lower than wild-type), which is in a good agreement with the apparent 16-fold discrimination displayed by population A in the screen (Fig. 1). For the other variants (K 281 N, K 281 T, K 281 S, D 282 E, N 316 L, K 317 N, K 317 G, K 317 L and K 317 D) there is a large combined effect of lower kcat/Km for pNP esters and high kcat/Km for MF that led to (kcat/Km)pNPC2/ (kcat/Km)MF ratios ranging from 4-to 14-fold lower than wild-type. Overall, the cleavage of MF was mostly positively affected by the mutations.

Gain-of-function by site-directed mutagenesis
In an attempt to improve the activity and selectivity levels a number of double and triple mutants were created by Quick-Change site-directed mutagenesis and expressed recombinantly in E. coli. As shown in the

Maximizing FAE activity of 3A6 by deletion
Although single mutations in key regions were sufficient enough to significantly alter hydrolytic activity and control substrate specificity, we attempted to delete the Phe 11 -Lys 29 and Gly 178 -Gly 211 regions, to check for their catalytic influence. Both regions, not found in homologous proteins, were selected based on structural alignments (Figs S3-S5). The first one is located on the N-terminus of the protein and its deletion caused a complete loss of enzyme activity and will not be discussed further. The second insert is located on a long loop with extremely low sequence conservation. Circular dichroism (CD) analysis of pure protein, named 3A6-I, revealed that the deletion variant exhibited a slightly modified CD spectrum com-pared with the 3A6 wild-type protein (Fig. S6) and moreover, it showed an excellent substrate discrimination (Table 1). Interestingly, compared with parental and saturation mutants, the 3A6-I protein was the only enzyme variant, which appeared to demonstrate a significant, 13-fold preference for longer substrates, with pNP butyrate being the preferred substrate over pNPC 2 (Table 1). This variant showed an unexpected residual activity towards the cinnamates, with a (kcat/Km)pNP/ (kcat/Km)MF ratio higher than 10 7 . It is therefore a CE-selective enzyme. Even though we do not know the exact role in vivo of this extension, it is clear that it has a substrate recognition role.

Substrate discrimination by random mutagenesis
The mutational strategy has therefore produced a number of enzymes with high activity levels and We first observed that the Km value for pNPC2 was 12-fold higher than that of the native enzyme, while maintaining that for MF (Table 1). Furthermore, mutant showed a 39-fold increase of (kcat/Km)pNP/(kcat/Km)MF ratio (from 17 to 1200). This result is explained by the~14-fold lower and~70fold higher kcat values for MF and pNPC2 respectively. These data suggest that there is a strong bias towards CE phenotype in the A8P4 mutant.
Above results unambiguously confirmed that mutations at specific hot spots of the polypeptide (i.e. K 281 , D 282 , N 316 and K 317 ) may play a more effective supportive role in substrate discrimination in the parental 3A6 protein com-pared with random mutations within the whole protein length, since only one mutant with substrate discrimination ability was identified by epPCR. Figure 2 summarized the effect of single to triple mutations and insert deletion on substrate preference.

Substrate preference and its association with activation energies
The experimental work on the in vitro evolution of CE and FAE activities was followed by calculating the differential binding (DDGB) and activation (DDGA) free energy profiles for the cleavage of pNPC2 and MF, respectively, by the 'parental' and 'mutant' 3A6 variants. Difference energy diagrams (energy of the variant minus energy of the parent 3A6) are shown in Fig. 3A and B, and were used to understand the thermodynamics of the changes in enzyme reaction specificity, since the changes in substrate hydrolysis and preference are brought about by the relative changes in the free energies of the transition-state barriers. First, we see that the changes in DDGB and DDGA from the native to double or triple mutants are not the sum of free energies of the single saturation mutants, either for binding or catalysis. Thus, the contribution of K 281 I, D 282 E, D 282 L, N 316 STOP, K 317 H and K 317 D is synergic rather than mutually independent for the cleavage of both substrates, each of them positively or negatively affecting free energy values.
The effects on mutants on DDGB and DDGA are rather complex. As shown, major differences are observed for the activation free energies of MF compared with that shown for pNPC2 ( Fig. 3A and B). This suggests that FAE phenotype is more mutation affected by introducing single point mutations in specific 'hot spot' residues (see also Table 2). This was further confirmed by plotting the DDGA for pNPC2 and MF against the (kcat/Km)pNPC2/(kcat/Km)MF ratio for each of the variants. Surprisingly, whereas the DDGA for pNPC2 do not varied significantly (-5.6 to 7.3 J mol -1 ), that of MF do with a sharp increase (from -13.7 to 33.1 J mol -1 ) at increasing the CE phenotype (Fig. 3C). Similar situation was found when analysing the DDGB, although at lower level (Fig. 3D). Data suggest that mutations producing FAE-selective enzymes, i.e. those with (kcat/Km)pNP/(kcat/Km)MF ratio lower than 17, may be more favourable energetically (decrease in DDG). The low dependence of pNPC2-associated free energy with cata-

Fig. 2.
Schematic representation of the complementary substrate preference of 3A6-like variants. The preferred substrate specificity of wild-type and variants are shown as the logarithm of the ratio of catalytic efficiencies (kcat/Km) towards pNPC2 and MF substrates. A value of zero thus represents an enzyme, which is non-discriminatory between the two substrates.
lytic efficiency is consistent with the fact that any of the mutations abolished the CE activity during the screening library tests (see Fig. 1 and Table 2).

Discussion
A detailed analysis of a newly identified FAE enzyme of the type A from metagenomic library is presented here. The recombinant 3A6 enzyme appeared to be a monomer of~37 kDa. Besides hydrolysing ester bonds, 3A6 efficiently catalysed the hydrolysis of pNP and cinnamate esters with a kcat/Km factor of~17. The ability to cleave both common and feruloylated esters is significantly different to that shown by homologous proteins known in bibliography and databases: those enzymes behave as common esterases and the capacity to hydrolyse cinnamates has not been reported in any of them. Here, creation of a number of complementary CE and FAEselective variants with improved activity phenotypes was accomplished using a combination of modelling-guided saturation mutagenesis and site-directed mutagenesis. Specifically, modelling and structural alignment were used to identify residues responsible for controlling discrimination of substrate targets with similar type of bonds.  lower than 1.0). Therefore, only four amino acids are required to produce both the most CE-and FAE-selective enzymes. This is a remarkable result which highlights the relative ease by which specificities of esterases can be interchanged, in a good agreement with previous studies (Levasseur et al., 2006). Since K 281 , D 282 , N 316 and K 317 are situated at specific loci within the catalytic core, their effect in promoting substrate promiscuity can be analysed in combination with the 3D model. K 281 I exerts a perturbation in the final access tunnel to the catalytic centre, generating a high (34-fold) or mild (1.4-fold) improvement in catalytic efficiency for FAE-and CE-like substrates respectively ( Fig. 4A and B). Saturation mutagenesis at the position 281 confirmed Ile as the best possible amino acid substitution for improving FAE phenotype. The Ile is a smaller residue making more accessible substrate channel that may correlate with higher values of kcat, whereas the substitution of a hydrophilic (Lys) by a hydrophobic (Ile) residue may correlate with enhanced binding of the substrates (lower Km values). This observation can also be extrapolated to the variant D 282 L (hydrophilic→hydrophobic); however, here, the slightly larger size of Asp than that of Leu appears to have more impact towards smaller substrates such as pNPC2 ( Fig. 4C and D). Lys 317 His appear to have a significant negative impact on the properties of the enzyme since the mutation was associated with a complete loss of FAE activity. The effect on pNP esters was mostly associated with a twofold decrease in substrate affinity while slightly affecting the reaction rates (kcat). Since it was suggested that the presence of at least one m/pmethoxy group in the cinnamate-like substrates is required for binding (Tarbouriech et al., 2005), one could argue that the mutation at His 317 may cause a partial unwinding of the loop and shortening the distance between binding residues and cinnamate-like substrates while maintaining the activity with common substrates (Fig. 4E and F). The mutation Asn 316 STOP produced a deletion of the C-terminal tail (25 amino acids) ( Fig. 4G and H). This fragment constitutes an a-helix located back to the catalytic cavity. Although, the deletion does not seem to be directly relevant to the catalytic core, we suggest that it may confer a higher flexibility, making catalysis more efficient, in particular, with shorter substrates. To further assess whether the higher flexibility is produced at expenses of lower structural stability, as suggested by the model, we studied the biophysical parameters of this protein variant after pre-incubation with different concentrations of guanidinium chloride (GdmCl) and at different temperatures. We showed that this variant tends to misfold to a large degree after pre-incubation with Ն 0.64 M GdmCl whereas the wildtype protein was mostly chemically stable (misfolding occurred Ն 2.1 M GdmCl) (Fig. S7).
Our results also show that the hybrid 3A6 protein can also be converted into a common CE enzyme through the deletion of 34-AA-long loop. The functional cost of the Gly 178 -Gly 211 insert was offset by a large change in the substrate channel that enables higher efficiency of exclusion of the shorter substrates (Fig. 4I). This configuration apparently provides an effective solution to re-channelling longer substrates to the active site. It is also likely to hamper the diffusion of shorter-chain pNP esters to and from the active site, explaining the observed difference in hydrolytic rates and binding efficiency and the inactivity with the cinnamate substrates. We assume that this additional insertion may have been acquired as a rudimentary cap in providing considerable structural and functional substrate variability, which may also corroborate their role in the phylogenetic separation of the 3A6-like proteins (Fig. S1). The present study suggests that mutations at specific amino acids were most effective for promoting substrate specificity compared with the random in vitro evolution. Whatever the case, independently of the mutagenesis method applied, the CE phenotype was present in all variants, in contrast to the gain or loss of FAE activity. This view is supported by the fact that after point mutations the activity could be fully abolished towards cinnamates, but not towards pNPC 2. Moreover, substrate discrimination appears to be associated to energy barriers. Free energy contribution analysis revealed that the energy barriers to affect CE-like activity were mutationindependent, whereas it is energetically more favourable (negative DDG) to create variants with improved FAE phenotype (Fig. 3C). In this context, it is possible to assume that the 3A6 enzyme may have a CE-like origin and that the newly evolved enzyme accumulated mutations in the vicinity of the original active site leading to an increase of its activity towards cinnamates (energetically favourable - Fig. 3C) without compromising its original esterase activity (energetically independent - Fig. 3C). We hypothesize that this scenario might be perhaps a consequence of the environmental conditions in vivo: earthworm gut microorganisms require an efficient enzymatic machinery to cope with a continuous flux of complex polymeric materials from the soil and from plant litter passing through the intestinal tract, thus demanding an instant response to adapt their enzymatic machinery to the changing environmental conditions. In this context, a substrate selection pressure applied may allow the creation/evolution of enzyme variants with broader substrate range but not necessarily high specificity (Levasseur et al., 2006). Our saturation mutagenesis experiments on 3A6 may have thus mimicked a possible scenario of natural evolution towards creating cinammate-selective enzymes from a 'common' carboxyl esterase and directly evidenced the close evolutionary relationship between both enzymes. Finally, the results obtained here are intriguing from both an academic and biotechnological point of view and also highlights the importance of uncultured microbial resources (Vieites et al., 2009) to access novel functionalities.

Experimental procedures
A full description of experimental procedures is available in Supporting information.

Protein samples
All hydrolases used in the present study (wild-type and variants) were cloned into pET-41 Ek/LIC vector, expressed with an N-terminal fusion to 6xHis tag and purified as described in Supporting information.

Enzyme characterization
Unlike indicated otherwise, hydrolytic activities were routinely measured and kinetic parameters determined as described by López-Cortés and colleagues (2007). Reaction conditions were: [E]o = 0-12 nM, [substrate] ranging from 0 to 50 mM, 100 mM Tris-HCl, pH 8.5, T = 40°C. Molecular mass of the 3A6 protein of 37.502 Da was considered for kinetic parameter calculations. Kinetic parameters were calculated from the Hanes-Woolf plot. Results shown are the average of three independent assays Ϯ the standard deviation.

Library construction and FAE screen
Earthworms were collected at the surface level (0-20 cm) from the Ecological Soil Station of the Lomonosov Moscow State University (Solnechnogorskiy District, Moscow Region, Russia). The worms were maintained in a terrarium at 12-15°C during 3 months and were fed with sterilized birch litter. The gut contents were collected after transferring worms onto a wet filter paper and keeping them at 4°C for 1 h. Approximately 0.5 g of the gut content was inoculated into the 1 l Erlenmeyer flask containing 500 ml of Getchinson medium (K2HPO3, 1.3 g; MgSO4¥7H2O, 0.3 g; CaCl2¥6H2O, 0.1 g; FeCl2¥6H2O, 0.01 g; NaNO3, 2.5 g; water 1 l; pH 7.2-7.4). The only carbon and energy source was the cellulose supplemented as a disk of filter paper of approximately 20 cm in diameter (Whatman 3MM), which was submerged in the medium. The enrichment was performed without shaking at 12°C until the filter paper was totally degraded (10 days). The DNA was extracted from 50 ml of this enrichment using G'NOME DNA Extraction Kit (Qbiogene). A fosmid library using CopyControl Genomic Library Production Kit was established in pCCFOS vector and E. coli EPI300-T1 R according to the protocols of the supplier (Epicentre). Fosmid clones, in total about 30 000, were picked with Qpix2 (Genetix, UK) and deposited in 384-microtiter plates containing Luria-Bertani (LB) medium with chloramphenicol (12.5 mg ml -1 ) and 15% (v/v) glycerol. To screen for FAE activity the clones were replicated onto large (22.5 ¥ 22.5 cm) square-shaped LB agar plates containing chloramphenicol (12.5 mg ml -1 ) (Qtray, Genetix, UK). In total, each plate contained 2304 clones. After overnight incubation at 37°C, the plates were overlaid with 20 ml of 5 mM EPPS buffer containing 0.4% (w/v) agarose, 0.456 mM phenol red and 320 ml of MF (120 mg ml -1 in dimethylformamide). A hydrolase-positive colony exhibiting a strong yellow halo after 2 min was picked, and the insert containing the hydrolase was sequenced after subcloning in pUC19 vector.

Rationale for mutagenesis
To understand structural backgrounds of substrate specificity we used four different mutagenesis approaches.
For site-directed mutagenesis, mutations were introduced into the gene 3A6 using the QuikChange mutagenesis kit (Stratagene) and the plasmid pUC19-3A6 as template with appropriate oligonucleotide pairs (s. details in Table S2). The resulting plasmids variants were then transferred into E. coli XL10 Gold and selected on the LB agar supplemented with 100 mg ml -1 ampicillin (Amp).
Fragment deletion in the 3A6 gene was carried out using a reverse PCR deletion, with pGEM-3A6 as template, PfuTurbo DNA polymerase and the pair of primers DGly 178 -Gly 211 Fwd and DGly 178 -Gly 211 Rev (Table S2). Conditions were as follows: 95°C -120 s, 30¥[95°C -45 s, 50°C -60 s, 72°C -120 s], 72°C -500 s. The PCR product was purified, diluted to a concentration of 5 ng ml -1 , further self-ligated with T4 DNA ligase in a total volume of 100 ml at room temperature, and transformed into E. coli TOP10. The resulting transformants were plated onto fresh LB agar plate containing 100 mg ml -1 Amp.
Saturation mutagenesis libraries were constructed using the QuikChange site-directed mutagenesis protocol (Stratagene), the pGEM-3A6 as emplate and appropriate pair of degenerated primers listed in Table S2. For each position subjected to the mutagenesis, the mutated plasmids were transformed in E. coli TOP10 (Invitrogen) and the resulting transformants plated onto fresh LB agar plate containing 100 mg ml -1 Amp.
Error-prone PCR mutagenesis (epPCR) was carried out using Taq polymerase (Sigma Chemical Co.) and pUC19-3A6 as template. The reaction was performed in 50 ml volume and contained 3% dimethyl sulfoxide (DMSO), 5 mM MnCl2, 1.5 mM MgCl2, 0.3 mM dNTPs, 2.5 U Taq polymerase, 5 ng of template and 4.5 pmol of oligonucleotides pet41Fwd and pet41Rev (Table S2). The concentration of template and MnCl2 was adjusted to achieve a mutation rate from 1 to 3 mutations per kb. The amplification program was as follows: 2 min at 95°C, 27 s at 94°C, 27 s at 53°C, followed by 28 cycles of 3 min at 74°C, and 10 min at 74°C. The amplified PCR products were purified from a 0.75% agarose gel using QIAEX II gel extraction kit from Qiagen, cloned into the plasmid pGEM (Promega) and transformed into E. coli TOP10 (Invitrogen) as recommended by the supplier, and the resulting transformants were plated onto LB agar plate containing 100 mg ml -1 Amp.
Clones from saturation mutagenesis and epPCR libraries were picked and growth in 96-well microtiter plates containing LB-100 mg ml -1 Amp. Plates were covered with gaspermeable films (Abgene) and incubated at 37°C with shaking at 300 r.p.m. for 16-20 h. The cultures were lysed in the plate wells using 50 ml of FastBreak reagent (Novagen) and 0.1 unit ml -1 of DN aseI, at room temperature for 30 min with shaking at 300 r.p.m. These lysates were used to determine CE-and FAE enzymatic activities in a BioTek Synergy HT spectrophotometer, with pNPC2 and MF as substrates respectively (López-Cortés et al., 2007).

Free energy calculations
The quantitative effects of mutations on kinetics are expressed as changes in free energy compared with that of the wild-type enzymes and were calculated as described elsewhere (Numata and Kimura, 2001;Bethel et al., 2006;Williams et al., 2006). Differences in the free energy change (DDG) caused by mutations were calculated using the equations: where R is the gas constant (8.31 J kmol -1 ), T is the absolute temperature (317 K) and DDGA and DDGB are the differential free activation and binding energies respectively. In order to analyse the mutual interplay of amino acids involved in pNPC2 and MF binding and cleavage, the free energy barriers to substrate specificity introduced by single, double and triple mutations were analysed. When the sum of the DDG values for single mutants is equal to that of the double/triple mutant [DDG(X + Y) = DDG(X) + DDG(Y)] the sites function independently.

Fig. S1
. Phylogenetic affiliation of 3A6 protein with related putative and experimentally analysed hydrolytic enzymes. Fig. S2. Temperature (A) and pH (B) optima of the wild-type 3A6 protein. Fig. S3. Overall three-dimensional structure of the 3A6 protein, as obtained by homology modelling. Fig. S4. Alignment of 3A6 with protein PDB 2C7B. Fig. S5. Multiple alignment of the C-terminal part of 3A6 esterase and related enzymes. Fig. S6. Far-UV CD spectra of 3A6 wild-type protein and 3A6-3I variant. Fig. S7. Stability effect by a single mutation Asn 316 STOP. Table S1. Annotation table of proteins homologous to the 3A6 protein sequence (BLAST search). Table S2. List of primers used in this study.
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Actin Crosslinking Toxins of Gram-Negative Bacteria

Actin crosslinking toxins produced by Gram-negative bacteria represent a small but unique class of bacterial protein toxins. For each of these toxins, a discrete actin crosslinking domain (ACD) that is a distant member of the ATP-dependent glutamine synthetase family of protein ligases is translocated to the eukaryotic cell cytosol. This domain then incorporates a glutamate-lysine crosslink between actin monomers, resulting in destruction of the actin cytoskeleton. Recent studies argue that the function of these toxins during infection is not destruction of epithelial layers, but rather may specifically target phagocytic cells to promote survival of bacteria after the onset of innate immune defenses. This review will summarize key experiments performed over the past 10 years to reveal the function of these toxins.

Overview of actin targeting toxins
Many toxins have been characterized that disrupt the eukaryotic actin cytoskeleton. These include fungal and marine small molecule toxins that bind to actin to prevent actin polymerization [1,2]. Other bacterial proteins toxins and effectors can alter the normal regulation of cytoskeletal assembly, by directly or indirectly modifying the activity of small Rho family GTPases [3]. Finally, toxins have been identified that covalently modify actin itself, for example by the addition of an ADP-ribosyl group that then prevents its incorporation into F-actin [4,5].
Studies over the past ten years have been directed toward characterizing a distinct mechanism to disrupt actin filament assembly by covalently crosslinking actin monomers. The crosslinks can be OPEN ACCESS introduced repetitively to form long chains of dysfunctional actin that cannot be incorporated into F-actin filaments, resulting in irreversible destruction of the host cell cytoskeleton [6].

Discovery of the actin crosslinking domain of MARTX Vc
An actin crosslinking toxin was first discovered in current pandemic strains of the human diarrheal pathogen Vibrio cholerae [6]. When added to mammalian cells in culture, V. cholerae was shown to secrete a factor that induced rapid cell rounding, ultimately resulting in loss of all polymerized actin [6,7]. When actin in V. cholerae-treated cells was monitored by western blotting as a potential target for covalent modification, it was noted that all the monomeric actin in the cell disappeared within 180 min of addition of bacteria and actin was detected as higher weight molecular forms. The change in the electrophoretic mobility of actin corresponded to the formation of dimers, trimers, and higher order oligomers, revealing that actin in V. cholerae-treated cells had been crosslinked into chains [6].
This actin crosslinking activity depended upon the gene rtxA, the largest gene of the V. cholerae genome [6,7] and actin crosslinking activity was found to be widely present among both clinical and environmental isolates of V. cholerae [8][9][10][11]. The protein encoded by rtxA, now referred to as the Multifunctional, Autoprocessing RTX toxin of V. cholerae (MARTX Vc ), is the founding member of a new family of toxins [12]. The MARTX toxins are a grouping of toxins that are all large in size (>350 kDa) and are secreted from the bacteria by dedicated Type I secretion systems. Conserved features of all MARTX toxins include N-and C-terminal glycine rich repeats and an autoprocessing cysteine protease. These conserved structural elements are proposed to function in toxin translocation to deliver one to five effector domains found in the central region of the toxins to the eukaryotic cell cytosol [12].
To identify the specific effector of MARTX Vc responsible for actin crosslinking, subfragments of V. cholerae rtxA gene were ectopically expressed in epithelial cells as fusions to GFP and GFP-positive cells were visualized for rounding. Cells transfected with DNA corresponding to amino acids 1963-2375 of the rtxA gene (according to the original annotation by Lin et al. [7]) resulted in cell rounding and cells were confirmed to contain crosslinked actin by western blotting. This region was thus designated the actin crosslinking domain, or ACD [13], and it is now recognized as an effector delivered by autoprocessing of the large MARTX Vc toxin [14].

Other proteins with actin crosslinking domains
Subsequent to the discovery of the ACD within MARTX Vc , two other MARTX toxins have been identified by protein sequence analyses that also carry ACD effectors. Genomic sequencing of Aeromonas hydrophila strain ATCC 7966T revealed this pathogen associated with disease in invertebrates and an emerging pathogen in humans has a gene for a MARTX toxin that includes an ACD effector [12,15]. By contrast, genomic sequencing of human pathogenic Biotype 1 V. vulnificus strains revealed they also encode a MARTX toxin [16,17], but this toxin was not associated with actin crosslinking and did not have an ACD [13,18]. However, an environmental V. vulnificus Biotype 2 strain isolated from a diseased eel was found to carry a gene for a second MARTX toxin on the pR99 virulence plasmid (MARTX R99 ) and this toxin included an ACD [19] (Figure 1). At this time, neither of these ACDs have been confirmed biochemically to be functional for actin crosslinking.
A second protein encoded by V. cholerae was also found to have an ACD [13]. This protein, VgrG-1, is a bacterial effector that is secreted from V. cholerae dependent upon a Type 6 secretion (T6S) system [20]. The first domain of the protein belongs to the VgrG family, a group of proteins required to form a structural element of the T6S apparatuses [21]. VgrG-1 is unusual among the Vgr proteins because it is an "evolved VgrG" with an attached second domain that is delivered to the cell cytosol and carries effector function, in this case, an ACD that has been demonstrated experimentally to have actin crosslinking activity [22] (Figure 1). Conserved MARTX repeat regions (grey) are predicted to converge at the eukaryotic plasma membrane to form a pore through which the centrally-located effectors are translocated. After translocation, the cysteine protease domain (CPD) initiates autoprocessing to release effectors, including the actin crosslinking domain (ACD-red), Rho-inactivation domain (RID-purple) [24], α/β hydrolase effector (α/β-green), Mcf conserved effector of unknown function (Mcf-orange), and Pastuerella multocida toxin conserved effector of unknown function (PMT C1/C2-brown) [12]. Note that MARTX R99 has a duplication of the Mcf effector. V. cholerae VgrG-1 is an effector of the V. cholerae VAS T6S system. The Vgr conserved domain (Vgr-pink) forms part of the translocation apparatus for transfer of the ACD across the phagosomal membrane after engulfment by phagocytes. Vibrio sp. AND4 hypothetical protein AND4_00045 is a putative stand-alone actin crosslinking toxin that is potentially an effector of an unknown transport apparatus. The final putative actin crosslinking toxin is hypothetical protein AND4_00045 identified in the draft genome of environmental Vibrio sp. AND4 isolated directly from the Andaman Sea in Thailand. Unlike the MARTX toxins or VgrG-1, this protein is a stand-alone actin crosslinking protein with no other associated domain for translocation [23]. Thus, if it is an effector used for pathogenesis of aquatic animals, it must be translocated directly to the eukaryotic cell cytosol, possibly by a Type III or T6S system also identified in the draft genome.

ACD directly crosslinks actin
Early models of the mechanism of actin crosslinking recognized that crosslinking could occur either directly, in which the ACD is the catalytic enzyme, or indirectly, by activation of an unknown host cell protein with this activity [6]. To demonstrate direct action, the ACD portion of MARTX Vc was purified as a fusion with the N-terminus of Anthrax Toxin Lethal Factor (LF N ). LF N has been shown previously to mediate cytosolic delivery of heterologous fusion proteins through pores formed by Anthrax Toxin Protective Antigen (PA) [25,26]. This approach facilitated pretesting the LF N ACD for biochemical activity in the cytosol of mammalian cells after delivery by PA, prior to initiating experiments to establish in vitro crosslinking [27].
Subsequent experiments showed that LF N ACD does crosslink actin both in vivo after delivery by PA, as well as in vitro when added to eukaryotic cell lysates. Further refinement of the crosslinking reaction revealed the in vitro reaction is catalytic and progresses to completion in less that 10 min. This crosslinking occurs under reaction conditions composed of only purified actin, ATP, and Mg 2+ , indicating that no other proteins or cellular co-factors are required for crosslinking of actin by the ACD; and thus, ACD itself is the catalytic enzyme [27].

Requirement of G-actin, ATP, and Mg 2+ for actin crosslinking
Polymerization of monomeric G-actin into F-actin filaments is dependent upon hydrolysis of ATP [28] and occurs in the presence of 2 mM Mg 2+ . Hence, the requirement of Mg 2+ and ATP during the in vitro crosslinking reaction was initially thought to indicate that actin must be polymerized to be crosslinked. However, it was shown that crosslinking in vitro progressed efficiently when actin was locked as monomeric G-actin by association with latrunculin or kabiramide C or by modification with tetramethyl red [29]. Similarly, actin crosslinking progressed in vivo after addition of MARTX Vcproducing V. cholerae to cells that were pretreated with cytochalasin or latrunculin to inhibit actin polymerization [27,30]. By contrast, stabilization of F-actin in vitro by the addition of phalloidin or in vivo using dolastatin 11 inhibited the efficiency of actin crosslinking [27,29]. These data indicated that G-actin, not F-actin, is the substrate for ACD in the crosslinking reaction. Furthermore, these data support that the requirement for ATP and Mg 2+ during ACD-mediated actin crosslinking is not to drive actin polymerization.
Interestingly, by contrast to studies with MARTX Vc , crosslinking of actin in macrophages due to VgrG-1 was completely blocked by cytochalasin. However, this inhibition was not due to the ACD of VgrG-1 requiring polymerized actin instead of monomeric actin; rather it revealed a requirement for the bacteria to be phagocytosed before the VgrG-1 effector could be delivered to the cytosol by T6S [21]. Next, to account for the requirement of ATP and Mg 2+ in the crosslinking reaction, it was considered if the ACD itself is an ATPase. During an in vitro crosslinking reaction, inorganic phosphate (P i ) is released in a dose dependent manner such that one molecule of ATP is used for each crosslink introduced. This ATP hydrolysis is not due to polymerization of actin, but is an essential aspect of the crosslinking reaction itself [29] (see Figure 2).

Most G-actin binding proteins do not inhibit the crosslinking reaction
It is well-recognized that in the living cell, G-actin does not exist in a free form and is typically bound to one of the several actin binding proteins (ABPs) [31]. Remarkably, prior association of actin with the common ABPs profilin, thymosin-β4, and gelsolin did not have any noticeable effect on actin crosslinking by ACD. Among the ABPs tested, only DNaseI completely blocked the formation of actin oligomers, and cofilin partially inhibited. These data show that in vivo, crosslinking of actin by MARTX Vc likely progresses without interference of most ABPs [29].
Furthermore, actin crosslinking can occur when actin is previously crosslinked to itself. As G-actin is the substrate for crosslinking, higher order oligomers could arise by continued addition of G-actin monomers to longer chains or by the joining of oligomers. It was found that actin dimers disappear during crosslinking at ~4 fold slower rate than the monomers. This result indicated that although ACD-crosslinked oligomers can be further joined to form higher order species, the efficiency of the reaction diminishes with increased size of oligomers [29]. Thus, the formation of long crosslinked actin chains may occur predominantly by addition of monomers to linked oligomers.

The Chemical Nature of the Crosslink
The chemical crosslink introduced into actin has been solved and shown to be an isopeptide linkage between glutamate 270 on one actin monomer with lysine 50 on a second actin monomer (Figure 2).  Actin-K50 Actin-E270 + ATP Actin-E270~ + ADP Actin-E270-K50-Actin +P i P No other crosslinks are introduced and mutation of these residues within actin prevented crosslinking entirely, both in vitro and in vivo [32]. The crosslinked residues occur in flexible loops of actin in regions isolated away from binding sites for latrunculin, kabiramide C, and ABP binding sites. Lysine 50 is a residue on the DNaseI binding loop accounting for the lack of crosslinking when actin was pre-associated with DNaseI [29,32].
As seen in the crystal structure of the dimerzied form of crosslinked actin (PDBID: 3CJC), the two actin molecules are tilted with respect to the normal alignment of polymerized actin. When added to a crosslinking reaction, the altered structure cannot facilitate addition of new monomers such that dimerized actin competes with monomeric actin in F-actin growth and halts further polymerization [32]. Thus, actin crosslinking ultimately rounds cells both by actively blocking growth of actin filaments and by depleting the G-actin pool.

The Catalytic Mechanism Is Similar to Glutamate Synthetases
Given the novel nature of the crosslink in actin, it is not surprising that little information regarding the mechanism of catalysis could be derived using bioinformatic analyses. Red boxes indicate residues identified by genetic analysis as important for crosslinking with the star indicating the glutamic acid required for phosphotransfer. Black arrows represent predicted beta-strands that might form a glutamine synthetase ligase-like active site (see Figure 4).  [34]) active site is composed of a beta-sheet (grey) that contributes amino acid side chains that bind three Mg 2+ ions (blue) and ATP (ADP in crystal structure, red). The active site shares conserved residues with ACD amino acids demonstrated to be important for crosslinking activity (magenta). The essential ACD glutamate residue (green) is likely required for phosphotransfer during the ligation reaction as shown in Figure 2. Model prepared using MacPyMol (Delano Scientific) based on information from Geissler et al. [33].
Protein alignments using the primary peptide sequence of the ACDs identified only that ACD proteins are highly conserved, but showed no homology to other known or hypothetical proteins, suggesting actin crosslinking by an ACD may be restricted to the Vibrionaceae bacteria ( Figure 3).
Without sequence-based information to inform research strategies, the catalytic mechanism was probed by a random genetic approach to identify essential amino acids within the ACD [33]. This analysis revealed that glutamic acid 1990 of MARTX Vc is absolutely essential for actin crosslinking while mutation of six other residues resulted in decreased actin crosslinking efficiency. No other important residues were identified. A structural alignment algorithm revealed these residues could be mapped against the conserved catalytic site of proteins in the glutamine synthetase family of ATP-dependent protein ligases. Modeling ACD using the crystal structure of γ-glutamylcysteine synthetase (GshA) from Escherichia coli [34] revealed that E1990 likely participates in the transfer of the γ-phosphate from ATP to actin glutamate 270 side chain to activate the ligation reaction with lysine 50, resulting in release of P i (Figure 2). Thus, actin crosslinking to link glutamate to lysine occurs by a ligation reaction similar to that required to attach ammonia to glutamate to form glutamine or cysteine to glutamate to form γ-glutamylcysteine [33]. Given the strong conservation of important residues among the five identified ACDs (Figure 3), it is reasonable to predict that all ACDs will crosslink actin by an identical mechanism.

Phagocytic Cells as the Primary Target of ACD
The predominance of work studying the action of actin crosslinking has been conducted using epithelial cells in culture. However, the role of these toxins in disease progression is unclear because, particularly for cholera disease, there is limited epithelial damage. Recent studies have been directed toward understanding whether actin crosslinking toxins more specifically target innate immune cells. MARTX Vc, in conjunction with other secreted factors, has been demonstrated to be important to prevent clearance of the bacteria from the small intestine [35,36]. This effect of toxin expression on the ability of the bacteria to persist suggests MARTX Vc effectively blocks clearance by slowing or completely deactivating phagocytosis of the bacteria. Consistent with this model, the virulence plasmid that encodes MARTX R99 promotes survival of V. vulnificus Biotype 2 strains in eel whole blood suggesting it may inactivate phagocytes [19]. Furthermore, the actin crosslinking Type VI secretion effector VgrG-1 from V. cholerae crosslinks actin within phagocytic macrophages and amoebae, but not epithelial cells [20,21]. Finally, A. hydrophilia has been demonstrated to evade phagocytosis, although this may be as much or more related to its T6S system and associated actin ADP-ribosylating effector [5,37]. Thus, it seems possible that the function of the actin crosslinking toxins could be to directly block bacterial engulfment by phagocytic cells to evade clearance from the site of infection. This has been experimentally demonstrated for the function of VgrG-1 proteins and infection data suggest other actin crosslinking toxins may function in similar capacity to facilitate colonization and disease [38].


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Disruption of Vps4 and JNK Function in Drosophila Causes Tumour Growth

Several regulators of endocytic trafficking have recently been identified as tumour suppressors in Drosophila. These include components of the endosomal sorting complex required for transport (ESCRT) machinery. Disruption of subunits of ESCRT-I and –II leads to cell-autonomous endosomal accumulation of ubiquitinated receptors, loss of apicobasal polarity and epithelial integrity, and increased cell death. Here we report that disruption of the ATPase dVps4, the most downstream component of the ESCRT machinery, causes the same array of cellular phenotypes. We find that loss of epithelial integrity and increased apoptosis, but not loss of cell polarity, require the activation of JNK signalling. Abrogation of JNK signalling prevents apoptosis in dVps4 deficient cells. Indeed double deficiency in dVps4 and JNK signalling leads to the formation of neoplastic tumours. We conclude that dvps4 is a tumour suppressor in Drosophila and that JNK is central to the cell-autonomous phenotypes of ESCRT-deficient cells.

Introduction
Correct receptor signalling and cell polarisation are required for proper organisation of epithelia. Signalling from ligandbound receptors is modulated by endocytosis [1,2], which mediates spatial restriction or downregulation of signalling [3][4][5] by sorting into intraluminal vesicles of multivesicular endosomes (MVEs) that fuse with lysosomes [6][7][8][9]. Both in the yeast Saccharomyces cerevisiae and in mammalian cells, Class E vacuolar protein sorting (Vps) proteins are important for sorting ubiquitinated receptors into the lumen of the MVEs. The class E vps yeast mutants accumulate vacuolar, endocytic and late-Golgi markers in an aberrant multilamellar endosome, the class E compartment [6,[9][10][11][12][13]. The majority of class E Vps proteins constitute the endosomal sorting complex required for transport (ESCRT) machinery, which is conserved in metazoans, including flies and mammals. ESCRT-I, -II and -III function cooperatively in endosomal receptor sorting and MVE biogenesis. Also important in this process is the ATPase Vps4, which functions to disassemble ESCRT-III multimers, thereby allowing the recycling of subunits [10,14,15]. The role of the ESCRT machinery in whole tissues has been particularly illuminated in the fruit fly Drosophila melanogaster in which disruption of ESCRT-I or -II causes cell-autonomous loss of polarity, cytoskeleton disruption and apoptosis. ESCRT-deficient cells also activate Notch signalling, which triggers the release of a cytokine thus activating proliferation in adjacent, normal cells [16][17][18][19]. In this paper, we focus on the cell autonomous effects caused by ESCRT deficiency.
Even though it is established that the ESCRTs control tissue organization, the mechanisms that mediate this activity remain unclear. Here we explore the effects of reducing the activity of Drosophila Vps4 (dVps4), the most downstream component of the ESCRT machinery. In the main, dVps4 activity was knocked down with a dominant negative construct carrying a point mutation in the catalytic site [14]. Supportive evidence was also obtained with a small genomic deletion and an RNAi hairpin construct. We find that several cell-autonomous effects observed when ESCRT function is disrupted are mediated by c-Jun Nterminal kinase (JNK), a member of the mitogen-activated protein kinase (MAPK) family, which has extensively been implicated in programmed cell death [20] and is misregulated in many types of mammalian cancers [21][22][23][24][25]. While reduction in either dVps4 activity or JNK signalling does not cause overgrowth, double deficiency leads to the formation of neoplastic tumours.

CG6842 encodes the Drosophila orthologue of Vps4
The Drosophila genome contains a single Vps4 homologue encoded by the CG6842 gene, here referred to as dvps4, which contains four exon-intron boundaries and is located between two genes of unknown function on the X-chromosome (Fig. 1A,B). The predicted dVps4 protein contains 442 amino acid residues and displays 75.1% identity to human Vps4b, 73.7% identity to human Vps4a and 58% identity to yeast Vps4p (Supporting Fig.  S1). There is no significant similarity to other proteins in the database. The dVps4 protein contains an AAA-ATPase domain and a Microtubule interacting and transport (MIT) domain ( Fig. 1C) [14,15]. For our initial studies of the function of dVps4 we constructed a small deficiency allele, dvps4 D7b . In this allele, the whole coding region and flanking sequences were deleted by FRTmediated recombination between two FRT-containing P-elements, f07379 and d09806 carried in trans (Fig. 1A, see Materials and Methods) [26]). An antibody raised against dVps4 recognised a protein with the predicted size on Western blots from lysates of wild type L1 control larvae, but not from L1 dvps4 D7b larvae, indicating that the antibody is specific and that dVps4 protein is indeed absent in the mutants (Fig. 1D). In the wild type, dVps4 protein was found to be present at all stages of development, from embryos to adult males and females (Fig. 1E), suggesting a pleiotropic role. We found that dvps4 D7b homozygous animals died at the first larval instar similar to tsg101/erupted (ept) and vps25 homozygous mutants [19,27]. This is likely because dvps4 is an essential gene although an essential contribution from the two neighbouring genes cannot be excluded (Fig. 1B). All attempts to rescue the dvps4 D7b mutants by expression of a dVps4 wild type construct failed. This was probably due to the dominant negative effect that is caused by overexpression of this construct (see below).

dVps4 disruption causes accumulation of ubiquitinated proteins and disruption of epithelial organisation
Patches of dVps4-deficient cells (homozygous dvps4 D7b ) were generated in developing eye imaginal discs. These cells showed an apparent loss of epithelial organisation and accumulated ubiquitin in punctuate structures ( Fig. 2A), phenotypes that are also seen as a result of disruption of the ESCRT-I component ept/tsg101, or of the ESCRT-II component vps25 [16,18,19,27]. dVps4 mutant cells also displayed a dramatic dysorganization of the actin cytoskeleton similar to that reported in dvps28 (ESCRT-I) mutant cells [17]. Mostly small (1-2 cells) dvps4 D7b mutant clones could be recovered, probably because the FRT-mediated recombination events used to create mutant patches is triggered in individual cells and that, in isolation, dvps4 D7b mutant cells undergo apoptosis faster than they can grow. In order to assess the contribution of dVps4 to the phenotype of dvps4 D7b homozygous cells, we constructed a Gal4-dependent transgene to express wild type dVps4 that could be used in a rescue experiment. Expression of this transgene in wing imaginal discs caused accumulation of endocytic cargo, polarity disruption and cell death (data not shown). These are characteristics of the loss of function phenotype and it is therefore likely that overexpressed wild type protein has a dominant negative effect on ESCRT activity. Since the dvps4 D7b mutant clones are so short lived and because of the possibility that the flanking genes might be affected by the deletion, we turned to RNAi-mediated knock-down [28] and a dominant negative form of dVps4, dVps4-DN as means of reducing dVps4 activity. Since both the RNAi and the DN constructs can be expressed under Gal4 control and since these constructs are likely to create a hypomorphic situation, large patches of deficient tissue can be generated thus overcoming the problem of growth from a single mutant cell. As a first step, heat-shock-inducible Gal4 was used to activate the UAS-dVps4-DN transgene ubiquitously, and egg chamber phenotypes were scored. As can be seen in Fig. 2B, this caused the accumulation of the endosome marker Hrs and conjugated ubiquitin on large structures within expressing cells. In addition we used phalloidin staining to visualize F-actin and found that the cytoskeleton was disrupted with tangles of abnormal accumulation. The same phenotype was seen in dvps4 D7b patches within the follicle epithelium and was also seen in wing imaginal disc cells expressing dVps4-IR driven by 1096-Gal4, a wing pouch driver (not shown). This phenotype was not observed in egg chambers only expressing the Hs-Gal4 driver upon heat shock (Fig. 2C). We conclude that, since dVps4-DN and dVps4-IR affect the endosomal distribution of ubiquitinated proteins, these constructs are likely to interfere with ESCRT activity as intended.
Having established that a reduction in dVps4 activity causes trafficking defects expected from ESCRT deficiency, we set out to ask if it also causes disruption of cell polarity and loss of epithelial integrity [16][17][18][19] as is seen following loss of ESCRT-I and -II components. Wing discs expressing dVps4-IR were stained with an antibody against Discs-large (Dlg) which labels the lateral membrane domains. Dlg was reduced in dVps4-IR expressing cells as compared to the situation in surrounding wild type tissue ( Fig. 2D-D999, where dVps4-IR-expressing cells are marked by GFP expression). This phenotype suggests loss of cell polarity and epithelial integrity. Larvae expressing dVps4-IR or dVps4-DN in the wing disc continued to grow beyond the normal time of growth termination, became oversized and died shortly after delayed pupation (Fig. 2E,F).
To investigate the ultrastructural defects of dVps4-deficient cells, we performed immuno-electron microscopy (EM) on wing imaginal discs expressing dVps4-DN or dVps4-IR. As a membrane marker we used CD8-GFP (detected with goldlabeled anti-GFP) expressed from a transgene. This transgene also allowed cells expressing the knockdown construct to be identified unambiguously within the tissue. Expression of either dVps4-IR or dVps4-DN caused the formation of enlarged tubular structures, which were not seen in control discs (Fig. 3A,B,C). Both CD8-GFP and ubiquitin accumulated on these structures, suggesting that they represent enlarged late endosomes. Interestingly, the Notch receptor also accumulated on these structures (data not shown), which could therefore be a site of Notch signalling. As could be seen upon closer examination, in addition to accumulating ubiquitin and being positive for CD8-GFP, these enlarged MVE-like structures contained internal vesicles (Fig. 3E,F,H,I). Perhaps residual dVps4 activity allows low-level endovesiculation to proceed. As in dvps4 D7b mutant tissue, an increased number of apoptotic cells were seen in the domain of dVps4-DN expression (asterisk, Fig. 3C). Apoptosis was also enhanced in response to dVps4-IR expression (data not shown), however this was less pronounced than with dVps4-DN. This might be because the DN construct inhibits dVps4 activity more thoroughly than dVps4-IR.
Larval overgrowth, epithelial disorganisation, and increased apoptosis are characteristic of mutants in tumour suppressor genes such as scribble (scrib), dlg, lethal giant larvae (lgl) [29] avalanche (avl) [30] and three other components of the ESCRT machinery, ept/tsg101 CD8-GFP; dVps4-IR and (C) dVps4-DN discs large dense structures were observed (arrows) containing intraluminal vesicles, often with irregular shape. These structures sometimes occupied most of the cell volume. In both cases GFP accumulation was observed (arrows in E and F) and also strong labeling against ubiquitin (arrows in H and I). In the 1096-Gal4; CD8-GFP; dVps4-DN discs an increased number of apoptotic cells were observed (asterisks in C), in contrast to the dVps4-IR discs, where less apoptotic cells were present (not shown). Scale bars; A-C, 3 mm; D-I, 500 nm. doi:10.1371/journal.pone.0004354.g003 [27], vps25 [16,18,19] and vps28 [17]. Therefore, dVps4 bears the hallmarks of a tumour suppressor.

dVps4 disruption causes upregulation of JNK, integrin and MMP1
As mentioned above, apoptosis is increased in cells expressing dVps4-DN or dVps4-IR, as well as in tsg101 and vps25 mutant cells [16,18,19,27]. Little is known so far about the signalling pathway that mediates this effect. One candidate pathway is that controlled by JNK since it is known to activate apoptosis in a variety of situations ( [31] and references therein). Indeed, in dVps4-IR-expressing cells, the levels of phosphorylated active JNK (pJNK) were found to be upregulated in comparison to control discs (Fig. 4A,B). Consistent with this observation, JNK downstream targets such as bPS integrin (myospheroid) and matrix metalloprotease 1 (MMP1) were ectopically expressed (Fig. 4E,H), a phenotype also seen in cells that express an activated form of Hemipterous, the Drosophila JNK kinase (Fig. 4C,F,I). Moreover, expression of dVps4-IR caused a similar disorganisation of the actin cytoskeleton as activated JNK (Fig. 4B,C,E,F) [32]. None of the above proteins were seen to be upregulated in the control discs expressing only CD8-GFP (Fig. 4A,D,G). Overall therefore, it appears that JNK signalling is upregulated following expression of dVps4-IR (or dVps4-DN; see below).
Since activation of JNK on its own produced similar effects to those caused by loss of dVps4, we reasoned that JNK signalling may be a key mediator of the defects observed in dVps4 deficient cells. To test this hypothesis we asked if the phenotypes caused by expression of dVps4-DN are suppressed by concomitant reduction of JNK signalling. Like dVps4-IR, dVps4-DN caused pronounced upregulation of MMP1 and integrin expression as well as a striking disruption of the actin cytoskeleton (Fig. 5A,D,G). Concomitant suppression of JNK signalling (by co-expression of a dominant negative from of JNK, basket DN (Bsk-DN) [33] brought back actin, MMP1 and integrin expression closer to wild type levels (Fig. 5C,F). Co-expression of the JNK phosphatase puckered (puc), a negative regulator of JNK signalling, had a similar, albeit weaker effect (data not shown). Actin, MMP1 and integrin were neither upregulated nor disrupted in discs expressing Bsk-DN alone (Fig. 5B,E). Therefore upregulation of actin, MMP1 and integrin expression in dVps4-DN-expressing cells is likely mediated by JNK signalling.
Another important consequence of reduced dVps4 activity is an increase in apoptosis. This was noticeable in dVps4-IR-expressing discs (not shown) and was striking in tissues expressing dVps4-DN ( Fig 5G). As expected, dVps4-DN-expressing discs were much reduced in size. Such size reduction required JNK signalling since co-expression of Bsk-DN restored growth in dVps4-DN-expressing discs. In fact, discs co-expressing dVps4-DN and Bsk-DN overgrew and reached a much larger size than control, wild type discs (Fig. 5J,K). A similar effect is seen in discs co-expressing dVps4-DN and puc. Overgrowth requires inhibition of both dVps4 and JNK signalling since discs expressing either Bsk-DN or puc alone are reduced in size (Fig. 5K, not shown). In order to test whether suppression of JNK signalling enables overgrowth of dVps4-deficient cells by suppressing apoptosis, anti-activated Caspase-3 stainings were performed. Only sporadic low levels of activated Caspase-3 were observed in discs expressing Bsk-DN alone (Fig. 5H). Importantly, high level staining detected in discs expressing dVps4-DN alone (Fig. 5G) was strongly suppressed by coexpression of Bsk-DN (Fig. 5I). We conclude that increased apoptosis caused by loss of dVps4 function is mediated by activation of JNK signalling. However, not all phenotypes caused by dVps4 deficiency require JNK signalling. As shown in Supporting Fig. S2, the downregulation of Dlg seen following expression of dVps4-DN is not suppressed by coexpression of Bsk-DN.

Discussion
In this study we have shown that reduced activity of the ESCRT-III regulator dVps4 causes the same array of cellular phenotypes as those seen in cells lacking ESCRT-I or -II components. These phenotypes include upregulation of MMP1 and integrin, dysregulation of the actin cytoskeleton, increased apoptosis, and loss of apico-basal polarity. In addition, we found that dVps4 knockdown also leads to activation of JNK signalling. In fact, JNK signalling appears to be a central mediator of many of the cell-autonomous phenotypes observed in dVps4-deficient cells. Indeed, all the above phenotypes, except for the loss of apico-basal  Importantly, apoptosis in dVps4-expressing tissue was strongly counteracted by downregulation of JNK signalling (Fig. 5). Overexpression of Bsk-DN, or of the JNK phosphatase puc, led to a reduction of Caspase-3 staining and increased cell survival in tissue expressing dVps4-DN. By contrast, blocking JNK signalling by expressing puc did not prevent apoptosis in vps25 null mutant cells [16]. The difference could be due to the extent of loss of ESCRT function in the two situations since dVps4-DN is likely to cause a hypomorphic situation. Our results thus show that JNK is central to the induction of apoptosis under conditions of partial loss of ESCRT activity. Strikingly, simultaneous reduction of dVps4 and JNK signalling leads to the formation of neoplastic tumours. dVps4 can therefore be considered a tumour suppressor gene. Importantly, such tumour suppressor activity is revealed only when activity is partially lost, perhaps because dVps4 has a pleiotropic role; complete loss of function would impede an essential cell biological activity and thus prevent growth while partial loss of function would unbridle a growth inhibiting mechanism.
Previous studies of vps25 and tsg101 Drosophila mutants have revealed a strong hyperplastic growth of tissue adjacent to the mutant cells [16,18,19,27]. Such an effect is quite distinct from the cell autonomous overgrowth reported in this manuscript, which requires inhibition of JNK signalling. Non-autonomous overgrowth triggered by vps25 and tsg101 loss of function has been attributed to upregulated cytokine secretion, following autonomous activation of the Notch signalling pathway in the mutant cells [16,18,19]. We have also observed an activation of Notch signalling and cell non-autonomous hyperplastic growth in tissues flanking dVps4-IR-expressing cells (data not shown), suggesting that non-autonomous stimulation of proliferation may be a common feature of ESCRT deficiency. We suggest that both the non-cell-autonomous and the cell autonomous effects of dVps4 disruption contribute to tumourigenesis.
In conclusion, we have shown that the ESCRT-III regulator dVps4 is crucial for attenuation of signalling pathways that mediate cell-autonomous apoptosis, actin rearrangement, integrin upregulation, MMP1 upregulation and loss of polarity. With exception of the latter, all these effects can be attributed, entirely or partially, to activation of JNK. Further studies aimed at clarifying the mechanistic relationship between dVps4 inactivation and JNK activation will be required in order to fully understand the tumour suppressor function of dVps4.

Generation of dvps4 D7b Drosophila mutants
The dvps4 D7b allele was generated by using P-transposase-based deletion by FRT-site recombination between the two transposon insertions f07379 id: AY515148 (WH p-element) and d09806 id: AY515149 (XP p-element) situated on opposite sides of the dvps4 gene locus [26] (a gift from Ruth Palmer) and Hs-FLP and P[w+]/ FM7c, w2.

Immunohistochemistry and microscopy
Antibodies against dVps4 were generated by immunising two rabbits with a mixture of the two peptides C-DLVTKA-TEEDRNKN and CSARSDNENDSVRRI, coupled to LPH. (BioGenes GmbH, Germany). Antibodies were affinity purified using the same two peptides coupled to Sulfo-Link (Pierce). Imaginal discs were fixed and stained according to standard protocols [34]. The following antibodies and their dilutions were used for immunohistochemisty: Mouse monoclonal antibodies against conjugated ubiquitin (FK2) (1:1000) (Affinity BioReagents, UK.), guinea pig anti-Hrs (1:1000) [35], mouse anti-Dlg 4F3

Electron microscopy
L3 larvae were fixed in 4% formaldehyde/0.1% gluteraldehyde in 0.1 M phosphate buffer at room temperature for 60 min, washed and the wing disc dissected out. This was embedded in 12% gelatin and infiltrated with 2.3 M sucrose, mounted on silver pins, and frozen in liquid nitrogen. Ultrathin cryosections were cut at 2110uC (EM FCS ultramicrotome; Leica) and collected with a 1:1 mixture of 2% methyl cellulose and 2.3 M sucrose. Sections were transferred to formvar/carbon-coated grids and labeled with antibodies against GFP, Notch or Ubiquitin, followed by Protein A conjugates essentially as described [36]. Sections were observed at 60-80 kV using a JEOL JEM-1230 electron microscope, equipped with a Morada camera.