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MODEL

Microbes and Infection xx (2014) 1e8 www.elsevier.com/locate/micinf

Original article

Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence Alaeddine Meghraoui, Lionel Schiavolin, Abdelmounaaim Allaoui*

Q3

Laboratoire de Bacte´riologie Mole´culaire, Faculte´ de Me´decine, Universite´ Libre de Bruxelles, Route de Lennik, 808, 1070 Bruxelles, Belgium Received 22 January 2014; accepted 31 March 2014

Abstract Infection of colonic epithelial cells by Shigella is associated with the type III secretion system, which serves as a molecular syringe to inject effectors into host cells. This system includes an extracellular needle used as conduit for secreted proteins. Two of these proteins, IpaB and IpaD, dock at the needle tip to control secretion and are also involved in the insertion of a translocation pore into host cell membrane allowing effector delivery. To better understand the function of IpaD, we substituted thirteen residues conserved among homologous proteins in other bacterial species. Generated variants were tested for their ability to surface expose IpaB and IpaD, to control secretion, to insert the translocation pore, and to invade host cells. In addition to a first group of seven ipaD variants that behaved similarly to the wild-type strain, we identified a second group with mutations V314D and I319D that deregulated secretion of all effectors, but remained fully invasive. Moreover, we identified a third group with mutations Y153A, T161D, Q165L and Y276A, that exhibited increased levels of translocators secretion, pore formation, and cell entry. Altogether, our results offer a better understanding of the role of IpaD in the control of Shigella virulence. Ó 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Keywords: Shigella; Needle tip; Type 3 secretion system; Virulence; IpaD

1. Introduction Shigella is the causative agent of shigellosis or bacillary dysentery, which is clinically manifested by bloody and mucoid diarrhoea resulting from the inflammation and destruction of the colonic mucosa. The emergence of new resistant strains and the absence of an efficient vaccine maintain shigellosis as a major public health issue [1]. The most recent data reports 125 million annual cases in Asia with 14.000 deaths [2]. Faecal-orally transmitted Shigella colonizes the gut lining, crosses the intestinal barrier through M cells, kills macrophages, and forces its entry into epithelial cells [3e5]. This virulence is governed by the Type 3 Secretion System (T3SS) that is shared by several Gram-negative bacteria such as Yersinia, Pseudomonas, Salmonella, and Burkholderia. The T3S apparatus (T3SA) is

* Corresponding author. E-mail address: [email protected] (A. Allaoui).

composed of a cytoplasmic bulb, a basal body and an extracellular needle. This machinery acts as a molecular syringe to inject effectors directly into host cell cytoplasm through a pore formed at the cell membrane [6e8]. As a result, these effectors subvert host cell physiology to permit bacterial internalization and then its dissemination to neighbouring cells [9]. In Shigella, the T3SA assembly is triggered upon favourable environmental conditions for invasion, but secretion is only activated following the sensing of a contact with a host cell [9]. T3SA assembly and secretion of effectors are hierarchically regulated and occur in an orchestrated fashion [10,11]. Indeed, upon assembly of the basal body, two proteins, MxiI and MxiH, forming respectively the inner rod component and needle structure, are secreted [7,12]. MxiH proteins polymerise helically, thus allowing the formation of a central channel [7,13] used as a secretion conduit facilitating the passage of partially unfolded proteins [6,14,15]. Once the needle length reaches approximately 50 nm, the substrate specificity switches from needle components to translocators

http://dx.doi.org/10.1016/j.micinf.2014.03.010 1286-4579/Ó 2014 Published by Elsevier Masson SAS on behalf of Institut Pasteur. Please cite this article in press as: Meghraoui A, et al., Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.010

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A. Meghraoui et al. / Microbes and Infection xx (2014) 1e8

[16]. Then, the host cell contact triggers the secretion of translocators IpaB/C, early effectors (e.g. IcsB and IpaA) and the anti-activator OspD1 [12,17]. IpaB and IpaC are stored in the cytoplasm in association with the chaperone IpgC, while OspD1 is bound to the transcriptional activator MxiE [17,18]. The release of IpaB, IpaC and OspD1 liberate both the coactivator IpgC and the activator MxiE, which become available to induce the transcription of late effector genes (e.g. IpaH) [17,19,20]. At the T3SA base, secretion of effectors is controlled by MxiC, which associates with the rod component MxiI to presumably form a plug at the entry gate [12]. At the needle tip, secretion is controlled by the IpaDeIpaB complex that prevents leakage of effectors prior to activation [21]. Cross talk between both devices has been highlighted, where the tip complex would sense the host cell contact and transmits a signal through MxiH/MxiI, thereby allowing secretion of MxiC and early effectors which is then followed by the late effector production and secretion [12,22,23]. Besides IpaB and IpaD proteins which form the needle tip complex, translocation into host cells requires the third translocator IpaC [9]. IpaD is hydrophilic and serves as a scaffold for the hydrophobic proteins IpaB and IpaC, which later form the translocation pore in the host cell membrane [8,21,24e27]. The maturation of the needle tip complex has been described to occur in a sequential process according to extracellular conditions. Prior to cell contact, IpaD and IpaB are surface exposed while IpaC is probably located underneath [26e30]. Another model has reported the exposure of only IpaD that promotes the presentation of IpaB upon conformational changes induced by deoxycholate (DOC) [31e33]. Regardless of the model, a final state is triggered by the contact between IpaB and membrane lipids, leading to the recruitment of IpaC at the needle tip to form the translocon together with IpaB [24,27,34]. In addition to its role as a tip complex component, IpaD was also reported to act intracellularly alongside MxiC as a probable co-activator of translocators secretion [35]. The structure of IpaD is composed of a coiled-coil domain formed between two long a-helices H3 and H7, an N-terminal domain composed of 3 a-helices, and a central domain located between H3 and H7 and consisting of a-helices and b-sheets. These domains are organized as two four-helix bundles that share the long coiled-coil domain [29]. This latter is required for IpaD oligomerisation, needle tip localisation and DOC binding [28,29,36], while the N-terminal portion is considered as a selfchaperoning domain that would prevent IpaD interactions within the bacterial cytoplasm [29]. Finally, the central domain of IpaD is involved in IpaB surface localisation and translocators insertion and would rotate from the coiled-coil [28,29,37]. SipD and BipD, two homologous proteins to IpaD, in Salmonella typhimurium and Burkholderia pseudomallei, respectively, share the same structural features [29,38]. In contrast, their counterparts, PcrV and LcrV, in Pseudomonas aeruginosa and Yersinia spp. differ by harbouring independent chaperones and by presenting a structurally unrelated N-terminal domain [39]. Our previous mapping of IpaD function by ten amino acid deletions shows that the tip complex composition and/or state determines late effector genes secretion, and that the central

domain is likely involved in the host cell contact [28]. Although it was hypothesised in the literature that IpaD could regulate the T3SA activity in an allosteric way, the required mechanism remains poorly understood [27,28]. By further investigating the role of IpaD in secretion control and upon host cell infection, we sought to identify point mutants impaired in secretion control and/or in host cell contact functions. For that, thirteen point mutations within IpaD were generated, by targeting conserved residues shared by the homologous SipD and BipD in Salmonella and Burkholderia, respectively. Our results revealed three distinct phenotypes based on the study of translocators surface exposure, secretion control, translocon formation and cell entry. 2. Materials and methods 2.1. Bacterial strains and growth conditions Bacteria used in this study are listed in Table S1. Shigella flexneri strains used are derivatives of M90T-SmR (serotype 5a) [40]. Plasmids were constructed in Escherichia coli TOP10 strain and suicide plasmids were constructed in E. coli DH5a strain (Invitrogen). Bacteria were grown in lysogeny broth (Sigma) or tryptic soy broth (Merck) at 37  C with shaking (180 rpm). Antibiotics were used at the following concentrations: ampicillin 100 mg/ml; streptomycin 100 mg/ ml; kanamycin 50 mg/ml and zeocin 50 mg/ml. 2.2. Construction of plasmids Plasmids and primers used in this study are listed in Tables S1 and S2, respectively. Plasmid pSL113 was constructed starting from the pSFDK3 suicide vector pGP704-ipaD::apha3 [41]. Plasmid pSFDK3 was amplified by inverse PCR using phosphorylated primers ipaD1as and ipaD8s, to introduce an NruI restriction site upstream of the aphA3 cassette and to remove codons 31 to 110 of the N-terminal domain, and then ligated to generate the pSL110 plasmid. Finally, the aphA3 cassette was replaced by the ble cassette (Zeocin resistance) by cloning ble DNA fragment from plasmid pMS05 [23] digested with EcoRV-SpeI into NruI and XbaI (downstream of the aphA3 cassette) to generate plasmid pSL113. The latter was then transferred to M90T-Sm by conjugal mating and transconjugants were selected for their resistance to zeocin and streptomycin. Clones in which a double-recombination event had exchanged the wild-type ipaD gene for the mutated copy carried by pSL113 were identified by screening for sensitivity to ampicillin. The structure of pWR100 derivatives carrying the ipaD mutation was further confirmed by PCR. The resulting strain was designated ipaD3. 2.3. Site directed mutagenesis Site-directed mutagenesis on ipaD gene was performed by PCR on plasmid pSL1 using primers listed in Table S2. They were designed according to Site-directed Mutagenesis Kit Protocol (Stratagen) to have mismatches corresponding to substitutions

Please cite this article in press as: Meghraoui A, et al., Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.010

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Y149A, Y153A, T161D, Q165L, K189A, K205A, W226A, Y276A, Y301A, N305A, V312A, V314D, and I319D. The constructed plasmids were screened by digestion as the use of each pair of primers creates a restriction site (Table S2). Protein expression was confirmed by western blot using rabbit polyclonal anti-IpaD antibodies and mutated ipaD genes were further confirmed by DNA sequencing. Constructed plasmids were then introduced by electroporation into the newly constructed Shigella ipaD3 mutated strain for further functional studies.

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at the corresponding concentrations (from 0.5 to 2  1010 bacteria/ml). Horse red blood cells (RBCs, Oxoid) were centrifuged at 2000 g for 10 min at 4  C and washed twice in cold PBS. In a 96-well plate, 50 ml of RBCs were mixed with bacterial preparations and centrifuged at 2000 g for 10 min. After 20 min of incubation at 37  C, the reaction was stopped by adding 100 ml of cold PBS. The mixture (RBC-bacteria) was resuspended and further centrifuged. 100 ml of supernatant was transferred in a new plate and haemoglobin release was evaluated with optical density measurement at 540 nm.

2.4. Analysis of protein production and secretion 2.7. Cell invasion assay Secretion phenotypes of Shigella wild-type or ipaD null mutant strains harbouring plasmids encoding ipaD variants were analysed as previously described [28]. For stationary secretion, overnight cultures were centrifuged at 6000 rpm for 5 min (min) at 4  C to recover pellets and supernatants that were analysed on SDS-PAGE either by Coomassie blue staining and/or by Western blot analysis. For exponential secretion, overnight cultures were diluted (1:100) and grown at 37  C to an OD600 z 1. Pellets were resuspended in Phosphate Buffered Saline (PBS) and incubated for 15 min at 37  C. Bacteria were pelleted and supernatants were recovered for Western blot analysis using rabbit polyclonal antibodies directed against IpaD, IpaB, IpaH9.8, IcsB, IcsA and MxiC and mouse monoclonal antibodies against IpaC [28,42,43] and DnaK (Enzo Life Sciences).

3T3 fibroblasts used for this assay were cultured in Dulbeco’s Modified Eagle’s Medium (DMEM, Sigma) supplemented with 10% fetal bovine serum and penicillinestreptomycin (Lonza) in a humidified incubator under 5% CO2. As described [5], Shigella strains were grown at 37  C until an OD600 z 0.4e0.6 was reached, washed once with PBS and resuspended in DMEM. Bacteria were centrifuged on semiconfluent 3T3 cells, at a multiplicity of infection of 100, seeded 24 h before infection in a 96-well plate in DMEM. After incubation (30 min at 37  C), infected cells were washed three times and treated for 1 h with gentamicin (50 mg/ml) to kill extracellular bacteria. Finally, cells were washed again and then lysed with 0.1% Triton X-100 (15 min, RT) and intracellular bacteria were serially diluted and plated on TSB agar for colony forming units counting.

2.5. IpaD and IpaB surface exposure 3. Results Exposure of IpaD and IpaB was monitored by Fluorescence Activated Cell Sorting (FACS) as described previously [28]. Briefly, cultures of Shigella strains were grown at 37  C until an OD600 z 1.2 was reached. Pellets corresponding to 1.2  108 bacteria/ml were washed twice with ice-cold washing buffer (PBS, 0.1% Triton X-100); then fixed with paraformaldehyde (2%). After 20 min of incubation at Room Temperature (RT), bacteria were first washed with washing buffer and then with PBS alone. The pellet was resuspended in 500 ml of the blocking solution (PBS with 4% Bovine Serum Albumin (BSA)) and incubated for 1 h (1 h) at 4  C, then incubated overnight at 4  C in 250 ml of the blocking solution containing the primary antibody (mouse anti-GST-IpaD131e332 or anti-His6-IpgC þ IpaB [28]). After washing, bacterial pellets were resuspended in 250 ml of goat CF647-conjugated anti-mouse IgG antibody (1:500, Sigma) and incubated for 1 h at 4  C. The same washing procedure was repeated and bacteria were resuspended in 500 ml of PBS. Flow cytometry analyses were performed by a four-colour FACS Calibur on samples diluted 1:10 in a 5 ml polystyrene tube (BD Falcon). 2.6. Haemolysis of red blood cells The contact-mediated haemolysis assay was performed as described previously [8] with slight modifications. Bacteria from overnight culture were diluted (1:100) and grown at 37  C until an OD600 z 0.4e0.6 was reached and resuspended

3.1. Generation of thirteen IpaD variants and analysis of the T3S effectors production In our previous study, almost all ten amino acid deletions within the coiled-coil domain impaired translocator surface localisation, thereby preventing secretion control, translocon formation and cell entry [28]. As these large truncations may have altered the conformation of IpaD, we used single amino acid substitution of conserved residues shared with SipD and BipD (Fig. 1). Mutation of the thirteen selected residues Y149A, Y153A, T161D, Q165L, K189A, K205A, W226A, Y276A, Y301A, N305A, V312A, V314D, and I319D were generated on plasmid pSL1 and then introduced into the ipaD3 mutant (ipaD) for further phenotypic analysis. ipaDþ refers to ipaD strain harbouring plasmid pSL1 encoding native IpaD, while ipaDY149A designates ipaD strain harbouring a plasmid with substitution of IpaD tyrosine 149 by alanine. The thirteen generated IpaD variants were subsequently tested for their ability to restore wildtype virulence properties (Table 1). The production of proteins representative of different classes of T3S substrates, such as IpaB and IpaC (translocators), IcsB (early effector), MxiC (intracellular regulator) and IpaH (late effector) was assayed by western blotting. As shown in Fig. 2, similarly to ipaDþ and to wild-type (WT) strains, all tested proteins were equally produced by the ipaD variant strains except for IpaH, whose production levels were high only in ipaD, ipaDV314D and ipaDI319D strains.

Please cite this article in press as: Meghraoui A, et al., Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.010

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Fig. 1. Map of IpaD showing protein domains and mutated residues. Amino acid sequence alignment of IpaD (Shigella flexneri), SipD (Salmonella typhimurium), and BipD (Burkholderia pseudomallei) showing conserved residues (highlighted in black) realised by Uniprot Clustal. Domains are coloured as in legend and IpaD mutated residues are indicated.

3.2. Generated ipaD variant strains show three distinct secretion patterns Prior to the T3SA activation (upon host cell contact or environmental sensing), the secretion of effectors is tightly Table 1 Summary of IpaD phenotypes including IpaB/D surface localization, secretion control, 3T3 cell entry and red blood cells haemolysis. Strainsa

Translocators localizationb

Secretion controlc

Cell entryd

Haemolysise

ipaDþ ipaD WT ipaDY149A ipaDY153A ipaDT161D ipaDQ165L ipaDK189A ipaDK205A ipaDW226A ipaDY276A ipaDY301A ipaDN305A ipaDV312A ipaDV314D ipaDI319D

þ  þ þ þ þ þ þ þ þ þ þ þ þ þ þ

þ  þ þ    þ þ þ  þ þ þ  

þ  þ þ þþ þþ þþ þ þ þ þþ þ þ þ þ þ

þ  þ NT þþ þþ þþ NT NT NT þþ NT NT NT þ 

NT: not tested. a ipaD strains correspond to ipaD expressing (þ) or not () ipaD or its derivatives (point mutations). b Surface exposition (þ) or not () of both IpaD and IpaB measured by FACS. c The secretion of ipaD strain is either controlled (þ) as the ipaDþ strain or constitutive () as the ipaD strain or presents an intermediate phenotype (). d ipaD strain is considered as invasive (þ) like the ipaDþ strain, non invasive () like the ipaD strain, or highly invasive (þþ). e ipaD strain is considered as haemolytic (þ) like the ipaDþ strain, nonhaemolytic () like the ipaD strain or highly haemolytic (þþ).

controlled, while inactivation of ipaB or ipaD gene lead to a constitutive secretion phenotype [41,44]. To investigate whether IpaD variants would affect the secretion control, proteins secreted by bacteria grown to stationary phase were analysed and revealed three groups of secretion patterns (Fig. 3A). The first group, including ipaDY149A, ipaDK189A, ipaDK205A, ipaDW226A, ipaDY301A, ipaDN305A and ipaDV312A strains, behaved similarly to the ipaDþ strain with weak leakage of T3S effectors. The second group, ipaDV314D and ipaDI319D strains, that produced high levels of IpaH, exhibited a constitutive secretion pattern almost similar to that of the ipaD strain. The third group composed of ipaDY153A, ipaDT161D, ipaDQ165L and ipaDY276A strains, showed an intermediate secretion pattern, with the early effector IpaA and

Fig. 2. Production of effectors by ipaD variant strains. Total extracts of overnight cultures of Shigella WT and ipaD strain either alone or complemented with native IpaD or its derivatives were collected, separated on SDS-PAGE and probed with antibodies directed against indicated proteins. A nonspecific band recognized by the anti-MxiC antibodies was used as a loading control. This result is representative of two independent experiments.

Please cite this article in press as: Meghraoui A, et al., Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.010

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consequence of an altered needle tip composition, all variants were tested by FACS for their ability to expose IpaB and IpaD at the bacterial surface. As expected, neither IpaB/IpaD were detected in the mxiH and ipaD mutants nor IpaB in the ipaB strain while both proteins were surface exposed in the wild-type and ipaDþ strains (Fig. S3A). Although IpaB was not surface exposed in the ipaD strain, IpaD was detected in the ipaB strain (Fig. S3A). These findings support the models of initial exposure of IpaB and IpaD and the dependence of IpaB on IpaD described previously [28e30]. The ipaDþ strain was used as a positive control for all FACS experiments. As shown in Fig. S3B and Table 1, surface exposure of IpaD and IpaB in all tested variants was similar to that of ipaDþ strain. Thus, our results suggest that the complete (ipaDV314D and ipaDI319D) or partial (ipaDY153A, ipaDT161D, ipaDQ165L and ipaDY276A) deregulation of secretion control are unlikely associated to a loss of IpaD and/ or IpaB needle tip localisation. 3.4. ipaDY153, ipaDT161D, ipaDQ165L and ipaDY276A strains exhibit enhanced cell invasion

Fig. 3. Effectors secretion by ipaD variant strains. Proteins of the supernatant prepared from overnight cultures of Shigella WT and ipaD strain either alone or complemented with native IpaD or its derivatives were separated on SDSPAGE and stained with Coomassie blue (A). The same cultures were run to recover proteins secreted by bacteria grown to exponential phase. Proteins of the supernatant were separated on SDS-PAGE and probed as in Fig. 2 (B). These results are representative of two independent experiments.

translocators being highly secreted (Fig. 3A). Proteins of the supernatant precipitated from bacteria grown to the stationary phase correspond to cumulated proteins that may form aggregates or be degraded [44]. For a better analysis, we monitored secretion of different classes of T3S effectors with bacteria grown to exponential phase. As shown in Fig. 3B, and similarly to the ipaD strain, mutations V314D and I319D abolished secretion control. In contrast, some mutants among the group that showed intermediate secretion phenotype exhibited an increased secretion of IpaD, IpaB, IpaC and/or IcsB (Fig. 3, Fig. S2B). For instance, the secretion of IpaB by ipaDY276A strain is almost three fold relative to ipaDþ(Fig. S2B). The increased secretion of translocators and early effectors in some mutants was not associated to their overproduction (Fig. 2). The remaining mutations Y149A, K189A, K205A, W226A, Y301A, N305A, and V312A presented the ipaDþ strain secretion pattern (Fig. 3). In conclusion, the secretion via the T3SA is partially deregulated in ipaDY153A, ipaDT161D, ipaDQ165L and ipaDY276A strains while it is totally constitutive in ipaDV314D and ipaDI319D strains. 3.3. Surface localisation of IpaD and IpaB at the needle tip is not affected in ipaD variant strains The exposure of both IpaD and IpaB at the needle tip is central for a functional T3SA [24,28,32]. To verify whether the deregulated secretion observed in some mutants is a

Constitutive protein secretion via the T3SA is not systematically an indicator of an avirulent phenotype, as short Cterminal deletions of IpaB lead to a secretion deregulation without impairing cell invasion [27]. However, we have previously shown that localisation of IpaB and IpaD is necessary but not sufficient to allow entry into host cells [28]. We therefore tested ipaD variant strains phenotype in 3T3 fibroblasts cell invasion. As shown in Fig. 4, ipaDY149A, ipaDK189A, ipaDK205A, ipaDW226A, ipaDY301A, ipaDN305A, ipaDV312A, ipaDV314D, and ipaDI319D strains restored the invasion defect of the ipaD strain to the level of ipaDþ strain. In contrast, ipaDY153A, ipaDT161D, ipaDQ165L and ipaDY276A strains, which partially affected the secretion of translocators and early effectors, exhibited a two-fold increase in cell invasion (Fig. 4). Thus, our results discriminate between constitutive secretion and cell invasion properties. Together, our finding define two

Fig. 4. Cell entry of ipaD variant strains. 3T3 fibroblast cells were infected with ipaD variant strains and cell entry was evaluated through colony forming units (CFU) counting. Percentages are relative to ipaDþ strain that was defined as invading cells at 100%. The shown values represent the average of two independent experiments performed each in triplicate (standard deviation). **p < 0,05 for comparison by a Student t test.

Please cite this article in press as: Meghraoui A, et al., Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.010

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new phenotypes that we designated i) Constitutive invasive variants (Civ) for ipaDV312A and ipaDV314D strains and ii) Enhanced virulence variants (Evv) for ipaDY153A, ipaDT161D, ipaDQ165L and ipaDY276A strains.

indicate that premature or faster secretion of translocators by the Evv strains induces an enhanced pore formation, which consequently increased cell invasion. 4. Discussion

3.5. ipaDY153A, ipaDT161D, ipaDQ165L and ipaDY276A strains exhibit an enhanced pore formation The loss of invasiveness due to the inactivation of ipaD is linked to a defect in the translocation pore formation in the cell membrane [25]. However, both features can be uncoupled as observed with ipaB deletion mutants that are able to invade cells without inserting a translocation pore [27]. To check whether or not the phenotype of the Civ and Evv strains is linked to pore formation, we performed a haemolysis assay in which RBCs were forced into contact with bacteria (see M&M). The ability to form the translocon was monitored by the quantification of haemoglobin release at different bacterial concentrations. As shown in Fig. 5, increased bacterial concentrations correlated with an increased RBCs lysis, suggesting that it is accompanied by an increased number of functional translocons, but RBCs lysis reached saturation at 1.5  1010 bacteria/ml. The ipaDV314D strain exhibited a haemoglobin release similar to the one induced by the ipaDþ strain, while it was decreased for the ipaDI319D strain to around 50% lysis in comparison to the 85% induced by the ipaDþ strain at 1010 bacteria/ml (Fig. 5). Therefore, the wildtype levels of invasion observed with ipaDV314D strain correlated with the levels of RBCs lysis. However, even if the ipaDI319D strain was able to exhibit wild-type levels of invasion, it was unable to lyse RBCs to the ipaDþ strain level. Furthermore, at lower bacterial concentrations, we revealed enhanced pore formation in Evv strains. Remarkably, at 0.5  1010 bacteria/ml, the haemoglobin release was higher (z82%) in ipaDQ165L strain compared to 55e65% in ipaDY153A, ipaDT161D, and ipaDY276A strains. Together, our results

Fig. 5. Haemolysis of ipaD variant strains. RBCs were forced into contact with Shigella WT, ipaD, ipaDþ, Evv and Civ strains. After 25 min of incubation at 37  C, the lysis capacity was evaluated by haemoglobin release measurement at 540 nm at different bacterial concentrations. 100% refers to haemolysis of ipaDþ strain at 2  1010 bacteria/ml. Values represent the average of triplicates (standard deviation) and the graph is representative of two independent experiments.

We report here a phenotypic characterization of thirteen IpaD variants obtained by single directed mutagenesis of selected residues conserved among homologous proteins in Salmonella and Bulkholderia. Compared to the ten amino acid deletion strategy or random insertions of 19 amino acids in PcrV of P. aeruginosa [28,45], the approach used in this study is probably less disruptive to IpaD structure. Indeed, the overall IpaD and IpaB surface localisation in our generated ipaD variant strains remained unaffected, which contributes to a better characterization of key virulence properties. In summary, in addition to the wild-type phenotype, our analysis pointed out two additional groups of mutants, Civ and Evv, with discrete dysfunctions in secretion control, cell invasion, and translocation pore formation. Our study revealed that the Evv strains tend to increase secretion of translocators IpaBCD and early effector IcsB. A comparable secretion profile was reported for “early variants” obtained with 10 amino acid deletion [28]. However, unlike deletion variants, an increase in pore formation and cell entry by the Evv strains was observed. It is tempting to hypothesize that Evv group mimics natural conformational change that normally occurs on IpaD through DOC binding [31,33], known to increase cell invasion by Shigella [32,36]. Interestingly, it has been shown that bile salts induce the leakage of proteins belonging to the translocators and early effectors classes [46]. As the fifth position of IpaD pentamer at the needle tip might be occupied by IpaB [29,39], IpaD conformational change may directly provoke a modified interaction between IpaB and IpaD at the needle tip, thereby modulating the closing and opening of the tip complex. However, our FACS analysis did not reveal a detectable change of their surface exposure, which suggests either a technical limitation or an exposure change that would occur transiently during bacterial growth. Yet, it is difficult to interpret our findings based on the distribution of mutated residues on IpaD structure (Fig. S1). In fact, no common point between the four substituted residues of the Evv group can be deduced, except that they all belong to the coiled-coil domain. Besides, Y153 is the only residue oriented opposite to the three others, T161, Q165 and Y276 (Fig. S1), but we cannot exclude their possible involvement in IpaD oligomerisation and IpaB binding through the coiled-coil [29,37,39]. In this case, the same protein will be faced in both interfaces of helices H3 and H7 involved in the pentamer formation. One may speculate that the Evv mutations represent an activation state of the T3SA that occurs in the appropriate environment (the intestinal lumen), a phenotype likely associated to the premature needle tip complex maturation, which consequently enhances the translocon formation and cell entry. Finally, these findings suggest that IpaD controls Shigella virulence through the modulation of the translocators/early effectors secretion.

Please cite this article in press as: Meghraoui A, et al., Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.010

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Secretion control of major T3S proteins was strongly affected in Civ strains; however, these two mutants kept their wild-type ability to invade cells. Similar results were previously reported with a Shigella strain expressing a C-terminal truncated IpaB that constitutively secretes proteins while keeping cell invasion unaffected [27]. We propose that, as far as the surface exposure of translocators is maintained, this feature is sufficient to permit bacterial entry under our experimental conditions, independently from the secretion state itself. Within the Civ group, the conserved hydrophobic residues V314 and I319 were replaced by the charged amino acid aspartate (D). A similar approach has previously been used by Gebus and coworkers to demonstrate the role of the C-terminal hydrophobic residues of PcrV in its oligomerisation and in mediating cell cytotoxicity [47]. Here, we demonstrate that mutations V314D and I319D retained IpaD ability to i) localise IpaB and IpaD at the bacterial surface, ii) invade cells, and iii) ensure a partial (ipaDI319D strain) or complete (ipaDV314D strain) pore formation but iv) not secretion control. Therefore, if a similar defect in oligomerization has taken place, it would affect the conformation of the pentameric structure of IpaD rather than the needle tip composition, probably through switching to the allosteric “open” state described for SipD [38]. Besides, we have reported that IpaB localisation determines the secretion of late effectors [28], but Civ strains, that have the same secretion phenotype, maintained IpaB surface exposure. This supports the hypothesis of a conformational change of IpaD, and therefore a change in the state of the tip complex. One consequence resulting from Civ strains might be a partial opening of the N-terminal domain similarly to the one of the central domain observed by Epler and co-workers (2012) [37]. In conclusion, the study of the Civ strains confirms that the lack of secretion control does not necessarily impair cell invasion. The secretion control is linked to T3S signal transmission from the tip complex to the base through MxiH/MxiI, which leads to internal activation of secretion [12,22,23,35]. We report here that Civ strains induced MxiC secretion, which probably reflect the signal transmission mechanism. Furthermore, these variants induced production and earlier secretion of the late effector IpaH. Thus, both signal transmission and transcriptional regulation mechanisms can explain the deregulated secretion observed in the Civ group. However, these features were not totally fulfilled in the Evv group, suggesting that signal transmission from the tip to the base is not sufficient or incomplete. This is supported by the reduced secretion of MxiC and IpaH by bacteria grown to exponential phase. Thus, the Evv group exhibited an intermediate secretion control that discriminate between early and late effectors. In conclusion, our study focused mainly on IpaD, but drove us to conclusions including the other tip complex partners and raised more additional questions about T3S function. A more global view of the pore formation function can be drawn through further studying the relation within IpaB/C/D complex and their effect on secretion signal transmission from the T3SA tip to the base. We also extend our knowledge about the link between major steps required for Shigella virulence as we found mutants that discriminate between IpaD functions in

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66 67 68 69 70 71 72 Acknowledgments 73 74 75 The present work was supported by grants from the ‘Fonds 76 National de la Recherche Scientifique e Fonds national Belge 77 de la Recherche Scientifique’ (FRS-FNRS; Convention 78 F.3.4556.11) and from the European Community’s Seventh 79 Framework Program: StopEnterics: FP7/2011-2015 under 80 grant agreement No. 261472. A.M. and L.S. were recipients of 81 82 a PhD fellowship from the Belgian Fonds pour la formation a` 83 la Recherche dans l’Industrie et dans l’Agriculture (FRIA) and 84 LS was a recipient of one year fellowship from FP7/201185 2015 program. A part of this work was also supported by 86 the Fonds Defay and by the Alice and David Van Buuren 87 Foundation. We thank Anne Op de Beeck for FACS facilities 88 and Maxime Lefe`vre for his contribution to construct plasmids 89 90 pML1 and pML2. Lastly, we thank N. El Hajjami and 91 A.Hachani for critical reading of the manuscript and C. Parsot 92 Q1 and L. Van Melderen for helpful discussions. 93 94 95 Appendix A. Supplementary data 96 97 98 Supplementary data related to this article can be found at 99 http://dx.doi.org/10.1016/j.micinf.2014.03.010 100 101 Q2 References 102 103 [1] Barry EM, Pasetti MF, Sztein MB, Fasano A, Kotloff KL, Levine MM. 104 Progress and pitfalls in Shigella vaccine research. Nat Rev Gastroenterol 105 Hepatol 2013;10:245e55. 106 [2] Bardhan P, Faruque AS, Naheed A, Sack DA. Decrease in shigellosis107 related deaths without Shigella spp. e specific interventions, Asia. 108 Emerg Infect Dis 2010;16:1718e23. 109 [3] Zychlinsky A, Kenny B, Menard R, Prevost MC, Holland IB, 110 Sansonetti PJ. IpaB mediates macrophage apoptosis induced by Shigella 111 flexneri. Mol Microbiol 1994;11:619e27. 112 [4] Sansonetti PJ, Phalipon A. M cells as ports of entry for enteroinvasive 113 pathogens: mechanisms of interaction, consequences for the disease 114 process. Semin Immunol 1999;11:193e203. 115 [5] Mounier J, Vasselon T, Hellio R, Lesourd M, Sansonetti PJ. Shigella 116 flexneri enters human colonic Caco-2 epithelial cells through the baso117 lateral pole. Infect Immun 1992;60:237e48. 118 [6] Tamano K, Aizawa S, Katayama E, Nonaka T, Imajoh-Ohmi S, Kuwae A, et al. Supramolecular structure of the Shigella type III 119 secretion machinery: the needle part is changeable in length and essential 120 for delivery of effectors. EMBO J 2000;19:3876e87. 121 [7] Blocker A, Jouihri N, Larquet E, Gounon P, Ebel F, Parsot C, et al. 122 Structure and composition of the Shigella flexneri “needle complex”, a 123 part of its type III secreton. Mol Microbiol 2001;39:652e63. 124 [8] Blocker A, Gounon P, Larquet E, Niebuhr K, Cabiaux V, Parsot C, et al. 125 The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC 126 into host membranes. J Cell Biol 1999;147:683e93. 127 [9] Schroeder GN, Hilbi H. Molecular pathogenesis of Shigella spp.: con128 trolling host cell signaling, invasion, and death by type III secretion. Clin 129 Microbiol Rev 2008;21:134e56. 130

secretion control, pore formation, and cell entry. In addition, our work highlights that increased secretion of translocators and early effectors correlates with increased virulence, which strongly suggests that this secretion state take place during early stages of Shigella infection.

Please cite this article in press as: Meghraoui A, et al., Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence, Microbes and Infection (2014), http://dx.doi.org/10.1016/j.micinf.2014.03.010

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[10] Buttner D. Protein export according to schedule: architecture, assembly, and regulation of type III secretion systems from plant- and animalpathogenic bacteria. Microbiol Mol Biol Rev 2012;76:262e310. [11] Diepold A, Wagner S. Assembly of the bacterial type III secretion machinery. FEMS Microbiol Rev; 2014. http://dx.doi.org/10.1111/15746976.12061. [12] Cherradi Y, Schiavolin L, Moussa S, Meghraoui A, Meksem A, Biskri L, et al. Interplay between predicted inner-rod and gatekeeper in controlling substrate specificity of the type III secretion system. Mol Microbiol 2013;87:1183e99. [13] Cordes FS, Komoriya K, Larquet E, Yang S, Egelman EH, Blocker A, et al. Helical structure of the needle of the type III secretion system of Shigella flexneri. J Biol Chem 2003;278:17103e7. [14] Akeda Y, Galan JE. Chaperone release and unfolding of substrates in type III secretion. Nature 2005;437:911e5. [15] Dohlich K, Zumsteg AB, Goosmann C, Kolbe M. A substrate-fusion protein is trapped inside the type III secretion system channel in Shigella flexneri. PLoS Pathog 2014;10:e1003881. [16] Botteaux A, Sani M, Kayath CA, Boekema EJ, Allaoui A. Spa32 interaction with the inner-membrane Spa40 component of the type III secretion system of Shigella flexneri is required for the control of the needle length by a molecular tape measure mechanism. Mol Microbiol 2008;70:1515e28. [17] Parsot C, Ageron E, Penno C, Mavris M, Jamoussi K, d’Hauteville H, Sansonetti P, Demers B. A secreted anti-activator, OspD1, and its chaperone, Spa15, are involved in the control of transcription by the type III secretion apparatus activity in Shigella flexneri. Mol Microbiol 2005;56:1627e35. [18] Menard R, Sansonetti P, Parsot C, Vasselon T. Extracellular association and cytoplasmic partitioning of the IpaB and IpaC invasins of S. flexneri. Cell 1994;79:515e25. [19] Mavris M, Page AL, Tournebize R, Demers B, Sansonetti P, Parsot C. Regulation of transcription by the activity of the Shigella flexneri type III secretion apparatus. Mol Microbiol 2002;43:1543e53. [20] Pilonieta MC, Munson GP. The chaperone IpgC copurifies with the virulence regulator MxiE. J Bacteriol 2008;190:2249e51. [21] Menard R, Sansonetti P, Parsot C. The secretion of the Shigella flexneri Ipa invasins is activated by epithelial cells and controlled by IpaB and IpaD. EMBO J 1994;13:5293e302. [22] Martinez-Argudo I, Blocker AJ. The Shigella T3SS needle transmits a signal for MxiC release, which controls secretion of effectors. Mol Microbiol 2010;78:1365e78. [23] Botteaux A, Sory MP, Biskri L, Parsot C, Allaoui A. MxiC is secreted by and controls the substrate specificity of the Shigella flexneri type III secretion apparatus. Mol Microbiol 2009;71:449e60. [24] Epler CR, Dickenson NE, Olive AJ, Picking WL, Picking WD. Liposomes recruit IpaC to the Shigella flexneri type III secretion apparatus needle as a final step in secretion induction. Infect Immun 2009;77:2754e61. [25] Picking WL, Nishioka H, Hearn PD, Baxter MA, Harrington AT, Blocker A, et al. IpaD of Shigella flexneri is independently required for regulation of Ipa protein secretion and efficient insertion of IpaB and IpaC into host membranes. Infect Immun 2005;73:1432e40. [26] Shen DK, Saurya S, Wagner C, Nishioka H, Blocker AJ. Domains of the Shigella flexneri type III secretion system IpaB protein involved in secretion regulation. Infect Immun 2010;78:4999e5010. [27] Roehrich AD, Martinez-Argudo I, Johnson S, Blocker AJ, Veenendaal AK. The extreme C terminus of Shigella flexneri IpaB is required for regulation of type III secretion, needle tip composition, and binding. Infect Immun 2010;78:1682e91. [28] Schiavolin L, Meghraoui A, Cherradi Y, Biskri L, Botteaux A, Allaoui A. Functional insights into the Shigella type III needle tip IpaD in secretion control and cell contact. Mol Microbiol 2013;88:268e82. [29] Johnson S, Roversi P, Espina M, Olive A, Deane JE, Birket S, et al. Selfchaperoning of the type III secretion system needle tip proteins IpaD and BipD. J Biol Chem 2007;282:4035e44.

[30] Veenendaal AK, Hodgkinson JL, Schwarzer L, Stabat D, Zenk SF, Blocker AJ. The type III secretion system needle tip complex mediates host cell sensing and translocon insertion. Mol Microbiol 2007;63:1719e30. [31] Dickenson NE, Zhang L, Epler CR, Adam PR, Picking WL, Picking WD. Conformational changes in IpaD from Shigella flexneri upon binding bile salts provide insight into the second step of type III secretion. Biochemistry 2011;50:172e80. [32] Olive AJ, Kenjale R, Espina M, Moore DS, Picking WL, Picking WD. Bile salts stimulate recruitment of IpaB to the Shigella flexneri surface, where it colocalizes with IpaD at the tip of the type III secretion needle. Infect Immun 2007;75:2626e9. [33] Stensrud KF, Adam PR, La Mar CD, Olive AJ, Lushington GH, Sudharsan R, et al. Deoxycholate interacts with IpaD of Shigella flexneri in inducing the recruitment of IpaB to the type III secretion apparatus needle tip. J Biol Chem 2008;283:18646e54. [34] Lafont F, Tran Van Nhieu G, Hanada K, Sansonetti P, van der Goot FG. Initial steps of Shigella infection depend on the cholesterol/sphingolipid raft-mediated CD44-IpaB interaction. EMBO J 2002;21:4449e57. [35] Roehrich AD, Guillossou E, Blocker AJ, Martinez-Argudo I. Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation. Mol Microbiol 2013;87:690e706. [36] Barta ML, Guragain M, Adam P, Dickenson NE, Patil M, Geisbrecht BV, et al. Identification of the bile salt binding site on IpaD from Shigella flexneri and the influence of ligand binding on IpaD structure. Proteins 2011;80:935e45. [37] Epler CR, Dickenson NE, Bullitt E, Picking WL. Ultrastructural analysis of IpaD at the tip of the nascent MxiH type III secretion apparatus of Shigella flexneri. J Mol Biol 2012;420:29e39. [38] Lunelli M, Hurwitz R, Lambers J, Kolbe M. Crystal structure of PrgISipD: insight into a secretion competent state of the type three secretion system needle tip and its interaction with host ligands. PLoS Pathog 2011;7:e1002163. [39] Blocker AJ, Deane JE, Veenendaal AK, Roversi P, Hodgkinson JL, Johnson S, et al. What’s the point of the type III secretion system needle? Proc Natl Acad Sci U S A 2008;105:6507e13. [40] Allaoui A, Mounier J, Prevost MC, Sansonetti PJ, Parsot C. icsB: a Shigella flexneri virulence gene necessary for the lysis of protrusions during intercellular spread. Mol Microbiol 1992;6:1605e16. [41] Menard R, Sansonetti PJ, Parsot C. Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol 1993;175:5899e906. [42] Kayath CA, Hussey S, El hajjami N, Nagra K, Philpott D, Allaoui A. Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector, IcsB. Microbes Infect 2010;12:956e66. [43] Goldberg MB, Barzu O, Parsot C, Sansonetti PJ. Unipolar localisation and ATPase activity of IcsA, a Shigella flexneri protein involved in intracellular movement. Infect Agents Dis 1993;2:210e1. [44] Parsot C, Menard R, Gounon P, Sansonetti PJ. Enhanced secretion through the Shigella flexneri Mxi-Spa translocon leads to assembly of extracellular proteins into macromolecular structures. Mol Microbiol 1995;16:291e300. [45] Sato H, Hunt ML, Weiner JJ, Hansen AT, Frank DW. Modified needle-tip PcrV proteins reveal distinct phenotypes relevant to the control of type III secretion and intoxication by Pseudomonas aeruginosa. PLoS One 2011;6:e18356. [46] Pope LM, Reed KE, Payne SM. Increased protein secretion and adherence to HeLa cells by Shigella spp. following growth in the presence of bile salts. Infect Immun 1995;63:3642e8. [47] Gebus C, Faudry E, Bohn YS, Elsen S, Attree I. Oligomerization of PcrV and LcrV, protective antigens of Pseudomonas aeruginosa and Yersinia pestis. J Biol Chem 2008;283:23940e9.

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Single amino acid substitutions on the needle tip protein IpaD increased Shigella virulence.

Infection of colonic epithelial cells by Shigella is associated with the type III secretion system, which serves as a molecular syringe to inject effe...
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