Portal de Periódicos da CAPES

Selective blockage of Serratia marcescens ShlA by nickel inhibits the pore‐forming toxin‐mediated phenotypes in eukaryotic cells

2019; Wiley; Volume: 21; Issue: 9 Linguagem: Inglês

10.1111/cmi.13045

1462-5822

Martina Lazzaro, Darío Krapf, Eleonora Garcı́a Véscovi,

Clostridium difficile and Clostridium perfringens research

Cellular MicrobiologyVolume 21, Issue 9 e13045 RESEARCH ARTICLEFree Access Selective blockage of Serratia marcescens ShlA by nickel inhibits the pore-forming toxin-mediated phenotypes in eukaryotic cells Martina Lazzaro, Corresponding Author Martina Lazzaro lazzaro@ibr-conicet.gov.ar Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Rosario, Argentina Correspondence Martina Lazzaro and Eleonora García Véscovi, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Ocampo y Esmeralda s/n, Predio CCT-CONICET-Rosario, 2000 Rosario. Argentina. Email: lazzaro@ibr-conicet.gov.ar; garciavescovi@ibr-conicet.gov.arSearch for more papers by this authorDarío Krapf, Darío Krapf Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Rosario, ArgentinaSearch for more papers by this authorEleonora García Véscovi, Corresponding Author Eleonora García Véscovi garciavescovi@ibr-conicet.gov.ar orcid.org/0000-0002-4431-8606 Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Rosario, Argentina Correspondence Martina Lazzaro and Eleonora García Véscovi, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Ocampo y Esmeralda s/n, Predio CCT-CONICET-Rosario, 2000 Rosario. Argentina. Email: lazzaro@ibr-conicet.gov.ar; garciavescovi@ibr-conicet.gov.arSearch for more papers by this author Martina Lazzaro, Corresponding Author Martina Lazzaro lazzaro@ibr-conicet.gov.ar Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Rosario, Argentina Correspondence Martina Lazzaro and Eleonora García Véscovi, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Ocampo y Esmeralda s/n, Predio CCT-CONICET-Rosario, 2000 Rosario. Argentina. Email: lazzaro@ibr-conicet.gov.ar; garciavescovi@ibr-conicet.gov.arSearch for more papers by this authorDarío Krapf, Darío Krapf Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Rosario, ArgentinaSearch for more papers by this authorEleonora García Véscovi, Corresponding Author Eleonora García Véscovi garciavescovi@ibr-conicet.gov.ar orcid.org/0000-0002-4431-8606 Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Rosario, Argentina Correspondence Martina Lazzaro and Eleonora García Véscovi, Instituto de Biología Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Científicas y Tecnológicas, Universidad Nacional de Rosario, Ocampo y Esmeralda s/n, Predio CCT-CONICET-Rosario, 2000 Rosario. Argentina. Email: lazzaro@ibr-conicet.gov.ar; garciavescovi@ibr-conicet.gov.arSearch for more papers by this author First published: 17 May 2019 https://doi.org/10.1111/cmi.13045Citations: 5AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Abstract Serratia marcescens is an opportunistic pathogen with increasing incidence in clinical settings. This is mainly attributed to the timely expression of a wide diversity of virulence factors and intrinsic and acquired resistance to antibiotics, including β-lactams, aminoglycosides, quinolones, and polypeptides. For these reasons, S. marcescens has been recently categorised by the World Health Organization as one priority to strengthen efforts directed to develop new antibacterial agents. Therefore, it becomes critical to understand the underlying mechanisms that allow Serratia to succeed within the host. S. marcescens ShlA pore-forming toxin mediates phenotypes that alter homeostatic and signal transduction pathways of host cells. It has been previously demonstrated that ShlA provokes cytotoxicity, haemolysis and autophagy and also directs Serratia egress and dissemination from invaded nonphagocytic cells. However, molecular details of ShlA mechanism of action are still not fully elucidated. In this work, we demonstrate that Ni2+ selectively and reversibly blocks ShlA action, turning wild-type S. marcescens into a shlA mutant strain phenocopy. Combined use of Ni2+ and calcium chelators allow to discern ShlA-triggered phenotypes that require intracellular calcium mobilisation and reveal ShlA function as a calcium channel, providing new insights into ShlA mode of action on target cells. 1 INTRODUCTION Serratia is a Gram-negative bacterium that can be isolated from diverse ambient niches: soil, water, and air. Strains of S. marcescens are wide-host-range pathogens, being able to infect plants, vertebrates, and invertebrates. In humans, it is classified as an opportunistic pathogen with reported increasing nosocomial infection incidence, being the aetiological agent of meningitis, pneumonia, and bacteraemia (Gastmeier, 2014). In 2017, S. marcescens was included by the World Health Organization in the list of antibiotic resistant priority pathogens, which highlighted research critical targets to develop alternative antimicrobial strategies (Lawe-Davies, 2017). In addition, S. marcescens was identified as one of the most abundant microbial species that colonise the gut microbiome in familial Crohn disease dysbiosis (Hoarau et al., 2016). We have previously reported that upon contact with eukaryotic cells, the expression of ShlA pore-forming toxin (PFT) by S. marcescens induces an autophagic response from the extracellular medium (Di Venanzio, Stepanenko, & García Véscovi, 2014). Serratia is able to invade, survive, and proliferate inside nonphagocytic cells, within a membrane bound compartment called Serratia containing vacuole (SeCV; Fedrigo, Campoy, Di Venanzio, Colombo, & García Véscovi, 2011). We have also demonstrated that, after intravacuolar proliferation, ShlA is responsible for provoking an increase in the cytosolic Ca2+ concentration that also remodels the actin cytoskeleton dynamics, promoting a nonlytic, exocytic-like egress of bacteria (Di Venanzio, Lazzaro, Morales, Krapf, & García Véscovi, 2017). It has been reported that cells maintain a resting intracellular Ca2+ concentration close to 100 nM (Maravall, Mainen, Sabatini, & Svoboda, 2000). In contrast, the endoplasmic reticulum, the Golgi apparatus, secretory vesicles, endosomes, autophagosomes, and lysosomes have luminal Ca2+ concentrations in the micromolar to millimolar range and function as intracellular calcium reservoirs (Dong, Wang, & Xu, 2010). Bacterial PFTs are able to modify the intracellular Ca2+ concentration, exerting a regulatory effect on eukaryotic phenotypes such as cell adhesion and cell death (Tran Van Nhieu, Dupont, & Combettes, 2018). Selectivity of PFTs pores depends on the nature of the constituent protein and its concentration: PFTs selectivity decreases as their concentration increases (Bischofberger, Iacovache, & Van Der Goot, 2012; Hotze & Tweten, 2012). In addition, the pores formed by members of the cholesterol-dependent cytolysins (CDCs) family are nonselective and cause a massive Ca2+ influx, resulting in several host-cell responses, such as the reseal of injured membrane sites, cytokine release, and cell death (Babiychuk & Draeger, 2015; Gonzalez, Bischofberger, Pernot, Van Der Goot, & Frêche, 2008). The toxins from the repeats-in-toxin family form small pores with a higher selectivity than CDCs and induce transient Ca2+ oscillations from internal stores (Gonzalez, Bischofberger, Pernot, Van Der Goot, & Frêche, 2008; Linhartova et al., 2010). ShlA haemolysin has been considered one of the main virulence factors of Serratia. ShlA is encoded by S. marcescens shlBA operon and secreted by a type Vb two-partner secretion system, composed by the ShlB translocator and by ShlA (Mazar & Cotter, 2007; Schiebel, Schwarz, & Braun, 1989). This PFT is a different type of haemolysin that does not present similarity in sequence or structure with repeats-in-toxin or CDC toxins. Haemolysins with homology with ShlA can be found encoded in the genomes of Proteus mirabilis, Proteus vulgaris, Haemophilus ducreyi, Edwardsiella tarda, or Erwinia chrysanthemi (Hertle, 2000). It has been demonstrated that ShlA exerts a cytotoxic action on different cell lines and that it can form pores in erythrocytes, fibroblasts, and epithelial cells. At sublytic concentrations, ShlA induces ATP depletion and K+ efflux from epithelial cells and fibroblasts. At higher concentrations, ShlA has been shown to be cytotoxic (Hertle, Hilger, Weingardt-Kocher, & Walev, 1999). It has been reported that the haemolysin is a critical virulence factor in several infection models. Marre, Hacker, and Braun (1989) have demonstrated that ShlA contributes to uropathogenicity in a rat model, and Kurz et al. (2003) identified shlBA as genes necessary for full in vivo virulence in Drosophila melanogaster and Caenorhabditis elegans. It has been reported that PFTs are common among pathogenic bacteria and that they represent 30% of the cytotoxic bacterial proteins. Considering that PFTs are the largest category of virulence factors, these toxins are important targets for the development of therapeutic antibacterial approaches (Los, Randis, Aroian, & Ratner, 2013). In this work, we demonstrate that Ni2+ is a potent inhibitor of the ShlA haemolysin, impeding haemolytic activity, cytotoxicity, autophagic response induction, and bacterial egress from invaded epithelial cells. Therefore, we herein show that, in the presence of Ni2+, wild-type Serratia behaves like a shlBA mutant. Moreover, we provide evidences that the inhibition of ShlA by Ni2+ is reversible. Our data indicate that ShlA is responsible for two distinct phenotypes on eukaryotic cells: those caused by intracellular Serratia, which are dependent of ShlA-mediated Ca2+ mobilisation, and those that are induced by extracellular Serratia, which do not necessarily involve intracellular Ca2+ concentration fluctuations. We postulate that the use of Ni2+ to selectively block ShlA is a useful tool to understand the PFT mechanism of action and dissect its relevance along Serratia infection steps in the host. Moreover, because blocking of ShlA action impedes Serratia pathogenic traits, we can foresee that structural insights of ShlA inhibition by Ni2+ will provide new tools for the rational design of ShlA inhibitors as antibacterial agents to combat Serratia infections. 2 RESULTS AND DISCUSSION We previously determined that, after intravacuolar replication, S. marcescens is able to promote its exit from the invaded epithelial cell in a ShlA-mediated process. In spite of being dependent on the expression of a PFT, Serratia exit strategy does not alter the integrity of the host cell and shows mechanistic hallmarks of an exocytic process (Di Venanzio, Lazzaro, Morales, Krapf, & García Véscovi, 2017). We have previously shown that the ShlA-mediated egress process was triggered by an increase of intracellular calcium concentration and blocked by addition of the intracellular Ca2+ chelator BAPTA-AM (Di Venanzio, Lazzaro, Morales, Krapf, & García Véscovi, 2017). We have therefore hypothesised that ShlA might act itself as a calcium channel or alternatively that it could modulate the activity of a calcium channel from the host cell. Considering that the widely distributed T-type calcium channels are blocked by Ni2+ (Díaz, Bartolo, Delgadillo, Higueldo, & Gomora, 2005; Kang et al., 2006; Lee, Gomora, Cribbs, & Perez-Reyes, 1999; Todorovic & Lingle, 1998), we analysed the possible role of Ni2+ as a blocker of Serratia exit from the invaded cell. To this aim, different concentrations of NiCl2 were added to the antibiotic-free culture medium of invaded Chinese hamster ovary (CHO) cells at 240 min p.i. Extracellular-released bacteria were monitored by CFU (colony-forming unit) enumeration at 360 min p.i. As shown in Figure 1a, the addition of 1-mM NiCl2 reduced 60% CFU levels from released bacteria, whereas lower Ni2+ concentrations did not affect bacterial egress. Moreover, this reduction in extracellular CFU was accompanied with an increase of 84% in intracellular CFU (Figure 1b,c). No significant differences in Serratia growth rates were observed in either LB or α-MEM when supplemented with up to 1 mM NiCl2 (Figure S1A, B). These results indicate that Ni2+ prevents ShlA-dependent Serratia egress probably through calcium mobilisation blockage. Figure 1Open in figure viewerPowerPoint Ni2+ impedes Serratia escape from eukaryotic invaded cells. (a) CHO cells were infected with wild-type Serratia at MOI = 10 (1,500,000 CFU per well). Different concentrations of NiCl2 were added to the CHO cells antibiotic-free culture medium at 240 min p.i., and extracellular-released bacteria were monitored by CFU enumeration at 360 min p.i. The percentage of CFU in the supernatant (SN) was calculated relative to the inoculum. The average ± SD for three independent experiments is shown (**p < 0.01). (b) CHO cells were infected with wild-type Serratia at MOI = 10 (1,500,000 CFU per well); 1 mM NiCl2 was added to the CHO cells antibiotic-free culture medium at 240 min p.i., and intracellular bacteria were monitored by CFU enumeration at 360 min p.i. The percentage of intracellular bacteria was calculated relative to the inoculum. The average ± SD for three independent experiments is shown (**p < 0.01). (c) CHO cells were infected with wild-type (wt) Serratia/pGFP at MOI = 10; 1 mM NiCl2 was added to the CHO cells antibiotic-free culture medium at 240 min p.i., and cells were fixed at 360 min p.i. and analysed by confocal microscopy. Representative confocal laser microscopy images are shown. Bars: 10 μm. (d) Non-invaded CHO cells were incubated with or without 1 mM NiCl2, and Fluo-4 AM was loaded 30 min after. Images were captured by confocal microscopy and the integrated density of GFP (Int Den) was quantified using the ImageJ software. Fold change in Int Den was calculated relative to the nontreated control. The average ± SD for three independent experiments is shown To examine the possibility of NiCl2 inhibiting a eukaryotic calcium channel that could be modulated by ShlA instead of directly blocking the PFT, we used the Ca2+ fluorophore Fluo-4 AM, which exhibits an increase in fluorescence upon intracellular Ca2+ binding. Non-invaded CHO cells were incubated with or without 1 mM NiCl2 for 30 min and further loaded with Fluo-4 AM. Images were captured by confocal microscopy 30 min after the fluorophore addition, maintaining NiCl2 concentration. As shown in Figure 1d, the presence of NiCl2 did not alter Fluo-4 AM fluorescence, suggesting that NiCl2 did not modify intracellular Ca2+ concentrations of the resting state. As a control, Fluo-4 AM fluorescence in the presence or absence of the Ca2+ ionophore A23187 was assessed. Non-invaded CHO cells treated with the ionophore showed an increase of 72% or 89% in the Fluo-4 AM fluorescence in the absence or presence of 1-mM NiCl2, respectively, compared with nontreated cells (Figure S1C). This result verified that CHO cells are responsive to Ca2+ variations. Collectively, these results suggest that Ni2+ is not altering the activity of a eukaryotic calcium channels and strongly indicate that it inhibits ShlA acting as a calcium channel. To gain further insight into this process, we investigated if other reported phenotypes mediated by ShlA could be blocked by Ni2+. We have previously shown that ShlA expression induces a localised mobilisation of intracellular Ca2+ as monitored with a calcium sensitive fluorescent probe (Di Venanzio, Lazzaro, Morales, Krapf, & García Véscovi, 2017). To assess whether Ni2+ inhibits this phenotype, invaded CHO cells were loaded with Fluo-4 AM at 270 min p.i., and images were captured by confocal microscopy 30 min after; 1 mM NiCl2 was added at 240 min p.i. and maintained during the microscopy, when indicated. As shown previously (Di Venanzio, Lazzaro, Morales, Krapf, & García Véscovi, 2017), Fluo-4 AM fluorescent signal was detected juxtaposed to 51% of wild-type SeCV, and the percentage of juxtaposition was reduced to 41% when the shlBA strain was used in the assay (Figure 2a,b, upper and lower panels). In cells incubated with wild-type Serratia and treated with NiCl2, juxtaposition was reduced to 29%, compared with nontreated cells (Figure 2a,b, middle panel), whereas no changes in the scores were detected by the addition of NiCl2 to shlBA mutant strain-invaded cells (Figure 2a). This result clearly demonstrates that Ni2+ blocks the intracellular Ca2+ mobilisation mediated by Serratia that expresses ShlA from the SeCV. Figure 2Open in figure viewerPowerPoint Ni2+ blocks the Ca2+ intracellular mobilisation mediated by ShlA. CHO cells were infected with wild-type/pmCherry (wt, red fluorescence) or shlBA/pmCherry (shlBA, red fluorescence) at MOI = 10. After 240 min, 1 mM NiCl2 was added, if indicated, and at 270 min p.i., Fluo-4 AM (green fluorescence) was added. Cells were analysed by in vivo confocal microscopy at 300 min p.i. (a) Percentage of SeCV with Fluo-4 AM juxtaposition was determined by fluorescence microscopy relative to total SeCV. The average ± SD for three independent experiments is shown (*p < 0.05). (b) Representative confocal laser microscopy images are shown. Arrows point at SeCV. Bars: 10 μm Hertle, Hilger, Weingardt-Kocher, and Walev (1999) have previously reported that epithelial cells are depleted of ATP after incubation with low concentrations of purified ShlA. At higher doses, ShlA caused irreversible vacuolation, and the cells were lysed. These effects were also observed with S. marcescens strains. They attributed this effect to the capacity of ShlA to induce ATP depletion and K+ efflux from epithelial cells (Hertle, Hilger, Weingardt-Kocher, & Walev, 1999). It was later demonstrated that K+ efflux mediated by PFT can promote the onset of the autophagic response (Kloft et al., 2009, 2010). We previously determined that upon contact with Serratia, an autophagic response is induced in epithelial cells prior to the bacterial internalisation and that this phenotype is ShlA dependent (Di Venanzio, Stepanenko, & García Véscovi, 2014; Fedrigo, Campoy, Di Venanzio, Colombo, & García Véscovi, 2011). Therefore, we examined whether Ni2+ could block the autophagic response induction. We first verified that Ni2+ does not affect the canonical autophagic response induced by amino acid starvation in CHO cells expressing EGFP-LC3 in EBSS (Earle's Balanced Salt Solution) medium with or without addition of 1 mM NiCl2 (Figure 3a). Next, CHO-EGFP-LC3 cells were challenged by coincubation with either wild-type or shlBA Serratia strains in the presence or absence of 1 mM NiCl2. At 120 min p.i., cells were fixed, stained, and microscopically inspected. As previously reported (Di Venanzio, Stepanenko, & García Véscovi, 2014), the autophagic phenotype (revealed by the EGFP-LC3 punctuated pattern as opposed to the homogeneous cytoplasmic distribution of the green fluorescence in control cells) was induced in 59% of the CHO-EGFP-LC3 cells when wild-type Serratia was used in the assay (Figure 3a,b, upper panel). When CHO-EGFP-LC3 cells coincubation was performed with shlBA mutant strain, no LC3 fluorescent puncta were detected (Figure 3a,b, lower panel). Treatment with 1 mM NiCl2 reduced the percentage of CHO-EGFP-LC3 cells challenged with wild-type Serratia that displayed the autophagic response to 25% (Figure 3a,b, middle panel). As shown in Figure 3a, the addition of BAPTA-AM did not alter the percentage of autophagic cells. No changes in the scores were detected by the addition of NiCl2 to cells that were challenged with the shlBA strain (Figure 3a). Therefore, we can conclude that Ni2+ inhibits ShlA-mediated autophagic response. The fact that ShlA-mediated induction of autophagy cannot be blocked by sequestration of intracellular Ca2+ argues with previous reports indicating that an increase in the intracellular concentration of Ca2+ could act as a potent inducer of autophagy (Gao, Ding, Stolz, & Yin, 2008; Grotemeier et al., 2010; Høyer-Hansen et al., 2007). Moreover, our previous result showing that ShlA-induced a noncanonical autophagic pathway, as it was not prevented by the PI3K III inhibitor wortmannin (Fedrigo, Campoy, Di Venanzio, Colombo, & García Véscovi, 2011), further supports our data. Considering that it has been suggested that K+ ions could also be implicated in autophagy regulation (Klein, Wörndl, Lütz-Meindl, & Kerschbaum, 2011; Williams et al., 2008) and that ShlA is able to cause K+ efflux (Hertle, Hilger, Weingardt-Kocher, & Walev, 1999), ShlA-promoted autophagy through K+ efflux cannot be discarded. Figure 3Open in figure viewerPowerPoint Ni2+ inhibits the autophagic response induced by ShlA. CHO cells expressing EGFP-LC3 (green fluorescence) were incubated with either wild-type Serratia (wt, red fluorescence) or shlBA mutant strain (red fluorescence) at MOI = 10 in the presence or absence of 1 mM NiCl2 or 40 μM BAPTA-AM. At 120 min p.i., cells were fixed, stained, and microscopically inspected. Non-invaded cells were incubated in EBSS medium in the presence or absence of 1-mM NiCl2 as controls of the autophagic response induced by amino acid starvation. (a) Percentage of autophagic cells was determined by fluorescence microscopy relative to total non-invaded cells. The average ± SD for three independent experiments is shown (*p < 0.05 and **p < 0.01). (b) Representative confocal laser microscopy images are shown. Bars: 10 μm The assays shown above allowed us to conclude that Ni2+ blocks the ShlA-dependent phenotypes caused by Serratia in nonlytic conditions. It has been demonstrated that ShlA is also responsible for lytic phenotypes, such as cultured cells cytotoxicity and haemolytic activity (Hertle, Hilger, Weingardt-Kocher, & Walev, 1999; Poole, Schiebel, & Braun, 1988). To examine whether ShlA-mediated cytotoxicity is inhibited by Ni2+, we measured CHO cells viability by monitoring NAD(P)H-dependent cellular oxidoreductase activity by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) reduction assay. CHO cells were incubated with wild-type or shlBA Serratia in the presence or absence of 1 mM NiCl2. At 180 min p.i., MTT was added. Non-infected cells were used for the negative control, and cells lysed by Triton X-100 treatment were used for the positive control. Both controls were not altered by addition of NiCl2 (Figure 4a). When CHO cells were incubated with wild-type Serratia, cytotoxicity levels reached 72%; in contrast, the shlBA mutant only caused 13% cytotoxicity (Figure 4a). The addition of 1 mM NiCl2 reduced the cytotoxicity caused by the wild-type strain to 18%, whereas no changes in the percentage of cytotoxicity caused by the shlBA mutant strain were detected by NiCl2 addition (Figure 4a). To test if cytotoxicity was due to ShlA-mediated Ca2+ mobilisation, we chelated intracellular Ca2+ by addition of BAPTA-AM. As shown in Figure 4a, the addition of BAPTA-AM did not alter the percentage of affected cells. These results show that ShlA-mediated cytotoxic effect can be blocked by Ni2+ and indicate that fluctuations in intracellular concentration of Ca2+ are not involved in this phenotype. Figure 4Open in figure viewerPowerPoint Ni2+ blocks ShlA-dependent phenotypes in lytic conditions. (a) CHO cells were incubated with wild-type (wt) or shlBA Serratia at MOI = 20; 1 mM NiCl2 or 40 μM BAPTA-AM was added, if indicated. At 180 min p.i., MTT was added. Non-infected cells were used for the negative control, and cells lysed by Triton X-100 treatment were used for the positive control. The average ± SD for four independent experiments is shown (**p < 0.01). (b) Wild-type (wt) or shlBA Serratia cultures were incubated with human red blood cells with or without 1 mM NiCl2 or 40 μM BAPTA-AM. The amount of released haemoglobin was determined by measuring absorbance at 562 nm. Fold change in haemolytic activity was calculated relative to the value obtained for the wild-type strain. The average ± SD for four independent experiments is shown (*p < 0.05 and **p < 0.01). (c) Wild-type (wt) or shlBA Serratia were grown overnight in the presence or absence of 1 mM NiCl2. Saturated cultures were centrifuged, and the supernatants were filtered, precipitated, and loaded into sodium dodecyl sulfate-polyacrylamide electrophoresis gels. Haemolysin was detected using ShlA antisera. The intensity of the band corresponding to flagellin was used as loading control. A representative image of the immunodetection assay is shown. Bars: 10 μm Next, to assess Ni2+ action on ShlA haemolytic activity, we performed human erythrocyte lysis assays in the presence or absence of 1 mM NiCl2. The addition of NiCl2 reduced 75% of the haemolytic activity caused by coincubation of the red blood cells with the wild-type strain, whereas no changes in the haemolytic activity were detected by the addition of NiCl2 when the shlBA strain was used (Figure 4b). Moreover, the addition of BAPTA-AM did not alter the haemolytic capacity of the wild-type strain (Figure 4b). Neither the stability of erythrocytes nor the osmotic lysis caused by incubation in H2O was affected by the addition of NiCl2 (Figure S1D). These results further substantiate that Ni2+ inhibits ShlA-mediated phenotypes and show that the haemolytic activity of ShlA is not dependent on intracellular Ca2+ fluxes. Finally, to discard a possible effect of Ni2+ on bacterial secretion of ShlA, wild-type or shlBA Serratia strains were grown overnight in the presence or absence of 1 mM NiCl2. Saturated cultures were centrifuged, and the supernatants were filtered, precipitated, and analysed by sodium dodecyl sulfate-polyacrylamide electrophoresis followed by immunodetection using anti-ShlA antibodies. Flagellin was used as loading control. As shown in Figure 4c, Ni2+ did not affect the levels of ShlA secreted to the extracellular medium. Altogether, these results allow us to conclude that Ni2+ inhibits either lytic or nonlytic effects mediated by ShlA. It has been previously proposed that the blockage of calcium channels by Ni2+ occurs by reversible occlusion of the formed pore (Mlinar & Enyeart, 1993). To evaluate this possibility for ShlA mode of action, CHO cells were incubated with wild-type or shlBA Serratia in the presence or absence of 1 mM NiCl2. At 180 min p.i., cells were washed repeatedly with PBS (phosphate-buffered saline). Medium supplemented with 50 μg ml−1 gentamicin with or without 1 mM NiCl2 was added, as indicated. At 240 min p.i., cytotoxicity by MTT reduction was evaluated. Wild-type Serratia provoked 83% cytotoxicity; in contrast, the shlBA mutant only caused 8% cytotoxicity to the CHO cells (Figure 5). The presence of Ni2+ during the entire assay diminished 35% of the cytotoxicity caused by the wild-type strain (Figure 5). Removal of Ni2+ restored cytotoxicity to 64% (Figure 5). These results show that the inhibition of ShlA caused by Ni2+ can be reverted, supporting a pore-occlusion mode of action. Figure 5Open in figure viewerPowerPoint The inhibition of ShlA caused by Ni2+ is reversible. (a) Scheme of the assay used to test reversibility of the ShlA inhibition by Ni2+. (b) CHO cells were incubated with wild-type (wt) or shlBA Serratia at MOI = 20; 1 mM NiCl2 or 40 μM BAPTA-AM was added, as indicated. At 180 min p.i., the cells were washed, and medium supplemented with 50 μg ml−1 gentamicin was added; 1 mM NiCl2 was added, as indicated. At 240 min p.i., MTT was added. Non-infected cells were used for the negative control, and cells lysed by Triton X-100 treatment were used for the positive control. The average ± SD for three independent experiments is shown (*p < 0.05 and **p < 0.01) 3 CONCLUDING REMARKS The results presented in this work allow us to conclude that Ni2+ inhibits ShlA-mediated phenotypes in eukaryotic cells. In the presence of 1-mM NiCl2, wild-type Serratia presents phenotypes that resemble the shlBA mutant strain. In addition, we can postulate that two different ShlA-dependent effects can be distinguished. One type that involves ShlA-mediated increase in intracellular Ca2+ concentration, which can be therefore inhibited using a Ca2+ chelator (see model in Figure 6, left panel). On the other hand, ShlA exerts effects on target cells that cannot be attributed to Ca2+ mobilisation and, consequently, cannot be blocked by Ca2+ sequestration. According to previous literature, it is conceivable that the latter phenotypes, such as autophagy induction, haemolytic, and cytotoxic activity, would be the result of the passage of other ions, such as K+ (Hertle, Hilger, Weingardt-Kocher, & Walev, 1999), enabled by ShlA (see model in Figure 6, right panel). Because all ShlA-dependent phenotypes tested in this work were inhibited by Ni2+, we can conjecture that this metal prevents by reversible occlusion the passage of Ca2+ (and possibly other ions such as K+) through the pore f