A protein that controls the onset of a Salmonella virulence program
2018; Springer Nature; Volume: 37; Issue: 14 Linguagem: Inglês
10.15252/embj.201796977
ISSN1460-2075
AutoresJinki Yeom, Mauricio H. Pontes, Jeongjoon Choi, Eduardo A. Groisman,
Tópico(s)Escherichia coli research studies
ResumoArticle1 June 2018free access Source Data A protein that controls the onset of a Salmonella virulence program Jinki Yeom Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA Search for more papers by this author Mauricio H Pontes Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA Yale Microbial Sciences Institute, West Haven, CT, USA Search for more papers by this author Jeongjoon Choi Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA Search for more papers by this author Eduardo A Groisman Corresponding Author [email protected] orcid.org/0000-0001-6860-7691 Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA Yale Microbial Sciences Institute, West Haven, CT, USA Search for more papers by this author Jinki Yeom Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA Search for more papers by this author Mauricio H Pontes Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA Yale Microbial Sciences Institute, West Haven, CT, USA Search for more papers by this author Jeongjoon Choi Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA Search for more papers by this author Eduardo A Groisman Corresponding Author [email protected] orcid.org/0000-0001-6860-7691 Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA Yale Microbial Sciences Institute, West Haven, CT, USA Search for more papers by this author Author Information Jinki Yeom1, Mauricio H Pontes1,2, Jeongjoon Choi1 and Eduardo A Groisman *,1,2 1Department of Microbial Pathogenesis, Yale School of Medicine, New Haven, CT, USA 2Yale Microbial Sciences Institute, West Haven, CT, USA *Corresponding author. Tel: +1 203 737 7940; Fax: +1 203 737 2630; E-mail: [email protected] EMBO J (2018)37:e96977https://doi.org/10.15252/embj.201796977 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The mechanism of action and contribution to pathogenesis of many virulence genes are understood. By contrast, little is known about anti-virulence genes, which contribute to the start, progression, and outcome of an infection. We now report how an anti-virulence factor in Salmonella enterica serovar Typhimurium dictates the onset of a genetic program that governs metabolic adaptations and pathogen survival in host tissues. Specifically, we establish that the anti-virulence protein CigR directly restrains the virulence protein MgtC, thereby hindering intramacrophage survival, inhibition of ATP synthesis, stabilization of cytoplasmic pH, and gene transcription by the master virulence regulator PhoP. We determine that, like MgtC, CigR localizes to the bacterial inner membrane and that its C-terminal domain is critical for inhibition of MgtC. As in many toxin/anti-toxin genes implicated in antibiotic tolerance, the mgtC and cigR genes are part of the same mRNA. However, cigR is also transcribed from a constitutive promoter, thereby creating a threshold of CigR protein that the inducible MgtC protein must overcome to initiate a virulence program critical for pathogen persistence in host tissues. Synopsis The anti-virulence protein CigR creates a threshold for a virulence program directed by the MgtC protein in the facultative intracellular pathogen Salmonella enterica serovar Typhimurium. The anti-virulence protein CigR binds to the virulence protein MgtC. CigR competes with the MgtC target AtpB for binding to MgtC. CigR inhibits the virulence functions of MgtC at early times inside macrophages. Introduction In the context of pathogenesis, the genes of a pathogen can be divided into three distinct groups: virulence genes, which promote virulence; anti-virulence genes, which decrease virulence; and genes that neither promote nor decrease virulence. Investigations over the last several decades have revealed the mechanism of action and contribution to pathogenesis of an increasing number of virulence genes. By contrast, little is known about how anti-virulence genes function. Anti-virulence genes have been identified in a wide variety of pathogens (Cunningham et al, 2001; Shimono et al, 2003; Ionescu et al, 2013). The interplay between virulence genes and anti-virulence genes is believed to regulate the pathogenicity of pathogens (Brown et al, 2016). Here, we report how an anti-virulence protein governs the onset of a Salmonella virulence program. Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen that causes gastroenteritis in humans and a typhoid fever-like disease in mice (Coburn et al, 2007; Fabrega & Vila, 2013). Salmonella can survive and replicate in acidic vacuoles within host phagocytic cells (Buchmeier & Heffron, 1991; Lee et al, 2013), where it senses low pH (Prost et al, 2007; Choi & Groisman, 2016) and expresses virulence genes via the PhoP/PhoQ two-component regulatory system (Alpuche Aranda et al, 1992; Guo et al, 1997; Bijlsma & Groisman, 2005; Retamal et al, 2009). Salmonella survival in host phagocytic cells is made possible by the precise timing at which virulence gene products are produced (Jones & Falkow, 1996; Marcus et al, 2000). Indeed, growth within acidic vacuoles requires the coordinated expression of several virulence factors (Eriksson et al, 2003). The mgtC gene mediates intramacrophage survival and proliferation within host tissues in several intracellular pathogens (Blanc-Potard & Groisman, 1997; Grabenstein et al, 2006; Rang et al, 2007; Alix & Blanc-Potard, 2008; Lee & Groisman, 2012; Belon et al, 2014; Pontes et al, 2015). In S. enterica serovar Typhimurium, the MgtC protein confers virulence in two distinct ways. On the one hand, MgtC inhibits Salmonella's own F1Fo ATP synthase (Lee et al, 2013), the machine responsible for the synthesis of the majority of adenosine triphosphate (ATP) in the cell (Senior, 1990). Thus, MgtC enables Salmonella to maintain its cytoplasmic pH near 7 when experiencing a mildly acidic pH inside macrophages (Lee et al, 2013) and to reduce transcription of ribosomal rRNA when cytosolic conditions prevent the assembly of functional ribosomes (Pontes et al, 2016). On the other hand, MgtC prevents degradation of PhoP (Yeom et al, 2017), the master regulator of Salmonella pathogenesis (Groisman et al, 1989; Miller et al, 1989), thereby advancing virulence gene expression. Thus, MgtC's actions commit Salmonella to low cytosolic ATP, slow growth, and expression of genes requiring large amounts of the regulator PhoP. We now report that Salmonella's commitment to the MgtC-dependent program requires not only the signals promoting expression of the mgtC gene, but also that MgtC protein amounts supersede those of CigR, an anti-virulence protein (Kidwai et al, 2013; Yin et al, 2016) that binds MgtC, thereby preventing MgtC from inhibiting the F1Fo ATP synthase. Surprisingly, the cigR and mgtC genes are located on the Salmonella pathogenicity island 3 (SPI-3; Blanc-Potard et al, 1999) and are part of the same transcription unit under MgtC-inducing conditions. However, the cigR gene is also transcribed constitutively and independently of MgtC, setting a threshold of CigR protein that MgtC must overcome to exert its virulence and metabolic functions. Results CigR is an inner membrane protein that binds to the inner membrane protein MgtC It was previously suggested that CigR is an effector protein secreted into host cells (Niemann et al, 2011, 2013). However, this does not appear to be the case because (i) CigR lacks the characteristics of effector proteins (Sato et al, 2011); (ii) CigR secretion was detected only in a Salmonella mutant lacking control of secretion and overexpressing a regulatory protein (Niemann et al, 2011); and (iii) CigR localizes to the inner membrane in wild-type Salmonella (Fig EV1A), in agreement with transmembrane domain predictions (TMHMM v. 2.0). Given that MgtC is also an inner membrane protein (Lee et al, 2013) and that inactivation of cigR renders Salmonella hypervirulent in mice and inside macrophages (Kidwai et al, 2013; Yin et al, 2016), we wondered whether CigR interacts with MgtC, which would implicate these two proteins in the same virulence pathway. Click here to expand this figure. Figure EV1. CigR is an inner membrane protein that interacts with the MgtC protein A. A Western blot analysis of inner membrane (IM), outer membrane (OM), and secreted fractions (SC) prepared from a strain specifying a chromosomally encoded CigR-HA protein (JY6). (Please note that the CigR-HA protein is fully functional.) Bacteria were grown in N-minimal medium (pH 7.7) with 10 μM MgCl2. NADH oxidase activity [100 × μmol of substrate oxidized (ΔOD340)/min/mg of protein] reflects the purity of the inner membrane preparations. B, C. Spots (B) and β-galactosidase activity (C) of Escherichia coli strain BTH101 harboring plasmids pKT25-CigR and pUT18/pUT18C-MgtC. Control strains carry the plasmid vector and pUT18/pUT18C-PmrB along with pKT25 or pKT25-CigR. Shown are the mean and SD from three independent experiments. D, E. Spots (D) and β-galactosidase activity (E) of E. coli strain BTH101 harboring plasmids pUT18/pUT18C-CigR and pKT15-MgtC. Control strains carry the plasmid vector and pKT25-PmrB along with pUT18/pUT18C or pUT18/pUT18C-CigR. Shown are the mean and SD from three independent experiments. F. Western blot analysis of outer membrane (OM), inner membrane (IM), and cytosolic fractions (SC) prepared from wild-type (14028s) and MgtC-N92T (EL551) Salmonella strains. NADH oxidase activity [100 × μmol of substrate oxidized (ΔOD340)/min/mg of protein] reflects the purity of the inner membrane preparations. G. β-galactosidase activity of E. coli strain BTH101 harboring plasmids pUT18-MgtC/MgtC-E84A/MgtC-N92T and pKT25-CigR. Control strains carry the pKT25 vector and pKT25-Zip along with pUT18 or pUT18-Zip. Shown are the mean and SD from three independent experiments. H. Western blot analysis of outer membrane (OM), inner membrane (IM), and cytosolic fractions (SC) prepared from wild-type (14028s) Salmonella harboring plasmids pUHE-cigR-FLAG, pUHE-D3cigR-FLAG, or pUHE-W133AcigR-FLAG. I. β-galactosidase activity of E. coli strain BTH101 harboring plasmids pUT18-CigR/CigR-D1/CigR-D3/CigR-W133A and pKT25-MgtC. Control strains carry the pKT25 vector and pKT25-Zip along with pUT18 or pUT18-Zip. Shown are the mean and SD from three independent experiments. Source data are available online for this figure. Download figure Download PowerPoint We determined that the CigR and MgtC proteins interact in a specific manner. First, bacterial two-hybrid analysis demonstrated CigR binding to MgtC but not to PmrB (Fig EV1B–E), an inner membrane protein used as a negative control (Wösten et al, 2000). Second, anti-HA antibodies pulled down MgtC-FLAG (see low-intensity band in center bottom panel of Fig 1A), and anti-FLAG antibodies pulled down CigR-HA (Fig 1A), in a strain expressing C-terminally FLAG-tagged MgtC and C-terminally HA-tagged CigR from their normal chromosomal locations. And third, CigR did not bind to YqjA-FLAG, an inner membrane protein used as a negative control (Fig 1A). In addition, CigR exhibited decreased binding to an MgtC variant with the N92T substitution (Fig 1B), which is defective in the ability to inhibit the F1Fo ATP synthase (Lee et al, 2013) but retains normal localization to the inner membrane (Fig EV1F). By contrast, CigR interacted with the MgtC variants with the E84A, C99A, or W226A amino acid substitutions (Lee et al, 2013), as it did with the wild-type MgtC protein (Fig 1B). Furthermore, the anti-HA antibody did not precipitate MgtC-FLAG from a strain expressing untagged CigR (Fig 1A), and the anti-FLAG antibody did not precipitate CigR in a strain expressing untagged MgtC (Fig 1A). In sum, the CigR protein specifically interacts with the MgtC protein in vivo. Figure 1. The AtpB and CigR proteins compete for binding to MgtC Western blot analysis of crude extracts prepared from JY6, EG16539, YS251, JY92, and JY95 Salmonella strains grown in N-minimal media pH 7.7 containing 10 μM MgCl2 for 6 h, followed by immunoprecipitation and detection with anti-HA and anti-FLAG antibodies, as indicated. Relevant bands are boxed. Western blot analysis of crude extracts prepared from wild-type (14028s), EL549, EL551, EL552, and EL553 Salmonella strains harboring a plasmid expressing CigR-FLAG grown in N-minimal media pH 7.7 containing 10 μM MgCl2 and 0.5 mM IPTG for 6 h. These strains express the wild-type MgtC protein or derivatives with the indicated amino acid substitutions. Immunoprecipitation and detection was carried out with anti-MgtC and anti-FLAG antibodies, as indicated. Relevant bands are boxed. Dissociation curves for CigR-HA to MgtC-FLAG, and AtpB-HA to MgtC-FLAG (from Fig EV3G and H; see Materials and Methods). The IC50 corresponds to the concentration at which half of the protein is dissociated from the MgtC-FLAG protein. Shown are the mean and SD from three independent experiments. Western blot analysis of crude extracts from strain EL481 harboring a plasmid expressing the cigR gene following growth in N-minimal media pH 7.7 containing 10 μM MgCl2 and IPTG (0.01, 0.1, and 1 mM) for 6 h. Immunoprecipitation and detection were carried out with anti-HA and anti-FLAG antibodies. Data are representative of three independent experiments, which gave similar results. Source data are available online for this figure. Source Data for Figure 1 [embj201796977-sup-0003-SDataFig1.pdf] Download figure Download PowerPoint CigR and AtpB compete for binding to MgtC We hypothesized that CigR competes with the F1Fo ATP synthase subunit AtpB for binding to MgtC because the MgtC N92T variant was defective for interaction with AtpB (Lee et al, 2013) and CigR (Figs 1B and EV1G). To test this hypothesis, we performed co-immunoprecipitation experiments using different amounts of in vitro synthesized MgtC-FLAG, AtpB-HA, and CigR-HA proteins reconstituted into proteoliposomes. As AtpB-HA amounts increased, binding between CigR and MgtC decreased (Fig EV2A, lower box). Likewise, an increase in CigR-HA amounts decreased the interaction between MgtC and AtpB (Fig EV2A, upper box). These results argue that CigR and AtpB compete with each other for binding to MgtC. Click here to expand this figure. Figure EV2. CigR competes with AtpB for interaction with MgtC Western blot analysis of proteoliposomes reconstituted with in vitro synthesized F1Fo ATP synthase containing AtpB-HA, MgtC-FLAG, and CigR-HA proteins. At the end of the reconstitution reaction, an aliquot (input) and fractions were immunoprecipitated with either anti-HA or anti-FLAG antibodies and analyzed using anti-HA and anti-FLAG antibodies. ×5 indicates that five times the amount of the protein is present in the reaction. Relevant bands are boxed. Western blot analysis of proteoliposomes reconstituted from in vitro synthesized F1Fo ATP synthase containing AtpB-HA and CigR-HA proteins. Proteoliposomes were prepared as described in Materials and Methods. The data are representative of two independent experiments, which gave similar results. Source data are available online for this figure. Download figure Download PowerPoint To examine the binding specificity of CigR and AtpB for MgtC, we calculated the half inhibitory concentration (IC50) values of CigR-HA and AtpB-HA for MgtC-FLAG using increasing amounts of competitor AtpB-HA and CigR-HA, respectively (Figs 1C and EV3). The IC50 values of CigR-HA and AtpB-HA for MgtC-FLAG are 8.9 and 22.3 μM, respectively (Fig 1C). Thus, MgtC binding to AtpB is stronger than to CigR (Figs 1C and EV3). Additionally, CigR does not appear to bind to AtpB because the intensity of the CigR-HA band was similar in the presence and absence of AtpB-FLAG following a pull-down with anti-FLAG antibodies (Fig EV2B). Competition between CigR and AtpB was also detected in vivo as expression of the cigR gene from a heterologous promoter reduced the interaction between the AtpB and MgtC proteins (Fig 1D). These experiments demonstrate that CigR binds MgtC directly and that this binding hinders MgtC binding to AtpB. The CigR protein determines whether Salmonella embarks on a pathway that alters normal bacterial physiology. Click here to expand this figure. Figure EV3. CigR and AtpB bind to MgtC with different apparent affinities A–F. (A, F) Western blot analysis of the purified AtpB-HA, CigR-HA, and MgtC-FLAG proteins to calculate apparent binding affinity (A) and to carry out competition experiments (F). Protein amounts were calculated based on known amounts of purified proteins. Proteins were loaded onto the same gel and detected using monoclonal antibodies against the HA and FLAG tags. (B–E) Western blot analysis of the MgtC-FLAG, CigR-HA, and AtpB-HA proteins synthesized using an in vitro transcription/translation system followed by immunoprecipitation and detection with the indicated antibodies. Kd values correspond to half of the amount of AtpB-HA or CigR-HA proteins that specifically bind to MgtC-FLAG. (B) Western blot analysis of proteins recovered following incubation of different amounts of AtpB-HA with a constant amount of MgtC-FLAG followed by pull-down with anti-FLAG antibodies. (C) Affinity of MgtC-FLAG and AtpB-HA from panel (B) calculated with purified proteins in panel (A). Shown are the mean and SD from three independent experiments. (D) Western blot analysis of proteins recovered following incubation of different amounts of CigR-HA and a constant amount of MgtC-FLAG followed by pull-down with anti-FLAG antibodies. (E) Affinity of CigR-HA and MgtC-FLAG bands in panel (D) was calculated with purified proteins in panel (A). Shown are the mean and SD from three independent experiments. G, H. In vitro competition between the AtpB-HA and CigR-HA proteins for the MgtC-FLAG protein was determined using a constant amount of MgtC-FLAG and pull-down with anti-FLAG antibodies. The amounts of CigR-HA and AtpB-HA were kept constant in (G) and (H), respectively. Source data are available online for this figure. Download figure Download PowerPoint The cigR gene decreases survival inside macrophages and increases both ATP levels and intracellular pH, in an mgtC-dependent manner If CigR exerts its anti-virulence function by targeting MgtC, a cigR mutant should exhibit the opposite behavior of an mgtC mutant. As predicted, a strain deleted for the cigR gene achieved four times the number of wild-type Salmonella inside the macrophage-like cell line J774A.1 by 20 h post-infection (Fig 2A). This is in contrast to the mgtC mutant, which survived five times less than the wild-type strain (Fig 2A; Blanc-Potard & Groisman, 1997; Rang et al, 2007), albeit not to the low levels as the phoP mutant (Fig 2A). The enhanced intramacrophage survival of the cigR mutant is due to inactivation of cigR (as opposed to the cigR mutation being polar on a downstream gene) because a plasmid harboring the cigR gene restored wild-type survival, whereas the plasmid vector did not (Fig 2A). Figure 2. The cigR and mgtC mutants display opposite behaviors Survival inside J774A.1 macrophages of wild-type (14028s), mgtC (EL4), cigR (JY12), mgtC cigR (JY6), and phoP (MS7953s) Salmonella without/with the indicated plasmids 20 h after infection. The mean and SD from two independent experiments are shown. Please note log10 scale of y-axis. ATP levels in wild-type (14028s), mgtC (EL4), cigR (JY12), mgtC cigR (JY6), and mgtB Salmonella, and in the cigR mutant harboring the plasmid vector or pcigR. Intracellular ATP levels correspond to picomoles of ATP per ml of cells at a given OD600. The mean and SD from three independent experiments are shown. Bacteria were grown in N-minimal media pH 7.7 containing 10 μM MgCl2 for 5 h. ATP hydrolysis rates in inverted vesicles prepared from wild-type (14028s), mgtC (EL4), and cigR (JY12) Salmonella. The reaction was initiated by adding ATP and monitored for 5 min. The mean and SD from two independent experiments are shown. Intracellular pH of wild type (14028s), mgtC (EL4), and cigR (JY12) Salmonella following bacterial growth in N-minimal medium pH 7.7 for 1 h and then switched to the same media at pH 4.6 for 1 h. Lines represent the average pH of seven independent replicates. Intracellular pH of wild type (14028s), mgtC (EL4), and cigR (JY12) Salmonella when inside macrophages. Bacteria grown in LB medium overnight were used to infect J774A.1 macrophages. pH measurements were carried out 6 h post-internalization. Numbers represent the average pH of four independent replicates. mRNA amounts of the PhoP-activated pagC and pcgL genes in wild type (14028s), mgtC (EL4), and cigR (JY12) Salmonella. The mean and SD from two independent experiments are shown. ATP levels present in wild type (14028s) and mgtC (EL4) Salmonella harboring no added plasmid, the plasmid vector, or plasmids expressing the cigR or mgtC genes. Bacteria were grown in N-minimal media pH 7.7 containing 10 μM MgCl2 with 200 μM IPTG for 4 h. ATP levels correspond to picomoles of ATP per ml of cells at given OD600. Shown are the mean and SD from three independent experiments. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test with each sample vs. wild type, N.S., not significant. Download figure Download PowerPoint The cigR mutant exhibited lower ATP levels (Fig 2B) and ATPase activity (Fig 2C) than wild-type Salmonella, the opposite phenotypes of those displayed by the mgtC mutant (Fig 2B and C; Lee et al, 2013). The plasmid harboring the cigR gene restored wild-type ATP levels to the cigR mutant, but the vector control did not (Fig 2B). [As previously reported (Lee et al, 2013), a mutant deleted for the mgtB gene, which is co-transcribed with mgtC (Snavely et al, 1991), retained wild-type ATP levels (Fig 2B).] Furthermore, the cytoplasmic pH of the cigR mutant was higher than that of wild-type Salmonella both during growth in defined laboratory media (Fig 2D) and inside macrophages (Fig 2E), unlike the lower pH displayed by the mgtC mutant in both conditions (Fig 2D and E; Lee et al, 2013). The cigR mutant produced more PhoP-activated mRNAs than wild-type Salmonella (Fig 2F). This result reflects that MgtC protects PhoP from degradation (Yeom et al, 2017). Thus, when CigR is absent, the higher amounts of free MgtC further PhoP-dependent gene transcription (Yeom et al, 2017). That CigR works primarily (if not exclusively) by inhibiting MgtC is supported by three additional lines of evidence. First, an mgtC cigR double mutant retained the behavior of the mgtC single mutant: It displayed lower intramacrophage survival (Fig 2A) and higher ATP levels (Fig 2B) than wild-type Salmonella. Second, a plasmid expressing the cigR gene from a heterologous promoter increased ATP levels in wild-type Salmonella but not in the mgtC mutant (Fig 2G). Taken together with the data presented above (Fig EV1A), these results indicate that the anti-virulence protein CigR exerts its effects by targeting the virulence protein MgtC. Moreover, they reinforce the notion that the inner membrane protein CigR operates inside Salmonella. The C-terminal domain of CigR is required for inhibition of MgtC A tBLASTN analysis using the deduced amino acid sequence of the S. enterica serovar Typhimurium cigR gene revealed the presence of cigR homologs in several enteric bacterial species (Appendix Fig S1). The C-terminal region of CigR, which harbors the single predicted transmembrane domain, is much more conserved (82~98% amino acid identity) than the rest of the protein (53~85% amino acid identity for the full-length CigR; Appendix Fig S1). Therefore, we hypothesized that the C-terminal region is crucial for CigR function. To test this hypothesis, we compared the behaviors of wild-type Salmonella and a strain deleted for the whole cigR open reading frame to those of strains with deletions or nucleotide substitutions in the part of the chromosomal copy of the cigR gene specifying the C-terminal region of CigR (Appendix Fig S1). We deleted the nucleotide sequence corresponding to three conserved domains (D1, D2, and D3), each four amino acids long (Appendix Fig S1). Because tryptophan residues are generally critical for protein function (Bogan & Thorn, 1998; Rasmussen et al, 2007), we also substituted the nucleotides specifying the single tryptophan at position 133 so that cigR specified an alanine residue at this position. Bacteria expressing the D3 and W133A CigR variants displayed the enhanced intramacrophage survival (Fig 3A), lower ATP levels during growth in laboratory media (Fig 3B), and higher pH inside macrophages (Fig 3C) that are characteristic of the cigR-deleted strain. The D3 and W133A CigR proteins localized to the inner membrane like wild-type CigR (Fig EV1H). By contrast, the D1 and D2 mutants retained a wild-type behavior (Fig 3A–C). Although bacteria expressing the D3 and W133A CigR variants exhibited the same phenotypes, the corresponding proteins differed in their ability to interact with the MgtC protein in vivo and in vitro. Specifically, the D3 variant interacted with MgtC like wild-type CigR or the D1 and D2 variants (Fig 3D and E). By contrast, the W133A CigR variant was defective in binding to MgtC (Figs 3D and E, and EV1I). Cumulatively, these data establish that W133 is required for CigR binding to MgtC and that one or more residues substituted in the D3 region are necessary for inhibition of the MgtC protein. Figure 3. The C-terminal domain of CigR is required for inhibition of MgtC A–C. Bacterial survival inside macrophages (A), ATP levels (B), and intracellular pH (C) of wild-type Salmonella (14028s) and mutants with deletions or nucleotide substitution in the cigR open reading frame (JY139, JY150, JY151, and JY152). (A) Salmonella survival inside J774A.1 macrophages 20 h after infection. The mean and SD from two independent experiments are shown. Please note log10 scale of y-axis. (B) ATP levels, corresponding to picomoles of ATP per ml of cells at a given OD600, of the strains listed in (A). Bacteria were grown for 4 h in N-minimal media pH 7.7 containing 10 μM MgCl2. The mean and SD from three independent experiments are shown. (C) Intracellular pH of the Salmonella strains listed in (A) was measured inside the macrophage-like cell line J774A.1. Numbers represent the average pH of four independent replicates, which gave similar bacterial colony counts when plated on LB agar plates. Data information: *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test with each sample vs. wild type, N.S., not significant. D. Western blot analysis of proteoliposomes reconstituted from in vitro synthesized MgtC-FLAG and CigR-HA proteins, followed by immunoprecipitation and detection with antibodies directed to the HA or FLAG epitopes, as indicated. At the end of the reconstitution reaction, an aliquot (input) and fractions immunoprecipitated with antibodies directed to the HA or FLAG epitopes were analyzed. Proteoliposomes were prepared as described in Materials and Methods. The data are representative of two independent experiments, which gave similar results. E. Western blot analysis of crude extracts prepared from wild-type Salmonella (14028s) harboring a plasmid expressing the CigR-FLAG protein grown in N-minimal media pH 7.7 containing 10 μM MgCl2 and IPTG (0.12 mM for wild type, 0.2 mM for D1 and D2, 0.3 mM for D3, and 0.4 mM for W) for 6 h. These strains express the wild-type CigR-FLAG protein or derivatives with the indicated amino acid substitution or domain mutations. Immunoprecipitation and detection were carried out with antibodies directed to the MgtC protein and FLAG epitope, as indicated. The data are representative of two independent experiments, which gave similar results. Source data are available online for this figure. Source Data for Figure 3 [embj201796977-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint The cigR gene is transcribed together with and independently of the mgtC gene Toxins and anti-toxins are typically encoded by adjacent genes and produced from the same transcript (Gerdes et al, 2005). Although the mgtC and cigR genes are separated by two genes (Fig 4A–C), we hypothesized that they are part of the same polycistronic mRNA because they exhibit comparable expression behavior in a different Salmonella strain (http://bioinf.gen.tcd.ie/cgi-bin/salcom.pl; Kröger et al, 2013). As hypothesized, similar RNA polymerase (RNAP) occupancy of the mgtC and cigR coding regions as well as of the intervening mgtB and mgtR genes was observed during growth in 10 μM Mg2+ (Fig EV4A), a condition promoting mgtCBR expression (Soncini et al, 1996). Moreover, reverse transcriptase PCR (RT–PCR) assays produced mgtC-cigR, mgtC-mgtB, mgtB-cigR, and cigR-cigR amplicons of the predicted sizes (Fig EV4B–F). These results establish that the mgtCBR operon is longer than previously reported (Alix & Blanc-Potard, 2008). Figure 4. Transcription of the cigR gene is partially PhoP-dependent Diagram of the mgtC-cigR chromosomal region showing the PhoP-dependent (green color) and PhoP-independent (red color) promoters. mRNA amounts of the mgtC, mgtB, and cigR genes produced by wild-type Salmonella (14028s) grown in N-minimal media pH 7.7 with 10 mM or 10 μM MgCl2 for 4 h. Sh
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