Analysis of the expression, secretion and translocation of the Salmonella enterica type III secretion system effector SteA

Analysis of the Expression, Secretion and Translocation of the Salmonella enterica Type III Secretion System Effector SteA Elena Cardenal-Mun ˜oz, Francisco Ramos-Morales* Departamento de Gene ´tica, Facultad de Biologı ´a, Universidad de Sevilla, Sevilla, Spain Abstract Many Gram-negative pathogens possess virulence-related type III secretion systems. Salmonella enterica uses two of these systems, encoded on the pathogenicity islands SPI-1 and SPI-2, respectively, to translocate more than 30 effector proteins into eukaryotic host cells. SteA is one of the few effectors that can be translocated by both systems. We investigated the conditions affecting the synthesis of this effector, its secretion to culture media and its translocation into host cells. Whereas steA was expressed under a wide range of conditions, some factors, including low and high osmolarity, and presence of butyrate, decreased expression. SteA was efficiently secreted to the culture media under both SPI-1 and SPI-2 inducing conditions. The kinetics of translocation into murine macrophages and human epithelial cells was studied using fusions with the 3xFLAG tag, and fusions with CyaA from Bordetella pertussis. Translocation into macrophages under non-invasive conditions was mainly dependent on the SPI-2-encoded type III secretion system but some participation of the SPI-1 system was also detected 6 hours post-infection. Interestingly, both type III secretion systems had a relevant role in the translocation of SteA into epithelial cells. Finally, a deletion approach allowed the identification of the N-terminal signal necessary for translocation of this effector. The amino acid residues 1–10 were sufficient to direct translocation into host cells through both type III secretion systems. Our results provide new examples of functional overlapping between the two type III secretion systems of Salmonella. Citation: Cardenal-Mun ˜oz E, Ramos-Morales F (2011) Analysis of the Expression, Secretion and Translocation of the Salmonella enterica Type III Secretion System Effector SteA. PLoS ONE 6(10): e26930. doi:10.1371/journal.pone.0026930 Editor: Dipshikha Chakravortty, Indian Institute of Science, India Received July 26, 2011; Accepted October 6, 2011; Published October 27, 2011 Copyright: ß2011 Cardenal-Mun ˜oz, Ramos-Morales. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grant SAF2010-15015 from the Spanish Ministry of Science and Innovation and the European Regional Development Fund, and grant P08-CVI-03487 from the Consejerı ´a de Economı ´a, Innovacio ´n y Ciencia, Junta de Andalucı ´a, Spain. ECM is the recipient of a predoctoral fellowship from the Junta de Andalucı ´a. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Introduction Many pathogenic Gram-negative bacteria rely on type III secretion systems (T3SS) for their interaction with the host. A type III secretion apparatus, also called injectisome or molecular needle [1], is a complex structure, functionally and structurally related to the flagella assembly system, consisting of at least 20 different subunits, that spans the inner membrane, the periplasmic space, and the outer membrane of the Gram-negative bacteria, and the cell membrane of the host [2]. This system allows delivery into the eukaryotic host cells of effector proteins that direct the different stages of the infection at the cellular level. Salmonella is a genus of Gram-negative bacteria that belongs to the family Enterobacteriaceae. Salmonellae are facultative intracellular parasites that can infect a wide variety of animals causing different diseases, from localized intestinal infection to severe systemic disease, depending on the Salmonella serovar and the host. Salmonella enterica possesses two distinct T3SS, T3SS1 and T3SS2, that are encoded by genes located in two different Salmonella pathogenicity islands: SPI-1 and SPI-2, respectively. Function and expression conditions for both systems are different (reviewed in [3]). It is suggested that T3SS2 is a more recently acquired trait because it is not present in another species of the genus: S. bongori [4]. The T3SS1 is necessary for the invasion of non-phagocytic cells [5,6], whereas the T3SS2 is induced after invasion and is essential for survival and replication within macrophages [7]. However, some overlap exists and effectors from both systems are involved in the biogenesis of the Salmonella-containing vacuole, a modified phagosome that is the intracellular niche for this pathogen [8,9,10,11] (reviewed in [12]). More than 30 effectors can be translocated by T3SS1 or T3SS2 into the host cell [13]. They manipulate a number of key host cellular functions, including signal transduction, membrane trafficking and pro-inflammatory immune responses. The related flagella system also contributes to virulence: aside from the effect of flagellar-based motility in the invasiveness of Salmonella [14], flagellin is translocated into the cytosol by the T3SS1 of Salmonellainfected macrophages and results in activation of the inflammasome and caspase-1-mediated cell death (pyroptosis) [15,16,17]. Many Salmonella effectors are encoded inside SPI-1 or SPI-2. Most of them are co-expressed with T3SS1 or with T3SS2 and are secreted only through their cognate secretion system. Interestingly, however, some of the effectors encoded outside the two main islands are known to be secreted by both T3SS1 and T3SS2, including SlrP [18,19,20], SopD [21], SpvC [22], SspH1 [23], SteA, and SteB [24] (reviewed in [13]) and the recently identified GtgE, SpvD, and SteE [25]. PLoS ONE | www.plosone.org 1 October 2011 | Volume 6 | Issue 10 | e26930 Translocation of Salmonella effectors can be studied infecting cultures of different cell lines. Invasive bacteria (expressing T3SS1) are used to infect non-phagocytic cell lines, like epithelial human HeLa cells. Phagocytic lines, like J774 and RAW264.7 macrophages, can also be infected in the same conditions but, since invasive Salmonella trigger a rapid form of cell death called pyroptosis in macrophages [26], infections of several hours requires the use of non-invasive bacteria. Interestingly, the expression of T3SS1 and T3SS2 and translocation of effectors change with the conditions used for cultivation of bacteria before infection, with the time post-infection, and with the host cell line [27]. While fractionation and immunoblot with antibodies raised against the effectors or against small tags can be used to analyze translocation, an alternative is the generation of fusions with a fragment of the gene cyaA from Bordetella pertussis encoding the catalytic domain of a calmodulin-dependent adenylate cyclase. This enzyme converts cellular ATP in cyclic AMP (cAMP) in the presence of calmodulin. Because calmodulin is present in eukaryotic host cells, but not in bacteria, translocation of one of these fusions would be detected as an increase in the level of cAMP in a culture of infected cells [28]. In vitro synthetic growth media can also be used to imitate, to some extent, the in vivo environments. Secretion of effectors to the supernatant of liquid bacterial cultures can then be analyzed by immunoblot. Optimal expression of T3SS1 can be achieved at the end of the exponential growth phase in bacteria cultured in rich media with low aeration and high concentration of NaCl [29,30]. T3SS2 expression is obtained in acidic minimal media with low phosphate and low magnesium concentration [29,31], conditions found in the Salmonella containing vacuole. T3SS2-dependent secretion can be increased by using strains lacking a complex of SPI-2-encoded proteins SsaM, SpiC, and SsaL, or exposing wildtype bacteria to pH 7.2 after growth at pH 5 [25,32]. SteA is a poorly characterized T3SS substrate that was identified in a screen to find genes encoding effectors in S. enterica serovar Typhimurium [24]. The screen was based on the CyaA system described above. A library of translational fusions between Salmonella chromosomal genes and a fragment of the gene cyaA from B. pertussis was used to infect cultures of J774 macrophages. When these cells were infected for 8 h under non-invasive conditions (stationary-phase bacteria), translocation of SteA depended on T3SS2. However, when using invasive bacteria, SteA was also translocated in a T3SS1-dependent manner into J774 infected for 1 h. A steA null mutant showed a three-fold disadvantage in mouse spleen colonization. SteA localized to the trans-Golgi network in both transfected and infected human epithelial HeLa cells and to Salmonella-induced membrane tubules containing trans-Golgi markers [24,33]. In this work we focus on SteA as a T3SS1 and T3SS2 effector and we study the conditions that modulate its expression, its secretion to culture media, and its translocation into eukaryotic cells. In addition, we identify the sequences in SteA that are important for secretion and translocation through both systems. Results Expression of steA under different conditions SteA is one of the few T3SS Salmonella effectors that are secreted by both T3SS1 and T3SS2. During infection Salmonella encounters different environments that require the expression of one or the other T3SS. Since both systems are optimally expressed in different conditions, it was interesting to study the expression of steA in relation with these conditions. For this purpose, two chromosomal steA fusions were generated: a lacZ fusion, and a 3xFLAG fusion. The lacZ fusion allowed the quantification of expression measuring b-galactosidase activities (Fig. 1). The initial comparison between cultures of the same strain grown overnight at 37uC with low aeration in rich medium (LB) with 0.3 M NaCl or in minimal medium with low phosphate, low pH, and low magnesium (LPM, pH 5.8)), indicated that the expression of steA was much higher in LPM (Fig. 1A). The first set of conditions induces optimal expression of the SPI-1 [29,30] genes whereas LPM mimics the environment that the bacteria find inside the eukaryotic cells in the Salmonella-containing vacuole (SCV) and induces the expression of the SPI-2 regulon [34]. This result indicates that steA is optimally expressed in coordination with the T3SS2. Key positive regulators of the expression of SPI-1 and SPI2 are the transcription factors HilA and SsrB, respectively. As seen in Fig. 1A, the expression of steA was only slightly reduced by a hilA mutation (measured under SPI-1-inducing conditions) or an ssrB mutation (under SPI-2-inducing conditions). Upon an oral infection Salmonella finds environmental conditions including acidity, hyperosmolarity and hypoxia and these factors regulate the expression of T3SS1 [35], therefore these conditions were tested in vitro using the LB medium. The same factors were also tested in LPM. High osmolarity and oxygen limitation are used in vitro to induce expression of SPI-1 genes. Expression of steA was induced in low oxygen (Fig. 1D and E) but was repressed by high osmolarity (Fig. 1B and C) and this repression was not due to osmotic shock, since the results were obtained in the presence of 1 mM proline used as osmoprotectant. SPI-1 genes are downregulated after Salmonella exposure to butyrate, which is one of the fermentation products found in the intestine. The same is true for steA (Fig. 1D and E). Most SPI-1 genes are presumably not expressed in the stomach due to acidity whereas the increase of pH in the intestine could induce their expression. Expression of steA, however, was not affected by changes in pH between 5 and 8.4, both in LB (Fig. 1D) or LPM (Fig. 1E). This result indicated that pH is not one of the factors inducing higher expression of steA in LPM. A general conclusion of these experiments is that some of the conditions (but not all) that lead to increased expression of SPI1 genes also up-regulate steA. The 3xFLAG fusion permitted detection of C-terminally tagged SteA by Western blotting against the FLAG epitope. This fusion was used to monitor the expression of steA at the protein level (Fig. 2). The results partially confirmed those obtained with the lac fusion: acidic pH slightly increased expression in LB with shaking, but the highest expression was obtained in LB with low aeration and in LPM (SPI-2 conditions). Good expression was also obtained in SPI-1 conditions (cultures in LB 0.3 M NaCl, without aeration). However, the optimal concentration of NaCl in LB cultures was 170 mM. It is interesting to note that the level of SteA protein decreased both at high and low osmolarity, in contrast with the results obtained with the lac fusion, suggesting some kind of posttranslational regulation. Secretion of SteA to the culture media Specific media and culture conditions have been used to attain optimal expression of T3SS1 or T3SS2 and to study in vitro secretion of different effectors using these systems. As previously mentioned, overnight cultures in LB with 0.3 M NaCl, without shaking (SPI-1 conditions), are optimal for expression of T3SS1, whereas maximal expression of T3SS2 is obtained in LPM at pH 5.8 (SPI-2 conditions). Secretion of SteA was tested in both conditions using the strain bearing the SteA-3xFLAG fusion. Culture supernatants were concentrated and analyzed by Western blot using anti-FLAG antibodies. Secretion was observed under both conditions (Fig. 3A and 3B) and, as Expression and Secretion of SteA PLoS ONE | www.plosone.org 2 October 2011 | Volume 6 | Issue 10 | e26930 expected, secretion under SPI-1 conditions was dependent on T3SS1 since it was no longer observed in a prgH background (PrgH is an essential component of T3SS1), and secretion under SPI-2 conditions was dependent on T3SS2 since it was no longer observed in a ssaV background (SsaV is an essential component of T3SS2). It was recently reported that some T3SS2 effectors are more efficiently secreted to the culture media from mutant strains lacking a complex of SPI-2-encoded proteins SsaM, SpiC and SsaL. In addition, wild-type bacteria exposed to pH 7.2 after growth at acidic pH also showed increased secretion of these effectors due to dissociation of the SsaM/SpiC/SsaL complex [32]. On these grounds, we tried these conditions for secretion of SteA, but neither of them improved secretion of this effector through the T3SS2 (Fig. 3C and 3D). Figure 1. Expression of steA in different conditions. Expression levels were monitored with a chromosomal steA::lacZ fusion. For optimal SPI-1inducing conditions, the S. enterica strains carrying the fusions were incubated overnight at 37uC without shaking in LB with 0.3 M NaCl. For optimal SPI-2-inducing conditions, the same strains were incubated overnight at 37uC with shaking in LPM. Means and standard deviations from duplicate experiments are represented. (A) b-galactosidase activities were measured in wild-type (wt) and hilA backgrounds from cultures in SPI-1-inducing conditions, and in wild-type and ssrB backgrounds from cultures in SPI-2-inducing conditions. SPI-1 (B) or SPI-2-inducing (C) cultures with different concentrations of NaCl were used to test the effect of osmolarity. 1 mM proline was added as osmoprotectant to prevent osmotic shock. The effects of pH, oxygen limitation and butyrate (But) on the expression of steA were tested under SPI-1 (D) and SPI-2-inducing conditions (E). doi:10.1371/journal.pone.0026930.g001 Expression and Secretion of SteA PLoS ONE | www.plosone.org 3 October 2011 | Volume 6 | Issue 10 | e26930 Translocation of SteA into RAW264.7 macrophages Next, we analyzed the kinetics of translocation of SteA into the eukaryotic host cells using well-established cell lines. First, we infected cultures of the murine macrophage-like cell line RAW264.7 with three strains of Salmonella producing SteA3xFLAG: one strain otherwise wild type, a prgH mutant, and an ssaV mutant. These bacteria were in non-invasive conditions (grown at 37uC in LB with shaking for 24 h prior to infection) in order to prevent induction of rapid death in the macrophages. 2 h, 6 h and 12 h post-infection, cells were lysed under mild conditions and the eukaryotic cytosol was separated from other components and intact bacteria (pellet) by centrifugation and filtration. The Figure 2. Levels of SteA protein in different conditions. Extracts from an S. enterica serovar Typhimurium 14028 (wild-type strain) derivative expressing 3xFLAG-tagged SteA were resolved by 4–15% gradient SDS-PAGE. Immunoblotting was performed with a monoclonal anti-FLAG antibody (upper panels). The conditions tested were: LB with different pH, high or low oxygen (LB cultures with or without shaking, respectively), LB with 10 mM butyrate (But), SPI-1 conditions (LB with 0.3 M NaCl, 16 h at 37uC, without shaking), SPI-2 conditions (LPM pH 5.8), and LB with different concentrations of NaCl, as indicated. Anti-GroEL antibodies were used as loading control (lower panels). Ponceau S red staining was used as loading control when testing the effect of osmolarity since GroEL expression is affected by changes in NaCl concentration. Molecular mass markers, in KDa, are indicated on the left. doi:10.1371/journal.pone.0026930.g002 Figure 3. Secretion of SteA to the culture media. The presence of SteA-3xFLAG in cell lysates (from 10 8 cells) and culture supernatants (from 2.5610 8 cells) was analyzed by immunoblotting with anti-FLAG antibodies as described in the Materials and Methods section. The immunoblots were probed for GroEL as a loading control for the cell lysates and to test unspecific leakage of proteins into the supernatants. All the strains used produced a 3xFLAG version of SteA and were derivatives of the wild-type strain 14028 (wt), a prgH mutant (lacking the T3SS1) and an ssaV mutant (lacking the T3SS2). Bacteria were grown at 37uC using the following conditions: (A) SPI-1 conditions (LB with 0.3 M NaCl, overnight without shaking); (B) SPI-2 conditions (LPM pH 5.8, 24 h with shaking); (C) SPI-2 conditions followed by shifting to pH 7.2 and 1 h incubation; (D) SPI-2 conditions in an ssaL background. doi:10.1371/journal.pone.0026930.g003 Expression and Secretion of SteA PLoS ONE | www.plosone.org 4 October 2011 | Volume 6 | Issue 10 | e26930 presence of SteA in the pellet and of translocated SteA in the eukaryotic cytosol was investigated by immunoblot with antiFLAG antibodies (Fig. 4A). Both fractions were also analyzed with anti-GroEL antibodies to have some measure of the survival and growth of bacteria and to get a control for the purity of the cytosol fraction. Anti-b-actin was used to control the relative number of host cells that were present at each time point. As seen in Fig. 4A, translocation of SteA was observed 6 h p.i. but was maximal 12 h after infection. An ssaV mutation abolished translocation 12 h p.i., indicating that in these conditions SteA is translocated specifically through the T3SS2. The decreases in the amount of b-actin observed in the infections with the wildtype strain and, to a lesser extent, with the prgH mutant, were indicative of the death of the infected cells. This was visually confirmed under the microscope (not shown). In fact, 24 h postinfection most of the cells infected with these strains were dead and it was impossible to evaluate the translocation of SteA (not shown). Translocation 6 h post-infection was only partially suppressed in the ssaV background, suggesting the possibility of some translocation through the T3SS1 in these conditions. In order to test this possibility we decided to use a sensitive method based on the generation of a fusion with the catalytic domain of CyaA from Bordetella pertussis. This protein is a calmodulindependent adenylate cyclase. Calmodulin is not present in bacteria but, upon translocation of a CyaA fusion into the host cell, the adenylate cyclase would be activated by host calmodulin to catalyze the production of cyclic AMP (cAMP). Plasmid pIZ1886 expressing a SteA-CyaA’ fusion was introduced in S. enterica serovar Typhimurium wild-type strain 14028 and isogenic prgH mutant (lacking T3SS1), ssaV mutant (lacking T3SS2), and prgH ssaV double mutant (lacking both T3SS). These strains were used to infect RAW264.7 cells and the translocation of SteACyaA’ was monitored 6 h post-infection by measuring the level of cAMP in the cell cultures (Fig. 4B). The results confirm a major role for T3SS2 in the translocation of SteA under these conditions but also support a role for T3SS1. Translocation of SteA into HeLa epithelial cells Immunoblot experiments similar to the experiments described in the previous section were also carried out infecting human epithelial HeLa cells. Since these cells are non-phagocytic, bacteria were in invasive conditions (grown overnight in LB with 0.3 M NaCl without shaking) for these experiments. The presence of SteA-3xFLAG in the pellet and the eukaryotic cytosol was analyzed 50 min and 4.5 h post-infection with antiFLAG antibodies (Fig. 5A). Translocation to the host cytosol was observed only in the wild-type background 4.5 h post-infection. Results with anti-GroEL, indicating that this method was not sensitive enough to detect bacterial proteins 50 min postinfection, prompted us to use the CyaA system described above to study translocation into HeLa cells. S. enterica serovar Typhimurium wild-type strain 14028 and isogenic prgH mutant, and ssaV mutant were used to infect HeLa cells under invasive conditions and the translocation of SteA-CyaA’ was monitored 15 min, 50 min, 2 h, 4 h, 8 h and 16 h post-infection. As seen in Fig. 5B, translocation from the wild-type strain was detected at the same level from 15 min to 16 h post-infection. Translocation of SteA into HeLa cells was dependent on the T3SS1, since infections with the prgH mutant gave low levels of cAMP. Surprisingly, the ssaV mutation significantly reduced translocation 15 min and 50 min post-infection, suggesting a role for T3SS2 at this short time. Figure 4. Translocation of SteA into RAW264.7 cells. (A) RAW264.7 cells were infected with Salmonella strains expressing SteA3xFLAG that were derivatives of the wild-type strain (wt), the prgH mutant (lacking T3SS1), or the ssaV mutant (lacking T3SS2). 2 h, 6 h and 12 h post-infection cells were lysed with 0.1% Triton and submitted to centrifugation. The pellets and the filtered and concentrated supernatants (cytosol) were analyzed by immunoblotting. The membranes were cut in three parts. The top was incubated with anti-GroEL antibodies as a control of contamination of the cytosol fraction with non-secreted bacterial proteins. The middle was incubated with antibodies against the cytosolic protein b-actin to measure the number of eukaryotic cells that was present in each condition. The bottom was incubated with anti-FLAG antibodies to detect the SteA protein that was present in the intracellular bacteria (pellet) and that was translocated into the cytosol of the eukaryotic cells (cytosol). (B) Macrophages RAW264.7 were infected with S. enterica serovar Typhimurium wild-type, prgH,ssaV,or prgH ssaV mutant strains, all of them harboring the plasmid pIZ1886 that expresses full length SteA in fusion with the catalytic domain of CyaA from B. pertussis. To analyze translocation, the level of cAMP was measured 6 h post-infection as described in the Materials and Methods section. It is expressed as % of the maximum level of cAMP that was measured per intracellular bacterium. doi:10.1371/journal.pone.0026930.g004 Expression and Secretion of SteA PLoS ONE | www.plosone.org 5 October 2011 | Volume 6 | Issue 10 | e26930 Expression of steA and the T3SS2 genes during HeLa cells infections The results obtained in the previous section prompted us to analyze the expression of steA and ssaV (as an indicator of the synthesis of the T3SS2) in the context of the infection of HeLa cells. Derivatives of the strain 14028 of S. enterica serovar Typhimurium carrying steA::lacZ or ssaV::lacZ fusions, were grown under SPI-1 inducing conditions (LB 0.3 M NaCl, low aeration). Under these conditions, expression of steA was high although not maximal, as seen above. Under the same conditions, as expected, ssaV was not expressed (less than 6 Miller units). Interestingly, very quickly after infection, expression increased three-fold for steA and ten-fold for ssaV (Fig. 6), giving support to the involvement of T3SS2 in the translocation of SteA at the beginning of the infection of HeLa cells under invasive conditions. Identification of secretion signals in SteA In order to investigate the signals relevant for secretion of SteA to the culture media, we generated several CyaA fusions expressed from plasmids. Different Nand C-terminal truncations were obtained and tested by immunoblotting with anti-CyaA antibodies. Fig. 7 shows the results obtained under SPI-1-inducing conditions. Fusions with the full-length protein and with fragments 1–20 and 1–10 were efficiently secreted. In contrast, the fusion with the amino acids 16–210 of SteA was not secreted. These results suggest that the signal necessary for secretion is contained in the first 10 amino acids of SteA. Analysis of translocation of SteA fragments into macrophages The different CyaA fusions used in the previous section for the secretion experiments were also tested in experiments of translocation into RAW264.7 cells. The results (data not shown) were consistent with the results of the secretion experiments and suggested that the first 10 amino acids of SteA were also sufficient for translocation of this effector into the host cell. Salmonella can enter phagocytic cells, like macrophages RAW264.7, by two different mechanisms: invasion and phagocyFigure 5. Translocation of SteA into HeLa cells. (A) HeLa cells were infected with Salmonella strains expressing SteA-3xFLAG grown under SPI-1 (invasive) conditions and the cells were processed 50 min or 4.5 h post-infection. Immunoblotting was carried out using anti-GroEL (top) and antiFLAG (bottom) antibodies on the same membranes. (B) Translocation of SteA-CyaA into HeLa cells. HeLa cells were infected with S. enterica serovar Typhimurium wild-type, prgH or ssaV mutant strains, harboring the plasmid pIZ1886 that expresses full length SteA in fusion with the catalytic domain of CyaA from B. pertussis. To analyze translocation, the level of cAMP was measured 15 min, 50 min, 2 h, 4 h, 8 h and 16 h post-infection as described in the Materials and Methods section. Data are expressed as % of the maximum cAMP level that was measured in this experiment. doi:10.1371/journal.pone.0026930.g005 Expression and Secretion of SteA PLoS ONE | www.plosone.org 6 October 2011 | Volume 6 | Issue 10 | e26930 tosis. Whereas invasion is T3SS1-dependent, phagocytosis is carried out without the participation of any T3SS. Therefore, different bacterial growth and infection conditions can be used to monitor the relevance of T3SS1 and T3SS2 in the translocation of SteA into phagocytic cells. Translocation of the CyaA fusions that were secreted to the medium (Fig. 7) and translocated into RAW264.7 (data not shown) was assessed in the mutants prgH and ssaV that are T3SS1and T3SS2-defective strains, respectively. Translocation of the three fusions measured 1 h after infection of macrophages with invasive bacteria was T3SS1-dependent (Fig. 8A). Translocation of the same fusions measured 12 h after infection of macrophages with non-invasive bacteria was T3SS2dependent (Fig. 8B). These results suggest that the same Nterminal motif is recognized by both Salmonella T3SS. Discussion For an efficient secretion, the synthesis of one effector should be coordinated with the expression of its cognate T3S apparatus. Examples of this coordination are found in Salmonella enterica [36,37], Shigella flexneri [38,39,40], and Yersinia enterocolitica [41]. This work focuses on the study of SteA, a Salmonella effector that was poorly characterized. It was described in 2005 as being able to translocate into macrophages J774 through T3SS1 and T3SS2 [24]. Since SteA is a Salmonella effector that can be secreted through two different T3SS, it was especially interesting to study expression, secretion and translocation of this effector under different conditions favoring the use of one or the other T3S apparatus. Our results (Fig. 1 and Fig. 2) indicate that steA is expressed under a wide range of conditions. This is consistent with the ability of this effector to be secreted through two systems that are expressed in different conditions. However, we have identified particular factors that increase or decrease expression. A comparison of different media showed that, in vitro, optimal expression is obtained in LPM at pH 5.8, a media that imitates the conditions found in the SCV and that is also optimal for the synthesis of T3SS2. Expression of steA, however, was not significantly affected by changes in pH between 5.0 and 8.4 in LB with 0.3 M NaCl without aeration (Fig. 1D) or in LPM (Fig. 1E). Bacteria grown in LB media with low aeration and high osmolarity until the end of the exponential growth phase are maximally invasive due to optimal expression of T3SS1. These conditions however are not the best for expression of steA because, although low aeration increases expression of this gene (Fig. 1D and E), hyperosmolarity does the opposite (Fig. 1B and C). This is not just a consequence of osmotic shock since the results were obtained in the presence of proline as osmoprotector. In addition, immunoblot experiments (Fig. 2) revealed that low osmolarity also decreases the amount of SteA, in spite of the expression of the translational steA-lacZ fusion. This result suggests that hypoosmolarity compromises the stability of the protein SteA. The suboptimal expression of SteA in the media that provides optimal invasivity may not be totally representative of the situation during the infection, since culture media are not able to perfectly mimic the in vivo conditions. In fact, our experiments measuring the level of expression of steA in the course of the infection of HeLa cells suggest that this expression significantly increases upon contact of Figure 6. Expression of steA and the T3SS2 during infection of HeLa cells. HeLa cells were infected with S. enterica serovar Typhimurium strains carrying a steA::lacZ fusion (left panel) or a ssaV::lacZ fusion (right panel) under invasive conditions. b-galactosidase activities were measured before infection (t = 0) and 15, 50, 120, and 240 min after infection. doi:10.1371/journal.pone.0026930.g006 Figure 7. Identification of the sequences necessary for secretion of SteA. Derivatives of S. enterica serovar Typhimurium strain 14028 carrying plasmids expressing full-length (1–210) or different fragments of SteA in fusion with CyaA from B. pertussis (numbers in brackets indicate aminoacids included) were grown under SPI-1 inducing conditions. The presence of the CyaA fusions in cell lysates (from 10 8 cells) and culture supernatants (from 2.5610 8 cells) was analyzed by immunoblotting with anti-CyaA antibodies as described in the Materials and Methods section (upper panels). AntiGroEL antibodies were used as loading control and to test unspecific leakage of proteins into the supernatants (lower panels). Molecular mass markers, in KDa, are indicated on the left. doi:10.1371/journal.pone.0026930.g007 Expression and Secretion of SteA PLoS ONE | www.plosone.org 7 October 2011 | Volume 6 | Issue 10 | e26930 Salmonella with the host cell and remains elevated several hours post-infection (Fig. 6). Another factor that decreases expression of steA is butyric acid (Fig. 1 and Fig. 2). This is one of the organic acids that are necessary to maintain the normal status of the intestinal epithelium [42]. In fact, treatment with butyric acid decreases invasion and colonization of cecal epithelial cells [43,44]. In a transcriptomic analysis 19 genes were found to be down-regulated with butyrate more than two-fold in S.enterica serovar Typhimurium and S. enterica serovar Enteritidis, 17 of these genes localized to SPI-1 and one of the other two genes was sopE2, a gene encoding a T3SS1 effector located outside the island [45]. In addition, the level of SipA, another effector encoded outside SPI-1, is also reduced after exposition to butyrate [46]. Therefore, in regard to butyrate, steA behaves like many T3SS1 related genes. We used culture media that are optimal for expression of either T3SS1 (LB 0.3 M NaCl) or T3SS2 (LPM pH 5.8) to characterize the secretion of SteA through these systems (Fig. 3). Efficient secretion was detected in both media and the secretion was dependent on the expected T3SS. It has been suggested that the complex SsaM-SpiC-SsaL regulates secretion through T3SS2 so that translocon proteins are secreted first and, once the translocon pores has been assembled and the pH of the cytosol is detected, the secretion of effectors begins [32]. As a consequence the effector SseJ is not secreted to the culture media unless the experiment is carried out in an ssaL background or the pH is shifted to 7.2 at the end of the experiment. This effect has also been observed for other effectors [25]. Our results, however, show that SteA can be efficiently secreted in LPM at pH 5.8 in a wild-type background, although a small increase in secretion can be observed in the ssaL mutant. T3SS1 is expressed at the beginning of the infection and is important for invasion. T3SS2 is expressed inside the Salmonella containing vacuole and is necessary for create and maintain an intracellular niche for survival and proliferation of the bacteria. However, as mentioned in the Introduction, some overlap exists between both systems. Our experiments studying the kinetics of translocation of SteA into eukaryotic host cells provide two additional examples of this overlapping. First, non-invasive Salmonella translocated SteA into RAW264.7 cells mainly through T3SS2 and this is clearly seen 12 h post-infection when the ssaV mutation completely abolishes translocation (Fig. 4A). However, although translocation 6 h post-infection is mainly T3SS2dependent, some translocation is observed in the ssaV mutant. That this translocation occurs through T3SS1 is confirmed because the double mutant shows no translocation (Fig. 4B). These results suggest that T3SS1 is expressed and is functional in the translocation of effectors inside macrophages for a certain period of time. The second example is observed in the experiments of translocation into HeLa cells using invasive bacteria. To interpret these experiments it should be taken into account that these cells are non-phagocytic. Therefore, entry of Salmonella depends on bacterial factors, mainly on the invasivity provided by T3SS1. This is consistent with the results of the kinetics of translocation into HeLa cells (Fig. 5B) showing that translocation is T3SS1-dependent throughout the experiment, from 15 min to 16 h. In this context it is difficult to ascertain the role of T3SS2, if any, in the translocation of effectors. Translocation for 4 h, 8 h and 16 h is measured for bacteria inside de host cells, since treatment with the antibiotic gentamicin is used to kill extracellular cells. Therefore, the T3SS1-deficient mutant prgH, that cannot enter the host cell, would not show translocation even if the secretion occurred through a different system. For shorter times, up to 2 h, extracellular bacteria are present in the protocol we used (see Materials and Methods), and we can detect translocation from outside the host cell through the cytoplasmic membrane. Interestingly, we observed a preponderant role for T3SS2 in the translocation of SteA into HeLa cells 15 min after the infection that decreases 50 min post-infection and is no longer detected 2 h Figure 8. Analysis of the translocation of fragments of SteA under invasive and non-invasive conditions. RAW264.7 cells grown in 24-well plates were infected with the wild-type, prgH,orssaV strains of S. enterica serovar Typhimurium expressing full-length SteA (1–210) or SteA fragments 1–10 or 1–20 in fusion with the catalytic domain of CyaA from B. pertussis. (A) Bacteria were grown for 16 h in LB with 0.3 M NaCl at 37uC without shaking and cAMP was measured 1 h post-infection. The results are expressed as % of the maximum level of cAMP measured in the experiment. (B) Bacteria were grown for 24 h in LB at 37uC with shaking and cAMP was measured 12 h post-infection. The results are expressed as % of the maximum level of cAMP per bacterium measured in the experiment. doi:10.1371/journal.pone.0026930.g008 Expression and Secretion of SteA PLoS ONE | www.plosone.org 8 October 2011 | Volume 6 | Issue 10 | e26930 post-infection (Fig. 5B). This result was unexpected for two reasons: (i) The bacteria were grown under invasive conditions that prevent expression of T3SS2 genes. This repression was confirmed using an ssa V::lacZ fusion (Fig. 6, right panel, time 0). However, as seen in Fig. 6, the expression of this fusion increased very quickly during infection of Hela cells, which is consistent with the translocation of SteA through this system at the beginning of the infection. It has been previously shown that SPI-1 and SPI-2 genes are expressed simultaneously inside HeLa cells [27]. It should be noted, however, that the shorter time after infection analyzed in that report was 2 h. Here, we show that ssaV is expressed as early as 15 min after infection of HeLa cells. This result increases a growing body of evidence showing expression of SPI-2 immediately after the entry of the bacteria into the small intestine and indicating that T3SS2 contributes to intestinal colonization [47,48,49,50]. A very recent report suggests that expression of SPI-2 before invasion of host cells reflects transcriptional priming that is needed later for intracellular survival [51]. Our results suggest that T3SS2 is not only expressed but also functional in the secretion of at least the effector SteA at that time. (ii) Even if 15 min post-infection SteA can be translocated through T3SS2, it is expected to be translocated also through T3SS1. The kinetics of SteA translocation in the ssaV single mutant suggested an irrelevant or minor contribution of T3SS1 to translocation of SteA 15 min post-infection, although translocation through T3SS1 increased with the time (Fig. 5B, compare 15 min, 50 min, and 2 h). A feature of T3SS is their ability to engage substrates in a certain order [52,53,54,55,56,57,58,59]. A sorting platform has been recently described for T3SS1 that ensures secretion of the translocases before the effectors [60]. This hierarchy of secretion could also extend to the establishment of an order of secretion between effector proteins based on differential affinities of the different effector-chaperone complexes. In this context, our results suggest that SteA is secreted through T3SS1 at a relatively late stage in the secretion process. Another factor that can contribute to a low level of secretion of SteA through T3SS1 at the beginning of the infection is the fact that this protein, unlike other T3SS1 effectors, is also a substrate of T3SS2. In should be noted, however, that in the conditions discussed above the detection of SteA in the host cytosol was also dependent on T3SS1. This result could be a consequence of the role of this system not only in translocation of proteins but also in host cell binding [56,61]. Due to low sequence similarity and lack of common features among different T3S signal sequences, attempts to use computational methods to identify these signals have only achieved limited accuracy [62,63,64,65,66]. As a result, experimental approaches based on the generation of fusions with truncated forms of effector proteins are necessary. Previous studies with other effectors have shown that some kind of secretion signal exists at the N-terminus [67,68,69,70,71]. In addition, for T3SS1 effectors like InvJ and SipB small sequences contained in the N-terminal 10 amino acids are enough to mediate secretion through this system [72,73]. Our experiments with truncated forms of SteA show that the Nterminal 10 amino acids of this protein are sufficient to direct secretion into culture media (Fig. 7) and translocation into eukaryotic host cells (Fig. 8) although the N-terminal 20 amino acids are necessary for optimal secretion and translocation, in particular through the T3SS2. Finally, the results obtained with prgH and ssaV mutants in macrophages suggest that the same N-terminal signal is recognized by T3SS1 and T3SS2 (Fig. 8). These results also support the idea of the universality of the secretion signals recognized by these systems. In fact, the ability of effectors from one species to be translocated by the T3SS of another bacterial species has been documented [74,75,76]. In addition, it has been shown that the Nterminal secretion sequence of the Salmonella T3SS1 effectors SopE and SptP can direct secretion through the related flagellar system [77]. In these cases, translocation through T3SS1 requires a chaperone-binding domain, located in the first 140 amino acids, in addition to the N-terminal secretion sequence. Although the existence of a similar chaperone mechanism for SteA cannot be ruled out, our experiments show that the N-terminal signal located in the first 10 amino acids is enough to direct specific translocation into RAW264.7 cells through T3SS1 and T3SS2. Additional experiments are needed to identify chaperones for SteA and the cognate chaperone-binding domain in the effector, if they exist. Materials and Methods Bacterial strains, bacteriophages and strain construction E. coli and S. enterica serovar Typhimurium strains used in this study are described in Table 1. Salmonella strains derive from the mouse-virulent strain ATCC 14028. Transductional crosses using phage P22 HT 105/1 int201 [78] were used for strain construction [79]. To obtain phage-free isolates, transductants were purified by streaking on green plates [80]. Phage sensitivity was tested by cross-streaking with the clear-plaque mutant P22 H5. Bacterial culture The standard culture medium for S. enterica was Luria-Bertani (LB) broth. Solid LB contained agar 1.5% final concentration. Antibiotics were used at the following concentrations: kanamycin (Km), 50 mgml 21 ; chloramphenicol (Cm), 20 mgml 21 ; ampicillin (Ap), 100 mgml 21 . For SPI-1-inducing conditions, Salmonella strains were grown overnight at 37uC in LB-0.3M NaCl medium in static conditions. For SPI-2-inducing conditions, cells from cultures in LB were washed and diluted 1:100 with minimal medium at pH 5.8 (LPM) containing 80 mM 2-(N-morpholino) ethanesulfonic acid (pH 5.8), 5 mM KCl, 7.5 mM (NH 4 ) 2 SO 4 , 0.5 mM K 2 SO 4 , 0.1% casamino acids, 38 mM glycerol, 337.5 mMK 2 HPO 4 -KH 2 PO 4 (pH 7.4) and 8 mM MgCl 2 , and incubated overnight at 37uC with shaking. For some experiments the concentration of NaCl or the pH of the medium were modified as indicated. 10 mM sodium butyrate (Sigma) was added to the medium in some experiments. DNA amplification with the polymerase chain reaction Amplification reactions were carried out in a Perkin Elmer Gene-Amp PCR System 2400 (Perkin Elmer Cetus). The final volume of reactions was 100 ml, and the final concentration of MgCl 2 was 1.5 mM. Reagents were used at the following concentrations: dNTPs, 300 mM; primers, 0.3 mM; and Taq polymerase (KAPA HiFi DNA Polymerase, Kapa Biosystems), 1 unit per reaction. The thermal program included the following steps: (i) initial denaturation, 5 min at 95uC; (ii) 25 cycles of denaturation (98uC, 20 s), annealing (57uC, 15 s), and extension (72uC, 30 s); and (iii) final incubation at 72uC for 5 min, to complete extension. Primers are listed in Table 2. PCR constructs were sequenced with an automated DNA sequencer (Stab Vida, Expression and Secretion of SteA PLoS ONE | www.plosone.org 9 October 2011 | Volume 6 | Issue 10 | e26930