Exchanges of conserved aspartate residues in the C- and N-terminal moieties of SiiE affect amounts of secreted SiiE

The giant adhesin SiiE possesses 53 BIg domains, most of which contain five conserved D or E residues that coordinate binding of two Ca²⁺ ions. We investigated the role of Ca^{2 +}-binding sites in BIg domains for secretion of SiiE. The Gaussia luciferase (GLuc) [17] was used as reporter for quantification of amounts of secreted SiiE (Fig 1, S1 Fig). Compared to Firefly luciferase, GLuc is ATP independent, and more robust and progressive [18]. To quantify the secretion, the reporter GLuc BIg50-53 was constructed by fusing GLuc to the C-terminal moiety of SiiE, i.e. BIg domains 50–53, the insertion and the C-terminal secretion signal (Fig 1A). Another construct was generated in which all five conserved D residues forming the type I and type II Ca²⁺-binding sites in BIg51 and BIg52 were exchanged to S (D/S exchange), termed GLuc BIg50-53Δ2. The number of deleted Ca²⁺-binding sites is indicated by Δn. A further reporter fusion consisting of GLuc and BIg47-53 was generated and D/S exchanges of various extent were introduced (S1C Fig).

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Fig 1. Role of Ca²⁺-binding sites for secretion of SiiE.

A) Schematic overview of fusion proteins analyzed in this study. GLuc fused to SiiE contains BIg50–BIg53. Conserved aspartate residues exchanged to serine are shown in blue and the position of the residues is indicated in subscript. GLuc Big50-53Δ2 is a mutant form lacking two Ca²⁺-binding sites. B) GLuc assay for secretion of fusion proteins. Salmonella WT and ΔsiiF strains harboring plasmids for synthesis of GLuc-SiiE fusions or empty vector were subcultured for 6 h. Samples were processed and GLuc activity determined as described in S1 Fig. Filled and open bars show total cell-associated GLuc activity (lysate) and secreted GLuc activity (supernatant), respectively. Experiments were performed in triplicates, one representative is shown. Statistical analysis was performed by one-way ANOVA with Bonferroni t-test and is indicated as follows: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001.

https://doi.org/10.1371/journal.ppat.1006418.g001

The synthesis and secretion of GLuc fusions with WT or mutant SiiE portions was compared using Salmonella WT and the siiF-deficient strain unable to form a functional T1SS. GLuc activities in the lysate and supernatant obtained after 6 h of subculture represent the cytosolic and surface-bound, or secreted portion of GLuc fusions, respectively (S1 Fig, Fig 1B). GLuc activities in lysates were similar for GLuc BIg50-53 and GLuc BIg50-53Δ2 reporters, indicating similar rates of synthesis and stability (Fig 1B). Secreted GLuc activity for the SiiE WT reporter was 46.7-fold lower in the ΔsiiF background, demonstrating SPI4-T1SS-dependent secretion of the reporter. In the SPI4-T1SS-proficient background, secreted GLuc activity for the GLuc BIg50-53Δ2 reporter was 122.4-fold lower than for the GLuc BIg50-53 reporter.

We also analyzed secretion of a GLuc fusion protein containing BIg47-53 (S1C and S1D Fig). Here, five D/S exchanges in GLuc BIg47-53Δ2 did not reduce secretion. Also, the exchanges resulting in GLuc BIg47-53Δ4 were without effect on the secretion of the reporter, while additional D/S exchanges for deletion of another two Ca²⁺-binding sites caused a five-fold reduced secretion of GLuc BIg47-53Δ6. The complete removal of 10 Ca²⁺-binding sites in GLuc BIg47-53Δ10 resulted in 73.7-fold reduced secretion, similar to levels of the WT reporter fusion in ΔsiiF background.

We conclude that Ca²⁺-binding sites in BIg SiiE are required for the secretion of SiiE. The removal of two Ca²⁺-binding sites in a secretion reporter with four BIg was sufficient to ablate T1SS-dependent secretion, while removal of at least 6 Ca²⁺-binding sites was required to affect secretion of a reporter fusion with 7 BIg.

The number of Ca²⁺-binding sites correlates with SiiE function

To analyze the role of Ca²⁺-binding sites in SiiE for SiiE-dependent virulence functions of Salmonella, we transferred mutant alleles with D/S exchanges of various extent into chromosomal siiE using λ Red recombineering (Fig 2A). The resulting strains synthesized mutant forms of SiiE with D/S exchanges resulting in removal of 2, 5, 6 or 10 Ca²⁺-binding sites (Fig 2B). No SiiE was detected for the ΔsiiE strain and compared to WT SiiE, variable amounts of mutant SiiE were observed. Compared to the WT, all mutant strains investigated contain lower amount of cell-associated SiiE. Since whole bacterial lysates were analyzed, one cannot distinguish between SiiE present in cytosol, or SiiE retained on the bacterial surface. Mutant forms of SiiE with reduced surface retention will lead to lower amounts of cell-associated SiiE, although levels of SiiE synthesis are comparable to WT SiiE.

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Fig 2. Role of Ca²⁺-binding sites in the C-terminal moiety for secretion and function of chromosomally encoded SiiE.

A) Schematic overview of the domain organization of SiiE and chromosomally encoded forms of SiiE with mutated Ca²⁺-binding sites. The positions of deleted Ca²⁺-binding sites is shown in blue and Δ1, Δ2, Δ4 and Δ10 indicate 1, 2, 4, or 10 mutated Ca²⁺-binding sites, respectively. Mutant alleles were generated using the λ Red-mediated replacement of I-SceI aph cassette by DNA fragments harboring sequence alterations. B) Western blot detection of SiiE in whole bacterial lysates of a 3.5 h subculture for analysis of protein synthesis. C) The amounts of SiiE retained on the bacterial surface of Salmonella WT and various mutant strains at 3.5 h and 6 h subculture was determined by dot blot analysis. Representative dot blots are shown. The loading of dot blots was normalized by parallel analysis of the signal for LPS. D) The amounts of secreted SiiE for Salmonella WT and various mutant strains at 3.5 h and 6 h subculture were determined by dot blot analysis. After TCA precipitation, the pellet was resuspended according to OD₆₀₀ of the culture and equal amounts of samples were loaded onto a nitrocellulose membrane. E) Comparison of activities of secreted GLuc-SiiE reporters obtained for plasmid-encoded GLuc conjugated SiiE by GLuc assay (S1 Fig) or chromosomally encoded AA exchanges by dot blot analysis depicted in D). F) SiiE-dependent invasion of polarized epithelial MDCK cells by Salmonella WT, ΔsiiF, and strains expressing various mutant alleles of siiE. Cells were infected with the indicated strains at a MOI of 5. Non-internalized bacteria were removed by washing and remaining bacteria were killed by addition of gentamicin for 1 h. Subsequently, cells were lysed and serial dilutions were plated onto agar plates for determination of colony-forming units (CFU). Invasion is depicted as percentage of the inoculum that was internalized by host cells. Experiments were performed in duplicates (C, D) or triplicates (E, F), and means and standard deviations are shown. Statistical analysis was performed by one-way ANOVA with Bonferroni t-test and is indicated as follows: n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ***, P < 0.001.

https://doi.org/10.1371/journal.ppat.1006418.g002

SiiE retention on the bacterial surface and secretion into culture supernatant was analyzed by dot blots of whole cells, and protein precipitated from culture supernatants, respectively (Fig 2C and 2D). Our previous work demonstrated that SiiE is mainly retained on the bacterial surface at 3.5 h of subculture, and predominantly released into the supernatant at 6 h and later of subculture [6]. Of the various mutant strains analyzed, only the strain producing SiiE BIg52Δ2 showed SiiE retention after 3.5 h of subculture similar to WT. After 6 h subculture levels of SiiE retention of WT and all mutant strains were as low as the negative control.

The number of mutated Ca²⁺-binding sites correlated with reduction of secreted SiiE at 3.5 and 6 h of subculture. With increasing numbers of D/S exchanges, lesser amounts of secreted SiiE were detected. Amounts of SiiE BIg47-52Δ10 were as low as the negative control, indicating a complete loss of secretion for this SiiE mutant (Fig 2D).

We compared secretion of WT and mutant SiiE quantified by dot blot analyses to GLuc activities of the GLuc-SiiE reporter (Fig 2E, S1C Fig). GLuc reporters for SiiE BIg52Δ2 and SiiE BIg47Δ1 BIg51-52Δ4 resulted in GLuc activities similar to GLuc-SiiE_WT. If introduced in chromosomal siiE, the mutations resulted in reduced amounts of secreted SiiE. Secretion of SiiE BIg47Δ2 BIg51-52Δ4 was highly reduced in both assays, while no secretion of SiiE BIg47-52Δ10 was detected in GLuc and dot blot assays. The data demonstrate that Ca²⁺-binding sites in BIg are important for the secretion of SiiE, and that amounts of secreted SiiE decreases with an increasing number of D/S exchanges in BIg domains.

We next determined the effect of deletion of Ca²⁺-binding sites on SiiE-dependent virulence functions, i.e. adhesion to polarized epithelial cell followed by SPI1-T3SS-mediated invasion (Fig 2F). Only SiiE BIg52Δ2 conferred invasion of MDCK cells at a level comparable to WT SiiE. All other mutant SiiE we investigated resulted in highly reduced invasion, comparable to the siiF-deficient strain that is unable to secrete SiiE.

We conclude that Ca²⁺-binding sites in the C-terminal part of SiiE are essential for secretion and function of the adhesin. Removal of Ca²⁺-binding sites in more than one BIg in this moiety results in loss of function.

The position of Ca²⁺-binding sites is critical for the function of SiiE

The C-terminal moiety of SiiE contains the signal for T1SS secretion, is secreted first and is likely to be exposed most distal to the cell envelope. We next tested the functional relevance of Ca²⁺-binding sites in the middle or N-terminal portions of SiiE. Strains were generated with mutations in chromosomal siiE resulting in deletion of Ca²⁺-binding sites in BIg2, BIg40, or BIg1-5 (Fig 3A). Since ¹¹⁷D of a previous BIg domain (BIg_(n-1)) forms a type I Ca²⁺-binding site with ⁴³D and ⁹⁷D of a subsequent BIg domain (BIg_(n)), ¹¹⁷D of BIg1 and BIg39 were exchanged instead of ¹¹⁷D of BIg2 and BIg40, resulting in SiiE BIg2Δ2 and SiiE BIg40Δ2, respectively. To control the precision of the Red recombineering method applied here and the absence of unwanted attenuating mutations, we used a siiE mutant strain and restored the WT sequence. This strain, termed WT_restored, showed SiiE-dependent phenotypes as the WT strain.

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Fig 3. Role of Ca²⁺-binding sites in N-terminal or central portions of SiiE.

A) Schematic overview of various mutant forms of SiiE with D/S exchanges in BIg2, BIg40 or BIg1-5 for deletion of 2 (Δ2) or 10 (Δ10) Ca²⁺-binding sites. B) Western blot for analyses of synthesis of mutant forms of SiiE. C) Amounts of retained SiiE after 3.5 h and 6 h of subculture. D) Secreted SiiE after 3.5 h and 6 h of subculture. E) SiiE-dependent invasion of polarized epithelial MDCK cells. Analyses of synthesis, surface retention and secretion and SiiE-dependent invasion of polarized cells were performed as described for Fig 2.

https://doi.org/10.1371/journal.ppat.1006418.g003

Strains expressing mutant chromosomal siiE were tested for protein synthesis. No expression could be detected for the negative control ΔsiiE. All mutant strains synthesized SiiE of correct size (Fig 3B). The deletion of Ca²⁺-binding sites only in BIg2 or BIg40 had no or only small effects on surface retention of SiiE (Fig 3C), secretion (Fig 3D), or SiiE-dependent invasion (Fig 3E). Secretion of SiiE BIg1-5Δ10 was slightly reduced, but there was still more secretion after 6 h of subculture than after 3.5 h of subculture. Interestingly, the level of SiiE retention was also reduced to approximately 50% of WT and maintained at the same level at 6 h of subculture. Destruction of all Ca²⁺-binding sites in BIg1-5 (BIg1-5Δ10) led to a 74.6-fold decreased invasion of polarized cells (Fig 3E), while the same extent of deletions in BIg47-52 (BIg47-52Δ10) resulted in 392.2-fold reduced invasion (Fig 2F). Compared to SiiE BIg47-52Δ10, reduction of retention and secretion is less pronounced for SiiE BIg1-5Δ10. If extracellular Ca²⁺ ions facilitate secretion of SiiE, the secretion might come to a halt earlier for BIg47-52Δ10 than for SiiE BIg1-5Δ10.

In addition to the conserved D or E residues involved in Ca²⁺ binding, 47 of 53 BIg domains possess a conserved tryptophan residue at position 74. To test a potential role of these conserved tryptophan residues in SiiE function, we performed W to F (W/F) exchanges in 1, 2, or 3 BIg in the C-terminal moiety of SiiE (S2A Fig). These mutations only resulted in minor changes of the amounts of SiiE retained and secreted at 3.5 h or 6 h of subculture (S2B, S2C and S2D Fig). Functionally, none of the mutant forms of SiiE with various degrees of W/F exchanges resulted in reduced invasion of polarized epithelial cells (S2E Fig), indicating that conserved ⁷⁴W residues in the C-terminal moiety of SiiE are neither important for secretion and retention of SiiE, nor for the SiiE-dependent adhesion and invasion.

T1SS substrate proteins are secreted in an unfolded state [19]. For example, secretion of E. coli HlyA was highly reduced if the protein was modified in a way that allowed folding in the cytosol [20]. To further investigate parameters known to affect secretion of T1SS substrate proteins, we tested if folding rate influences secretion as for HlyA. We fused the C-terminal portion of SiiE harboring the secretion signal to MalE. Distinct point mutations in the MalE portion of the fusion protein led to different folding rates as previous established by Bakkes et al. [20]. Secretion of various MalE-SiiE fusion proteins was analyzed at 3.5 h and 6 h of subculture. Similar amounts of fusion proteins were detected in the culture supernatant at both time points (S3 Fig). This indicates that the rate of intracellular folding did not affect SiiE secretion, supporting that binding of extracellular Ca²⁺ ions by the secreted portion of BIg domains is more important.

Molecular dynamics simulations for roles of type I and type II Ca²⁺-binding sites

In order to assess the role of Ca²⁺ ions for the conformational stability, molecular dynamics (MD) simulations of WT and mutant SiiE were performed. We focused on BIg domains 50–52 for the following reasons: (i) a high-resolution crystal structure is available for this portion of SiiE, (ii) the fragment is sufficiently small to allow for extensive MD simulations, and (iii) the role of Ca²⁺-binding sites in BIg50-52 constructs was experimentally investigated (Fig 1). The role of Ca²⁺ ions was assessed from inspection of the tilt and twist angles defining the relative orientation of BIg 51 and 52 (Fig 4A–4D). The percentage of tilted or twisted structures detected over the simulation time is summarized in Fig 4E. A comparison of WT and mutant forms revealed that mutation of both type I and type II sites caused an enhanced tilting of the structure and thus a less extended domain arrangement compared to the WT structure. The stronger effect was observed for the type I site, which is consistent with the direct location in the domain interface. Notably, the concomitant mutation of both sites had an additive effect resulting in the highest portion of tilted structures among all systems investigated. Mutation of type I and type II site did not only affect tilting, but also resulted in an enhanced twisting of the domain pair (Fig 4E). However, in contrast to tilting, type I and type II site had a similar effect on domain twisting and there was no additive effect upon mutation of both sites. These data support the role of Ca²⁺-binding sites in increasing the rigidity of SiiE.

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Fig 4. Effect of mutations of type I and II sites on the geometry of the BIg51-52 domain pair.

Schematic presentation of the tilt and twist angles analyzed. A, B) Tilt angle between the BIg51 (green) and BIg52 (orange) domains in the crystal structure (A), and a tilted conformation observed during the MD simulations (B). The tilt angle (depicted as black line) was defined between the Cα-atoms of residues ⁶P and ⁹S of BIg51 and ⁵⁰V of BIg52. C, D) Twist angle between the BIg51 (green) and BIg52 (orange) domains in the crystal structure (C), and a twisted conformation observed during the MD simulations (D). The twist angle (depicted as black line) was defined as torsion angle between the Cα-atoms of residues ⁹S and ⁹¹I of BIg51 and ⁷E and ⁵⁰V of BIg52. E) Effect of mutation of the type I and II site on the geometry of the BIg51-52 domain pair. Structures were defined as tilted or twisted, if the respective interdomain angle deviates by more than 15 degree from the conformation present in the crystal structure. See A)-D) for the definition of the interdomain angles. All simulations were performed for BIg50-52. BIg50 has been omitted in the presentation for reasons of clarity. Ca²⁺ ions are indicated by spheres.

https://doi.org/10.1371/journal.ppat.1006418.g004

To assess the relative Ca²⁺ binding affinities of the type I and type II sites, steered molecular dynamics (SMD) simulations were performed. The setup is schematically depicted in Fig 5A and 5B. The Ca²⁺ ions were independently removed from both sites and 10 simulations were performed for each site. For the type II site, all 10 work plots display an overall similar shape (Fig 5C). Up to a distance of ~ 15 Å, the work linearly increases reflecting the disruption of the interactions between the Ca²⁺ ion and its protein ligands. For larger distances there is only a marginal further increase of the work, indicating that dissociation is almost complete at a distance of ~ 15 Å. The work required in the 10 SMD runs of the type II site ranges from 246–329 kcal x mol^-1. For the type I sites, the qualitative appearance of the curves is similar (Fig 5D); however, less work is required for the removal of the ion from this site. The resulting work ranges from 134–212 kcal x mol^-1, which is significantly lower than for the type II site.

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Fig 5. Steered molecular dynamics simulations for displacement of Ca²⁺ ions in SiiE.

A) Setup for pulling the Ca²⁺ ion out of the type II site. A spring-like restraint (black zigzag line) was defined between the BIg51 center of mass (green circle) and the Ca²⁺ ion. The exit pathway of the ion from the binding site is schematically shown as dashed line. SiiE BIg domains 50, 51, and 52 are colored in blue, green, and orange, respectively. B) Setup for pulling the Ca²⁺ ion out of the type I site. In contrast to the setup in panel (A), the spring-like restraint was defined between the BIg52 center of mass (orange circle) and the Ca²⁺ ion. C, D) Work required to pull the Ca²⁺ ion out of the type II site (C) or the type I site (D). Results of 10 independent simulations are shown in different colors.

https://doi.org/10.1371/journal.ppat.1006418.g005

Role of Ca²⁺-binding sites in SiiE for conformation, stability and thermal-induced aggregation properties of SiiE

SiiE variants with altered type I and type II Ca²⁺-binding sites in the C-terminal moiety of SiiE (SiiE_Cterm) were characterized in detail in in vitro experiments. These variants encompass BIg domains 48–53, the insertion and the C-terminal segment that includes the secretion signal (Fig 6A). We have previously shown that recombinant protein production of a fragment that covers BIg domains 50 to 52 in E. coli and subsequent purification of the fragment without the addition of any Ca²⁺ ions leads to a protein sample in which all Ca²⁺-binding sites are fully occupied [8]. This observation was corroborated not only by the final electron density map of the solved crystal structure, but also by analyzing in detail the anomalous scattering signal and via X-ray fluorescence measurements [8]. Here, we now used inductively coupled plasma—atom emission spectroscopy (ICP-AES) to verify the presence and/or absence of Ca²⁺ ions in protein variants that were produced following a similar purification protocol as previously described for the BIg domains 50 to 52 protein fragment [8]. In case of WT SiiE_Cterm the occurrence of 8 to 10 Ca²⁺-binding sites was expected. In the ICP-AES experiment the protein was analysed at a concentration of 6.5 mg x ml^-1 (88.1 μM), which results in a theoretical maximum calcium content of 28–35 μg x ml^-1 (705.2–881.5 μM). The experimentally determined calcium concentration was 23 μg x ml^-1 which amounts to 81.8% to 65.44% of the expected theoretically maximum content (Table 1, S4 Fig). Variant SiiE_Cterm with type I and type II Ca²⁺-binding sites mutated (SiiE_Cterm BIg48-52Δ8_{type I + II}) was measured at a concentration of 4.2 mg x ml^-1 (57.63 μM). For this variant, a calcium signal below the 0.5 μg x ml^-1 calibration standard and outside the calibration range was detected (S4 Fig). Thus, no calcium binding is observed for variant SiiE_Cterm BIg48-52Δ8_{type I + II} (Table 1). These measurements show that in case of the WT protein the Ca²⁺-binding sites are almost fully occupied in the recombinantly produced protein sample whereas the substitution of defined aspartic residues against serines in the SiiE_Cterm BIg48-52Δ8_{type I + II} results in a protein that is devoid of any calcium binding. Conversely, variants with some of the Ca²⁺-binding sites disrupted should display reduced Ca²⁺ binding if one assumes the absence of any cooperativity between the binding sites.

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Fig 6. Role of Ca²⁺-binding sites in SiiE for folding, thermal stability and aggregation properties of SiiE.

A) Schematic overview of the constructs containing the C-terminal region of SiiE which were used to examine the impact of D/S exchanges on the folding and stability of the protein in vitro. B) Circular dichroism (CD) spectra in the far UV region of SiiE_Cterm WT and mutant forms with mutation of type I, type II, or type I and type II Ca²⁺-binding sites. C) Thermal scanning CD analysis of the stability of the SiiE_Cterm variants. D) Electrophoretic motility on native PAGE of SiiE_Cterm WT and Ca²⁺-binding site mutants. E) The aggregation behavior of SiiE_Cterm WT and variant forms was investigated by incubating samples of each protein at increasing temperatures and analysis on native PAGE.

https://doi.org/10.1371/journal.ppat.1006418.g006

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Table 1. Analysis of calcium content of SiiE_Cterm WT and BIg48-52Δ8_{type I + II} by ICP-AES.

https://doi.org/10.1371/journal.ppat.1006418.t001

To experimentally address individual roles of type I and type II Ca²⁺-binding sites for the conformational stability of SiiE, we performed circular dichroism (CD) measurements. Highly similar FarUV spectra were recorded for all variants, namely the SiiE_Cterm construct with WT sequence, mutations of type I, type II and of both type I and type II Ca²⁺-binding sites (Fig 6B). Estimation of the secondary structure content using the BeStSel server suggests that the SiiE_Cterm wild-type variant consist of around 50% β-sheets, 10% turns and 40% others (e.g. random coil). These values are close to those derived from the available SiiE BIg-domain crystal structure (BIg domains 50–52, PDB entry: 2YN5). This suggests that the so far structurally uncharacterized domains (BIg domains 48–49 and BIg domain 53) display similar secondary structures as domains 50 to 52. Most importantly, however, the highly similar spectra and concomitant results from the secondary structure analysis of the variants studied here demonstrate that the secondary structure composition of the proteins is not altered, thus excluding pronounced mis- or unfolding, when mutating the type I and/or type II Ca²⁺-binding sites (S4 Table). The thermal scanning CD measurements revealed distinct effects of type I and type II site mutations on the conformational stability of SiiE_Cterm (Fig 6C). All protein variants exhibit a decrease in ellipticity upon heating, which suggests that instead of a thermally induced unfolding an increased secondary structure and/or β-sheet formation via aggregation occurs. However, the magnitude of this transition is lower for SiiE_Cterm WT and SiiE_Cterm BIg48-52Δ4_{type II} than for SiiE_Cterm BIg48-52Δ4_{type I} and SiiE_Cterm BIg48-52Δ8_{type I + II}. Also, T_onset of these structural changes is higher for WT and Δ4_{type II} variants at 72°C and 69°C, respectively, than for Δ4_{type I} and Δ8_{type I + II} variants at 50°C and 45°C, respectively. Thus, while the thermal scanning CD curve of SiiE_Cterm BIg48-52Δ4_{type II} resembles that of SiiE_Cterm WT, the spectra of proteins with mutations of type I and both type I and II sites indicated that the proteins are more prone to aggregation.

To further investigate the conformational stability, the SiiE_Cterm variants were subjected to native PAGE (Fig 6D and 6E). All variants covering the C-terminal moiety of SiiE migrated as a single band under mild conditions (Fig 6D). To further investigate the aggregation behavior, individual samples of each variant were incubated at increasing temperatures and subsequently analyzed by native PAGE. SiiE_Cterm WT was resistant to aggregation up to 80°C and SiiE_Cterm BIg48-52Δ4_{type II} behaved similar to WT protein, although pronounced aggregation was detected at a slightly lower temperature (Fig 6E). A ladder-like pattern indicates that SiiE_Cterm BIg48-52Δ8_{type I + II} started to form oligomers and aggregated already at 50°C. Aggregation of SiiE_Cterm BIg48-52Δ4_{type I} resembled SiiE_Cterm BIg48-52Δ8_{type I + II}, although slightly delayed (Fig 6D). The analysis of the aggregation behavior therefore reflects the results of the thermal scanning CD measurements. Faster migrating protein species are visible in the native PAGE of the Δ4_{type I} and Δ8_{type I + II} mutants. To control whether unwanted proteolysis might have caused the occurrence of these so-called lower bands, SDS-PAGE analysis was performed for SiiE_Cterm samples after incubation at various temperatures (S5 Fig). Neither WT SiiE_Cterm nor any of the mutant forms indicate a temperature-dependent occurrence of proteolytic fragments. The increased migration behavior thus results from a partial collapse of the expected linear overall structure of SiiE into a more globular domain arrangement as the result of the removal of the type I Ca²⁺-binding sites that are located in the interface between BIg domains.

Next, the conformation and compactness of the SiiE_Cterm variants was probed by limited proteolysis using α-Chymotrypsin and Proteinase K (S6 Fig). Multi-domain proteins with flexible domain surface loops and/or interdomain linkers are expected to be more prone to proteolytic cleavage than proteins with very rigid domain architecture. Resistance against proteolytic cleavage by α-Chymotrypsin was clearly reduced for SiiE_Cterm BIg48-52Δ8_{type I + II} in comparison to WT protein or protein with only type I or type II binding site mutations. This suggests that both types of Ca²⁺-binding sites help to stabilize the fold against proteolytic degradation. The effect is possibly enhanced by the close spatial proximity of the two binding sites [8]. Resistance against proteolytic cleavage by Proteinase K was also reduced most for SiiE_Cterm BIg48-52Δ8_{type I + II} similarly to the proteolysis using α-Chymotrypsin. However, the stability of the individual type I or type II Ca²⁺-binding site mutants was also decreased, although to a lesser extent than for the variant with both binding sites mutated.

Type I Ca²⁺-binding sites are more important for secretion than type II Ca²⁺-binding sites

Finally, we set out to functionally dissect the roles of type I and type II Ca²⁺-binding sites in SiiE. We generated mutant alleles by site-directed mutagenesis for single aa exchanges in BIg51 and BIg52 (S7 Fig), or exchanges of all residues of either the type I or the type II Ca²⁺-binding sites within BIg52, within BIg47-52, or BIg1-5 (Fig 7).

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Fig 7. Functional dissection of type I and type II Ca²⁺-binding sites.

SiiE was mutated in 5 consecutive BIg domains in the N-terminal (BIg1-5) or C-terminal (BIg47-52) region in 10 Ca²⁺-binding sites (D10), 5 type I Ca²⁺-binding sites only (Δ5_typeI), or 5 type II Ca²⁺-binding sites only (Δ5_typeII). A) Schematic overview of D/S exchanges introduced for deletion of Ca²⁺-binding sites in BIg1-5 or BIg 47–52. B) Western blot for analyses of synthesis of mutant forms of SiiE. C) Amounts of SiiE retained on the bacterial surface at 3.5 h, 6 h, 8 h and 24 h of subculture. D) Amounts of SiiE secreted in culture supernatant after 3.5 h and 6 h of subculture. E) SiiE-dependent invasion of polarized epithelial MDCK cells. Analyses of synthesis, surface retention and secretion and SiiE-dependent invasion of polarized cells were performed as described for Fig 2.

https://doi.org/10.1371/journal.ppat.1006418.g007

Mutation of single aspartate residues or D/S exchanges in single Ca²⁺-binding sites in chromosomal siiE did not affect SiiE synthesis, surface retention, secretion, or SiiE-dependent invasion (S7B, S7C, S7D and S7E Fig). In contrast, if either type I or type II Ca²⁺-binding sites are missing within the five C-terminal BIg domains 47–52, invasion is reduced to the level of the negative control (Fig 7E). For both mutants, retention (Fig 7C) and secretion (Fig 7D) was reduced. This reduction was more pronounced for SiiE BIg47-52Δ5_{type I} than for SiiE BIg47-52Δ5_{type II}. Retention was fully abolished for SiiE BIg47-52Δ5_{type I}, as well as for BIg47-52Δ10 for all time points tested, while secretion was reduced to 50% of WT SiiE. For SiiE BIg47-52Δ5_{type II} retention after 3.5 h of subculture was reduced to 40% of WT SiiE and after 6 h and later time points no surface retention was detected, similar to WT SiiE.

Removal of type I or type II Ca²⁺-binding sites in BIg1-5 had only minor effects on retention and secretion of SiiE. In contrast to SiiE BIg1-5Δ5_{type II}, SiiE BIg1-5Δ5_{type I} was retained at late time points at a level similar to BIg1-5Δ10 (8 and 24 h). SiiE BIg1-5Δ5_{type II} was also retained at late time points, but to a lesser extent than SiiE BIg1-5Δ5_{type I} or SiiE BIg1-5Δ10. The mutation of five type I sites in BIg1-5 resulted in highly (43.5-fold) reduced invasion, while removal of five type II Ca²⁺-binding sites in the same moiety (BIg1-5Δ5_{type II)} did not reduce invasion of polarized epithelial cells (Fig 7E). These results suggest distinct roles of type II Ca²⁺-binding sites in N- and C-terminal portions of SiiE.

To further analyze the role of type II Ca²⁺-binding sites for function of SiiE, we removed type II Ca²⁺-binding sites by D/S exchanges in BIg31-35, BIg 36–40, BIg 41–45, or BIg 46–50. The invasion of MDCK cells of strains expressing these mutant forms of siiE was compared to invasion by strains with WT SiiE, SiiE BIg1-5Δ5_{type II} and SiiE BIg47-52Δ5_{type II} (Fig 8). We observed that strains producing SiiE with type II sites removed in BIg31-35, BIg 36–40, BIg 41–45, BIg 46–50 or BIg47-52 all exhibited reduced invasion compared to strains with SiiE WT or SiiE BIg1-5Δ5_{type II}. The reduction of invasion was pronounced if BIg domains in the C-terminal region were affected, and smallest reduction of invasion was observed for the strain with SiiE BIg30-35Δ5_{type II}.

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Fig 8. The position of type II Ca²⁺-binding sites is critical for SiiE function.

The type II Ca²⁺-binding sites of 5 consecutive BIg domains (Δ5_typeII) were removed by D/S exchanges in various positions of SiiE as indicated (A). B) SiiE-dependent invasion of polarized epithelial MDCK cells by Salmonella strains expressing WT and mutant alleles of SiiE. Analysis of SiiE-dependent invasion of polarized cells was performed as described for Fig 2.

https://doi.org/10.1371/journal.ppat.1006418.g008

The fact that SiiE BIg1-5Δ5_{type II}, but not SiiE BIg47-52Δ5_{type II} still mediates binding to, and invasion of MDCK cells indicates that type II Ca²⁺-binding sites are more important for the correct local conformation of the protein, which might be necessary for proper binding. We conclude that in SiiE BIg1-5Δ5_{type II} the C-terminal part is correctly folded and can mediate binding to the host cell, while this is not the case for SiiE BIg47-52Δ5_{type II}.

Bacterial strains and culture conditions

Salmonella enterica serovar Typhimurium (S. Typhimurium) NCTC 12023 was used as wild-type strain in this study and all mutant strains are isogenic to this strain. The characteristics of strains used in this study are listed in Table 2. Bacterial strains were routinely grown in LB broth or on LB agar containing antibiotics if required for selection of specific markers. The Ca²⁺ concentration in LB media is not defined. Carbenicillin or kanamycin were used at 50 μg x ml^-1, and tetracycline or chloramphenicol were added to a final concentration of 20 or 10 μg x ml^-1 respectively, if required for the selection of phenotypes or maintenance of plasmids.

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Table 2. Bacterial strains used in this study.

https://doi.org/10.1371/journal.ppat.1006418.t002

Cell culture

Madin-Darby Canine Kidney Epithelial (MDCK) cells are an immortalized cell line initially derived from renal tube of a cocker spaniel. MDCK Pf subclone used for the generation of polarized epithelial cell monolayers was kindly provided by Department of Nephroplogy, FAU Erlangen-Nürnberg. MDCK cells were used for the generation of polarized epithelial cell layer. Cell culture conditions were previously described by Wagner et al. [6]. Briefly, cells were cultures in MEM with Earle’s salts, 4 mM Glutamax, non-essential amino acids and 10% heat-inactivated fetal calf serum. The Ca²⁺ concentration in this medium is 1.8 mM.

Cloning

The synthetic DNA fragments (GeneArt or IDT) were subcloned in blunt end restriction sites of the pJET1.2 vector backbone. The synthetic DNA fragment SiiE-BIg49_half-52_D-S was first cloned into pWRG454 (pWSK29::P_siiAGlucM43LM110L::siiE-BIg50-53) via HindIII, NheI digest and ligation. To obtain the whole SiiE-BIg49-52_D-S, the second synthetic DNA fragment SiiE-BIg47-49_{half D-S} was cloned into pWSK29::P_siiAGlucM43LM110L::siiE-BIg49_half-52_D-S via HindIII digestion and ligation. Orientation was checked by PstI, ClaI diagnostic digest and subsequent sequencing confirmed the construct.

Site-directed mutagenesis

The Q5 site-directed mutagenesis kit (NEB) was used to create plasmid with exchanges in codons for single conserved aspartate residues or exchanges for type I or type II Ca²⁺-binding sites in SiiE. Primers included new recognition sites for restriction enzymes through silent mutations. After performing colony PCR, the PCR fragment was digested with an appropriate restriction enzyme to confirm the silent mutations. Clones have also been confirmed by sequencing.

Construction of chromosomal exchanges of conserved aspartate residues

The construction of scar-less in-frame deletions in siiE using the I-SceI site was previously described by Blank et al. [32] and the protocol modified by Hoffmann et al. [33] was applied here. Briefly, the I-SceI aph resistance cassette was amplified from pWRG717 using primers with 20 bp homology and 40 bp 5’ overlap. The resistance cassette was inserted by Red-mediated recombination within the desired region. The plasmid pWRG730 was used instead of pKD46 and features a heat-inducible promoter for red genes. For preparing competent cells of WT [pWRG730], cells were grown in LB Cm¹⁰ to OD₆₀₀ of 0.4–0.5 in a baffled flask in a shaking water bath at 150 rpm at 30°C. Subsequently, cells were immediately transferred to another water bath pre-heated to 42°C and incubated for 12.5 min at 100 rpm. After incubation of heat-induced cells on ice for 15 min, competent cells were prepared as described before [34]. After DpnI-digestion and purification, the PCR product was electroporated into Salmonella and transformants were selected on LB Km²⁵ agar plates. Transformants were checked by colony PCR and confirmed clones were streaked on LB Cm¹⁰ plates with or without 100 ng x ml^-1 anhydrotetracycline (AHT, Sigma-Aldrich). Clones with the highest inhibition on AHT containing plates at 30°C were selected for further procedures.

PCR fragments were amplified from plasmids containing exchanges of conserved aspartate codons in siiE or synthetic DNA were used. These fragments contain 5’ overlaps which are homolog to the insertion site of the chromosomally integrated I-SceI aph cassette. The strain containing the I-SceI aph cassette and harboring pWRG730 was then transformed by electroporation with either purified PCR product or synthetic DNA fragments. Serial dilutions from 10⁻¹ to 10⁻⁴ were plated on LB Cm¹⁰ plates containing 100 ng x ml^-1 AHT and incubated overnight at 30°C. The next day, large colonies were re-streaked on LB Cm¹⁰ AHT¹⁰⁰ plates. Clones were verified by testing sensitivity to kanamycin and by colony PCR.

Gaussia luciferase assay

The Gaussia luciferase assay was performed as previously described by Wille et al. [17].

Quantification of SiiE retention

Bacterial strains were diluted 1:31 in LB from O/N cultures and grown at 37°C for 3.5 h. Aliquots of 1 ml of bacterial culture were collected, cells pelleted and resuspended in 1 ml of sterile LB. After an additional washing step with sterile LB, optical density was measured and adjusted to OD₆₀₀ of 1 in 500 μl of 3% PFA in PBS. After fixation of bacterial cells for 15 min at RT, cells were pelleted (10,000 x g; 5 min) and resuspended in 500 μl PBS. Five microliters of bacterial suspensions were spotted on a nitrocellulose membrane which has been pre-wetted with PBS and dried again before adding bacteria. After drying of the spots, membranes were blocked with 5% dry milk powder in TBS/T (TBS; 0.1% Tween20) for at least 30 min. For detection of SiiE on the bacterial surface, antiserum against the C-terminal moiety of SiiE [35] was diluted 1:10,000 in blocking solution and applied to the membrane. LPS was detected using antiserum against Salmonella O-antigen (Becton-Dickinson) at the same dilution. After incubation O/N at 4°C, membranes were washed thrice with TBS/T and bound primary antibodies were detected with anti-rabbit IRDye 800CW (LI-COR) at a dilution of 1:20,000 in PBS/T (PBS; 0.1% Tween20). Subsequently, membranes were incubated for 1 h at RT in the dark and washed thrice with PBS/T. Membranes were rinsed in PBS and signals were quantified using the Odyssey Imaging System (LI-COR Biotechnology).

Quantification of secreted SiiE

Bacterial strains were diluted 1:31 in LB from an O/N culture and grown at 37°C for 6 h. The OD₆₀₀ was measured. Aliquots of 2 ml of bacterial culture were taken and pelleted. Supernatants were filter sterilized (0.2 μm Millex filter units, Millipore). 200 μl of 100% TCA was added to 1.8 ml of filtered sterilized supernatant and incubated O/N at 4°C. The precipitated proteins were pelleted by centrifugation at 4°C for 45 min. After two washing steps with 1 ml ice-cold acetone (14,000 x g, 30 min, 4°C) the precipitate was air dried and afterwards resuspended in 25 μl PBS per OD₆₀₀ of 1. Dot blot analysis was carried out as described before without detection of LPS.

Determination of SiiE by Western blot analysis

For Western blot analysis, O/N cultures were diluted 1:31 in LB and grown for 3.5 h at 37°C. OD₆₀₀ was measured, and 150 μl were pelleted in 2 min at 4°C at 16.000 x g. The pellet was resuspended in OD₆₀₀ x 50 μl in 1 x SDS sample buffer and incubated at 100°C for 5 min. 15 μl of each sample were loaded onto 3–8% gradient gels (NuPage) and electrophoretically separated for 1 h at 150 V. Semi-dry blotting was performed using a 0.2 μm nitrocellulose membrane with 64 mA/blot for 4 h. After blocking with 5% milk/TBS/T, the membrane was incubated first with a primary antibody against SiiE and subsequently with a secondary HRP-conjugated antibody against rabbit IgG. The signals were determines using ECL reagent (ThermoScientific) and the ChemiDoc system (BioRad).

Invasion assay

Invasion assay was performed as previous described by Wagner et al. [6]. Briefly, O/N cultures of Salmonella strains were diluted 1:31 in LB and grown for 3.5 h in test tubes with aeration in a roller drum. The cultures were diluted in MEM medium to obtain a multiplicity of infection (MOI) of 5 and this inoculum as added to the MDCK cells. After infection for 25 min cells were washed three times with PBS to remove non-internalized bacteria, and medium was replaced by medium containing 100 μg x ml^-1 gentamicin to kill remaining extracellular bacteria. After incubation for 1 h, cells were washed again with PBS, lysed by addition of 0.5% sodium desoxychlate in PBS, and colony forming units were determined by plating serial dilutions of the lysates onto agar plates.

Protein production of SiiE_Cterm variants for in vitro characterization

All SiiE_Cterm variants (p4033, p4034, p4462, p4463, S1 Table) were cloned into the pGEX-6P-1 vector (GE Healthcare) and expressed as GST fusion constructs in E. coli BL21 (DE3) (Novagen). The bacteria were chemically transformed with the expression plasmid and transformants selected on LB agar plates containing 100 μg x ml^-1 ampicillin. The expression was done in terrific broth containing 100 μg x ml^-1 ampicillin. A starter culture was inoculated with a single colony and grown over night at 37°C and 180 rpm. On the next day, the main expression cultures were inoculated to an OD₆₀₀ of 0.08 and incubated at 37°C and 180 rpm. At OD₆₀₀ 0.6 to 0.7 the temperature was reduced to 20°C and at OD₆₀₀ 1.0 to 1.2 the protein expression was induced by adding 0.5 mM IPTG. After induction, the proteins were expressed for 20 h at 20°C and 180 rpm, the bacteria harvested by centrifugation and the pellets stored at -80°C until used for purification. Briefly, the GST-tagged proteins were captured by Glutathione Sepharose affinity column (GE Healthcare) using standard buffers given in the manual. The GST tag was cleaved by adding GST tagged HRV 3C protease at a mass ratio of 1 to 250 (protease-to-fusion protein) and the tag, undigested fusion proteins and the protease were extracted by a second Glutathione Sepharose purification. As a final purification step, the proteins were separated by size exclusion chromatography using a HiLoad 26/60 Superdex 200 pg column (GE Healthcare) and a buffer with 25 mM Tris-HCl, 150 mM NaCl, pH 8.0. No calcium was added to the buffers during the chromatographic purification. The protein was concentrated to 8 mg x ml^-1, frozen in liquid nitrogen and stored at -80°C until use.

Inductively coupled plasma—atom emission spectroscopy

The calcium content of the SiiE_Cterm variants was analysed by inductively coupled plasma—atom emission spectroscopy. All proteins were purified as described above, but without the second Glutathione Sepharose step after tag cleavage. The purified SiiE_Cterm proteins were concentrated and dialysed against the same batch of buffer (5 mM Tris-HCl, pH 8.0, ratio of sample-to-buffer volume 1:100). Calcium standard for ICP (Sigma-Aldrich) was diluted to 0.5, 5 and 50 μg x ml^-1 with the same buffer. The ICP-AES analyses were performed using a Ciros CCD (Spectro Analytical Instruments GmbH). Three individual measurements of the same sample were conducted for each variant and the mean calculated.

Limited proteolysis assays

Limited proteolysis was performed in order to investigate the influence of the mutations on the conformation and compactness of the proteins [36]. The proteolysis experiments were conducted at 20°C and 550 rpm in a benchtop shaker. The assay was done in a buffer containing 25 mM Tris-HCl, 150 mM NaCl, pH 8.0 and the SiiE protein concentration was adjusted to 1 mg x ml^-1. 10 μg α-Chymotrypsin or 0.5 μg Proteinase K (Proti-Ace & Proti-Ace 2 Kit, Hampton Research, Aliso Viejo, USA) were added per mg of SiiE protein. Aliquots of 18 μl were taken prior (0 min), and at various time points after protease addition (1 min, 5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 7 h, O/N). The samples were mixed with 6 μl 4 x SDS-PAGE loading buffer and boiled at 95°C for 5 min to stop the cleavage reaction. The heat-treated samples were briefly spun down and stored at -20°C. The 10 μl of each sample were analyzed by SDS-PAGE using 15% polyacrylamide gels. Gels were stained with Coomassie Blue.

Circular dichroism spectroscopy

The conformation and stability of SiiE variants were probed using circular dichroism (CD) spectroscopy. All measurements were done with a Jasco J-815 spectropolarimeter (Jasco, Tokyo, Japan) using a cuvette with a 0.1 cm path length. CD spectra were recorded at 20°C in the far UV region between 185 and 260 nm in 10 mM potassium phosphate buffer, pH 8.0. Protein concentrations of 15 μM were used for SiiE_Cterm variants. The band width was set to 1.0 nm, the scan speed to 20 nm x min⁻¹, data integration time to 1 sec, data pitch to 0.1 nm and sensitivity to standard. Each measurement was averaged across ten accumulations and the protein spectra corrected for the sample buffer.

The stability of the proteins was compared by thermal scanning analysis. Changes in the secondary structure composition were investigated between 20°C and 96°C by monitoring the CD signal at wavelengths of 222 nm for SiiE_Cterm proteins. A band width of 1.0 nm, data integration time of 8 sec, heat rate of 1°C x min^-1, sampling rate of one data point per 0.2°C and standard sensitivity was used for all thermal scanning experiments. Conversion of the data to concentration- and length-independent mean residue weight (MRW) ellipticities [θ]_MRW was done as described previously [37]. The secondary structure analysis of the CD-spectra and of the Ig-domain structure of SiiE BIg domains 50 to 52 (PDB entry: 2YN5) was done with single spectrum analysis and the secondary structure and beta-sheet decomposition for PDB-structures tools of the BeStSel server, respectively (http://bestsel.elte.hu/ssfrompdb.php) [38]. The wavelength range between 190 nm and 250 nm of the CD spectra was used for estimation of the secondary structure content.

Native PAGE

Native polyacrylamide gel electrophoresis (PAGE) was used to analyze the aggregation tendency of the SiiE variants. All protein samples were adjusted to 0.3125 mg x ml^-1 and 20 μl samples of each protein were incubated at various temperatures (20, 30, 41.7, 50, 60, 70, 80 and 90°C) for 5 min, briefly spun down and chilled on ice for 1 min. 5 μl of 5 x native PAGE-buffer (0.25% (w/v) Bromophenol blue, 4.5% (w/v) sucrose) were added to each sample to achieve a final protein concentration of 0.25 mg x ml^-1 and 15 μl (3.75 μg protein) were loaded per sample. Native PAGE was done using 7.5% native polyacrylamide gels and native PAGE running buffer (50 mM Tris, 384 mM glycine). The gel runs were done at 5 mA per gel for 5 h and 6–8°C in the cold room and gels were stained with Coomassie Blue. For the SDS-PAGE analysis, 4 μl of 4 x SDS sample buffer were added to 16 μl of each sample, the mixture boiled at 95°C for 5 min and briefly centrifuged. 10 μl of each of the samples were analyzed on 15% acrylamide gels. The gel runs were done at 200 V for 60 min.

Bioinformatics

The crystal structure of SiiE wild-type BIg domains 50–52 (PDB code 2YN5, chain A) was used for all computational studies. Based on the wild-type system, three mutants were modelled that lack either the type I, the type II, or both types of Ca²⁺-binding sites. Mutants were generated by replacing the respective coordinating aspartate and glutamate residues by serine.

All systems were neutralized by adding an appropriate amount of sodium counter ions. Each system was placed in a periodic TIP3P water box [39] extending at least 12 Å in all directions from the solute. All simulations were done with Amber 14 [40] using the ff99SB force field [41]. Long-range electrostatics were calculated with the particle mesh Ewald (PME) approximation [42]. Shake was used to constrain hydrogen atoms during equilibration and simulation [43].

Minimization, equilibration and MD calculations were carried out with the pmemd module of AMBER. Minimizations were run for 10,000 steps and switched from steepest descent to conjugate gradient after 500 cycles. During equilibration the system was gradually heated from 30 K to 310 K in 60 ps with backbone restraints of 2.0 kcal/mol Ų and then relaxed at 310 K with backbone restraints of 0.2 kcal/mol Ų for another 20 ps. Two independent production runs of 300 ns were generated for each system using the weak-coupling algorithm [44] and a Berendsen barostat in an NPT ensemble.

For the SMD simulations ten restart files containing atomic coordinates and velocities were taken from wild-type MD simulation (at intervals of 10 ns). Ca²⁺ ions were pulled from type I and type II sites of SiiE individually by a harmonic potential with the spring energy constant of 50 kcal/mol Ų. The center of this potential was moved away from the center of mass (CoM) of the backbone atoms of one BIg domain with a constant velocity of 0.2 Å/ps. In this way the force was evenly distributed on the protein and no positional restraints had to be used.