Abstract
Type VII secretion systems (T7SS) are found in many bacteria and secrete proteins involved in virulence and bacterial competition. In Staphylococcus aureus the small ubiquitin-like EsaB protein has been previously implicated as having a regulatory role in the production of the EsxC substrate. Here we show that in the S. aureus RN6390 strain, EsaB does not genetically regulate production of any T7 substrates or components, but is indispensable for secretion activity. Consistent with EsaB being a core component of the T7SS, loss of either EsaB or EssC are associated with upregulation of a common set of iron acquisition genes. However, a further subset of genes were dysregulated only in the absence of EsaB. In addition, fractionation revealed that although an EsaB fusion to yellow fluorescent protein partially localised to the membrane, it was still membrane-localised when the T7SS was absent. Taken together our findings suggest that EsaB has T7SS-dependent and T7SS-independent roles in S. aureus.
INTRODUCTION
Protein secretion systems are nanomachines employed by bacteria to transport protein substrates across their cell envelopes. Gram-negative bacteria produce a number of different secretion machineries that export proteins involved in a wide variety of processes including signalling, nutrient scavenging, host interaction and virulence (1). The type VII secretion system (T7SS) is found in some Gram-negative and many Gram-positive bacteria, and is particularly common among organisms of the actinobacteria and firmicutes phyla (2). The T7SS was initially described in the pathogenic mycobacteria Mycobacterium tuberculosis and Mycobacterium bovis, where the ESX-1 T7SS was shown to be essential for virulence, due to the secretion of two major T-cell antigens EsxA (formerly known as ESAT-6) and EsxB (formerly known as CFP-10) (3-5). EsxA and EsxB are founding members of the WXG100 protein family that appear to be exclusively linked to T7SSs, and all characterised T7 systems are associated with at least one family member. The presence of a membrane-bound ATPase of the SpoIIIE/FtsK family (termed EccC in actinobacteria and EssC in firmicutes) is another hallmark of all T7SSs (6). In Mycobacteria, three further membrane proteins EccB, EccD and EccE assemble with EccC to form a large 1.5 MDa core complex (7, 8). This complex further associates with a membrane-bound mycosin serine protease, MycP, that is essential for T7 protein secretion and for stability of the membrane complex (9).
Staphylococcus aureus, an opportunistic pathogen of humans and animals, also elaborates a T7SS that is distantly related to the T7SSs found in mycobacteria (10). Mutational analysis has indicated that it plays an important role in persistence in mouse models of infection, intraspecies competition and potentially iron homeostasis (10-15). In commonly-studied strains of S. aureus such as Newman, USA300 and RN6390, the secretion system is encoded by the 12 gene ess locus (10, 12, 16). The first six genes at this locus encode core components of the secretion machinery, including the WXG100 protein EsxA and the SpoIIIE/FtsK ATPase EssC (Fig 1A,B). However, S. aureus and other firmicutes lack homologues of EccB, EccD, EccE and MycP and instead have an apparently unrelated set of membrane-bound secretion components (EsaA, EssA and EssB in S. aureus) (12, 17-19). The sixth component of the S. aureus T7SS is EsaB, which is predicted to be a small cytoplasmic protein of 80 amino acids that is structurally related to ubiquitin (20). In S. aureus strains Newman and USA300, a transposon insertion in esaB does not abolish secretion of T7 substrates but is linked with an increase in RNA transcripts covering the gene encoding the substrate EsxC (11). By contrast, in-frame deletion of esaB abolished EsxA and EsxC secretion in strain RN6390 but did not detectably affect production of these substrate proteins (12). Similarly, inactivation of yukD, which encodes the Bacillus subtilis esaB homologue, also abolished T7 secretion (17, 18).
In this study, we have addressed the role of EsaB in S. aureus T7 secretion using strain RN6390. We show that EsaB does not regulate esxC transcripts or those of other ess-encoded genes. Instead our findings show that EsaB behaves as a core component of the T7SS. Interestingly, however, RNA-Seq analysis identified a subset of genes from the AirSR regulon that showing altered regulation in the absence of EsaB, suggesting that it may play additional, T7SS-independent roles in S. aureus physiology.
METHODS
Bacterial strains and growth conditions
S. aureus strain RN6390 (NCTC8325 derivative, rbsU, tcaR, cured of (ϕ11, (ϕ12, (ϕ13; (21)) and the isogenic ΔesaB and Δesx (ΔesxA – esaG) strains (12) were employed in this study. S. aureus strains were cultured in Tryptic Soy Broth (TSB) at 37°C with shaking unless otherwise stated. For calculation of cell numbers we estimated by dilution analysis that one unit at OD 600nm corresponds to 6x108 CFU for strain. When required, chloramphenicol (Cml, final concentration 10 μg/ml) was added for plasmid selection. E. coli strain JM110 (Stratagene) was used for cloning purposes and BL21(DE3) (22) for EsaB overproduction and purification. E. coli was grown in Luria-Bertani (LB) medium at 37°C with agitation. When appropriate, ampicillin was used for plasmid selection (final concentration 125 μg/ml).
Genetic constructs
All plasmids used in this study are listed in Table 1. The esaB gene with its own RBS was PCR amplified from S. aureus RN6390 genomic DNA using primers EsaB-fw and EsaB-rev (Table S1). The 0.3 kb HpaI/EcoRI restriction fragment was cloned into pRAB11 under control of the tetracycline inducible promoter, giving pRAB11-esaB. Clones were selected in E. coli and verified by DNA sequencing. Plasmid pRAB11-esaB-YFP was generated by cloning the 0.3 kb HpaI/EcoRI restriction fragment into pRAB11-YFP (15). Clones were selected in E. coli and verified by DNA sequencing. Nucleotide variants of esaB were generated by the Quickchange site-directed mutagenesis protocol (Stratagene) using pRAB11-esaB or pRAB11-esaB-YFP as a template and primers listed in Table S1. Modified plasmids were digested using DpnI for at least 1h at 37°C and transformed into E. coli. Single point mutations were verified by DNA sequencing.
RNA isolation and RT-PCR
For RNA-Seq analysis, three biological repeats of the S. aureus esaB strain was grown aerobically in TSB up to an OD600 of 1 at which point mRNA was prepared (in three technical replicates). This experiment was carried out alongside the RN6390 and essC strains (15) and followed identical methodology.
For RT-PCR, the indicated S. aureus strains were grown aerobically in TSB and harvested at an OD600 of 1. At this point, the mRNA was extracted using the SV total RNA Isolation Kit (Promega) with some minor modifications. Cell samples were stabilized in 5% phenol/95% ethanol on ice for at least 30 min and then centrifuged at 2770 g from 10 min. Cells were then resuspended in 100 μl of TE buffer containing 500 μg ml-1 lysostaphin and 50 μg ml-1 lysozyme and incubated at 37°C from 30 min. Subsequently, the manufacturer’s instructions were followed. Isolated RNA was subjected to a second DNase treatment using the DNA-free kit (Ambion). RNA was stored at -80°C until use. RT-PCR to probe transcription of genes in the indicated strains was carried out using 500 ng of mRNA as template with the indicated primers (Table S1). PCR products were visualized on 1% agarose gels.
Purification of 6His-EsaB and generation of polyclonal antisera
The EsaB coding sequence (UniProt code ESAB_STAAM) was PCR amplified from a synthetic gene (codon optimized for Escherichia coli K12 (Genscript)) using the primers EsaB-pET1 and EsaB-pET2 (Table S1) and cloned into the NdeI/XhoI site of a modified pET15b vector (Novagen). The plasmid produces an N-terminal His6-tagged protein with a TEV (tobacco etch virus) protease cleavage site. The protein was expressed and purified as described previously (23), except the tag-free EsaB was not collected in the flow-through of the negative purification but required a 30mM imidazole elution. The final size exclusion chromatography step used a 24ml HR 30/100 GL Superdex75 column (GE healthcare), equilibrated with 20 mM Tris pH 7.8, 100 mM NaCl and was calibrated with molecular mass standards (thyroglobulin, 670 kDa; γ-globulin, 158 kDa; serum albumin, 67 kDa; ovalbumin; 44 kDa, myoglobin, 17 kDa; and vitamin B12, 1 kDa). 2 mg purified EsaB (retaining a Gly–Ala–Ser–Thr sequence at the N-terminus after the cleavage step) was utilized as antigen to immunize rabbits for polyclonal antibody production in a standard three injections protocol (Seqlab, Goettingen, Germany).
Secretion assays, subcellular fractionation and western blotting
The indicated strains were grown overnight in TSB, diluted 1/100 in fresh medium and grown up to mid-log phase, at which point whole cells and supernatant fractions were harvested as described previously (12). Briefly, cells and supernatant were separated by a 10 min centrifugation step at 2770 g. Cells were washed twice with PBS, adjusted to and OD600 of 1 and digested using 50 μg/ml of lysostaphin by incubation at 37°C for 30 min. Supernatants were filtered using a 0.22 μm filter and TCA-precipitated in the presence of 50 μg/ml deoxycholate, as described. For S. aureus subcellular fractionation, cells were grown to mid-log phase with shaking and treated as previously described (12). Briefly, cells were harvested by centrifugation and resuspended in TSM buffer (50 mM Tris-HCl pH 7.6, 0.5 M sucrose, 10 mM MgCl2). Lysostaphin was added to a final concentration of 50 μg ml-1 and cells were incubated at 37°C for 30 min to digest the cell wall. At this point, protoplasts were sedimented to recover the cell wall (supernatant fraction). Protoplasts were disrupted by sonication and the membrane was obtained after an ultracentrifugation step at 227 000 g for 30 min and at 4°C. The supernatant was retained as the cytoplasmic fraction. Samples were boiled for 10 min prior to separation in bis-Tris gels and subsequent western blotting.
Polyclonal antisera were used at the following dilutions: α-EsxA 1:2500 (12), α-EsxB 1:1000 (15), α-EsxC 1:2000 (12), α-EsaB 1:500, α-TrxA 1:20000 (24) and α-SrtA (Abcam) 1:3000. Anti-GFP antibody was obtained from Roche and used according to manufacturer’s instructions.
RESULTS
EsaB does not regulate the level of esxC transcripts in strain RN6390
A previous study has shown that a transposon insertion in the esaB gene results in an increase in esxC transcripts in the Newman and USA300 strain backgrounds, and a concomitant increase in the EsxC polypeptide, implicating it as a regulator (11). To investigate whether loss of esaB by in-frame deletion affects the level of esxC mRNA in strain RN6390, we isolated mRNA from the parental strain and the isogenic esaB mutant, prepared cDNA and undertook reverse transcriptase PCR with primers covering either esxA (the first gene at the ess locus, included as a negative control) or esxC (Fig 1A). It can be seen (Fig 1C) that the level of transcripts for each of these genes was qualitatively similar in the wild type and esaB backgrounds.
To examine this quantitatively, we undertook RNA-Seq analysis on RNA prepared from three biological repeats of the RN6390 and esaB strains grown aerobically in TSB to an OD600 of 1. Note that these experiments were performed at the same time as the RN6390 vs essC RNA-Seq analysis described in (15) and used the same RN6390 dataset. Fig 1D shows that the level of esxC transcripts were indistinguishable between the wild type and esaB strains. Analysis of the transcript levels of the other genes at the ess locus indicates that in general they were also not significantly altered by the loss of esaB although there was a small increase in the level of essB. We conclude that there is no evidence that esaB regulates the level of esxC transcripts in RN6390.
We next examined the entire transcript profile of the esaB mutant to investigate the transcriptional/post-transcriptional response to the loss of this small protein. We found 101 genes de-regulated in the esaB mutant compared to the parental strain (using a cut off of logFC > 2 or < -2 and qvalue < 0.05, as applied previously (15)), Fig 2A. Of these, 43 were upregulated by the loss of esaB whereas 58 were downregulated when esaB was absent – these genes are listed in Table 2. Interestingly, almost all of the genes that were differentially regulated in the essC mutant (15) were also similarly regulated in the esaB strain (Fig 4B), although there was a substantive subset of genes that were differentially expressed in the esaB mutant but not the essC strain (Table 2). It can be seen that almost all of the iron acquisition genes, including those for heme acquisition, staphyloferrin synthesis and uptake and ferrichrome import were commonly upregulated by loss of either esaB or essC (Table 2). Furthermore six of the eight downregulated genes from the essC strain were also down regulated in the esaB strain (note that one of the two genes unaffected in the esaB dataset is essC itself, which appears downregulated in the essC dataset because it has been deleted). The finding that almost the entire subset of genes differentially regulated in the absence of essC is also similarly altered by loss of esaB strongly suggests that EsaB is, like EssC, a core component that is essential for activity of the secretion machinery in strain RN6390.
As mentioned above, a subset of transcripts were differentially expressed in the esaB but not the essC strain. These include downregulated genes required for anaerobic nitrate respiration (narGHJ/narK), some secreted proteases (sspA/B/C, aur), capsular polysaccharide synthesis (capG/F/hysA), lactose metabolism (lacB/C/D) and antimicrobial peptide synthesis (epiA/C/D/P). Many of these genes are under control of the essential two component regulatory system AirSR (formerly YhcSR) (25-28). These findings suggest that EsaB may have additional roles in the cell in addition to its requirement for T7 protein secretion.
EsaB is present at low amounts in cells of S. aureus RN6390
To explore the biological role of EsaB in T7 secretion, we firstly overproduced recombinant EsaB with a cleavable His-tag in E. coli, and following cleavage of the tag the protein was further purified by gel filtration chromatography (Fig 3A, B). The purified protein, which eluted with an estimated molecular mass of approximately 12.8 kDa, is close to the expected size of a monomer (9.1 kDa + 0.3 kDa retained following cleavage of the tag = 9.4 kDa). This is in agreement with structural analysis of the B. subtilis EsaB homologue, YukD, which also appears to be monomeric (20).
Polyclonal antisera were raised against purified EsaB and the antibody was affinity purified against the EsaB antigen, before being used to detect the protein in whole cells of S. aureus. Fig 3C shows that although the purified antiserum could clearly recognize purified EsaB, it did not detected a band of the expected size of EsaB in whole cells. Probing a dilution series of purified EsaB indicated that the antibody was able to cross-react with as little as 25ng of protein, which is equivalent to 1.6 x 1011 EsaB molecules. Since the antibody was unable to detect EsaB in whole cells from 9.6 x 108 colony forming units that were loaded onto the SDS gel, we conclude that are less than 170 molecules of EsaB per cell.
Since we were unable to detect native EsaB in S. aureus cell extracts, we constructed a series of tagged variants for which commercial antisera were available. To this end we introduced His6, Myc, hemagglutinin (HA) and Strep epitopes onto the N-terminus of EsaB, and His6, Myc, HA, mCherry or FLAG epitopes onto the C-terminus, but in each case were unable to detect the tagged protein (not shown). We also introduced His6 and His9 epitopes into two predicted loop regions internal to the EsaB sequence but again were unable to detect tagged EsaB (not shown). The only tag we introduced that allowed detection of EsaB was a C-terminal yellow fluorescent protein (YFP) tag. Fig 3D shows that basal production of either native (untagged) EsaB or EsaB-YFP from plasmid vector pRAB11 was sufficient to restore secretion of the T7SS extracellular protein EsxA and of substrates EsxB and EsxC to the culture supernatant. Blotting the same cell samples for the presence of the YFP fusion protein (Fig 3E) showed that it migrated at close to the predicted mass (37 kDa) of the EsaB fusion. There was no evidence for degradation of the fusion protein even after prolonged exposure of the immunoblot (Fig 3E). We conclude that the YFP-tagged variant of EsaB probably retains functionality.
EsaB-YFP partially localizes to the cell membrane
EsaB is predicted to be a soluble cytoplasmic protein (10), and is known to share structural homology with ubiquitin (20). Interestingly, a domain sharing the same fold is also associated with the actinobacterial T7SS, being found at the cytoplasmic N-terminus of EccD (29), indicating that ubiquitin-like proteins are essential features of all T7SSs. To determine the subcellular location of EsaB-YFP, we blotted secreted and whole cell samples of the esaB mutant strain producing plasmid-encoded EsaB-YFP with the YFP antiserum. Fig 4A shows that EsaB-YFP was associated exclusively with the cellular fraction.
We next fractionated these cells to obtain cytoplasm, cell wall and membrane fractions. Immunoblotting with antisera to control proteins known to localize to the cell membrane (SrtA) and cytoplasm (TrxA) indicated that the fractionation had been largely successful, although some SrtA was found in the cell wall fraction (Fig 4B). Blotting these same fractions for the presence of EsaB-YFP showed that the protein localised to both the cytoplasm and membrane fractions. Some degradation of the fusion protein was also noted in these experiments which may result from the activation of proteases during fractionation. When unfused YFP was produced in the wild type strain it did not localise to the membrane (Fig 4C), indicating that membrane binding was unlikely to be mediated through the YFP portion of the fusion.
Next we tested whether EsaB-YFP localised to the membrane through interactions with membrane components of the T7SS. To this end we repeated the fractionation in a strain carrying a chromosomal deletion in all twelve genes at the ess locus (Fig 1A). However this did not alter the localization of EsaB-YFP, which was still detected in both cytoplasm and membrane fractions (Fig 4B). It may be that EsaB-YFP localises to the membrane through interaction with additional membrane proteins, consistent with additional, T7SS-independent roles for EsaB suggested through the RNA-Seq analysis. Alternatively, we cannot rule out that the membrane localization arises as an artifact of the C-terminal YFP tag, since this tag is known to influence protein behaviour (e.g. (30)).
Mutagenesis of conserved hydrophilic and hydrophobic patches on EsaB
An alignment of EsaB homologues encoded across firmicutes (Fig 5A) identifies a number of highly conserved amino acids. Many of these are hydrophilic and fall on one face of the predicted structure of EsaB including T8 (S. aureus numbering) which is highly conserved as either threonine or serine, and the invariant K56. The presence of an invariant lysine is particularly intriguing since there are a number of highly conserved lysine residues on the structurally-related protein ubiquitin, that are used to assemble polyubiquitin chains (31). To probe potential roles of these conserved residues we mutated each of T8, D10, L21, K30, K52, K56, L66, G74 and D75 to alanine on plasmid-encoded EsaB and assessed whether the variant EsaB proteins were able to restore T7 secretion activity to the esaB deletion strain.
Fig 5C shows that alanine substitutions of each of these conserved residues was tolerated by EsaB with the exception of T8A, which completely abolished EsaB activity. To test whether other side chain substitutions were permissive at T8, we subsequently constructed EsaB T8S, T8E, T8H, T8K and T8R substitutions. As seen in Fig 5D, in addition to T8A the T8R substitution also abolished EsaB activity, but the other substitutions resulted in active protein.
Ubiquitin has a conserved hydrophobic patch (Fig 6A, left) that forms a common site of interaction with many different binding partners (32). Analysis of the predicted structure of EsaB (Fig 6A, right) shows that there are some hydrophobic residues on the surface potentially at positions approximating the hydrophobic patch region of ubiquitin. To assess whether these hydrophobic residues may be involved in EsaB function, we firstly mutated V7, I44, I71 and L77 to alanine residues. Fig 6B shows that these substitutions did not detectably affect T7 secretion indicating that the function of EsaB had not been compromised. We next substituted each of these residues for a positively-charged lysine. This more drastic change of amino acid side-chain was still tolerated at positions 44 and 77, but inactivated EsaB when substituted for V7 or I71.
Finally we attempted to assess whether any of the inactivating substitutions E7K, T8A, T8R or I71K altered the subcellular location of EsaB-YFP. However, when we introduced each of these substitutions into EsaB-YFP we found that they destabilised the protein as it was almost undetectable in whole cells (Fig 6D), precluding further analysis. We are therefore unable to determine whether these substitutions directly alter EsaB function or have an indirect effect by disrupting folding.
DISCUSSION
In this work we have investigated the role of EsaB in Type VII secretion. EsaB proteins are conserved in firmicutes that produce the T7SS and are encoded at the same loci. Previous work had implicated EsaB in the regulation of esxC transcripts (11), although this cannot be a conserved role for EsaB proteins as they are found in all S. aureus strains, including the subset that do not encode esxC (16). Here we show that EsaB does not regulate esxC in strain RN6390, nor any of the other genes encoded at the ess locus. Instead, deletion of esaB is associated with upregulation of genes involved in iron acquisition, mirroring the upregulation of iron-acquisition genes seen when the core T7 component, EssC, is absent (15). This supports the notion that EsaB is a core component of the secretion machinery in RN6390, and in agreement with this, deletion of esaB prevented export of the T7-dependent extracellular proteins EsxA, EsxB and EsxC. This conclusion is also in agreement with related studies in B. subtilis, where the EsaB homologue YukD was shown to be essential for secretion of the WXG100 protein YukE (17, 18).
The precise role of EsaB in T7 secretion is unclear. Structural analysis of B. subtilis YukD shows that it shares a very similar fold to ubiquitin but that it lacks the ability to be conjugated with other proteins (20). Interestingly, a ubiquitin-like domain is also associated with the actinobacterial T7SS, being found at the cytoplasmic N-terminus of the polytopic EccD membrane component (29), suggesting that EsaB-like components are essential features of all T7SSs. Ubiquitin interacts directly with many different protein binding partners (32), and it is therefore likely that EsaB interacts with one or more components of the T7SS, potentially regulating activity. Post-translational regulation of the S. aureus T7SS has been suggested because in some growth conditions the secretion machinery is present but there is no or very little substrate secretion (12, 19). Other protein secretion systems are also post-translationally regulated, for example the flagellar type III secretion system is regulated through interaction of the FliI component with the second messenger cyclic di-GMP (33), and Type VI secretion systems are regulated by phosphorylation (34). In this context, EsaB proteins contain a highly conserved threonine (or serine) residue close to their N-termini which we considered as a potential site for phosphorylation. Intriguingly, substitution of EsaB T8 for alanine abolished the function of EsaB, although introduction of either the phospho-mimetic glutamate at this position or a positively charged lysine did not affect EsaB activity.
The low cellular levels of EsaB precluded further analysis of the native protein, but a C-terminal fusion of EsaB with YFP partially localised to the cell membrane. We reasoned that binding of EsaB-YFP to membranes was mediated through interaction with one or more of the T7SS membrane proteins. However, some EsaB-YFP was still membrane associated when it was analysed in a strain lacking all of the core T7 components, suggesting that it may interact with additional membrane proteins potentially unrelated to the T7SS. Support for this suggestion comes from RNA-Seq analysis of the esaB mutant strain. In addition to a common set of genes showing similar regulation in the esaB and essC strains, a further subset of genes were uniquely deregulated in the esaB mutant. Many of the genes in this EsaB-specific subset are part of the AirSR regulon (25-28). The AirSR two component system responds to oxidation signals via a redox-active [2Fe-2S] cluster in the sensor kinase AirS to regulate diverse sets of genes involved in anaerobic respiration, lactose metabolism and capsule biosynthesis. In future it will be interesting to further decipher the roles of EsaB in T7 protein secretion and S. aureus physiology.
ACKNOWLEDGEMENTS
This study was supported by the Wellcome Trust (through Investigator Award 10183/Z/15/Z to TP and through Clinical PhD studentship support to CPH through grant 104241/z/14/z), the Biotechnology and Biological Sciences Research Council and the Medical Research Council (through grants BB/H007571/1 and MR/M011224/1, respectively). We thank Dr Sarah Murdoch for calculating the relationship between OD 600nm and CFU for RN6390. Dr Francesca Short is thanked for her assistance with RNA-Seq data analysis and Professor Nicola R. Stanley-Wall for her advice with RNA extraction. The authors declare no conflicts of interest.