The type VII secretion system of Staphylococcus aureus secretes a nuclease toxin that targets competitor bacteria

Abstract

The type VII protein secretion system (T7SS) plays a critical role in the virulence of human pathogens including Mycobacterium tuberculosis and Staphylococcus aureus. Here, we report that the S. aureus T7SS secretes a large nuclease toxin, EsaD. The toxic activity of EsaD is neutralized during its biosynthesis through complex formation with an antitoxin, EsaG, which binds to its C-terminal nuclease domain. The secretion of EsaD is dependent on a further accessory protein, EsaE, that does not interact with the nuclease domain, but instead binds to the EsaD N-terminal region. EsaE has a dual cytoplasmic/membrane localization, and membrane-bound EsaE interacts with the T7SS secretion ATPase, EssC, implicating EsaE in targeting the EsaDG complex to the secretion apparatus. EsaD and EsaE are co-secreted, whereas EsaG is found only in the cytoplasm and may be stripped off during the secretion process. Strain variants of S. aureus that lack esaD encode at least two copies of EsaG-like proteins, most probably to protect themselves from the toxic activity of EsaD secreted by esaD⁺ strains. In support of this, a strain overproducing EsaD elicits significant growth inhibition against a sensitive strain. We conclude that the T7SS may play unexpected and key roles in bacterial competitiveness.

Fig 1. EsaD is a substrate of the T7SS.

a. The ess locus – genes coding for core components of the secretion machinery are in green, secreted components yellow and proteins investigated as part of this study in white. b. and c. EsaD is not required for secretion of EsxA and EsxC – (b) the RN6390 wild-type or isogenic deletion strains, as indicated, were cultured in TSB medium to OD₆₀₀ of 2 or (c) the indicated strains harbouring pRAB11 (empty) or pRAB11-EsxC were cultured in TSB medium to OD₆₀₀ of 0.5, then supplemented with ATC (50ng/ml; to induce plasmid-encoded gene expression) until OD₆₀₀ of 2. Cells were pelleted and the supernatant (sn) was retained as the secreted protein fraction. Samples of the supernatant and whole cells (an equivalent of 200μl of culture supernatant and 10μl of cells adjusted to OD1) were separated on 12 % bis-Tris gels and immunoblotted with the indicated antisera (with TrxA serving as a cytoplasmic control). Note that the samples were run on the same gel but intervening lanes have been spliced out (unspliced version is shown in Fig S9). (d) and (e). EsaD is secreted in an essC-dependent manner. The indicated S. aureus strains harbouring pRAB11 (empty) or pRAB11-EsaD(H528A)-HA were treated as described in (c) except that 250ng/ml ATC was used to induce EsaD(H528A)-HA production and an equivalent of 250μl of supernatant and 10μl of cells adjusted to OD₆₀₀ of 1 were loaded f. Cells of the wild type S. aureus strain, RN6390, harbouring pRAB11 or pRAB11-EsaD(H528A)-HA from (d) were fractionated into cytoplasmic (cyt) and membrane (m) fractions. Samples of each fraction (20μl aliquot of cyt, and 2mg of membrane) were separated on 12% bis-Tris gels and immunoblotted using either anti-HA, anti-EssB (membrane protein control) or anti-TrxA (cytoplasmic control) antisera.

Fig 2. EsaDG form a nuclease toxin-antitoxin pair.

a. EsaD is toxic to E. coli. E. coli BL21(DE3) harbouring pT7.5 (empty vector), pT7.5-esaD(V584Y) or pT7.5-esaDG was cultured to OD₆₀₀ 0.5, supplemented with 1 mM IPTG (time zero) and OD₆₀₀ measured at 1 hr intervals (n=3 biological replicates, error bars are ± SD). b. – d. EsaG interacts with the nuclease domain of EsaD. b. Interactions between pT25-EsaG and EsaD variants fused to pT18 assessed by β-galactosidase activity assay in E. coli BTH101. BTH101 harbouring pT25 and pT18 was the negative control. Error bars are ± SD (n=3 biological replicates). Student’s t-test gives p values < 0.00001 for EsaD/EsaG and EsaD_421-614/EsaG relative to the negative control. Inset shows the same strain/plasmid combinations on MacConkey maltose plates. c. and d. Top two panels: S. aureus RN6390 carrying c. pRAB11 (empty vector), pRAB11-EsaG-HA or pRAB11-EsaD(H528A)-His-EsaG-HA, or e. pRAB11 (empty), pRAB11-EsaG-HA, pRAB11-EsaD_421-614(H528A)-His or pRAB11-EsaD_421-614(H528A)-His-EsaG-HA was cultured to OD₆₀₀ of 0.5, then supplemented with ATC (500ng/ml). Cells were harvested at OD₆₀₀ 3, lysed and histidine-tagged EsaD purified. Cell lysate (load) and eluted fractions (20μl of each) were analysed by western blot with anti-His and anti-HA antisera. Bottom two panels show repeat experiments of: c. the EsaD(H528A)-His-EsaG-HA co-purification or d. the EsaD_421-614(H528A)-His-EsaG-HA co-purification. Samples of load (10µl), flow through (20µl), final wash (30µl) and elution fraction (30µl) were analysed using the same antisera. Coomassie-stained samples of the load and elute fractions are shown in Fig S4. e. E. coli M15[prep4] harbouring pQE70 alone (empty) pQE70-EsaG-EsaG-EsaD_421-614-His or pQE70-EsaG-EsaG-EsaD_421-614(H528A)-His were cultured to OD₆₀₀ of 0.5 and supplemented with 1mM IPTG. An aliquot was harvested after 4 hours’ induction and resuspended in lysis buffer. His-tagged EsaD nuclease domain (wild type or H528A variant) was purified in the presence of 8M urea, refolded and eluted as described in Methods. 10µl of each sample were separated (12% bis-Tris gel) and stained using coomassie instant blue. f. EsaD is a Mg²⁺-dependent DNase. Plasmid DNA was incubated with purified EsaD_421-614-His, EsaD_421-614(H528A)-His or buffer alone with either 50mM MgCl₂ or ZnCl₂, after which the DNA was analysed by agarose gel electrophoresis.

Fig 3. EsaE is co-secreted with EsaD and together with EsaG they form a ternary complex.

a – c. EsaE interacts with EsaD. Interactions between a. pT25-EsaD(H528A) and the indicated fusions to pT18 or c. pT25-EsaE and EsaD_1-420 or EsaD_421-614(H528A) fused to pT18 as well as pT18-EsaE and full length EsaD(H528A) fused to pT25 assessed by β-galactosidase activity assay in BTH101. BTH101 harbouring pT25 and pT18 was the negative control. Error bars are ± SD (n=3 biological replicates). Student’s t-test gives p values < 0.00001 for EsaD(H528A)/EsaE, EsaD(H528A)/EsaG, EsaD/EsaE, and EsaD_1-420/EsaE relative to the negative control. Inset shows the same strain and plasmid combinations on MacConkey maltose plates. b. Top two panels - S. aureus RN6390 carrying pRAB11 (empty), pRAB11-EsaE-HA or pRAB11-EsaD(H528A)-His-EsaE-HA was cultured to OD₆₀₀ of 0.5 supplemented with 500ng/ml ATC and harvested at OD₆₀₀ of 3. Cells were lysed and histidine-tagged EsaD(H528A) was purified. Cell lysate (load) and eluted fractions (20μl of each) were analysed by western blot with anti-His and anti-HA antisera. Coomassie-stained samples of these fractions are shown in Fig S4. Bottom two panels show repeats of EsaD(H528A)-His-EsaE-HA co-purification. Samples of load (10µl), flow through (20µl), final wash (30µl) and elution fraction (30µl) were analysed using the same antisera. d and e. EsaE is co-secreted with EsaD. S. aureus RN6390 harbouring pRAB11 (empty) and either d. pRAB11-EsaE-His or e. pRAB11-EsaD-His-EsaE-HA was cultured to OD₆₀₀ of 0.5 supplemented with 250ng/ml ATC and harvested at OD₆₀₀ of 3. Samples of supernatant and cells (equivalent to 250μl supernatant and 10μl cells adjusted to OD₆₀₀ of 1) were separated on 12% bis-Tris gels and immunoblotted with the indicated antisera. f. EsaE, EsaD and EsaG form a ternary complex. E. coli M15[pRep4] carrying pQE70 (empty) or pQE70- EsaE-HA-EsaD(H528A)-Myc-EsaG-His was cultured to OD₆₀₀ of 0.5, supplemented with 2 mM IPTG for 4 hours, harvested and lysed. HA-tagged EsaE was purified and 10μl, 25μl and 35μl of the elution fractions were analysed by western blot with anti-HA, anti-His and anti-myc antibodies, respectively. Coomassie-stained samples of these fractions are shown in Fig S10. g. Samples of load (10µl), flow through (20µl), final wash (30µl) and elution fraction (30µl) from the EsaE-HA-EsaD(H528A)-Myc-EsaG-His co-purification experiment shown in f. were analysed by western blotting with the same antisera.

Fig 4. EsaE is a membrane-associated protein that interacts with multimeric EssC.

a. A proportion of EsaE is bound to the membrane. The S. aureus wild type strain, RN6390, harbouring pRAB11 (empty) or pRAB11-EsaE-His was cultured in TSB medium to OD₆₀₀ of 0.5, supplemented with ATC (250ng/ml) and harvested at OD₆₀₀ of 2. Cells were fractionated into cell wall (cw), cytoplasmic (cyt) and membrane (m) fractions. An aliquot of the membrane fraction was washed with 0.2 M Na₂CO₃ (m+). Samples of each fraction (20μl aliquot of cw and cyt; 2mg of membrane) were separated on 12% bis-Tris gels and immunoblotted using either anti-His or anti-sortase A (SrtA) antisera. b and c. EsaE crosslinks to a multimeric form of EssC. b. Whole cells of the S. aureus wild type (RN6390), or c. the wild type and the isogenic essC deletion strain, as indicated, harbouring pRAB11 (empty) or pRAB11-EsaE-His were cultured in TSB medium to OD₆₀₀ of 0.5 supplemented with ATC (250ng/ml) and at OD₆₀₀ of 2, cells were incubated with paraformaldehyde (PFA) as described under Methods. Following quenching, cells were lysed and membrane fractions prepared, and membrane protein (1mg for samples from the wild type strain, 10mg for samples from the essC strain) loaded on b. a bis-Tris gel containing 12% acrylamide or c. SDS-gels containing 5 % acrylamide (bottom panel in part C showing EsaE-His monomer is 12% bis-Tris gel) and analysed by western blot with the indicated antisera. d. Model for EsaD synthesis and secretion. Following synthesis of EsaD (shown in green), 1 EsaE binds to the N terminal region of the protein and 2 EsaG binds to the nuclease domain to prevent activity against the DNA of the producing cell. 3 the ternary complex is targeted to the secretion machinery facilitated by the interaction of EsaE with multimeric EssC. 4 EsaG is released from EsaD during the transport step and 5 the EsaD-EsaE complex is secreted out of the cell via the T7SS.

Fig 5. Secreted EsaD kills sensitive strains of S. aureus.

a. EsaG homologues are encoded in S. aureus strains that lack esaD. DUF600-family proteins encoded at the ess loci in S. aureus strains NCTC8325 (parental strain of RN6390), MRSA252, ST398 and EMRSA15. Genes encoding DUF600 proteins are shaded in purple, essC is shaded green and esaD grey. The two genes shaded in brown are highly conserved across all strains and define the 3’ boundary of the ess locus. b. and c. Interactions between pT25-EsaD(H528A) and DUF600 proteins; b. from strains ST398, MRSA252 and EMRSA15, and c. from strain NCTC8325. In each case the DUF600 reading frame was fused to pT18 and interaction with full length EsaD fused to pT25 assessed by β-galactosidase activity assay in E. coli BTH101. BTH101 harbouring pT25 and pT18 was the negative control. Error bars are ± SD (n=3 biological replicates). Student’s t-test gives p values < 0.00001 relative to the negative control. Insets shows the same strain and plasmid combinations on MacConkey maltose plates. d. In vitro growth competition assays between the indicated attacker and prey strains in liquid medium. In each case the attacker strain (COL or COLΔess) overproduced EsaD-HA or EsaD(H528A)-HA along with EsaG-His as described in Methods, and was incubated with either RN6390, RN6390Δ00268-00278 or RN6390 pEsaG-His as prey, as indicated. To the right, the three prey strains incubated with COL pEsaD-HA-EsaG-His as attacker are replotted next to each other to allow a more direct comparison. In all experiments five biological replicates of each attacking strain was used against a single culture of prey. Bars represent the average value of c.f.u. of prey bacteria at the end of the experiment. Asterisks indicate significant differences in c.f.u. * p value < 0.05; ** p value < 0.005, *** p value < 0.0005. Comparison of RN6390 with RN6390Δ00268-00278 survival when COL pEsaD-HA-EsaG-His was used as attacker was not significant (p = 0.069). Error bars are ± SD (n = 5 biological replicates).