Characterization of EssB, a protein required for secretion of ESAT-6 like proteins in Staphylococcus aureus
© Chen et al.; licensee BioMed Central Ltd. 2012
Received: 24 July 2012
Accepted: 21 September 2012
Published: 25 September 2012
Staphylococcus aureus secretes EsxA and EsxB, two small polypeptides of the WXG100 family of proteins. Genetic analyses have shown that production and secretion of EsxA and EsxB require an intact ESAT-6 Secretion System (ESS), a cluster of genes that is conserved in many Firmicutes and encompasses esxA and esxB . Here, we characterize EssB, one of the proteins encoded by the ESS cluster. EssB is highly conserved in Gram-positive bacteria and belongs to the Cluster of Orthologous Groups of protein COG4499 with no known function.
By generating an internal deletion in essB , we demonstrate that EssB is required for secretion of EsxA. We use a polyclonal antibody to identify EssB and show that the protein fractionates with the plasma membrane of S. aureus . Yet, when produced in Escherichia coli, EssB remains mostly soluble and the purified protein assembles into a highly organized oligomer that can be visualized by electron microscopy. Production of truncated EssB variants in wild-type S. aureus confers a dominant negative phenotype on EsxA secretion.
The data presented here support the notion that EssB may oligomerize and interact with other membrane components to form the WXG100-specific translocon in S. aureus .
KeywordsESAT-6 secretion ESS WXG100 EssB Type 7 secretion Staphylococcus aureus
EssB is required for the secretion of EsxA by S. aureusUSA300
Subcellular localization of EssB
We wondered whether EssB is itself secreted or localizes to a particular subcellular compartment (cytosol/membrane). A culture of S. aureus USA300 was centrifuged to separate cells from the extracellular milieu. As expected Hla, but not EssB, was found in the extracellular medium (Figure 2C; lane M). Further fractionation was achieved by subjecting lysed cellular extracts to sedimentation at 100,000 × g . As a control for subcellular fractionation, samples were examined by immunoblot for the ribosomal protein L6 (S, soluble) and membrane protein SrtA (I, insoluble). EssB was identified in the membrane sediment along with SrtA (Figure 2C), suggesting that EssB may either be inserted into the lipid bilayer or associated with one or more proteins in the membrane. This finding is in good agreement with a recent report suggesting that YukC the B. subtilis homologue of EssB (Figure 1) belongs to the membrane proteome of B. subtilis .
The TMHMM algorithm (http://www.cbs.dtu.dk/services/TMHMM-2.0) was used to perform sequence-based prediction of EssB, which identified a string of hydrophobic residues amino acids 229–251 (W229VAIGMTTLSVLLIAFLAFLYFS251) at the center of the EssB polypeptide. Hereafter we refer to the segment of hydrophobic amino acids within EssB as the Putative Trans Membrane Domain (PTMD).
Deleting essBaffects the production of several ESS factors
Recombinant EssB is soluble and prone to multimerization
Visualization of purified EssB protein by transmission electron microscopy suggested that the sample is homogenous. Small dense structures could be seen throughout the field and at larger magnification they revealed a clear rod-shaped organization of the molecule (Figure 4D). A similar analysis was performed for affinity purified EssBΔM. Transmission electron micrography revealed that overall the protein preparation was homogeneous (not shown), however the rod-shaped structure of EssB is lost in this variant (Figure 4E). Together, these results suggest that the PTMD segment is required for the multimerization of EssB and that the rod-shaped structure may be an energetically favorable conformation in the cytoplasm of E. coli . Interestingly, the structure for a so-called “cytoplasmic component of EssB” has been deposited in the databank and made publicly available (http://www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?Dopt=s&uid=99898, http://www.rcsb.org/pdb/explore/explore.do?pdbId=4ANN). This component encompasses the first 215 amino acids of EssB and behaves as a soluble monomer quite like EssBN examined in this study.
Truncated EssB variants display a dominant negative phenotype in S. aureus
When transformed into wild-type S. aureus USA300, plasmid produced EssB and variants fractioned as before following 100,000 × g ultracentrifugation (Figure 5C). Briefly, EssB, EssBNM and EssBMC were found in the sediment, EssBΔM remained soluble and EssBC fractionated equally in the soluble and insoluble compartments (Figure 5C). Expression of EssBNM led to some degradation of EssB (Figure 5C, black asterisk). As before, very little EssBN immunoreactive material could be detected in S. aureus USA300 cells (Figure 5C, white arrow). Interestingly, its production caused a reduction of wild-type EssB (Figure 5C, blue arrow). EssB was also unstable in the merodiploid strain expressing EssBMC (Figure 5C; purple arrow). Not surprisingly, destabilization of EssB by either EssBN or EssBMC led to altered expression and secretion of EsxA (Figure 5D). Sedimentable variants encompassing the PTMD, EssBNM and EssBMC, caused a dominant-negative phenotype on the activity of wild-type EssB and as a result expression or secretion of EsxA were altered. On the contrary, EssBΔM lacking PTMD remained soluble and did not interfere with EssB function. Taken together, these data suggest that EssB variants that sediment with staphylococcal membranes interfere with the stability or function of endogenous EssB and as a consequence EsxA production and secretion are also affected. Thus, EssB is part of the secretion machine and its multimerization and possible association with other Ess components enables the secretion of EsxA.
Secreted proteins are generally tagged with topogenic sequences for recognition by a specific secretion machine and transport across the plasma membrane. Over a third of all proteins synthesized by a bacterial cell carry leader peptides, the topogenic signal for recognition by the Sec machine . The corresponding sec genes are scattered on the chromosome although their gene products assemble specifically at the membrane to mediate the faithful secretion of a variety of polypeptides. Bacteria have also evolved highly specialized secretion systems for the transport of specific proteins across lipid bilayers and organized the genes encoding machine components and their substrates into clusters whose expression is controlled by adjacent transcriptional units [25, 26]. The S. aureus ESS cluster represents one such dedicated secretion pathway. ESS genes are encoded within an eleven gene cluster and when deleted impair the production or secretion of small proteins with the WXG amino acid signature. Here, we have begun the characterization of EssB, one of the proteins of the staphylococcal ESS cluster (Figure 1).
Bioinformatic searches revealed that EssB is found in Gram-positive bacteria that harbor ESS gene clusters closely related to the staphylococcal ESS pathway (Figure 1). The protein belongs to the Cluster of Orthologous Groups of protein COG4499 and is annotated as a predicted membrane protein homologous to B. subtilis YukC (Figure 1). COG4499 protein members are all arranged in a single architecture meaning that the entire protein defines a single domain that is never truncated nor fused with another protein domain. By performing a Position-Specific Iterative BLAST (PSI-BLAST) in NCBI (under default conditions within two iterations), an obvious homology can be identified between the EssB/YukC family of proteins and the TraF proteins from Gram-positive conjugative plasmids . This is interesting (yet perplexing) because it has been proposed that the specialized secretory apparatus ESX-1 of M. smegmatis that lacks an EssB/YukC/TraF homologue carries out DNA transfer .
By raising a polyclonal antibody against EssB, we find that the protein sediments with S. aureus membranes in a manner similar to SrtA, a well-characterized membrane embedded protein . Residues 229–251 roughly define a hydrophobic sequence reminiscent of a transmembrane spanning segment (PTMD). Interestingly, recombinant EssB behaves as a soluble oligomer in E. coli with a rod-shaped like structure and the PTMD sequence appears to be necessary and sufficient for this oligomerization process. Obviously, this conformation may simply represent an energetically favorable state for an otherwise membrane-spanning. Nonetheless, recombinant EssBNM and EssBMC are more prone to multimerization than intact EssB suggesting that the full-length sequence limits or regulates the oligomerization of the protein. Protein translocators of other secretion systems such as the Tat or holin pathways undergo regulated multimerization to facilitate pore function in the membrane [30, 31]. In S.aureus , the presence of the PTMD targets EssBNM and EssBMC to the membrane. This targeting appears to affect the function of endogenous EssB in wild-type staphylococci. On the contrary, EssBΔM (lacking PTMD) is soluble. It is unable to complement the essB mutant and it displays no dominance over wild-type for EsxA secretion. As such, none of the truncated EssB variant could complement wild-type EssB for secretion. Further studies are needed to determine whether the PTMD sequence serves as an autonomous membrane-spanning domain or whether it provides a mean to associate with another integral membrane protein encoded within the ESS cluster.
Deletion of essB in strain USA300 leads to loss of EsxA secretion and EsxA remains in the cell. Because overproduction of EssB is not toxic in E. coli , we do not believe that this protein alone is capable of forming a pore for the passage of secreted substrates. Interestingly, two other proteins EsaB and EsaD also accumulate in the essB mutant. While the exact role of EsaB is still unknown, it does not appear to be a secreted substrate , and thus the reason for this increase is unclear but it points to additional biochemical interactions within proteins of the ESS cluster. Recent evidence suggests that EsaD is a membrane protein also required for EsxA secretion . Perhaps EssB interacts physically with EsaD to either complete or regulate formation of the translocon. Future studies are needed to address this possibility and determine whether EssB is an integral or peripheral element of the ESS translocon.
The ESS pathway is an alternate and conserved secretion system of several Gram-positive bacteria. Here, we show that EssB is found in the membrane of S. aureus and deletion of the corresponding gene abrogates secretion of EsxA. We show that a hydrophobic segment in the middle of the protein referred as PTMD is required for targeting to the plasma membrane. We observe that recombinant EssB harboring PTMD folds into an oligomeric rod-shaped structure that allows the protein to remain soluble in E. coli. Interestingly, truncated EssB variants harboring an intact PTMD display a dominant negative phenotype over wild type EssB for secretion of EsxA. The data indicate that EssB is an essential component of the ESS translocon and likely interacts with itself and other machine components. Together, this study provides the first genetic and biochemical characterization of the ESS translocon in S. aureus .
S. aureus and Escherichia coli cultures were grown at 37° in tryptic soy (TS) with 0.2% serum or Luria Bertani (LB) broth or agar, respectively. Chloramphenicol and ampicillin were used at 10 and 100 μg/l for plasmid selection, respectively.
Bacterial strains and plasmids
Oligonucleotides used in this study
Cloning of the essB deletion mutant in pKOR1 for allelic replacement
Same as above
Same as above
Same as above
Gene expression in E. coli or S. aureus using pET15b or pWWW412, respectively
Same as above
Same as above
Same as above
essB(1–252)- BamH I-R
Same as above
Same as above
essB (Δ224-252)- EcoR I-F
Same as above
essB (Δ224-252)- EcoR I-R
Same as above
Production of GST hybrids using pGEX-2TK
Same as above
Same as above
Same as above
Strains and plasmids used in this study
S. aureus sau1 hsdR laboratory strain used for passaging plasmid DNA
Community-acquired methicillin resistant S. aureus
NARSA repository 
USA300 carrying an internal deletion of essB
E. coli K12 fhuA2 Δ (argF-lacZ)U169 phoA glnV44 Φ80 Δ (lacZ)M15 gyrA96 recA1 relA1 endA1 thi-1 hsdR17 for cloning
E. coli B F- dcm ompT hsdS (rB-mB-) gal for recombinant protein production
pOS1 derivative carrying the constitutive hprK promoter, CmR
temperature sensitive plasmid for allelic replacement, CmR
Vector carrying an N-terminal hexa-histidine repeat followed by a thrombin cleavage site and T7 transcription start; AmpR
Vector carrying GST followed by thrombin cleavage and phosphorylation sites, and a taq promoter; AmpR
pWWW412 expressing wild-type essB codons 1-444
p essB N
pWWW412 expressing codons 1–223 of essB
p essB NM
pWWW412 expressing codons 1–252 of essB
p essB C
pWWW412 expressing codons 253–444 of essB
p essB CM
pWWW412 expressing codons 220–444 of essB
p essB ΔM
pWWW412 expressing essB lacking codons 224-252
p his - essB
pET15b expressing histidine tagged essB codons 1-444
p his - essB NM
pET15b expressing histidine tagged essB codons 1- 252
p his - essB CM
pET15b expressing histidine tagged essB codons 220- 444
p his - essB ΔM
pET15b expressing histidine tagged essB lacking codons 224-252
p gst - essB N
pGEX-2TK expressing GST fused to codons 1–223 of essB
p gst - essB C
pGEX-2TK expressing GST fused to codons 253–444 of essB
Culture and bacterial fractionation, and western blot experiments
Bacterial strains were grown overnight from isolated colonies in TS broth supplemented with 0.2% serum at 37°C with shaking. Cultures were diluted 1:100 in fresh broth and allowed to shake at 37°C until they reached an absorbance of 1 at 600 nm (A600nm) corresponding to exponentially growing bacteria. For whole culture lysates (samples labeled T, for total culture extracts as shown in Figures 2A and 3), cultures (6 ml) were incubated in the presence of lysostaphin (100 μg/ml) for 30 min at 37°C. To separate proteins in the culture medium (M) from those in the bacterial cell (C), cultures (6 ml) were centrifuged (10,000 × g for 10 min) and the supernatant was transferred to a new tube prior to lysostaphin treatment of intact cells. For subcellular localization of EssB (Figures 1A and 5 top panel), cultures were centrifuged to separate medium and cells. Staphylococci were washed, and peptidoglycan digested with lysostaphin. Staphylococcal extracts were subjected to ultracentrifugation at 100,000 × g for 40 min at 4°C. The supernatant, containing soluble proteins (S), was transferred to a new tube. The sediment containing insoluble membrane proteins (I), was suspended in 6 ml PBS buffer. Proteins in all samples were precipitated with 10% trichloroacetic acid on ice for 30 min. Precipitates were sedimented by centrifugation at 15,000 × g , washed, dried and solubilized in 100 μl of 0.5 M Tris–HCl (pH 8.0)/4% SDS and heated at 90°C for 10 min. Proteins were separated on SDS/PAGE and transferred to poly(vinylidene difluoride) membrane for immunoblot analysis with appropriate polyclonal antibodies. Immunoreactive signals were revealed by using a secondary antibody coupled to IRDye© 680. Quantification of western blots was conducted using a Li-Cor Biosciences Odyssey imager. Briefly, cells were grown to the same optical density. All strains reached similar density in the same time period suggesting that either deletion or cis -expression of genes did not affect growth of bacteria. Signal intensity of immune reactive signals for EsxA, EssB, EsaB and EsaD was compared to that obtained for WT, WT/vector, essB /p essB or WT/p essB sample extracts for Figures 2, 3, 5 A, B, C and D, respectively. Immune reactive signals (as shown in Figure 3) were averaged in three independent experiments and the data was analyzed in pairwise comparisons between WT/vector and variant strains with the unpaired two-tailed Student’s t -test and found to be statistically significant.
Protein and polyclonal antibody purification
Briefly, recombinant EssB, EssBNM, EssBMC, EssBΔM, tagged with N-terminal hexa-histidine were purified using Ni-NTA Agarose (Qiagen) following manufacturer’s recommendations. Recombinant EssBN and EssBC fused to glutathione S-transferase were purified by using sepharose-immobilized glutathione (Glutathione SepharoseTM 4B, GE Healthcare) following manufacturer’s recommendations. Bound proteins were incubated in 50 mM Tris–HCl buffer (pH 7.5) containing 300 mM NaCl (buffer A) with thrombin (10 U/mg, GE Healthcare) at 4°C for 12 h to cleave the hexa-histidine and gluthathione S-transferase moieties, respectively. Released proteins were dialyzed in buffer B (50 mM Tris–HCl [pH 8.0] containing 150 mM NaCl and 1 mM DTT) and stored at 4°C for use within the next 48 hours. A 100-μl volume of each recombinant protein (~100 μg) was loaded onto a SuperdexTM 75 10/300 GL (GE Healthcare) in buffer B at 4°C. The chromatography was performed at a flow rate of 0.5 ml/min, and fractions of 0.5 ml were collected and analyzed by SDS-PAGE. The gel filtration column was calibrated by running a set of protein standards (Aldolase, 158 kDa; Conalbumin, 75 kDa; Ovalbumin, 43 kDa and Myoglobin, 17 kDa). Rabbit polyclonal antibodies raised against full-length EssB were purified prior to use in immunoblot experiments as described earlier .
Transmission electron microscopy (TEM) and image processing
Purified recombinant proteins EssB and EssBΔM were prepared as described above, dialyzed in Buffer B (without DTT) and diluted to approximately 10 to 50 μg/ml. Proteins were bound to glow discharged, carbon coated (Edwards Auto 306 Evaporator) copper grids (400 mesh), washed, and subsequently negatively stained using 2% uranyl acetate (Electron Microscopy Services). Images were recorded using a Tecnai F30 (Philips/FEI) transmission electron microscope (Field emission gun, 300-kV accelerating voltage, with a magnification of 49,000 to 75,000×) and a high performance CCD camera with a 4k × 4k resolution. Images were captured using Gatan DigitalMicrograph software and processed using Adobe Photoshop (Adobe, San Jose, CA, USA). Images of single protein were selected manually.
ESAT-6 secretion system
Type 7 secretion system
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Transmission electron microscopy microscopy
Insoluble membrane proteins.
Acknowledgements and funding
The authors thank Olaf Schneewind for careful reading of the manuscript, Khaled Aly and members of the Schneewind and Missiakas laboratory for suggestions and discussions. The authors are grateful for comments provided by the referees and help with BLAST analyses. Mark Anderson acknowledges support by the Biodefense Training Grant in Host-Pathogen Interactions T32 AI065382 at the University of Chicago and American Heart Association award 11PRE7600117. This work was supported by the National Institute of Allergy and Infectious Diseases, Infectious Diseases Branch (award AI 75258) to DM.
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