Structure ReportCrystal structure of the pilotin from the enterohemorrhagic Escherichia coli type II secretion system
Introduction
Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important pathogenic E. coli strain that spreads via contaminated food sources. The bacteria colonize intestinal epithelial cells and cause hemorrhagic colitis, and the illness caused is sometimes called Hamburger disease (Sherman et al., 2010). EHEC is especially dangerous since it may lead to a potentially lethal disease, hemolytic uremic syndrome (Melton-Celsa et al., 2012), in particular in young children. EHEC infection and damage to the host depends on a large number of virulence factors (Farfan and Torres, 2012). A major role is played by the large pO157 plasmid which encodes the type II secretion system (T2SS), a metalloprotease StcE (secreted protease of C1-esterase inhibitor), hemolysin, a subtilisin-like serine protease and other virulence factors (Burland et al., 1998). The metalloprotease StcE, which is secreted by the T2SS, is important for early steps in colonization of epithelial cells by EHEC (Grys et al., 2005, Lathem et al., 2002, Paton and Paton, 2002, Yu et al., 2012). Another known substrate of the T2SS from EHEC is a metal binding protein YodA that is also involved in colonization process via an as yet unknown mechanism (Ho et al., 2008). Furthermore, EHEC deletion mutants of the T2SS are defective in colonization in vivo, which underscores the importance of the T2SS role in the infection process (Ho et al., 2008).
The T2SS is a sophisticated multi-protein machinery that transports folded proteins from the periplasm across the outer membrane of Gram-negative bacteria into the extracellular milieu (Douzi et al., 2012, Korotkov et al., 2012, McLaughlin et al., 2012). The T2SS spans two membranes and consists of multiple copies of at least 12 different proteins. In the cytoplasm, the secretion ATPase GspE interacts with the inner membrane platform consisting of GspL, GspM, GspF, and GspC. This platform interacts with GspG, which is the most abundant subunit of a helical subassembly called the pseudopilus. The outer membrane channel is formed by the secretin GspD. Secretins are also channels for secreted proteins, fimbriae or phages in a number of other systems, including the type III secretion system (T3SS), the type IV pilin system (T4PS) and the filamentous phage assembly system (Korotkov et al., 2011).
The biogenesis of secretins in the outer membrane requires in several cases lipoprotein chaperones called pilotins (Koo et al., 2012). The related T2SS secretins PulD from Klebsiella oxytoca (KoGspDPulD) and OutD from Dickeya dadantii, formerly Erwinia chrysanthemi, (DdGspDOutD) rely, respectively, on their cognate pilotins PulS (KoGspSPulS) and OutS (DdGspSOutS) for outer membrane targeting (Hardie et al., 1996, Shevchik et al., 1997). These pilotins have an outer membrane lipoprotein-sorting signal that directs them to the outer membrane via interactions with proteins of the Lol sorting pathway (Collin et al., 2011). In addition to pilotins, some secretins require additional accessory proteins for stability (Ast et al., 2002, Gauthier et al., 2003, Schuch and Maurelli, 2001, Strozen et al., 2011).
The pilotins KoGspSPulS and DdGspSOutS have been shown to interact with the C-terminal 60 residues of their secretins, the so-called S-domain, and protect secretin monomers from proteolysis (Daefler et al., 1997, Shevchik et al., 1997). However, the absence of pilotin or deletion of S-domain does not prevent the multimerization of KoGspDPulD and DdGspDOutD, but then these multimers assemble in the inner membrane of a bacterium causing phage shock response (Guilvout et al., 2011, Guilvout et al., 2006, Shevchik and Condemine, 1998). Interestingly, KoGspDPulD can also spontaneously form multimers in liposomes in vitro (Guilvout et al., 2008).
In EHEC, the T2SS cluster on the pO157 plasmid contains the etpO gene that encodes a protein with approximately 40% amino acid sequence identity to some, but not all, other T2SS pilotins. Here we report the crystal structure of this pilotin, which we call EHEC GspS (Korotkov et al., 2012). Based on extensive extra density in a hydrophobic groove of EHEC GspS, we suggest a possible binding site of EHEC GspS for the S-domain of the EHEC secretin GspD.
Section snippets
Protein expression, purification and crystallization
The gene fragment corresponding to the residues 16–106 of EHEC GspS (EtpO) was cloned into a modified pET-28b vector (Novagen) for expression as a fusion with maltose-binding protein (MBP). The construct has an N-terminal hexahistidine tag followed by MBP, a tobacco etch virus (TEV) protease cleavage site and GspS. The protein was expressed in BL21 (DE3) cells (Novagen) in LB media for 4 h at 30 °C. The harvested cells were resuspended in buffer containing 20 mM HEPES pH 7.5, 300 mM NaCl and lysed
Data collection, structure determination and analysis
A native dataset was collected at beamline 8.2.1 of the Berkeley Center for Structural Biology, the Advanced Light Source, at 100 K using a wavelength of 0.9794 Å. The native crystal was exposed at a low dose in an (unsuccessful) attempt to utilize anomalous signal from sulfur for phasing because Se-Met crystals were not available at that time. A dataset from SeMet-labeled crystal was collected at beamline 9–2 of the Stanford Synchrotron Radiation Lightsource at 100 K using a wavelength 0.97915 Å.
Overall structure of GspS and putative secretin binding site
In order to obtain protein suitable for crystallization, we constructed a soluble variant of GspS from enterohemorrhagic E. coli O157:H7 that encoded residues 13–110 of mature protein and hence lacked the signal sequence and the N-terminal lipidation residue. Because this variant failed to crystallize, the construct was optimized to include only residues 16–106 based on secondary structure prediction analysis (Cole et al., 2008). The resultant crystals yielded the structure of the EHEC pilotin
Structural and functional comparisons
While this manuscript was in preparation, structures of related pilotins have been reported: K. oxytoca KoGspSPulS and D. dadantii DdGspSOutS (Gu et al., 2012, Tosi et al., 2011), which allows us to analyze all available structures. The EHEC GspS structure could be superimposed onto KoGspSPulS with an r.m.s.d. of 0.96 Å and 43% sequence identity over 90 amino acid residues, and onto DdGspSOutS with an r.m.s.d. of 1.08 Å and 39% sequence identity over 89 residues (Fig. 1C). The disulfide bridge
Conclusions
Since EHEC can cause severe foodborne disease, and can even be life threatening, the spread of antibiotic resistant variants of pathogenic bacteria is a source of growing concern. At the same time, the importance of commensal human microorganisms raises questions about safety of broad-spectrum anti-microbial drugs. Therefore, much attention has recently been given to the concept of devising alternative therapies based on targeting virulence factors (Cegelski et al., 2008). Increasing our
Accession numbers
The structure factors and coordinates have been deposited in the Protein Data Bank under accession number 3SOL.
Acknowledgments
We thank Stewart Turley for assistance during data collection and Steve Moseley for providing E. coli O157:H7 DNA. We thank the staff of the Berkeley Center for Structural Biology and the Stanford Synchrotron Radiation Light source for support during data collection. The Berkeley Center for Structural Biology is supported in part by the National Institutes of Health, National Institute of General Medical Sciences, and the Howard Hughes Medical Institute. The Advanced Light Source is supported
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Present address: Department of Molecular & Cellular Biochemistry, University of Kentucky, Lexington, KY 40536, United States.