Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi
Introduction
Outer membrane protein OmpF is a member of non-specific general diffusion porin family of Gram-negative bacteria, such as Salmonella and Escherichia coli (Nakae, 1976, Nikaido, 1994) whose main function is to facilitate the transport of hydrophilic solutes with molecular mass up to 600 Da across the outer membrane (Jap and Walian, 1990, Nikaido, 1993). Salmonella typhi is an obligatory human pathogen that causes typhoid which continues to be a major health problem in developing countries (Crump et al., 2004). Among the newer generation vaccines against typhoid, S. typhi Ty21a vaccine and Vi polysaccharide have proven to be safe (DeRoeck et al., 2007, Ochiai et al., 2007). The S. typhi Ty21a vaccine is orally administered live attenuated vaccine licensed for use in persons 2 years of age or older but requires 3–4 immunisations to induce long-term (at least 6–7 years) protective immunity in two thirds of the immunised individuals (Levine et al., 1999). Interestingly, it has been found that highly immunogenic live oral Salmonella vaccine would ideally be suited as a carrier of genes that express protective antigens cloned from other antigens (Aggarwal et al., 1990, Formal et al., 1981, Wu et al., 1989) and such hybrid recombinant Salmonella vaccines are expected to invoke protective immunity against both the carrier strains as well as the foreign antigens (Fraillery et al., 2007, Hone et al., 1992). In this context, outer membrane proteins (OMPs) of Salmonella have been shown to elicit a protective immunity (Isibasi et al., 1988, Udhayakumar and Muthukkaruppan, 1987). It has also been shown that the Salmonella antiOmpF and antiOmpC antibodies reached maximum bactericidal titres during the secondary response, antiOmpF antibodies being less immunogenic than antiOmpC antibodies (Secundino et al., 2006). S. typhi porins (OmpC) has been shown to display heterologous epitopes on the cell surface (Puente et al., 1995) which can be exploited as vaccine candidate carrying antigens of other disease causing organisms in their loops, making it possible for a double protective therapy.
In addition to their immunological properties as potent surface antigens, porins also act as entry port for various antibiotics (Nikaido, 2003). The bacteria uses either one of the following mechanism to develop antibiotic resistance using porins: loss/reduction of porins, by expression of other porins not involved in antibiotic translocation and by expression of porins with mutations in the key residues involved in the uptake of antibiotics (Delcour, 2009, Pages et al., 2008). Much of the biophysical and mechanistic studies in determining the pathway of antibiotic translocation through porins have focussed on E. coli OmpF, as its structural and functional properties are well understood (Cowan et al., 1992, Danelon et al., 2006). The influx of antibiotics through porins is not just a passive diffusion but involves interactions with key residues in the porin channel and it has been shown in E. coli OmpF that any mutations in these key residues alter the pore properties in terms of diffusion of antibiotics (Bredin et al., 2002, Hajjar et al., 2010b). Hence the crystal structure of porins from different bacterial sources are pre-requisite to understand the specific atomic details, electrostatic pore potential and favourable channel properties involved in antibiotic translocation. Also, structure of porins from pathogenic species like Salmonella will help in designing specific vaccines and improved antibiotics therapy. However, structure determination of membrane proteins is still a bottleneck due to difficulties in producing large amounts of protein and crystallisation. Refolding of porins from inclusion bodies (IBs) and their structure determination was successful in the case of Rhodopseudomonas blastica porin (Schmid et al., 1996) and OpCA from Neisseria meningitidis (Prince et al., 2001) where the refolded proteins showed structural similarity to their native structures. OmpF from E. coli had been overexpressed and refolded in the presence of detergents (Miedema et al., 2004, Visudtiphole et al., 2005). However, this is the first report of crystallisation and structure determination of in vitro refolded OmpF from a human pathogen.
Here we report the crystal structure of OmpF from S. typhi Ty21a at 2.8 Å resolution (PDB: 3NSG).
Section snippets
Overexpression, purification and crystallisation
Genomic DNA was isolated from the vaccine strain of S. typhi Ty21a (Germanier and Furer, 1975). Primers for ompF gene were designed for the mature S. typhi OmpF (SwissProt Accession: Q56113). PCR amplified product was cloned into NdeI and BamHI sites of pET20b (Novagen). E. coli GJ1158 (Bhandari and Gowrishankar, 1997), a salt-inducible overexpression host, was transformed with ompF/pET20b. Protein was expressed into cytoplasmic inclusion bodies (IBs). Purification, solubilisation and refolding
Structure of S. typhi OmpF
The structure of S. typhi Ty21a rfOmpF obeys the construction principle of other general diffusion porins and each monomer barrel has a 16-stranded anti-parallel β-sheet defining an aqueous channel that spans the outer membrane (Fig. 1). S. typhi and E. coli OmpF share higher percentage of similarity at sequence (57.6% identity) (Fig. 2) and structure with the RMSD of 1 Å (Cα). There are eight short beta hairpin turns (T1–T8) at the periplasmic end of the barrel and eight long loops (L1–L8) at
Conclusions
The structure of S. typhi OmpF reported here is from overexpressed inclusion bodies, refolded in vitro in the presence of a zwitterionic detergent. The significantly different packing observed here suggests the possibility of understanding complexes between OmpF and other biologically relevant molecules that can interact at the porin surface. The variations in the exposed surface loops can be used to design OmpF as a potential multivalent vaccine carrier. The asymmetry seen in the pore and the
Acknowledgments
We acknowledge staff of BM14, ESRF, Grenoble, France, DBT, India and Dr. M. Yogavel, and Dr. G. Jasmita for the data collection. Funding and facilities provided by DBT Programme Support, DBT Nanotechnology project, DBT CoE in Bioinformatics and UGC-SAP at School of Biotechnology, MKU are acknowledged. DB and PDK were recipient of fellowship from UGC and CSIR.
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