Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Biophysical characterization of the catalytic domain of guanine nucleotide exchange factor BopE from Burkholderia pseudomallei
Introduction
Burkholderia pseudomallei is the aetiological agent of melioidosis, a severe emerging infection of humans and animals that is endemic in southeast Asia and tropical Australia and that has the potential to spread worldwide [1]. B. pseudomallei infections can mimic other disorders and melioidosis is difficult to diagnose without relatively sophisticated laboratory facilities, so the true incidence of the disease is currently uncertain. Melioidosis has a range of clinical manifestations, including rapidly fatal septicaemia, pneumonia, skin and soft tissue abscesses, and osteomyelitis or septic arthritis. Infection is usually via contaminated soil, dust or water [2], [3], [4].
Asymptomatic infection is common in areas where the infection is endemic and progression to disease depends on the condition of the host [3]. Between the fatal and asymptomatic extremes, the infection may be chronic or run a relapsing course. B. pseudomallei is closely related to Burkholderia mallei, the pathogen that causes glanders, a disease of horses and other solipeds. B. mallei can also affect humans and is often fatal if left untreated [5]. Due to the severity of the infection, aerosol infectivity and worldwide availability, both B. pseudomallei and B. mallei are considered to be potential bioweapons [6].
The molecular mechanisms of B. pseudomallei pathogenesis are not well-characterized, although B. pseudomallei contains at least three loci encoding putative type III secretion systems (TTSS) [7]. One of these is homologous to the inv/spa/prg TTSS of Salmonella typhimurium [7], [8], [9]. TTSSs are central to the virulence of many Gram-negative pathogens, including Salmonella, Shigella, Yersinia, enteropathogenic Escherichia coli and the four major genera of plant pathogenic bacteria [10]. TTSS resemble molecular syringes for the injection of multiple bacterial effector proteins into the host cell cytoplasm that modify host cell physiology to the benefit of the pathogen. The dimensions of the type III needle [11], [12] and available experimental evidence [13], [14] indicate that, in order to be secreted, proteins must be unfolded or only partially folded.
Among the potential effector proteins identified in a recent analysis of the B. pseudomallei genome sequence [9], BopE [15] shares sequence homology with the translocated effector proteins SopE [16] and SopE2 [17] of Salmonella. SopE and SopE2 play an important role in Salmonella invasion of non-phagocytic intestinal epithelial cells. SopE is a potent guanine nucleotide exchange factor (GEF) for the mammalian Rho GTPases Cdc42 and Rac1 in vitro and in vivo, whereas SopE2 efficiently activates Cdc42 but not Rac1 [18], [19]. SopE also catalyses nucleotide exchange in Rab5, a GTPase involved in intracellular vesicle transport [20].
In addition to the SopE/SopE2/BopE family from pathogenic bacteria, the major family of eukaryotic GEFs for Rho GTPases comprises proteins with a catalytic Dbl homology (DH) domain and an adjacent pleckstrin homology (PH) domain. The structures of SopE [21] and DH GEFs [22] are entirely different, although there are similarities in the respective catalytic mechanisms [23].
Here we report over-expression, purification and biophysical properties of the catalytic domain of BopE (BopE78–261), to our knowledge the first effector protein from B. pseudomallei to be characterized in this way. The data indicate that the protein is monomeric in aqueous solution, adopts a single conformation that is predominantly α-helical, stable over a wide range of pH and able to fold independently.
Section snippets
Materials
The expression vector pGEX-2T and column chromatography products for protein purification and size exclusion chromatography analysis were from Amersham Biosciences. The E. coli expression hosts BL21 (DE3) and BL21 Star™ (DE3) were from Stratagene and Invitrogen. Electrophoresis products were from Invitrogen and Bio-Rad. Ampicillin and isopropyl-β-d-thiogalactoside (IPTG) were from Melford Laboratories. Human plasma thrombin was from Calbiochem. All other chemicals were of analytical grade.
Expression and purification of recombinant BopE78–261
Sequence comparison of B. pseudomallei BopE with Salmonella SopE and SopE2
BopE has been identified [9] as a homologue of the Salmonella effector proteins SopE and SopE2 (Fig. 1). Overall, BopE has approximately 16% and 17% sequence identity with SopE and SopE2, respectively. Within the catalytic domain (comparing residues 78–240 of SopE and SopE2 with residues 78–240 of BopE), the sequence identity/similarity with SopE and SopE2 is approximately 25%/40% and 24%/39%.
Expression and purification
BopE78–261 was expressed with an N-terminal GST tag in order to improve solubility and facilitate
Discussion
The molecular mechanisms of B. pseudomallei pathogenesis are not well understood. A number of putative type III-secreted effector proteins have been identified recently by analysis of the B. pseudomallei genome sequence [9]. One of these proteins, BopE, is a homologue of the potent guanine nucleotide exchange factors SopE [16] and SopE2 [17] from Salmonella enterica (Fig. 1). SopE and SopE2 catalyse nucleotide exchange in mammalian Rho GTPases, contributing to disruption of the host cell
Acknowledgments
We thank Margaret Nutley and Professor Alan Cooper of the BBSRC/EPSRC Biological Microcalorimetry Facility, University of Glasgow, for excellent calorimetry service and advice. This work was supported at Bath by The Wellcome Trust (grant 060998) and at IAH by BBSRC. The Wellcome Trust is acknowledged for purchase of the 600 MHz NMR spectrometer (grant 051902) used in this study. C.W. is supported by a PhD studentship from EPSRC.
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