Membrane targeting and translocation of bacterial hydrogenases

Periplasmic or membrane-bound bacterial hydrogenases are generally composed of a small subunit and a large subunit. The small subunit contains a peculiar N-terminal twin-arginine signal peptide, whereas the large subunit lacks any known targeting signal for export. Genetic and biochemistry data support the assumption that the large subunit is cotranslocated with the small subunit across the cytoplasmic membrane. Indeed, the signal peptide carried by the small subunit directs both the small and the large subunits to the recently identified Mtt/Tat pathway, independently of the Sec machinery. In addition, the twin-arginine signal peptide of hydrogenase is capable of directing protein import into the thylakoidal lumen of chloroplasts via the homologous deltapH-driven pathway, which is independent of the Sec machinery. Therefore, the translocation of hydrogenase shares characteristics with the deltapH-driven import pathway in terms of Sec-independence and requirement for the twin-arginine signal peptide, and with protein import into peroxisomes in a "piggyback" fashion.     

Lassa virus glycoprotein signal peptide displays a novel topology with an extended endoplasmic reticulum luminal region

Lassa virus glycoprotein C (GP-C) is translated as a precursor (preGP-C) into the lumen of the endoplasmic reticulum (ER) and cotranslationally cleaved into the signal peptide and immature GP-C before GP-C is proteolytically processed into its subunits, GP-1 and GP-2, which form the mature virion spikes. The signal peptide of preGP-C comprises 58 amino acids and contains two distinct hydrophobic domains. Here, we show that each hydrophobic domain alone can insert preGP-C into the ER membrane. Furthermore, we demonstrate that the native signal peptide only uses the N-terminal hydrophobic domain for membrane insertion, exhibiting a novel type of a topology for signal peptides with an extended ER luminal part, which is essential for proteolytic processing of GP-C into GP-1 and GP-2.     

The corticotropin-releasing factor receptor type 2a contains an N-terminal pseudo signal peptide

The corticotropin-releasing factor receptor type 2a (CRF(2(a)) receptor) belongs to the family of G protein-coupled receptors. The receptor possesses a putative N-terminal signal peptide that is believed to be cleaved-off after mediating the endoplasmic reticulum targeting/insertion process, like the corresponding sequence of the homologous CRF(1) receptor. Here, we have assessed the functional significance of the putative signal peptide of the CRF(2(a)) receptor and show that it is surprisingly completely incapable of mediating endoplasmic reticulum targeting, despite meeting all sequence criteria for a functional signal by prediction algorithms. Moreover, it is uncleaved and forms part of the mature receptor protein. Replacement of residue Asn(13) by hydrophobic or positively charged residues converts the sequence into a fully functional and cleaved signal peptide demonstrating that conventional signal peptide functions are inhibited by a single amino acid residue. Deletion of the domain leads to an increase in the amount of immature, intracellularly retained receptors demonstrating that the sequence has adopted a new function in receptor trafficking through the early secretory pathway. Taken together, our results identify a novel hydrophobic receptor domain in the family of the heptahelical G protein-coupled receptors and the first pseudo signal peptide of a eukaryotic membrane protein. Our data also show that the extreme N termini of the individual CRF receptor subtypes differ substantially.     

Application of immuno-mass spectrometry to analysis of a bacterial virulence factor

Here we describe a novel antibody-based assay that combines specificity of antibody with precision of mass spectral analysis. The assay is carried out in three steps using a single antigen capture and transfer reagent. The first step of the assay involves antibody immobilization. The second step is antigen capture and washing to remove unbound proteins. The third step involves the analysis of the captured antigens by surface enhanced laser desorption ionization time-of-flight mass spectrometry. The assay is facilitated by the ability of a single nonviable bacterial preparation expressing immunoglobulin-binding proteins that enable antibody immobilization, specific capture of fluid-phase antigen, and direct sample transfer to a protein chip for mass spectral analysis. Proof-of-concept studies using a model Streptococcus pyogenes virulence factor, the secreted cysteine protease SpeB, are presented.     

Membrane localisation of a UDP-sugar hydrolase, in Salmonella, is by an uncleaved N-terminal signal peptide

Abstract Most isolates of Salmonella contain two unrelated UDP-sugar hydrolases, one of which, encoded by the ushB gene, is inner membrane-associated. Previous studies showed that this enzyme contains a typical N-terminal signal peptide; the evidence also indicated, however, that this peptide is not cleaved, and serves to anchor the UshB protein in the inner membrane. In this report, we present strong evidence that this is indeed the case by using ushB'-'blaM fusions to demonstrate that this signal peptide is capable of localising β-lactamase to the inner membrane. We also present evidence that UshB is located on the exterior (periplasmic) side of the membrane, and hence has an 'N-terminus inside/C-terminus outside' membrane orientation, consistent with a role in the degradation of external substrates.     

Overlapping transport and chaperone-binding functions within a bacterial twin-arginine signal peptide

The twin-arginine translocation (Tat) pathway is a protein targeting system present in many prokaryotes. The physiological role of the Tat pathway is the transmembrane translocation of fully-folded proteins, which are targeted by N-terminal signal peptides bearing conserved SRRxFLK 'twin-arginine' amino acid motifs. In Escherichia coli the majority of Tat targeted proteins bind redox cofactors and it is important that only mature, cofactor-loaded precursors are presented for export. Cellular processes have been unearthed that sequence these events, for example the signal peptide of the periplasmic nitrate reductase (NapA) is bound by a cytoplasmic chaperone (NapD) that is thought to regulate assembly and export of the enzyme. In this work, genetic, biophysical and structural approaches were taken to dissect the interaction between NapD and the NapA signal peptide. A NapD binding epitope was identified towards the N-terminus of the signal peptide, which overlapped significantly with the twin-arginine targeting motif. NMR spectroscopy revealed that the signal peptide adopted a α-helical conformation when bound by NapD, and substitution of single residues within the NapA signal peptide was sufficient to disrupt the interaction. This work provides an increased level of understanding of signal peptide function on the bacterial Tat pathway.     

Functional characterization of bacterial signal peptide OmpA in a baculovirus-mediated expression system

Signal sequences are evolutionarily conserved and are often functionally interchangeable between prokaryotes and eukaryotes. However, we have found that the bacterial signal peptide, OmpA, functions incompletely in insect cells. Upon baculovirus-mediated expression of chloramphenicol acetyltransferase (CAT) in insect cells, OmpA signal peptide led to the cytosolic accumulation of the CAT molecules in an aglycosylated, signal-peptide cleaved form, in addition to the secretion of the glycosylated CAT. When green fluorescent protein (GFP) was used as another reporter, the GFP molecules expressed from the OmpA-GFP construct was distributed primarily in the cytosol as aggresome-like structures. These results together suggest that, subsequent to the cleavage of OmpA signal peptide in the ER, some of the processed proteins are returned to the cytoplasm. Since the prototypical insect signal peptide, melittin, did not result in this ER-to-cytosol dislocation of the reporter proteins, we proposed a model explaining the dislocation process in insect cells, apparently selective to the OmpA-directed secretory pathway bypassing the co-translational transport.     

The bacterial virulence factor NleA undergoes host-mediated O-linked glycosylation

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form “attaching and effacing” lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection.     

Not just an antibiotic target: Exploring the role of type I signal peptidase in bacterial virulence

The looming antibiotic crisis has prompted the development of new strategies towards fighting infection. Traditional antibiotics target bacterial processes essential for viability, whereas proposed antivirulence approaches rely on the inhibition of factors that are required only for the initiation and propagation of infection within a host. Although antivirulence compounds have yet to prove their efficacy in the clinic, bacterial signal peptidase I (SPase) represents an attractive target in that SPase inhibitors exhibit broad-spectrum antibiotic activity, but even at sub-MIC doses also impair the secretion of essential virulence factors. The potential consequences of SPase inhibition on bacterial virulence have not been thoroughly examined, and are explored within this review. In addition, we review growing evidence that SPase has relevant biological functions outside of mediating secretion, and discuss how the inhibition of these functions may be clinically significant.     

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.     

Identification of Lassa virus glycoprotein signal peptide as a trans-acting maturation factor

Lassa virus glycoprotein is translated as a precursor (pre-GP-C) into the lumen of the endoplasmic reticulum and is cotranslationally cleaved into the signal peptide and GP-C, before GP-C is proteolytically processed into its subunits GP1 and GP2. The signal peptide of pre-GP-C comprises 58 amino acids. The substitution of Lassa virus pre-GP-C signal peptide with another signal peptide still mediates translocation and the release of signal peptide but abolishes the proteolytic cleavage of GP-C into GP1 and GP2. Remarkably, cleavage of GP-C from these hybrid pre-GP-C substrates was restored on coexpression of the wild-type pre-GP-C signal peptide, indicating that the signal peptide functions as a trans-acting factor to promote Lassa virus GP-C processing. To our knowledge, this is the first report on a signal peptide that is essential for proteolytic processing of a secretory pathway protein.     

Substrate based peptide aldehyde inhibits bacterial type I signal peptidase

Bacterial type I signal peptidase is a potential target for the development of novel antibacterial agents. In this study we demonstrate that a substrate based peptide aldehyde inhibits signal peptidases with a lower IC₅₀ value than the lipopeptides described to date. The length of the core lipopeptide could be reduced by removing several amino acids from both termini. Conversion of this peptide to an aldehyde resulted in a molecule with an IC₅₀ value of 0.09 μM when tested against Saccharomyces aureus SPase I, SpsB.     

Structure-based design of an agonistic peptide targeting Fas

A small agonistic peptide FRAP-4 (WEWT, Fas reactive peptide-4) that binds to the human Fas molecule was discovered using our computer screening strategy named the Amino acid Complement Wave (ACW) method, which is based on the complementarities of interacting amino acids between comprehensive testing peptides and a target protein surface pocket. In silico docking studies demonstrated the specific interaction of FRAP-4 with the main Fas ligand (FasL) binding domain in the Fas molecule. An octamer of this peptide produced by carboxyl terminal linkages of polylysine branches (MAP), (FRAP-4)₈-MAP, effectively induced apoptosis in human ovarian cancer cell line NOS4 cells that was associated with the activation of caspases-8, -9 and -3, and the cleavage of PARP. Alanine substitution of the N-terminal W in FRAP-4 resulted in complete loss of FasL-mimetic action of (FRAP-4)₈-MAP, suggesting that the aromatic functionality at the N-terminal position W appears to play an essentially important role in Fas binding ability. These observations indicate that the FasL-mimetic peptide should serve as an excellent starting point for the design of effective compounds with FasL-mimetic activity. Furthermore, the ACW method for the structure-based design of optimized small peptides against receptor molecules such as Fas could open new avenues for the development of peptide mimetic and nonpeptidic organic forms to generate novel effective pharmaceuticals.     

Optimisation of signal peptide for recombinant protein secretion in bacterial hosts

Escherichia coli—the powerhouse for recombinant protein production—is rapidly gaining status as a reliable and efficient host for secretory expression. An improved understanding of protein translocation processes and its mechanisms has inspired and accelerated the development of new tools and applications in this field and, in particular, a more efficient secretion signal. Several important characteristics and requirements are summarised for the design of a more efficient signal peptide for the production of recombinant proteins in E. coli. General approaches and strategies to optimise the signal peptide, including the selection and modification of the signal peptide components, are included. Several challenges in the secretory production of recombinant proteins are discussed, and research approaches designed to meet these challenges are proposed.     

EPIYA motif is a membrane-targeting signal of Helicobacter pylori virulence factor CagA in mammalian cells

Helicobacter pylori contributes to the development of peptic ulcers and atrophic gastritis. Furthermore, H. pylori strains carrying the cagA gene are more virulent than cagA-negative strains and are associated with the development of gastric adenocarcinoma. The cagA gene product, CagA, is translocated into gastric epithelial cells and localizes to the inner surface of the plasma membrane, in which it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motif. Tyrosine-phosphorylated CagA specifically binds to and activates Src homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) at the membrane, thereby inducing an elongated cell shape termed the hummingbird phenotype. Accordingly, membrane tethering of CagA is an essential prerequisite for the pathogenic activity of CagA. We show here that membrane association of CagA requires the EPIYA-containing region but is independent of EPIYA tyrosine phosphorylation. We further show that specific deletion of the EPIYA motif abolishes the ability of CagA to associate with the membrane. Conversely, reintroduction of an EPIYA sequence into a CagA mutant that lacks the EPIYA-containing region restores membrane association of CagA. Thus, the presence of a single EPIYA motif is necessary for the membrane localization of CagA. Our results indicate that the EPIYA motif has a dual function in membrane association and tyrosine phosphorylation, both of which are critically involved in the activity of CagA to deregulate intracellular signaling, and suggest that the EPIYA motif is a crucial therapeutic target of cagA-positive H. pylori infection.     

The rsmA-like gene rsmA(Xoo) of Xanthomonas oryzae pv. oryzae regulates bacterial virulence and production of diffusible signal factor

The plant-pathogenic prokaryote Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial leaf blight, one of the most destructive diseases of rice. A nonpolar mutant of the rsmA-like gene rsmA(Xoo) of the Xoo Chinese strain 13751 was constructed by homologous integration with a suicide plasmid. Virulence tests on a host plant, namely the hybrid rice cultivar Teyou 63, showed that the mutant had lost its virulence almost completely, whereas tests on a nonhost, namely castor-oil plant (Ricinus communis), showed that the mutant had also lost the ability to induce a hypersensitive response in the nonhost. In addition, the rsmA(Xoo) mutant produced significantly smaller amounts of the diffusible signal factor, extracellular endoglucanase, amylase and extracellular polysaccharide, but showed significantly higher glycogen accumulation, bacterial aggregation and cell adhesion. The expression of most hrp genes, genes encoding AvrBs3/PthA family members, rpfB, xrvA, glgA, eglXoB and XOO0175 (encoding an α-amylase) was down-regulated in the rsmA(Xoo) mutant. All phenotypes and expression levels of the tested genes in the rsmA(Xoo) mutant were restored to their levels in the wild-type by the presence of rsmA(Xoo) in trans. These results indicate that rsmA(Xoo) is essential for the virulence of Xoo.