Bacterial Signal Peptide Research Articles

An Interplay between Mitochondrial and ER Targeting of a Bacterial Signal Peptide in Plants.

Protein targeting is essential in eukaryotic cells to maintain cell function and organelle identity. Signal peptides are a major type of targeting sequences containing a tripartite structure, which is conserved across all domains in life. They are frequently included in recombinant protein design in plants to increase yields by directing them to the endoplasmic reticulum (ER) or apoplast. The processing of bacterial signal peptides by plant cells is not well understood but could aid in the design of efficient heterologous expression systems. Here we analysed the signal peptide of the enzyme PmoB from methanotrophic bacteria. In plant cells, the PmoB signal peptide targeted proteins to both mitochondria and the ER. This dual localisation was still observed in a mutated version of the signal peptide sequence with enhanced mitochondrial targeting efficiency. Mitochondrial targeting was shown to be dependent on a hydrophobic region involved in transport to the ER. We, therefore, suggest that the dual localisation could be due to an ER-SURF pathway recently characterised in yeast. This work thus sheds light on the processing of bacterial signal peptides by plant cells and proposes a novel pathway for mitochondrial targeting in plants.

Bacterial Signal Peptides- Navigating the Journey of Proteins.

In 1971, Blobel proposed the first statement of the Signal Hypothesis which suggested that proteins have amino-terminal sequences that dictate their export and localization in the cell. A cytosolic binding factor was predicted, and later the protein conducting channel was discovered that was proposed in 1975 to align with the large ribosomal tunnel. The 1975 Signal Hypothesis also predicted that proteins targeted to different intracellular membranes would possess distinct signals and integral membrane proteins contained uncleaved signal sequences which initiate translocation of the polypeptide chain. This review summarizes the central role that the signal peptides play as address codes for proteins, their decisive role as targeting factors for delivery to the membrane and their function to activate the translocation machinery for export and membrane protein insertion. After shedding light on the navigation of proteins, the importance of removal of signal peptide and their degradation are addressed. Furthermore, the emerging work on signal peptidases as novel targets for antibiotic development is described.

A conserved signal-peptidase antagonist modulates membrane homeostasis of actinobacterial sortase critical for surface morphogenesis

Most Actinobacteria encode a small transmembrane protein, whose gene lies immediately downstream of the housekeeping sortase coding for a transpeptidase that anchors many extracellular proteins to the Gram-positive bacterial cell wall. Here, we uncover the hitherto unknown function of this class of conserved proteins, which we name SafA, as a topological modulator of sortase in the oral Actinobacterium Actinomyces oris. Genetic deletion of safA induces cleavage and excretion of the otherwise predominantly membrane-bound SrtA in wild-type cells. Strikingly, the safA mutant, although viable, exhibits severe abnormalities in cell morphology, pilus assembly, surface protein localization, and polymicrobial interactions-the phenotypes that are mirrored by srtA depletion. The pleiotropic defect of the safA mutant is rescued by ectopic expression of safA from not only A. oris, but also Corynebacterium diphtheriae or Corynebacterium matruchotii. Importantly, the SrtA N terminus harbors a tripartite-domain feature typical of a bacterial signal peptide, including a cleavage motif AXA, mutations in which prevent SrtA cleavage mediated by the signal peptidase LepB2. Bacterial two-hybrid analysis demonstrates that SafA and SrtA directly interact. This interaction involves a conserved motif FPW within the exoplasmic face of SafA, since mutations of this motif abrogate SafA-SrtA interaction and induce SrtA cleavage and excretion as observed in the safA mutant. Evidently, SafA is a membrane-imbedded antagonist of signal peptidase that safeguards and maintains membrane homeostasis of the housekeeping sortase SrtA, a central player of cell surface assembly.

Bacterial signal peptides: structure, optimization, and applications

Bacterial signal peptides are N-terminal tags that direct proteins for export through one of various transport pathways. These signal peptides are highly important as they are the key determinants of transport, ensuring that the correct protein ends up at the correct pathway. While these peptides consist of three domains with well conserved biochemical properties, there still remains a large amount of diversity between the signal sequences for different proteins, transport pathways, and bacterial species. Recent advancements have allowed us to predict signal sequences and manipulate them in an attempt to optimize export efficiency. This knowledge can then be exploited in the field of recombinant protein production wherein bacterial species can be used to produce and secrete proteins of interest. By fusing the protein with an optimized signal peptide, the yield or rate of export can be improved. This review focuses on signal peptides for two primary transport pathways (Sec and Tat) in E. coli specifically, with an emphasis on applications and the production of recombinant proteins.

Integrative proteomic and glycoproteomic profiling of Mycobacterium tuberculosis culture filtrate.

Despite being the subject of intensive research, tuberculosis, caused by Mycobacterium tuberculosis, remains at present the leading cause of death from an infectious agent. Secreted and cell wall proteins interact with the host and play important roles in pathogenicity. These proteins are explored as candidate diagnostic markers, potential drug targets or vaccine antigens, and more recently special attention is being given to the role of their post-translational modifications. With the purpose of contributing to the proteomic and glycoproteomic characterization of this important pathogen, we performed a shotgun analysis of culture filtrate proteins of M. tuberculosis based on a liquid nano-HPLC tandem mass spectrometry and a label-free spectral counting normalization approach for protein quantification. We identified 1314 M. tuberculosis proteins in culture filtrate and found that the most abundant proteins belong to the extracellular region or cell wall compartment, and that the functional categories with higher protein abundance factor were virulence, detoxification and adaptation, and cell wall and cell processes. We could identify a group of proteins consistently detected in previous studies, most of which were highly abundant proteins. In culture filtrate, 140 proteins were predicted to contain one of the three types of bacterial N-terminal signal peptides. Besides, various proteins belonging to the ESX secretion systems, and to the PE and PPE families, secreted by the type VII secretion system using nonclassical secretion signals, were also identified. O-glycosylation was identified in 46 proteins, many of them lipoproteins and cell wall associated proteins. Finally, we provide proteomic evidence for 33 novel O-glycosylated proteins, aiding to the glycoproteomic characterization of relevant antigenic membrane and exported proteins. These findings are expected to collaborate with the research on pathogen derived biomarkers, virulence factors and vaccine candidates, and to provide clues to the understanding of the pathogenesis and survival strategies adopted by M. tuberculosis.

A comprehensive in silico characterization of bacterial signal peptides for the excretory production of Anabaena variabilis phenylalanine ammonia lyase in Escherichia coli.

Anabaena variabilis double mutant (C503S/C565S) phenylalanine ammonia-lyase (PAL) is an appealing enzyme in the enzyme-replacement therapy of phenylketonuria. Yet abundant production of this enzyme has been of concern for industrial production. In this study, we have characterized 1175 bacterial signal peptides (SPs) and identified the most efficient ones for the excretory production of mutant AvPAL. Analysis by SignalP 4.1 revealed that more than 61% of SPs had a D-score greater than 0.7, denoting to be highly efficient. The optimum length of a bacterial SP was 25-30. The preferable net positive charge and the second residue of N-region were + 2 and Lys/Arg, respectively. Highly efficient SPs possessed 3-5 Leus in their H-region and A/L/VXA-FF cleavage site. Highly efficient SPs were from Escherichia coli, corroborating the necessity of an agreement between SPs and the host. Physiochemical characterization of mutant AvPAL conjugates via ProtParam and PROSOII, revealed that ~ 99.5% of proteins would not be entraped in inclusion bodies. Secretory pathways were identified by EffectiveDB and more than 98% of SPs were cleavable. Chimeras were modeled using the I-TASSER program, being evaluated by the Ramachandran plots. The mRNA secondary structure of mutant AvPAL upon linkage to SPs was assessed using the mfold program. It was shown that the linkage of a SP does not affect mutant AvPAL's stability at the protein or mRNA level. AllergenFP tool demonstrated that chimeras were not allergen. SPs, including FMF4_ECOLX, E2BB_ECOLX, and LPTA_ECOLI exhibited the highest propensity for secretion and appropriate features in all analyses.

Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions

BackgroundTumor necrosis factor-related apoptosis-inducing ligand (TRAIL) selectively eliminates tumor cells. However, the short biological half-life of this molecule limits its potential use in the clinic. Our aim was to construct a recombinant strain of nonpathogenic Lactococcus lactis bacteria as a vector for effective and prolonged human TRAIL production. Herein, we examined the expression and secretion conditions leading to the production of biologically active protein in vitro.ResultsThe human soluble TRAIL-cDNA (hsTRAIL-cDNA) with optimized codons was designed to fit the codon usage pattern (codon bias) of the L. lactis host. This cDNA construct was synthesized and cloned in lactococcal plasmid secretion vector pNZ8124 under the control of the nisin-induced PnisA promoter. The pNZ8124-hsTRAIL plasmid vector was transformed into the L. lactis NZ9000 host strain cells by electroporation. Secretion of the protein occurred at the neutral pH during induction, with optimized concentration of the inducer and presence of serine proteases inhibitor. Using Western blotting and amino acid sequencing method we found that TRAIL was secreted in two forms, as visualized by the presence of two distinct molecular size bands, both deprived of the usp45 protein, the bacterial signal peptide. By the use of MTS assay we were able to prove that hsTRAIL present in supernatant from L. lactis (hsTRAIL+) broth culture was cytotoxic to human HCT116 colon cancer cells but not to normal human fibroblasts. Flow cytometry analysis revealed TRAIL-induced apoptosis of cancer cells.ConclusionsWe designed recombinant L. lactis bacteria, which efficiently produce biologically active, anti-tumorigenic human TRAIL in vitro. Further studies in tumor-bearing NOD-SCID mice will reveal whether the TRAIL-secreting L. lactis bacteria can be used as a safe carrier of this protein, capable of inducing effective elimination of human colon cancer cells in vivo.

A Hinged Signal Peptide Hairpin Enables Tat-Dependent Protein Translocation

The Tat machinery catalyzes the transport of folded proteins across the bacterial cytoplasmic membrane and the thylakoid membrane in plants. Using fluorescence quenching and cross-linking approaches, we demonstrate that the Escherichia coli TatBC complex catalyzes insertion of a pre-SufI signal peptide hairpin that penetrates about halfway across the membrane bilayer. Analysis of 512 bacterial Tat signal peptides using secondary structure prediction and docking algorithms suggest that this hairpin interaction mode is generally conserved. An internal cross-link in the signal peptide that blocks transport but does not affect binding indicates that a signal peptide conformational change is required during translocation. These results suggest, to our knowledge, a novel hairpin-hinge model in which the signal peptide hairpin unhinges during movement of the mature domain across the membrane. Thus, in addition to enabling the necessary recognition, the interaction of Tat signal peptides with the receptor complex plays a critical role in the transport process itself.

Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxinH35L in Escherichia coli

Secretion of heterologous proteins into Escherichia coli cell culture medium offers significant advantages for downstream processing over production as inclusion bodies; including cost and time savings, and reduction of endotoxin. Signal peptides play an important role in targeting proteins for translocation across the cytoplasmic membrane to the periplasmic space and release into culture medium during the secretion process. Alpha toxinH35L (ATH35L) was selected as an antigen for vaccine development against Staphylococcus aureus infections. It was successfully secreted into culture medium of E. coli by using bacterial signal peptides linked to the N-terminus of the protein. In order to improve the level of secreted ATH35L, we designed a series of novel signal peptides by swapping individual domains of modifying dsbA and pelB signal peptides and tested them in a fed-batch fermentation process. The data showed that some of the modified signal peptides improved the secretion efficiency of ATH35L compared with E. coli signal peptides from dsbA, pelB and phoA proteins. Indeed, one of the novel signal peptides improved the yield of secreted ATH35L by 3.5-fold in a fed-batch fermentation process and at the same time maintained processing at the expected site for signal peptide cleavage. Potentially, these new novel signal peptides can be used to improve the secretion efficiency of other heterologous proteins in E. coli. Furthermore, analysis of the synthetic signal peptide amino acid sequences provides some insight into the sequence features within the signal peptide that influence secretion efficiency.

A Bacterial Signal Peptide Increases Mucociliary Clearance in Explanted Mouse Trachea

Objective: Bacterial signal peptides are known to trigger innate immunity responses by activation of formyl peptide receptors (FPRs) present in immune cells, e.g. leukocytes. Members of the FPR-family are also found in the murine vomeronasal organ where they are candidates for chemosensory recognition of bacterial pathogens. Here, we investigated the effects of bacterial signal peptides on mucociliary clearance in the murine trachea.

Efficient targeting of recombinant proteins to the thylakoid lumen in Chlamydomonas reinhardtii using a bacterial Tat signal peptide

Interest in the exploitation of microalgae for biotechnological applications has increased over the last decade, and microalgae are now viewed as offering a sustainable alternative to traditionally used host chassis. A number of recombinant proteins have been expressed in genetically modified algal strains, with the green alga Chlamydomonas reinhardtii being a particularly popular host strain. While nuclear transformation is possible with this organism, chloroplast transformation offers more reliable expression, and several proteins have been expressed in the stroma. Here, we present the first utilisation of the thylakoid lumen for recombinant protein production in microalgae. A bacterial export signal peptide was used to efficiently translocate two recombinant proteins, a fluorescent reporter protein (pHRed) and a biopharmaceutical model substrate (scFv) into the thylakoid lumen. This approach expands the algal chloroplast genetic toolkit and offers a means of expressing proteins that are difficult to express in the stroma for reasons of toxicity, stability or a requirement for disulphide bonding.

The sensing of bacteria: emerging principles for the detection of signal sequences by formyl peptide receptors.

The ability to detect specific chemical signatures released by bacteria and other microorganisms is a fundamental feature of immune defense against pathogens. There is increasing evidence that chemodetection of such microorganism-associated molecular patterns (MAMPs) occurs at many places in the body including specific sets of chemosensory neurons in the mammalian nose. Formyl peptide receptors (FPRs) are a unique family of G protein-coupled receptors (GPCRs) that can detect the presence of bacteria and function as chemotactic receptors. Here, we highlight the recent discovery of a vast family of natural FPR agonists, the bacterial signal peptides (or signal sequences), thus providing new insight into the molecular mechanisms of bacterial sensing by human and mouse FPRs. Signal peptides in bacteria are formylated, N-terminal protein signatures required for directing the transfer of proteins through the plasma membrane. After their cleavage and release, signal peptides are available for FPR detection and thus provide a previously unrecognized MAMP. With over 170 000 predicted sequences, bacterial signal peptides represent one of the largest families of GPCR ligands and one of the most complex classes of natural activators of the innate immune system. By recognizing a conserved three-dimensional peptide motif, FPRs employ an unusual detection mechanism that combines structural promiscuity with high specificity and sensitivity, thus solving the problem of detecting thousands of distinct sequences yet maintaining selectivity. How signal peptides are released by bacteria and sensed by GPCRs and how these processes shape the responses of other cells and whole organisms represents an important topic for future research.

Protein fusion tags for efficient expression and purification of recombinant proteins in the periplasmic space of E. coli.

Disulfide bonds occurred in majority of secreted protein. Formation of correct disulfide bonds are must for achieving native conformation, solubility and activity. Production of recombinant proteins containing disulfide bond for therapeutic, diagnostic and various other purposes is a challenging task of research. Production of such proteins in the reducing cytosolic compartment of E. coli usually ends up in inclusion bodies formation. Refolding of inclusion bodies can be difficult, time and labor consuming and uneconomical. Translocation of these proteins into the oxidative periplasmic compartment provides correct environment to undergo proper disulfide bonds formation and thus achieving native conformation. However, not all proteins can be efficiently translocated to the periplasm with the help of bacterial signal peptides. Therefore, fusion to a small well-folded and stable periplasmic protein is more promising for periplasmic production of disulfide bonded proteins. In the past decades, several full-length proteins or domains were used for enhancing translocation and solubility. Here, protein fusion tags that significantly increase the yields of target proteins in the periplasmic space are reviewed.

Crystal Structures of SecYEG in Lipidic Cubic Phase Elucidate a Precise Resting and a Peptide-Bound State

The bacterial SecYEG translocon functions as a conserved protein-conducting channel. Conformational transitions of SecYEG allow protein translocation across the membrane without perturbation of membrane permeability. Here, we report the crystal structures of intact SecYEG at 2.7-Å resolution and of peptide-bound SecYEG at 3.6-Å resolution. The higher-resolution structure revealed that the cytoplasmic loop of SecG covers the hourglass-shaped channel, which was confirmed to also occur in the membrane by disulfide bond formation analysis and molecular dynamics simulation. The cytoplasmic loop may be involved in protein translocation. In addition, the previously unknown peptide-bound crystal structure of SecYEG implies that interactions between the cytoplasmic side of SecY and signal peptides are related to lateral gate opening at the first step of protein translocation. These SecYEG structures therefore provide a number of structural insights into the Sec machinery for further study.

A Novel Dual Expression Platform for High Throughput Functional Screening of Phage Libraries in Product like Format.

High throughput screenings of single chain Fv (scFv) antibody phage display libraries are currently done as soluble scFvs produced in E.coli. Due to endotoxin contaminations from bacterial cells these preparations cannot be reliably used in mammalian cell based assays. The monovalent nature and lack of Fc in soluble scFvs prevent functional assays that are dependent on target cross linking and/or Fc functions. A convenient approach is to convert scFvs into scFv.Fc fusion proteins and express them in mammalian cell lines for screening. This approach is low throughput and is only taken after primary screening of monovalent scFvs that are expressed in bacteria. There is no platform at present that combines the benefits of both bacterial and mammalian expression system for screening phage library output. We have, therefore, developed a novel dual expression vector, called pSplice, which can be used to express scFv.Fc fusion proteins both in E.coli and mammalian cell lines. The hallmark of the vector is an engineered intron which houses the bacterial promoter and signal peptide for expression and secretion of scFv.Fc in E.coli. When the vector is transfected into a mammalian cell line, the intron is efficiently spliced out resulting in a functional operon for expression and secretion of the scFv.Fc fusion protein into the culture medium. By applying basic knowledge of mammalian introns and splisosome, we designed this vector to enable screening of phage libraries in a product like format. Like IgG, the scFv.Fc fusion protein is bi-valent for the antigen and possesses Fc effector functions. Expression in E.coli maintains the speed of the bacterial expression platform and is used to triage clones based on binding and other assays that are not sensitive to endotoxin. Triaged clones are then expressed in a mammalian cell line without the need for any additional cloning steps. Conditioned media from the mammalian cell line containing the fusion proteins are then used for different types of cell based assays. Thus this system retains the speed of the current screening system for phage libraries and adds additional functionality to it.

Recognition of Bacterial Signal Peptides by Mammalian Formyl Peptide Receptors: A NEW MECHANISM FOR SENSING PATHOGENS

Formyl peptide receptors (FPRs) are G-protein-coupled receptors that function as chemoattractant receptors in innate immune responses. Here we perform systematic structure-function analyses of FPRs from six mammalian species using structurally diverse FPR peptide agonists and identify a common set of conserved agonist properties with typical features of pathogen-associated molecular patterns. Guided by these results, we discover that bacterial signal peptides, normally used to translocate proteins across cytoplasmic membranes, are a vast family of natural FPR agonists. N-terminally formylated signal peptide fragments with variable sequence and length activate human and mouse FPR1 and FPR2 at low nanomolar concentrations, thus establishing FPR1 and FPR2 as sensitive and broad signal peptide receptors. The vomeronasal receptor mFpr-rs1 and its sequence orthologue hFPR3 also react to signal peptides but are much more narrowly tuned in signal peptide recognition. Furthermore, all signal peptides examined here function as potent activators of the innate immune system. They elicit robust, FPR-dependent calcium mobilization in human and mouse leukocytes and trigger a range of classical innate defense mechanisms, such as the production of reactive oxygen species, metalloprotease release, and chemotaxis. Thus, bacterial signal peptides constitute a novel class of immune activators that are likely to contribute to mammalian immune defense against bacteria. This evolutionarily conserved detection mechanism combines structural promiscuity with high specificity and enables discrimination between bacterial and eukaryotic signal sequences. With at least 175,542 predicted sequences, bacterial signal peptides represent the largest and structurally most heterogeneous class of G-protein-coupled receptor agonists currently known for the innate immune system.

Structure of Signal Peptide Peptidase A with C-Termini Bound in the Active Sites: Insights into Specificity, Self-Processing, and Regulation

Bacterial signal peptide peptidase A (SppA) is a membrane-bound enzyme that utilizes a serine/lysine catalytic dyad mechanism to cleave remnant signal peptides within the cellular membrane. Bacillus subtilis SppA (SppABS) oligomerizes into a homo-octameric dome-shaped complex with eight active sites, located at the interface between each protomer. In this study, we show that SppABS self-processes its own C-termini. We have determined the crystal structure of a proteolytically stable fragment of SppABSK199A that has its C-terminal peptide bound in each of the eight active sites, creating a perfect circle of peptides. Substrate specificity pockets S1, S3, and S2' are identified and accommodate C-terminal residues Tyr331, Met329, and Tyr333, respectively. Tyr331 at the P1 position is conserved among most Bacillus species. The structure reveals that the C-terminus binds within the substrate-binding grooves in an antiparallel β-sheet fashion. We show, by C-terminal truncations, that the C-terminus is not essential for oligomeric assembly. Kinetic analysis shows that a synthetic peptide corresponding to the C-terminus of SppABS competes with a fluorometric peptide substrate for the SppABS active site. A model is proposed for how the C-termini of SppA may function in the regulation of this membrane-bound self-compartmentalized protease.

SIGLOCPRED: an algorithm to predict bacterial signal peptides and OMPS.

There is a growing interest in Biological investigation to determine the location of proteins, to identify new potentially accessible drug targets. Signal peptide directs the transport of the protein to its location. Bacterial OMPs are essential for their survival in the host organism. SIGLOCPRED a signal peptide predictor for the bacterial proteins as well as OMP prediction has been developed. The signal peptide prediction is done based on the influence of the flanking residues on the signal peptide cleavage. A dataset of proteins with confirmed outer membrane location has being created, and the probable OMP polypeptide sequence is predicted. Since the algorithm uses confirmed datasets the prediction is more reliable and efficient. SIGLOCPRED is as efficient as many of the existing signal peptide predictors and can also predict OMPs in addition.

A phylum level analysis reveals lipoprotein biosynthesis to be a fundamental property of bacteria

Bacterial lipoproteins are proteins that are post-translationally modified with a diacylglyceride at an N-terminal cysteine, which serves to tether these proteins to the outer face of the plasma membrane or to the outer membrane. This paper reviews recent insights into the enzymology of bacterial lipoprotein biosynthesis and localization. Moreover, we use bioinformatic analyses of bacterial lipoprotein signal peptide features and of the key biosynthetic enzymes to consider the distribution of lipoprotein biosynthesis at the phylum level. These analyses support the important conclusion that lipoprotein biosynthesis is a fundamental pathway utilized across the domain bacteria. Moreover, with the exception of a small number of sequences likely to derive from endosymbiont genomes, the enzymes of bacterial lipoprotein biosynthesis appear unique to bacteria, making this pathway an attractive target for the development of novel antimicrobials. Whilst lipoproteins with comparable signal peptide features are encoded in the genomes of Archaea, it is clear that these lipoproteins have a distinctive biosynthetic pathway that has yet to be characterized.

Kinetics of Precursor Interactions with the Bacterial Tat Translocase Detected by Real-time FRET

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.