Type 1 protein secretion in bacteria, the ABC-transporter dependent pathway (review)

The relatively simple type 1 secretion system in gram-negative bacteria is nevertheless capable of transporting polypeptides of up to 800 kDa across the cell envelope in a few seconds. The translocator is composed of an ABC-transporter, providing energy through ATP hydrolysis (and perhaps the initial channel across the inner membrane), linked to a multimeric Membrane Fusion Protein (MFP) spanning the initial part of the periplasm and forming a continuous channel to the surface with an outer membrane trimeric protein. Proteins targeted to the translocator carry an (uncleaved), poorly conserved secretion signal of approximately 50 residues. In E. coli the HlyA toxin interacts with both the MFP (HlyD) and the ABC protein HlyB, (a half transporter) triggering, via a conformational change in HlyD, recruitment of the third component, TolC, into the transenvelope complex. In vitro, HlyA, through its secretion signal, binds to the nucleotide binding domain (NBD or ABC-ATPase) of HlyB in a reaction reversible by ATP that may mimic initial movement of HlyA into the translocation channel. HlyA is then transported rapidly, apparently in an unfolded form, to the cell surface, where folding and release takes place. Whilst recent structural studies of TolC and MFP-like proteins are providing atomic detail of much of the transport path, structural analysis of the HlyB NBD and other ABC ATPases, have revealed details of the catalytic cycle within an NBD dimer and a glimpse of how the action of HlyB is coupled to the translocation of HlyA.     

Protein secretion in the absence of ATP: the autotransporter,two-partner secretion and chaperone/usher pathways of Gram-negative bacteria (Review)

Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In Gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these ‘self-sufficient’ pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.     

The accessory protein TagV is required for full Type VI secretion system activity in Serratia marcescens

The bacterial Type VI secretion system (T6SS) is a dynamic macromolecular structure that promotes inter- and intra-species competition through the delivery of toxic effector proteins into neighbouring cells. The T6SS contains 14 well-characterised core proteins necessary for effector delivery (TssA-M, PAAR). In this study, we have identified a novel accessory component required for optimal T6SS activity in the opportunistic pathogen Serratia marcescens, which we name TagV. Deletion of tagV, which encodes an outer membrane lipoprotein, caused a reduction in the T6SS-dependent antibacterial activity of S. marcescens Db10. Mutants of S. marcescens lacking the core component TssJ, a distinct outer membrane lipoprotein previously considered essential for T6SS firing, retained a modest T6SS activity that could be abolished through deletion of tagV. TagV did not interact with the T6SS membrane complex proteins TssL or TssM, but is proposed to bind to peptidoglycan, indicating that the mechanism by which TagV promotes T6SS firing differs from that of TssJ. Homologues of tagV were identified in several other bacterial genera, suggesting that the accessory function of TagV is not restricted to S. marcescens. Together, our findings support the existence of a second, TssJ-independent mechanism for T6SS firing that is dependent upon the activity of TagV proteins.     

Type II secretion system: A magic beanstalk or a protein escalator

Type II protein secretion systems (T2SS) are molecular machines that promote specific transport of folded periplasmic proteins in Gram-negative bacteria, across a dedicated channel in the outer membrane. Secreted substrates, released to the milieu or displayed on the cell surface, contribute to bacterial adaptation to a range of habitats, from deep-sea waters to animal and plant tissues. The past decade has seen remarkable progress in structural, biochemical and functional analysis of T2SS and related systems, bringing new mechanistic insights into these dynamic complexes. This review focuses on recent advances in the field, and discusses open questions regarding the secretion mechanism. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Comparative analysis of twin-arginine (Tat)-dependent protein secretion of a heterologous model protein (GFP) in three different Gram-positive bacteria

In contrast to the general protein secretion (Sec) system, the twin-arginine translocation (Tat) export pathway allows the translocation of proteins across the bacterial plasma membrane in a fully folded conformation. Due to this feature, the Tat pathway provides an attractive alternative to the secretory production of heterologous proteins via the Sec system. In this study, the potential for Tat-dependent heterologous protein secretion was compared in the three Gram-positive bacteria Staphylococcus carnosus, Bacillus subtilis, and Corynebacterium glutamicum using green fluorescent protein (GFP) as a model protein. In all three microorganisms, fusion of a Tat signal peptide to GFP resulted in its Tat-dependent translocation across the corresponding cytoplasmic membranes. However, striking differences with respect to the final localization and folding status of the exported GFP were observed. In S. carnosus, GFP was trapped entirely in the cell wall and not released into the supernatant. In B. subtilis, GFP was secreted into the supernatant, however, in an inactive form. In contrast, C. glutamicum effectively secreted active GFP. Our results clearly demonstrate that a comparative evaluation of different Gram-positive host microorganisms is a crucial step on the way to an efficient Tat-mediated secretory production process for a desired heterologous target protein. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This paper is dedicated to Hermann Sahm on the occasion of his 65th birthday.     

High-level production of Serratia proteamaculans metalloprotease using a recombinant ABC protein exporter-mediated secretion system in Pseudomonas fluorescens

Serratia proteamaculans metalloprotease (SPP) was successfully secreted by a heterologous ABC protein exporter, the Pseudomonas fluorescens TliDEF, in recombinant host strains. Escherichia coli and P. fluorescens cells containing the SPP-encoding gene showed the extracellular protease activity only when the TliDEF-encoding gene cluster was coexpressed. Recombinant P. fluorescens produced an approximately 34.8-fold higher amount of extracellular SPP than did E. coli. The use of a more nutrient-rich medium and controlled dissolved oxygen conditions was effective in increasing SPP secretion in P. fluorescens batch fermentation (an 8.7-fold increase from 41.8 U/mL to 365.2 U/mL). Therefore, SPP, which could not be secreted without an ABC protein exporter, was produced in large quantities by applying the heterologous TliDEF exporter in P. fluorescens. The results also suggest that the use of the ABC protein exporter in P. fluorescens could be an efficient production platform for an industrially promising type I secretion pathway-dependent enzyme.     

The Type 1 secretion pathway — The hemolysin system and beyond

Type 1 secretion systems (T1SS) are wide-spread among Gram-negative bacteria. An important example is the secretion of the hemolytic toxin HlyA from uropathogenic strains. Secretion is achieved in a single step directly from the cytosol to the extracellular space. The translocation machinery is composed of three indispensable membrane proteins, two in the inner membrane, and the third in the outer membrane. The inner membrane proteins belong to the ABC transporter and membrane fusion protein families (MFPs), respectively, while the outer membrane component is a porin-like protein. Assembly of the three proteins is triggered by accumulation of the transport substrate (HlyA) in the cytoplasm, to form a continuous channel from the inner membrane, bridging the periplasm and finally to the exterior. Interestingly, the majority of substrates of T1SS contain all the information necessary for targeting the polypeptide to the translocation channel — a specific sequence at the extreme C-terminus. Here, we summarize our current knowledge of regulation, channel assembly, translocation of substrates, and in the case of the HlyA toxin, its interaction with host membranes. We try to provide a complete picture of structure function of the components of the translocation channel and their interaction with the substrate. Although we will place the emphasis on the paradigm of Type 1 secretion systems, the hemolysin A secretion machinery from E. coli, we also cover as completely as possible current knowledge of other examples of these fascinating translocation systems. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

A NanoLuc luciferase-based assay enabling the real-time analysis of protein secretion and injection by bacterial type III secretion systems

The elucidation of the molecular mechanisms of secretion through bacterial protein secretion systems is impeded by a shortage of assays to quantitatively assess secretion kinetics. Also the analysis of the biological role of these secretion systems as well as the identification of inhibitors targeting these systems would greatly benefit from the availability of a simple, quick and quantitative assay to monitor principle secretion and injection into host cells. Here, we present a versatile solution to this need, utilizing the small and very bright NanoLuc luciferase to assess the function of the type III secretion system encoded by Salmonella pathogenicity island 1. Type III secretion substrate–NanoLuc fusions are readily secreted into the culture supernatant, where they can be quantified by luminometry after removal of bacteria. The NanoLuc-based secretion assay features a very high signal-to-noise ratio and sensitivity down to the nanolitre scale. The assay enables monitoring of secretion kinetics and is adaptable to a high throughput screening format in 384-well microplates. We further developed a split NanoLuc-based assay that enables the real-time monitoring of type III secretion-dependent injection of effector–HiBiT fusions into host cells stably expressing the complementing NanoLuc–LgBiT.     

革兰氏阴性细菌蛋白分泌系统研究进展

革兰氏阴性细菌由于具有复杂的双层膜结构,其蛋白质分泌能力较差.这使得革兰氏阴性细菌的典型菌株——大肠杆菌作为最常用的受体细胞在生物制药工程和其他生物技术产品生产中受到一定的限制.因此,革兰氏阴性细菌蛋白分泌系统的研究具有重要意义.本文详细地归纳了革兰氏阴性细菌已知的蛋白分泌系统,分别从分泌系统的分泌过程、分泌蛋白类别、...     

Dual location of MAR-binding, filament-like protein 1 in Arabidopsis, tobacco, and tomato

Matrix attachment region-binding filament-like protein 1 (MFP1) is a plant-specific long coiled-coil protein that binds double-stranded DNA. While originally identified as a component of the tobacco nuclear matrix, it was subsequently shown that the majority of MFP1 resides in mature chloroplast where it is located at the stroma side of the thylakoids and is able to bind to nucleoids. On the other hand, a 90 kDa MFP1-like protein from onion has been convincingly shown to be an intrinsic component of the onion meristematic nuclear matrix. Here, we have expanded the analysis of the subcellular location of MFP1 by using high-resolution confocal immunofluorescence microscopy and immunogold electron microscopy. Two different antisera raised against MFP1 from two species were used on isolated nuclei and chloroplasts from tomato, tobacco, and Arabidopsis. Our data show that both antibodies detect a signal in both compartments in all three species. An Arabidopsis MFP1 T-DNA insertional mutation abolishes both nuclear and chloroplast signals, indicating that the nuclear and plastidic antigens are derived from the same gene. We therefore suggest that MFP1 is a protein with a dual location, in both nuclei and chloroplasts, consistent with prior findings in onion and the dicot species investigated here.     

The Tat pathway in bacteria and chloroplasts (Review)

Both in prokaryotic organisms and in chloroplasts, a specialized protein transport pathway exists which is capable of translocating proteins in a fully folded conformation. Transport is mediated in both instances by signal peptides harbouring a twin-arginine consensus motif (twin-arginine translocation (Tat) pathway). The Tat translocase comprises the three functionally different membrane proteins TatA, TatB, and TatC. While TatB and TatC are involved in the specific recognition of the substrate, TatA might be the major pore-forming component. Current evidence suggests that a functional Tat translocase is assembled from separate TatBC and TatA assemblies only on demand, i.e., in the presence of transport substrate and a transmembrane H⁺-motive force.     

Enhancement of protein secretion in Pichia pastoris by overexpression of protein disulfide isomerase

A potential vaccine candidate, Necator americanus secretory protein (Na-ASP1), against hookworm infections, has been expressed in Pichia pastoris. Na-ASP1, a 45 kDa protein containing 20 cysteines, was directed outside the cell by fusing the protein to the preprosequence of the alpha-mating factor of Saccharomyces cerevisiae. Most of the protein produced by single copy clones was secreted outside the cell. However, increasing gene copy number of Na-ASP1 protein in P. pastoris saturated secretory capacity and therefore, decreased the amount of secreted protein in clones harboring multiple copies of Na-ASP1 gene. Overexpression of the endoplasmic reticulum (ER) resident, homologous chaperone protein, protein disulfide isomerase (PDI) was able to increase the secretion of (Na-ASP1) protein in high copy clones. The effect of PDI levels on secretion of Na-ASP1 protein was examined in clones with varying copy number of PDI gene. Increase in secreted Na-ASP1 secretion is correlated well with the PDI copy number. Increasing levels of PDI also increased overall Na-ASP1 protein production in all the clones. Nevertheless, there was still accumulation of intracellular Na-ASP1 protein in P. pastoris clones over-expressing Na-ASP1 and PDI proteins.     

Structural determinants of protein translocation in bacteria: conformational flexibility of SecA IRA1 loop region

Bacteria employ the SecA motor protein to push unfolded proteins across the cytoplasmic membrane through the SecY protein‐conducting channel complex. The crystal structure of the SecA–SecY complex shows that the intramolecular regulator of ATPase1 (IRA1) SecA domain, made up of two helices and the loop between them, is partly inserted into the SecY conducting channel, with the loop between the helices as the main functional region. A computational analysis suggested that the entire IRA1 domain is structurally autonomous, and was the basis to synthesize peptide analogs of the SecA IRA1 loop region, to the aim of investigating its conformational preferences. Our study indicates that the loop region populates a predominantly flexible state, even in the presence of structuring agent. This provides indirect evidence that the SecA loop–SecY receptor docking involves loop‐mediated opening of the SecY channel. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

A positive role of mammalian Tip41-like protein,TIPRL, in the amino-acid dependent mTORC1-signaling pathway through interaction with PP2A

Target of rapamycin complex 1 (TORC1) has a key role in cellular regulations in response to environmental conditions. In yeast, Tip41 downregulates TORC1 signaling via activation of PP2A phosphatase. We show here that overexpression of TIPRL, a mammalian Tip41, suppressed dephosphorylation of mechanistic TORC1 (mTORC1) substrates under amino acid withdrawal, and knockdown of TIPRL conversely attenuated phosphorylation of those substrates after amino acid refeeding. TIPRL associated with the catalytic subunit of PP2A (PP2Ac), which was required for the TIPRL action on mTORC1 signaling. Collectively, unlike yeast TIP41, TIPRL has a positive effect on mTORC1 signaling through the association with PP2Ac.     

Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.     

细菌VI型分泌系统调节因素的研究进展

细菌的VI型分泌系统(type VI secretion system,T6SS)是一种新发现的分泌系统,在病原菌对宿主黏附、侵入及杀伤等方面均发挥了重要作用。目前的研究主要集中在T6SS在细菌致病、细菌间竞争等作用方面。然而对于其调控因素的研究尚处于初级阶段。对于大多数细菌而言,T6SS的表达并不是恒定的。现已发现温度、渗透压、抗生素、离子等环境因素均可调节T6SS。此外,在分子层面,H-NS蛋白、RpoN转录因子、c-di-GMP等也可发挥对T6SS的调节作用。在这些调控因素的调节下,细菌可以适时地开启或关闭其T6SS的表达,从而更好地感知并适应环境。对T6SS调控因素的研究对于充分认识细菌致病性并进行有效控制至关重要。本文将对调节T6SS的环境因素与调节因子做一综述。