Protein secretion systems in Fusobacterium nucleatum: Genomic identification of Type 4 piliation and complete Type V pathways brings new insight into mechanisms of pathogenesis

Recent genomic analyses of the two sequenced strains F. nucleatum subsp. nucleatum ATCC 25586 and F. nucleatum subsp. vincentii ATCC 49256 suggested that the major protein secretion systems were absent. However, such a paucity of protein secretion systems is incongruous with F. nucleatum pathogenesis. Moreover, the presence of one or more such systems has been described for every other Gram-negative organism sequenced to date. In this investigation, the question of protein secretion in F. nucleatum was revisited. In the current study, the absence in F. nucleatum of a twin-arginine translocation system (TC #2.A.64.), a Type III secretion system (TC #3.A.6.), a Type IV secretion system (TC #3.A.7.) and a chaperone/usher pathway (TC #1.B.11.) was confirmed. However, contrary to previous findings, our investigations indicated that a Type I protein secretion system was also absent from F. nucleatum. In contrast, members of the holin family (TC #1.E) and the machinery required for a Type 4 piliation/fimbriation system (TC #3.A.15.2.) were identified using a variety of bioinformatic tools. Furthermore, a complete range of proteins resembling members of the Type V secretion pathway, i.e., the Type Va (autotransporter; TC #1.B.12.), Type Vb (two-partner secretion system; TC #1.B.20.) and Type Vc (YadA-like trimeric autotransporter; TC #1.B.42.), was found. This work provides new insight into the protein secretion and virulence mechanisms of F. nucleatum.     

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.     

Bacterial hemoglobins and flavohemoglobins: versatile proteins and their impact on microbiology and biotechnology

In response to oxygen limitation or oxidative and nitrosative stress, bacteria express three kinds of hemoglobin proteins: truncated hemoglobins (tr Hbs), hemoglobins (Hbs) and flavohemoglobins (flavo Hbs). The two latter groups share a high sequence homology and structural similarity in their globin domain. Flavohemoglobin proteins contain an additional reductase domain at their C-terminus and their expression is induced in the presence of reactive nitrogen and oxygen species. Flavohemoglobins detoxify NO in an aerobic process, termed nitric oxide dioxygenase reaction, which protects the host from various noxious nitrogen compounds. Only a small number of bacteria express hemoglobin proteins and the best studied of these is from Vitreoscilla sp. Vitreoscilla hemoglobin (VHb) has been expressed in various heterologous hosts under oxygen-limited conditions and has been shown to improve growth and productivity, rendering the protein interesting for biotechnology industry. The close interaction of VHb with the terminal oxidases has been shown and this interplay has been proposed to enhance respiratory activity and energy production by delivering oxygen, the ultimate result being an improvement in growth properties.     

Type II secretion: from structure to function

Gram-negative bacteria use the type II secretion system to transport a large number of secreted proteins from the periplasmic space into the extracellular environment. Many of the secreted proteins are major virulence factors in plants and animals. The components of the type II secretion system are located in both the inner and outer membranes where they assemble into a multi-protein, cell-envelope spanning, complex. This review discusses recent progress, particularly newly published structures obtained by X-ray crystallography and electron microscopy that have increased our understanding of how the type II secretion apparatus functions and the role that individual proteins play in this complex system.     

Size and conformation limits to secretion of disulfide-bonded loops in autotransporter proteins

Autotransporters are a superfamily of virulence factors typified by a channel-forming C terminus that facilitates translocation of the functional N-terminal passenger domain across the outer membrane of Gram-negative bacteria. This final step in the secretion of autotransporters requires a translocation-competent conformation for the passenger domain that differs markedly from the structure of the fully folded secreted protein. The nature of the translocation-competent conformation remains controversial, in particular whether the passenger domain can adopt secondary structural motifs, such as disulfide-bonded segments, while maintaining a secretion-competent state. Here, we used the endogenous and closely spaced cysteine residues of the plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli to investigate the effect of disulfide bond-induced folding on translocation of an autotransporter passenger domain. We reveal that rigid structural elements within disulfide-bonded segments are resistant to autotransporter-mediated secretion. We define the size limit of disulfide-bonded segments tolerated by the autotransporter system demonstrating that, when present, cysteine pairs are intrinsically closely spaced to prevent congestion of the translocator pore by large disulfide-bonded regions. These latter data strongly support the hairpin mode of autotransporter biogenesis.     

Following nature's roadmap: folding factors from plasma cells led to improvements in antibody secretion in S. cerevisiae

Therapeutic protein production in yeast is a reality in industry with an untapped potential to expand to more complex proteins, such as full‐length antibodies. Despite numerous engineering approaches, cellular limitations are preventing the use of Saccharomyces cerevisiae as the titers of recombinant antibodies are currently not competitive. Instead of a host specific approach, the possibility of adopting the features from native producers of antibodies, plasma cells, to improve antibody production in yeast. A subset of mammalian folding factors upregulated in plasma cells for expression in yeast and screened for beneficial effects on antibody secretion using a high‐throughput ELISA platform was selected. Co‐expression of the mammalian chaperone BiP, the co‐chaperone GRP170, or the peptidyl‐prolyl isomerase FKBP2, with the antibody improved specific product yields up to two‐fold. By comparing strains expressing FKBP2 or the yeast PPIase Cpr5p, the authors demonstrate that speeding up peptidyl‐prolyl isomerization by upregulation of catalyzing enzymes is a key factor to improve antibody titers in yeast. The findings show that following the route of plasma cells can improve product titers and contribute to developing an alternative yeast‐based antibody factory.     

Type III secretion system-associated pilus of Pseudomonas syringae as an epitope display tool

Type III secretion system-associated pili found in several plant pathogenic bacteria are required for injection of virulence proteins from bacteria into the plant cells. The possibility to use the type III secretion pilus of Pseudomonas syringae as an epitope display tool was studied. The advantage of the type III secretion pilus, compared with conventional fimbrial epitope display tools, is that the pilin subunits of the type III secretion pilus can auto-assemble into intact pili in vitro. Various peptides were inserted into the type III secretion pilin subunit, and secretion, assembly and surface properties of the modified pili were monitored. It was concluded that the outwards-projecting N-terminal region of the pilin can bear even 43 amino acids insertion. The three-dimensional structure of the epitope, however, can restrict the use of the pilus as an epitope display tool: a beta-hairpin structure was poorly tolerated.     

Type VI secretion system effector proteins: Effective weapons for bacterial competitiveness

The Type VI secretion system (T6SS) is a protein translocation nanomachine widespread among Gram‐negative bacteria and used as a means to deliver effectors directly into target bacterial or eukaryotic cells. These effectors have a wide variety of functions within target cells that ultimately help the secreting cell gain a competitive fitness advantage. Here, we discuss the different ways in which these effectors can be delivered by the T6SS and the diverse mechanisms by which they exert their noxious action upon recipient cells. We also highlight the existence of roles for T6SS effectors beyond simply the killing of neighbouring cells.     

Secretory delivery of recombinant proteins in attenuated Salmonella strains: potential and limitations of Type I protein transporters

Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems.     

Take five — Type VII secretion systems of Mycobacteria

Mycobacteria use type VII secretion (T7S) systems to secrete proteins across their complex cell envelope. Pathogenic mycobacteria, such as the notorious pathogen Mycobacterium tuberculosis, have up to five of these secretion systems, named ESX-1 to ESX-5. At least three of these secretion systems are essential for mycobacterial virulence and/or viability. Elucidating T7S is therefore essential to understand the success of M. tuberculosis and other pathogenic mycobacteria as pathogens, and could be instrumental to identify novel targets for drug- and vaccine-development. Recently, significant progress has been achieved in the identification of T7S substrates and a general secretion motif. In addition, a start has been made with unraveling the mechanism of secretion and the structural analysis of the different subunits. This review summarizes these recent findings, which are incorporated in a working model of this complex machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

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.     

Protein secretion by the mouse blastocyst: Stimulatory effect on secretion into the blastocoel by transforming growth factor-α

We have previously demonstrated that newly synthesized proteins are secreted into the mouse blastocoel [Dardik and Schultz (1991): Biol Reprod 45:328–333]. In the present study we examine the effect of transforming growth factor-α (TGF-α) on these proteins. We observe that TGF-α stimulates secretion of these newly synthesized proteins into the blastocoel and apical medium, which faces the zona pellucida, by about 65%. Although one-dimensional gel electrophoretic analysis does not reveal any marked differences in the patterns of newly synthesized proteins secreted into the blastocoel in response to TGF-α, zymography reveals a marked stimulation in the secretion of several gelatinases into the blastocoel and apical medium. These results suggest additional functions for TGF-α in mouse preimplantation development. © 1993 Wiley-Liss, Inc.     

空肠弯曲菌Ⅵ型分泌系统研究进展

Ⅵ型分泌系统(Type Ⅵ Secretion System,T6SS)是一种倒置于细胞膜上的类噬菌体样结构,能够输送效应蛋白并在定殖和生态位建立中发挥作用.近年来,在空肠弯曲菌(Campylobacter jejuni)中发现了T6SS同源基因且能够表达组装成结构完整的T6SS,但T6SS对空肠弯曲菌的毒力影响尚不清...     

Type III secretion: the bacteria-eukaryotic cell express

Type III secretion (T3S) is an export pathway used by Gram-negative pathogenic bacteria to inject bacterial proteins into the cytosol of eukaryotic host cells. This pathway is characterized by (i) a secretion nanomachine related to the bacterial flagellum, but usually topped by a stiff needle-like structure; (ii) the assembly in the eukaryotic cell membrane of a translocation pore formed by T3S substrates; (iii) a non-cleavable N-terminal secretion signal; (iv) T3S chaperones, assisting the secretion of some substrates; (v) a control mechanism ensuring protein delivery at the right place and time. Here, we review these different aspects focusing in open questions that promise exciting findings in the near future.     

Bacillus subtilisα-amylase: the rate limiting step of secretion is growth phase-independent

When Bacillus subtilis alpha-amylase was expressed under the control of sacR in a degU32(Hy) strain, the production of exoenzyme occurred during both the exponential and stationary phases of growth. In each phase, pulse-chase experiments showed that the rate-limiting step of the secretion process was the release of the processed form of the protein in each physiological context. The rate of this event was slightly slower (t(1/2) = 3.2 min) during the stationary phase than during the exponential phase (t(1/2) = 2 min). The effectors which possibly control the efficiency of the release stage, the level of PrsA or the calcium binding properties of the cell wall, remained unchanged throughout growth phases.     

Artificial signal peptide prediction by a hidden markov model to improve protein secretion via Lactococcus lactis bacteria

A hidden Markov model (HMM) has been utilized to predict and generate artificial secretory signal peptide sequences. The strength of signal peptides of proteins from different subcellular locations via Lactococcus lactis bacteria correlated with their HMM bit scores in the model. The results show that the HMM bit score +12 are determined as the threshold for discriminating secreteory signal sequences from the others. The model is used to generate artificial signal peptides with different bit scores for secretory proteins. The signal peptide with the maximum bit score strongly directs proteins secretion.     

Tools to evaluate the conformation of protein products

Production of recombinant proteins is a process intensively used in the research laboratory. In addition, the main biotechnology market products are recombinant proteins and monoclonal antibodies. The biological (and clinical) properties of the protein product strongly depend on the conformation of the polypeptide. Therefore, assessment of the correct conformation of the produced protein is crucial. There is no single method to assess every aspect of protein structure or function. Depending on the protein, the methods of choice vary. There are general methods to evaluate not only mass and primary sequence of the protein, but also higher-order structure. This review outlines the principal techniques for determining the conformation of a protein from structural (biophysical methods) to functional (in vitro binding assays) analyses.     

Type VI secretion system in Pseudomonas aeruginosa: secretion and multimerization of VgrG proteins

Pseudomonas aeruginosa is a Gram-negative bacterium causing chronic infections in cystic fibrosis patients. Such infections are associated with an active type VI secretion system (T6SS), which consists of about 15 conserved components, including the AAA+ ATPase, ClpV. The T6SS secretes two categories of proteins, VgrG and Hcp. Hcp is structurally similar to a phage tail tube component, whereas VgrG proteins show similarity to the puncturing device at the tip of the phage tube. In P. aeruginosa, three T6SSs are known. The expression of H1-T6SS genes is controlled by the RetS sensor. Here, 10 vgrG genes were identified in the PAO1 genome, among which three are co-regulated with H1-T6SS, namely vgrG1a/b/c. Whereas VgrG1a and VgrG1c were secreted in a ClpV1-dependent manner, secretion of VgrG1b was ClpV1-independent. We show that VgrG1a and VgrG1c form multimers, which confirmed the VgrG model predicting trimers similar to the tail spike. We demonstrate that Hcp1 secretion requires either VgrG1a or VgrG1c, which may act independently to puncture the bacterial envelope and give Hcp1 access to the surface. VgrG1b is not required for Hcp1 secretion. Thus, VgrG1b does not require H1-T6SS for secretion nor does H1-T6SS require VgrG1b for its function. Finally, we show that VgrG proteins are required for secretion of a genuine H1-T6SS substrate, Tse3. Our results demonstrate that VgrG proteins are not only secreted components but are essential for secretion of other T6SS substrates. Overall, we emphasize variability in behavior of three P. aeruginosa VgrGs, suggesting that, although very similar, distinct VgrGs achieve specific functions.