Of linkers and autochaperones: an unambiguous nomenclature to identify common and uncommon themes for autotransporter secretion.

Autotransporter (AT) proteins provide a diverse array of important virulence functions to Gram-negative bacterial pathogens, and have also been adapted for protein surface display applications. The 'autotransporter' moniker refers to early models that depicted these proteins facilitating their own translocation across the bacterial outer membrane. Although translocation is less autonomous than originally proposed, AT protein segments upstream of the C-terminal transmembrane ß-barrel have nevertheless consistently been found to contribute to efficient translocation and/or folding of the N-terminal virulence region (the 'passenger'). However, defining the precise secretion functions of these AT regions has been complicated by the use of multiple overlapping and ambiguous terms to define AT sequence, structural, and functional features, including 'autochaperone', 'linker' and 'junction'. Moreover, the precise definitions and boundaries of these features vary among ATs and even among research groups, leading to an overall murky picture of the contributions of specific features to translocation. Here we propose a unified, unambiguous nomenclature for AT structural, functional and conserved sequence features, based on explicit criteria. Applied to 16 well-studied AT proteins, this nomenclature reveals new commonalities for translocation but also highlights that the autochaperone function is less closely associated with a conserved sequence element than previously believed.

Assembly of ß-barrel proteins into bacterial outer membranes.

Membrane proteins with a ß-barrel topology are found in the outer membranes of Gram-negative bacteria and in the plastids and mitochondria of eukaryotic cells. The assembly of these membrane proteins depends on a protein folding reaction (to create the barrel) and an insertion reaction (to integrate the barrel within the outer membrane). Experimental approaches using biophysics and biochemistry are detailing the steps in the assembly pathway, while genetics and bioinformatics have revealed a sophisticated production line of cellular components that catalyze the assembly pathway in vivo. This includes the modular BAM complex, several molecular chaperones and the translocation and assembly module (the TAM). Recent screens also suggest that further components of the pathway might remain to be discovered. We review what is known about the process of ß-barrel protein assembly into membranes, and the components of the ß-barrel assembly machinery. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

Structure and function of BamE within the outer membrane and the ß-barrel assembly machine.

Insertion of folded proteins into the outer membrane of Gram-negative bacteria is mediated by the essential ß-barrel assembly machine (Bam). Here, we report the native structure and mechanism of a core component of this complex, BamE, and show that it is exclusively monomeric in its native environment of the periplasm, but is able to adopt a distinct dimeric conformation in the cytoplasm. BamE is shown to bind specifically to phosphatidylglycerol, and comprehensive mutagenesis and interaction studies have mapped key determinants for complex binding, outer membrane integrity and cell viability, as well as revealing the role of BamE within the Bam complex.

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.

Transcription of the plasmid-encoded toxin gene from enteroaggregative Escherichia coli is regulated by a novel co-activation mechanism involving CRP and Fis.

Enteroaggregative Escherichia coli (EAEC) is a major cause of diarrhoea in developing countries. EAEC 042 is the prototypical strain. EAEC 042 secretes the functionally well-characterized Pet autotransporter toxin that contributes to virulence through its cytotoxic effects on intestinal epithelial cells. Following a global transposon mutagenesis screen of EAEC 042, the transcription factors, CRP and Fis, were identified as essential for transcription of the pet gene. Using both in vivo and in vitro techniques, we show that the pet promoter is co-dependent on CRP and Fis. We present a novel co-activation mechanism whereby CRP is placed at a non-optimal position for transcription initiation, creating dependence on Fis for full activation of pet. This study complements previous findings that establish Fis as a key virulence regulator in EAEC 042.

Soluble flagellin, FliC, induces an Ag-specific Th2 response, yet promotes T-bet-regulated Th1 clearance of Salmonella typhimurium infection.

Clearance of disseminated Salmonella infection requires bacterial-specific Th1 cells and IFN-γ production, and Th1-promoting vaccines are likely to help control these infections. Consequently, vaccine design has focused on developing Th1-polarizing adjuvants or Ag that naturally induce Th1 responses. In this study, we show that, in mice, immunization with soluble, recombinant FliC protein flagellin (sFliC) induces Th2 responses as evidenced by Ag-specific GATA-3, IL-4 mRNA, and protein induction in CD62L(lo) CD4(+) T cells without associated IFN-γ production. Despite these Th2 features, sFliC immunization can enhance the development of protective Th1 immunity during subsequent Salmonella infection in an Ab-independent, T-cell-dependent manner. Salmonella infection in sFliC-immunized mice resulted in augmented Th1 responses, with greater bacterial clearance and increased numbers of IFN-γ-producing CD4(+) T cells, despite the early induction of Th2 features to sFliC. The augmented Th1 immunity after sFliC immunization was regulated by T-bet although T-bet is dispensable for primary responses to sFliC. These findings show that there can be flexibility in T-cell responses to some subunit vaccines. These vaccines may induce Th2-type immunity during primary immunization yet promote Th1-dependent responses during later infection. This suggests that designing Th1-inducing subunit vaccines may not always be necessary since this can occur naturally during subsequent infection.

A generalised module for the selective extracellular accumulation of recombinant proteins.

BACKGROUND: It is widely believed that laboratory strains of Escherichia coli, including those used for industrial production of proteins, do not secrete proteins to the extracellular milieu. RESULTS: Here, we report the development of a generalised module, based on an E. coli autotransporter secretion system, for the production of extracellular recombinant proteins. We demonstrate that a wide variety of structurally diverse proteins can be secreted as soluble proteins when linked to the autotransporter module. Yields were comparable to those achieved with other bacterial secretion systems. CONCLUSIONS: The advantage of this module is that it relies on a relatively simple and easily manipulated secretion system, exhibits no apparent limitation to the size of the secreted protein and can deliver proteins to the extracellular environment at levels of purity and yields sufficient for many biotechnological applications.

The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response.

Invasive nontyphoidal Salmonella (NTS), including Salmonella typhimurium (STm), are major yet poorly-recognized killers of infants in sub-Saharan Africa. Death in these children is usually associated with bacteremia, commonly in the absence of gastrointestinal symptoms. Evidence from humans and animal studies suggest that severe infection and bacteremia occur when specific Ab is lacking. Understanding how Ab responses to Salmonella are regulated will help develop vaccines against these devastating infections. STm induces atypical Ab responses characterized by prominent, accelerated, extrafollicular T-independent (TI) Ab against a range of surface antigens. These responses develop without concomitant germinal centers, which only appear as infection resolves. Here, we show STm rapidly induces a population of TI B220(+)CD5(-) B1b cells during infection and TI Ab from B1b cells targets the outer membrane protein (Omp) porins OmpC, OmpD and OmpF but not flagellin. When porins are used as immunogens they can ablate bacteremia and provide equivalent protection against STm as killed bacterial vaccine and this is wholly B cell-dependent. Furthermore Ab from porin-immunized chimeras, that have B1b cells, is sufficient to impair infection. Infecting with porin-deficient bacteria identifies OmpD, a protein absent from Salmonella Typhi, as a key target of Ab in these infections. This work broadens the recognized repertoire of TI protein antigens and highlights the importance of Ab from different B cell subsets in controlling STm infection. OmpD is a strong candidate vaccine target and may, in part, explain the lack of cross-protection between Salmonella Typhi and STm infections.

The essential ß-barrel assembly machinery complex components BamD and BamA are required for autotransporter biogenesis.

Autotransporter biogenesis is dependent upon BamA, a central component of the ß-barrel assembly machinery (BAM) complex. In this report, we detail the role of the other BAM components (BamB-E). We identify the importance of BamD in autotransporter biogenesis and show that BamB, BamC, and BamE are not required.

SadA, a trimeric autotransporter from Salmonella enterica serovar Typhimurium, can promote biofilm formation and provides limited protection against infection.

Salmonella enterica is a major cause of morbidity worldwide and mortality in children and immunocompromised individuals in sub-Saharan Africa. Outer membrane proteins of Salmonella are of significance because they are at the interface between the pathogen and the host, they can contribute to adherence, colonization, and virulence, and they are frequently targets of antibody-mediated immunity. In this study, the properties of SadA, a purported trimeric autotransporter adhesin of Salmonella enterica serovar Typhimurium, were examined. We demonstrated that SadA is exposed on the Salmonella cell surface in vitro and in vivo during infection of mice. Expression of SadA resulted in cell aggregation, biofilm formation, and increased adhesion to human intestinal Caco-2 epithelial cells. Immunization of mice with folded, full-length, purified SadA elicited an IgG response which provided limited protection against bacterial challenge. When anti-SadA IgG titers were enhanced by administering alum-precipitated protein, a modest additional protection was afforded. Therefore, despite SadA having pleiotropic functions, it is not a dominant, protective antigen for antibody-mediated protection against Salmonella.

The efficiency of nuclear plasmid DNA delivery is a critical determinant of transgene expression at the single cell level.

BACKGROUND: The nuclear envelope that encloses the nucleus is a significant barrier to non-viral vectors and shrouds the relationship between the trafficking of plasmid DNA to the nucleus and expression of an encoded transgene. Here, we use a novel single cell approach to quantify nuclear import of plasmid DNA following non-viral transfection and correlate this with reporter gene expression. METHODS: Through the fractionation of intact nuclei from HeLa cells, the intranuclear copy number of plasmid DNA was quantified after transfection with either polyethylenimine (PEI) or LipofectAMINE2000 (LFA). Importantly, the use of a reporter protein that is incorporated into chromatin and retained in isolated nuclei permits analysis of gene expression by flow cytometry to be compared with nuclear plasmid delivery. RESULTS: PEI was found to mediate a greater and more rapid nuclear accumulation of plasmid DNA compared to LFA, but reporter gene expression was shown to be higher for LFA than PEI when an equivalent number of plasmids were in the nucleus. Sorting of the extracted nuclei according to the level of reporter expression demonstrated that reporter expression was dependent upon the number of plasmids delivered into the nucleus, with both threshold and saturation in expression evident with few or many nuclear plasmids. CONCLUSIONS: Our findings demonstrate formally that although the efficiency of plasmid nuclear delivery is a critical determinant of the level of transgene expression, intranuclear events also influence the transcriptional activity of the transgene, and must be taken into consideration when attempting to maximize the efficiency of non-viral vectors.

Dynein light chain association sequences can facilitate nuclear protein import.

Nuclear localization sequence (NLS)-dependent nuclear protein import is not conventionally held to require interaction with microtubules (MTs) or components of the MT motor, dynein. Here we report for the first time the role of sequences conferring association with dynein light chains (DLCs) in NLS-dependent nuclear accumulation of the rabies virus P-protein. We find that P-protein nuclear accumulation is significantly enhanced by its dynein light chain association sequence (DLC-AS), dependent on MT integrity and association with DLCs, and that P-protein-DLC complexes can associate with MT cytoskeletal structures. We also find that P-protein DLC-AS, as well as analogous sequences from other proteins, acts as an independent module that can confer enhancement of nuclear accumulation to proteins carrying the P-protein NLS, as well as several heterologous NLSs. Photobleaching experiments in live cells demonstrate that the MT-dependent enhancement of NLS-mediated nuclear accumulation by the P-protein DLC-AS involves an increased rate of nuclear import. This is the first report of DLC-AS enhancement of NLS function, identifying a novel mechanism regulating nuclear transport with relevance to viral and cellular protein biology. Importantly, this data indicates that DLC-ASs represent versatile modules to enhance nuclear delivery with potential therapeutic application.

Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome.

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome.

Roles of periplasmic chaperone proteins in the biogenesis of serine protease autotransporters of Enterobacteriaceae.

The serine protease autotransporters of Enterobacteriaceae (SPATEs) represent a large family of virulence factors. The prevailing model for autotransporter secretion comprises entry to the periplasm via the Sec apparatus, followed by an obscure series of steps in which the C terminus of the periplasmic species inserts into the outer membrane as a beta-barrel protein, accompanied by translocation of the passenger domain to the bacterial cell surface. Little is known about the fate of the autotransporter proteins in the periplasm, including whether accessory periplasmic proteins are involved in translocation to the external milieu. Here we studied the role of the major periplasmic chaperones in the biogenesis of EspP, a prototype SPATE protein produced by Escherichia coli O157:H7. The yeast two-hybrid approach, secretion analysis of chaperone mutant strains, and surface plasmon resonance analysis (SPR) revealed direct protein-protein interactions between the periplasmic SurA and DegP chaperones and either the EspP-beta or EspP passenger domains. The secretion of EspP was moderately reduced in the surA and skp mutant strains but severely impaired in the degP background. Site-directed mutagenesis of highly conserved aromatic amino acid residues in the SPATE family resulted in approximately 80% reduction of EspP secretion. Synthetic peptides containing aromatic residues derived from the EspP passenger domain blocked DegP and SurA binding to the passenger domain. SPR suggested direct protein-protein interaction between periplasmic chaperones and the unfolded EspP passenger domain. Our data suggest that translocation of AT proteins may require accessory factors, calling into question the moniker "autotransporter."

Molecular basis for the folding of ß-helical autotransporter passenger domains.

Bacterial autotransporters comprise a C-terminal ß-barrel domain, which must be correctly folded and inserted into the outer membrane to facilitate translocation of the N-terminal passenger domain to the cell exterior. Once at the surface, the passenger domains of most autotransporters are folded into an elongated ß-helix. In a cellular context, key molecules catalyze the assembly of the autotransporter ß-barrel domain. However, how the passenger domain folds into its functional form is poorly understood. Here we use mutational analysis on the autotransporter Pet to show that the ß-hairpin structure of the fifth extracellular loop of the ß-barrel domain has a crucial role for passenger domain folding into a ß-helix. Bioinformatics and structural analyses, and mutagenesis of a homologous autotransporter, suggest that this function is conserved among autotransporter proteins with ß-helical passenger domains. We propose that the autotransporter ß-barrel domain is a folding vector that nucleates folding of the passenger domain.

The ß-Barrel Assembly Machinery Complex.

The outer membranes of gram-negative bacteria contain integral membrane proteins, most of which are of ß-barrel structure, and critical for bacterial survival. These ß-barrel proteins rely on the ß-barrel assembly machinery (BAM) complex for their integration into the outer membrane as folded species. The central and essential subunit of the BAM complex, BamA, is a ß-barrel protein conserved in all gram-negative bacteria and also found in eukaryotic organelles derived from bacterial endosymbionts. In Escherichia coli, BamA docks with four peripheral lipoproteins, BamB, BamC, BamD and BamE, partner subunits that add to the function of the BAM complex in outer membrane protein biogenesis. By way of introduction to this volume, we provide an overview of the work that has illuminated the mechanism by which the BAM complex drives ß-barrel assembly. The protocols and methodologies associated with these studies as well as the challenges encountered and their elegant solutions are discussed in subsequent chapters.

Reconstitution of a nanomachine driving the assembly of proteins into bacterial outer membranes.

In biological membranes, various protein secretion devices function as nanomachines, and measuring the internal movements of their component parts is a major technological challenge. The translocation and assembly module (TAM) is a nanomachine required for virulence of bacterial pathogens. We have reconstituted a membrane containing the TAM onto a gold surface for characterization by quartz crystal microbalance with dissipation (QCM-D) and magnetic contrast neutron reflectrometry (MCNR). The MCNR studies provided structural resolution down to 1 Å, enabling accurate measurement of protein domains projecting from the membrane layer. Here we show that dynamic movements within the TamA component of the TAM are initiated in the presence of a substrate protein, Ag43, and that these movements recapitulate an initial stage in membrane protein assembly. The reconstituted system provides a powerful new means to study molecular movements in biological membranes, and the technology is widely applicable to studying the dynamics of diverse cellular nanomachines.

A mortise-tenon joint in the transmembrane domain modulates autotransporter assembly into bacterial outer membranes.

Bacterial autotransporters comprise a 12-stranded membrane-embedded ß-barrel domain, which must be folded in a process that entraps segments of an N-terminal passenger domain. This first stage of autotransporter folding determines whether subsequent translocation can deliver the N-terminal domain to its functional form on the bacterial cell surface. Here, paired glycine-aromatic 'mortise and tenon' motifs are shown to join neighbouring ß-strands in the C-terminal barrel domain, and mutations within these motifs slow the rate and extent of passenger domain translocation to the surface of bacterial cells. In line with this, biophysical studies of the autotransporter Pet show that the conserved residues significantly quicken completion of the folding reaction and promote stability of the autotransporter barrel domain. Comparative genomics demonstrate conservation of glycine-aromatic residue pairings through evolution as a previously unrecognized feature of all autotransporter proteins.

From self sufficiency to dependence: mechanisms and factors important for autotransporter biogenesis.

Autotransporters are a superfamily of proteins that use the type V secretion pathway for their delivery to the surface of Gram-negative bacteria. At first glance, autotransporters look to contain all the functional elements required to promote their own secretion: an amino-terminal signal peptide to mediate translocation across the inner membrane, a central passenger domain that is the secreted functional moiety, and a channel-forming carboxyl terminus that facilitates passenger domain translocation across the outer membrane. However, recent discoveries of common structural themes, translocation intermediates and accessory interactions have challenged the perceived simplicity of autotransporter secretion. Here, we discuss how these studies have led to an improved understanding of the mechanisms responsible for autotransporter biogenesis.

A bioinformatic strategy for the detection, classification and analysis of bacterial autotransporters.

Autotransporters are secreted proteins that are assembled into the outer membrane of bacterial cells. The passenger domains of autotransporters are crucial for bacterial pathogenesis, with some remaining attached to the bacterial surface while others are released by proteolysis. An enigma remains as to whether autotransporters should be considered a class of secretion system, or simply a class of substrate with peculiar requirements for their secretion. We sought to establish a sensitive search protocol that could identify and characterize diverse autotransporters from bacterial genome sequence data. The new sequence analysis pipeline identified more than 1500 autotransporter sequences from diverse bacteria, including numerous species of Chlamydiales and Fusobacteria as well as all classes of Proteobacteria. Interrogation of the proteins revealed that there are numerous classes of passenger domains beyond the known proteases, adhesins and esterases. In addition the barrel-domain-a characteristic feature of autotransporters-was found to be composed from seven conserved sequence segments that can be arranged in multiple ways in the tertiary structure of the assembled autotransporter. One of these conserved motifs overlays the targeting information required for autotransporters to reach the outer membrane. Another conserved and diagnostic motif maps to the linker region between the passenger domain and barrel-domain, indicating it as an important feature in the assembly of autotransporters.