The patatin-like protein PlpD forms structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded ß-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal ß-barrel domain. Challenging the current dogma, we find that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring ß-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.

ß-barrel membrane proteins fold via hybrid-barrel intermediate states.

Gram-negative bacteria and eukaryotic organelles of bacterial origin contain outer membrane proteins that possess a transmembrane "ß-barrel" domain. The conserved ß-barrel assembly machine (BAM) and the sorting and assembly machine (SAM) are required for the folding and membrane insertion of ß-barrels in Gram-negative bacteria and mitochondria, respectively. Although the mechanisms by which ß-barrels are folded are incompletely understood, advances in cryo-electron microscopy (cryo-EM) have recently yielded unprecedented insights into their folding process. Here we highlight recent studies that show that both bacterial and mitochondrial ß-barrels fold via the formation of remarkable "hybrid-barrel" intermediate states during their interaction with the folding machinery. We discuss how these results align with a general model of ß-barrel folding.

Molecular Machines that Facilitate Bacterial Outer Membrane Protein Biogenesis.

Almost all outer membrane proteins (OMPs) in Gram-negative bacteria contain a ß-barrel domain that spans the outer membrane (OM). To reach the OM, OMPs must be translocated across the inner membrane by the Sec machinery, transported across the crowded periplasmic space through the assistance of molecular chaperones, and finally assembled (folded and inserted into the OM) by the ß-barrel assembly machine. In this review, we discuss how considerable new insights into the contributions of these factors to OMP biogenesis have emerged in recent years through the development of novel experimental, computational, and predictive methods. In addition, we describe recent evidence that molecular machines that were thought to function independently might interact to form dynamic intermembrane supercomplexes. Finally, we discuss new results that suggest that OMPs are inserted primarily near the middle of the cell and packed into supramolecular structures (OMP islands) that are distributed throughout the OM.

In Vivo Disulfide-Bond Crosslinking to Study ß-Barrel Membrane Protein Interactions, Dynamicity, and Folding Intermediates.

Membrane-embedded ß-barrels are the major building blocks of the Gram-negative outer membrane and are involved in antibiotic resistance, virulence, and the maintenance of bacterial cell physiology. The increased frequency of multidrug resistant Gram-negative infections warrants the sharing of accessible methods for the study of ß-barrels. One such method is "in vivo disulfide-bond crosslinking" which is a highly informative and cost-effective approach to study the structure, topology, dynamicity, and function of ß-barrels in situ. The approach can also be used to identify and finely map both stable or transient interactions between ß-barrels and other interacting proteins. In this chapter, I describe the conceptual basis of in vivo disulfide-bond crosslinking and the potential pitfalls in experimental design. I also provide a general protocol for high-efficiency in vivo disulfide-bond crosslinking and modified protocols as examples for how the method can be adapted to different scenarios.

The patatin-like protein PlpD forms novel structurally dynamic homodimers in the Pseudomonas aeruginosa outer membrane.

Members of the Omp85 superfamily of outer membrane proteins (OMPs) found in Gram-negative bacteria, mitochondria and chloroplasts are characterized by a distinctive 16-stranded ß-barrel transmembrane domain and at least one periplasmic POTRA domain. All previously studied Omp85 proteins promote critical OMP assembly and/or protein translocation reactions. Pseudomonas aeruginosa PlpD is the prototype of an Omp85 protein family that contains an N-terminal patatin-like (PL) domain that is thought to be translocated across the OM by a C-terminal ß-barrel domain. Challenging the current dogma, we found that the PlpD PL-domain resides exclusively in the periplasm and, unlike previously studied Omp85 proteins, PlpD forms a homodimer. Remarkably, the PL-domain contains a segment that exhibits unprecedented dynamicity by undergoing transient strand-swapping with the neighboring ß-barrel domain. Our results show that the Omp85 superfamily is more structurally diverse than currently believed and suggest that the Omp85 scaffold was utilized during evolution to generate novel functions.

Small Molecule Antibiotics Inhibit Distinct Stages of Bacterial Outer Membrane Protein Assembly.

Several antibacterial compounds have recently been discovered that potentially inhibit the activity of BamA, an essential subunit of a heterooligomer (the barrel assembly machinery or BAM) that assembles outer membrane proteins (OMPs) in Gram-negative bacteria, but their mode of action is unclear. To address this issue, we examined the effect of three inhibitors on the biogenesis of a model E. coli OMP (EspP) in vivo. We found that darobactin potently inhibited the interaction of a conserved C-terminal sequence motif (the "ß signal") with BamA, but had no effect on assembly if added at a postbinding stage. In contrast, Polyphor peptide 7 and MRL-494 inhibited both binding and at least one later step of assembly. Taken together with previous studies that analyzed the binding of darobactin and Polyphor peptide 7 to BamA in vitro, our results strongly suggest that the two compounds inhibit BAM function by distinct competitive and allosteric mechanisms. In addition to providing insights into the properties of the antibacterial compounds, our results also provide direct experimental evidence that supports a model in which the binding of the ß signal to BamA initiates the membrane insertion of OMPs. IMPORTANCE There is a clear need to develop novel broad-spectrum antibiotics to address the global problem of antimicrobial resistance. Multiple compounds that have strong antibacterial activity have recently been described that appear to inhibit the activity of the barrel assembly machinery (BAM), an essential complex that catalyzes the assembly (i.e., folding and membrane insertion) of outer membrane proteins (OMPs) in all Gram-negative bacteria. We analyzed the effects of three of these compounds on OMP biogenesis in vivo and found that they inhibited different stages of the assembly process. Because these compounds have distinct modes of action, they can be used in combination to reduce the emergence of resistant strains. As a corollary, we obtained evidence that these compounds will be valuable tools in future studies on BAM function.

Function of the Omp85 Superfamily of Outer Membrane Protein Assembly Factors and Polypeptide Transporters.

The Omp85 protein superfamily is found in the outer membrane (OM) of all gram-negative bacteria and eukaryotic organelles of bacterial origin. Members of the family catalyze both the membrane insertion of ß-barrel proteins and the translocation of proteins across the OM. Although the mechanism(s) by which these proteins function is unclear, striking new insights have emerged from recent biochemical and structural studies. In this review we discuss the entire Omp85 superfamily but focus on the function of the best-studied member, BamA, which is an essential and highly conserved component of the bacterial barrel assembly machinery (BAM). Because BamA has multiple functions that overlap with those of other Omp85 proteins, it is likely the prototypical member of the Omp85 superfamily. Furthermore, BamA has become a protein of great interest because of the recent discovery of small-molecule inhibitors that potentially represent an important new class of antibiotics.

Cryo-EM structures reveal multiple stages of bacterial outer membrane protein folding.

Transmembrane ß barrel proteins are folded into the outer membrane (OM) of Gram-negative bacteria by the ß barrel assembly machinery (BAM) via a poorly understood process that occurs without known external energy sources. Here, we used single-particle cryo-EM to visualize the folding dynamics of a model ß barrel protein (EspP) by BAM. We found that BAM binds the highly conserved "ß signal" motif of EspP to correctly orient ß strands in the OM during folding. We also found that the folding of EspP proceeds via "hybrid-barrel" intermediates in which membrane integrated ß sheets are attached to the essential BAM subunit, BamA. The structures show an unprecedented deflection of the membrane surrounding the EspP intermediates and suggest that ß sheets progressively fold toward BamA to form a ß barrel. Along with in vivo experiments that tracked ß barrel folding while the OM tension was modified, our results support a model in which BAM harnesses OM elasticity to accelerate ß barrel folding.

BamA forms a translocation channel for polypeptide export across the bacterial outer membrane.

The ß-barrel assembly machine (BAM) integrates ß-barrel proteins into the outer membrane (OM) of Gram-negative bacteria. An essential BAM subunit (BamA) catalyzes integration by promoting the formation of a hybrid-barrel intermediate state between its own ß-barrel domain and that of its client proteins. Here we show that in addition to catalyzing the integration of ß-barrel proteins, BamA functions as a polypeptide export channel. In vivo structural mapping via intermolecular disulfide crosslinking showed that the extracellular "passenger" domain of a member of the "autotransporter" superfamily of virulence factors traverses the OM through the BamA ß-barrel lumen. Furthermore, we demonstrate that a highly conserved residue within autotransporter ß-barrels is required to position the passenger inside BamA to initiate translocation and that during translocation, the passenger stabilizes the hybrid-barrel state. Our results not only establish a new function for BamA but also unify the divergent functions of BamA and other "Omp85" superfamily transporters.

The virulence domain of Shigella IcsA contains a subregion with specific host cell adhesion function.

Shigella species cause bacillary dysentery, especially among young individuals. Shigellae target the human colon for invasion; however, the initial adhesion mechanism is poorly understood. The Shigella surface protein IcsA, in addition to its role in actin-based motility, acts as a host cell adhesin through unknown mechanism(s). Here we confirmed the role of IcsA in cell adhesion and defined the region required for IcsA adhesin activity. Purified IcsA passenger domain was able block S. flexneri adherence and was also used as a molecular probe that recognised multiple components from host cells. The region within IcsA's functional passenger domain (aa 138-148) was identified by mutagenesis. Upon the deletion of this region, the purified IcsAΔ138-148 was found to no longer block S. flexneri adherence and had reduced ability to interact with host molecules. Furthermore, S. flexneri expressing IcsAΔ138-148 was found to be significantly defective in both cell adherence and invasion. Taken together, our data identify an adherence region within the IcsA functional domain and provides useful information for designing therapeutics for Shigella infection.

Unprecedented Abundance of Protein Tyrosine Phosphorylation Modulates Shigella flexneri Virulence.

Evidence is accumulating that protein tyrosine phosphorylation plays a crucial role in the ability of important human bacterial pathogens to cause disease. While most works have concentrated on its role in the regulation of a major bacterial virulence factor, the polysaccharide capsule, recent studies have suggested a much broader role for this post-translational modification. This prompted us to investigate protein tyrosine phosphorylation in the human pathogen Shigella flexneri. We first completed a tyrosine phosphoproteome, identifying 905 unique tyrosine phosphorylation sites on at least 573 proteins (approximately 15% of all proteins). This is the most tyrosine-phosphorylated sites and proteins in a single bacterium identified to date, substantially more than the level seen in eukaryotic cells. Most had not previously been identified and included proteins encoded by the virulence plasmid, which is essential for S. flexneri to invade cells and cause disease. In order to investigate the function of these phosphorylation sites in important virulence factors, phosphomimetic and ablative mutations were constructed in the type 3 secretion system ATPase Spa47 and the master virulence regulator VirB. This revealed that tyrosine residues phosphorylated in our study are critical for Spa47 and VirB activity, and tyrosine phosphorylation likely regulates their functional activity and subsequently the virulence of this major human pathogen. This study suggests that tyrosine phosphorylation plays a critical role in regulating a wide variety of virulence factors in the human pathogen S. flexneri and serves as a base for future studies defining its complete role.

A small conserved motif supports polarity augmentation of Shigella flexneri IcsA.

The rod-shaped enteric intracellular pathogen Shigella flexneri and other Shigella species are the causative agents of bacillary dysentery. S. flexneri are able to spread within the epithelial lining of the gut, resulting in lesion formation, cramps and bloody stools. The outer membrane protein IcsA is essential for this spreading process. IcsA is the initiator of an actin-based form of motility whereby it allows the formation of a filamentous actin 'tail' at the bacterial pole. Importantly, IcsA is specifically positioned at the bacterial pole such that this process occurs asymmetrically. The mechanism of IcsA polarity is not completely understood, but it appears to be a multifactorial process involving factors intrinsic to IcsA and other regulating factors. In this study, we further investigated IcsA polarization by its intramolecular N-terminal and central polar-targeting (PT) regions (nPT and cPT regions, respectively). The results obtained support a role in polar localization for the cPT region and contend the role of the nPT region. We identified single IcsA residues that have measurable impacts on IcsA polarity augmentation, resulting in decreased S. flexneri sprading efficiency. Intriguingly, regions and residues involved in PT clustered around a highly conserved motif which may provide a functional scaffold for polarity-augmenting residues. How these results fit with the current model of IcsA polarity determination is discussed.

The passenger-associated transport repeat promotes virulence factor secretion efficiency and delineates a distinct autotransporter subtype.

Autotransporters are a superfamily of virulence factors secreted by Gram negative bacteria. They are comprised of an N-terminal passenger domain that is translocated across the outer membrane and a C-terminal domain that inserts into the outer membrane forming a ß-barrel anchor. It is still poorly understood how the passenger is efficiently translocated in the absence of external energy inputs. Several mechanisms have been proposed in solution of this problem, yet due to the vast diversity of size, sequence and function of the passenger, it is not clear how widely these mechanisms are employed. In this study we functionally characterize a conserved repeat found in many passengers that we designate the Passenger-associated Transport Repeat (PATR). Using the autotransporter IcsA from the enteropathogen Shigella flexneri, we identified conserved PATR residues that are required for efficient export of the passenger during growth and infection. Furthermore, PATR-containing autotransporters are significantly larger than non-PATR autotransporters, with PATR copy number correlating with passenger size. We also show that PATR-containing autotransporters delineate a subgroup that associates with specific virulence traits and architectures. These results advance our understanding of autotransporter composition and indicate that an additional transport mechanism is important for thousands of these proteins.

Lipopolysaccharide surface structure does not influence IcsA polarity.

Shigella species are the causative agents of human bacillary dysentery. These bacteria spread within the lining of the gut via a process termed actin-based motility whereby an actin 'tail' is formed at the bacterial pole. The bacterial outer membrane protein IcsA initiates this process, and crucially is precisely positioned on the bacterial polar surface. Lipopolysaccharide (LPS) O-antigen surface structure has been implicated as an augmenting factor of polarity maintenance due to the apparent dysregulation of IcsA polarity in O-antigen deficient strains. Due to Shigellae having long and short O-antigen chains on their surfaces, it has been proposed that O-antigen chain lengths are asymmetrically distributed to optimize IcsA exposure at the pole and mask exposure laterally. Additionally, it has been proposed that LPS O-antigen restricts IcsA diffusion from the pole by maintaining minimal membrane fluidity. This study utilizes minicells and quantitative microscopy providing data refuting the models of asymmetric masking and membrane diffusion, and supporting a model of symmetric masking of IcsA. We contend that IcsA surface distribution is equivalent between wild-type and O-antigen deficient strains, and that differences in cellular IcsA levels have confounded previous conclusions.

LPS unmasking of Shigella flexneri reveals preferential localisation of tagged outer membrane protease IcsP to septa and new poles.

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP(HA) was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP(HA) was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP(HA) was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP(HA) and IcsA showed that IcsP(HA) preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP(HA) in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP(HA) was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP(HA) detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.