Pseudomonas aeruginosa MipA-MipB envelope proteins act as new sensors of polymyxins.

Due to the rising incidence of antibiotic-resistant infections, the last-line antibiotics, polymyxins, have resurged in the clinics in parallel with new bacterial strategies of escape. The Gram-negative opportunistic pathogen Pseudomonas aeruginosa develops resistance to colistin/polymyxin B by distinct molecular mechanisms, mostly through modification of the lipid A component of the LPS by proteins encoded within the arnBCDATEF-ugd (arn) operon. In this work, we characterized a polymyxin-induced operon named mipBA, present in P. aeruginosa strains devoid of the arn operon. We showed that mipBA is activated by the ParR/ParS two-component regulatory system in response to polymyxins. Structural modeling revealed that MipA folds as an outer-membrane ß-barrel, harboring an internal negatively charged channel, able to host a polymyxin molecule, while the lipoprotein MipB adopts a ß-lactamase fold with two additional C-terminal domains. Experimental work confirmed that MipA and MipB localize to the bacterial envelope, and they co-purify in vitro. Nano differential scanning fluorimetry showed that polymyxins stabilized MipA in a specific and dose-dependent manner. Mass spectrometry-based quantitative proteomics on P. aeruginosa membranes demonstrated that ∆mipBA synthesized fourfold less MexXY-OprA proteins in response to polymyxin B compared to the wild-type strain. The decrease was a direct consequence of impaired transcriptional activation of the mex operon operated by ParR/ParS. We propose MipA/MipB to act as membrane (co)sensors working in concert to activate ParS histidine kinase and help the bacterium to cope with polymyxin-mediated envelope stress through synthesis of the efflux pump, MexXY-OprA.IMPORTANCEDue to the emergence of multidrug-resistant isolates, antibiotic options may be limited to polymyxins to eradicate Gram-negative infections. Pseudomonas aeruginosa, a leading opportunistic pathogen, has the ability to develop resistance to these cationic lipopeptides by modifying its lipopolysaccharide through proteins encoded within the arn operon. Herein, we describe a sub-group of P. aeruginosa strains lacking the arn operon yet exhibiting adaptability to polymyxins. Exposition to sub-lethal polymyxin concentrations induced the expression and production of two envelope-associated proteins. Among those, MipA, an outer-membrane barrel, is able to specifically bind polymyxins with an affinity in the 10-µM range. Using membrane proteomics and phenotypic assays, we showed that MipA and MipB participate in the adaptive response to polymyxins via ParR/ParS regulatory signaling. We propose a new model wherein the MipA-MipB module functions as a novel polymyxin sensing mechanism.

Architecture and genomic arrangement of the MurE-MurF bacterial cell wall biosynthesis complex.

Peptidoglycan (PG) is a central component of the bacterial cell wall, and the disruption of its biosynthetic pathway has been a successful antibacterial strategy for decades. PG biosynthesis is initiated in the cytoplasm through sequential reactions catalyzed by Mur enzymes that have been suggested to associate into a multimembered complex. This idea is supported by the observation that in many eubacteria, mur genes are present in a single operon within the well conserved dcw cluster, and in some cases, pairs of mur genes are fused to encode a single, chimeric polypeptide. We performed a vast genomic analysis using >140 bacterial genomes and mapped Mur chimeras in numerous phyla, with Proteobacteria carrying the highest number. MurE-MurF, the most prevalent chimera, exists in forms that are either directly associated or separated by a linker. The crystal structure of the MurE-MurF chimera from Bordetella pertussis reveals a head-to-tail, elongated architecture supported by an interconnecting hydrophobic patch that stabilizes the positions of the two proteins. Fluorescence polarization assays reveal that MurE-MurF interacts with other Mur ligases via its central domains with KDs in the high nanomolar range, backing the existence of a Mur complex in the cytoplasm. These data support the idea of stronger evolutionary constraints on gene order when encoded proteins are intended for association, establish a link between Mur ligase interaction, complex assembly and genome evolution, and shed light on regulatory mechanisms of protein expression and stability in pathways of critical importance for bacterial survival.

Genomic erosion and horizontal gene transfer shape functional differences of the ExlA toxin in Pseudomonas spp.

Two-partner secretion (TPS) is widespread in the bacterial world. The pore-forming TPS toxin ExlA of Pseudomonas aeruginosa is conserved in pathogenic and environmental Pseudomonas. While P. chlororaphis and P. entomophila displayed ExlA-dependent killing, P. putida did not cause damage to eukaryotic cells. ExlA proteins interacted with epithelial cell membranes; however, only ExlA Pch induced the cleavage of the adhesive molecule E-cadherin. ExlA proteins participated in insecticidal activity toward the larvae of Galleria mellonella and the fly Drosophila melanogaster. Evolutionary analyses demonstrated that the differences in the C-terminal domains are partly due to horizontal movements of the operon within the genus Pseudomonas. Reconstruction of the evolutionary history revealed the complex horizontal acquisitions. Together, our results provide evidence that conserved TPS toxins in environmental Pseudomonas play a role in bacteria-insect interactions and discrete differences in CTDs may determine their specificity and mode of action toward eukaryotic cells.

ExlA Pore-Forming Toxin: Localization at the Bacterial Membrane, Regulation of Secretion by Cyclic-Di-GMP, and Detection In Vivo.

ExlA is a highly virulent pore-forming toxin that has been recently discovered in outlier strains from Pseudomonas aeruginosa. ExlA is part of a two-partner secretion system, in which ExlA is the secreted passenger protein and ExlB the transporter embedded in the bacterial outer membrane. In previous work, we observed that ExlA toxicity in a host cell was contact-dependent. Here, we show that ExlA accumulates at specific points of the outer membrane, is likely entrapped within ExlB pore, and is pointing outside. We further demonstrate that ExlA is maintained at the membrane in conditions where the intracellular content of second messenger cyclic-di-GMP is high; lowering c-di-GMP levels enhances ExlB-dependent ExlA secretion. In addition, we set up an ELISA to detect ExlA, and we show that ExlA is poorly secreted in liquid culture, while it is highly detectable in broncho-alveolar lavage fluids of mice infected with an exlA+ strain. We conclude that ExlA translocation is halted at mid-length in the outer membrane and its secretion is regulated by c-di-GMP. In addition, we developed an immunological test able to quantify ExlA in biological samples.

The bacterial toxin ExoU requires a host trafficking chaperone for transportation and to induce necrosis.

Pseudomonas aeruginosa can cause nosocomial infections, especially in ventilated or cystic fibrosis patients. Highly pathogenic isolates express the phospholipase ExoU, an effector of the type III secretion system that acts on plasma membrane lipids, causing membrane rupture and host cell necrosis. Here, we use a genome-wide screen to discover that ExoU requires DNAJC5, a host chaperone, for its necrotic activity. DNAJC5 is known to participate in an unconventional secretory pathway for misfolded proteins involving anterograde vesicular trafficking. We show that DNAJC5-deficient human cells, or Drosophila flies knocked-down for the DNAJC5 orthologue, are largely resistant to ExoU-dependent virulence. ExoU colocalizes with DNAJC5-positive vesicles in the host cytoplasm. DNAJC5 mutations preventing vesicle trafficking (previously identified in adult neuronal ceroid lipofuscinosis, a human congenital disease) inhibit ExoU-dependent cell lysis. Our results suggest that, once injected into the host cytoplasm, ExoU docks to DNAJC5-positive secretory vesicles to reach the plasma membrane, where it can exert its phospholipase activity.

Self-association of MreC as a regulatory signal in bacterial cell wall elongation.

The elongasome, or Rod system, is a protein complex that controls cell wall formation in rod-shaped bacteria. MreC is a membrane-associated elongasome component that co-localizes with the cytoskeletal element MreB and regulates the activity of cell wall biosynthesis enzymes, in a process that may be dependent on MreC self-association. Here, we use electron cryo-microscopy and X-ray crystallography to determine the structure of a self-associated form of MreC from Pseudomonas aeruginosa in atomic detail. MreC monomers interact in head-to-tail fashion. Longitudinal and lateral interfaces are essential for oligomerization in vitro, and a phylogenetic analysis of proteobacterial MreC sequences indicates the prevalence of the identified interfaces. Our results are consistent with a model where MreC's ability to alternate between self-association and interaction with the cell wall biosynthesis machinery plays a key role in the regulation of elongasome activity.

Exolysin (ExlA) from Pseudomonas aeruginosa Punctures Holes into Target Membranes Using a Molten Globule Domain.

Bacteria employ several mechanisms, and most notably secretion systems, to translocate effectors from the cytoplasm to the extracellular environment or the cell surface. Pseudomonas aeruginosa widely employs secretion machineries such as the Type III Secretion System to support virulence and cytotoxicity. However, recently identified P. aeruginosa strains that do not express the Type III Secretion System have been shown to express ExlA, an exolysin translocated through a two-partner secretion system, and are the causative agents of severe lung hemorrhage. Sequence predictions of ExlA indicate filamentous hemagglutinin (FHA-2) domains as the prevalent features, followed by a C-terminal domain with no known homologs. In this work, we have addressed the mechanism employed by ExlA to target membrane bilayers by using NMR, small-angle X-ray scattering, atomic force microscopy, and cellular infection techniques. We show that the C-terminal domain of ExlA displays a "molten globule-like" fold that punctures small holes into membranes composed of negatively charged lipids, while other domains could play a lesser role in target recognition. In addition, epithelial cells infected with P. aeruginosa strains expressing different ExlA variants allow localization of the toxin to lipid rafts. ExlA homologs have been identified in numerous bacterial strains, indicating that lipid bilayer destruction is an effective strategy employed by bacteria to establish interactions with multiple hosts.

Baseplate Component TssK and Spatio-Temporal Assembly of T6SS in Pseudomonas aeruginosa.

The Gram-negative bacteria use the contractile multi-molecular structure, called the Type VI Secretion System (T6SS) to inject toxic products into eukaryotic and prokaryotic cells. In this study, we use fluorescent protein fusions and time-lapse microscopy imaging to study the assembly dynamics of the baseplate protein TssK in Pseudomonas aeruginosa T6SS. TssK formed transient higher-order structures that correlated with dynamics of sheath component TssB. Assembly of peri-membrane TssK structures occurred de novo upon contact with competing bacteria. We show that this assembly required presence of TagQ-TagR envelope sensors, activity of PpkA kinase and anchoring to the inner membrane via TssM. Disassembly and repositioning of TssK component was dependent on PppA phosphatase and indispensable for repositioning and deployment of the entire contractile apparatus toward a new target cell. We also show that TssE is necessary for correct elongation and stability of TssB-sheath, but not for TssK assembly. Therefore, in P. aeruginosa, assembly of the TssK-containing structure relays on the post-translational regulatory envelope module and acts as spatio-temporal marker for further recruitment and subsequent assembly of the contractile apparatus.

Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system.

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.

Structural and Functional Characterization of the Type Three Secretion System (T3SS) Needle of Pseudomonas aeruginosa.

The type three secretion system (T3SS) is a macromolecular protein nano-syringe used by different bacterial pathogens to inject effectors into host cells. The extracellular part of the syringe is a needle-like filament formed by the polymerization of a 9-kDa protein whose structure and proper localization on the bacterial surface are key determinants for efficient toxin injection. Here, we combined in vivo, in vitro, and in silico approaches to characterize the Pseudomonas aeruginosa T3SS needle and its major component PscF. Using a combination of mutagenesis, phenotypic analyses, immunofluorescence, proteolysis, mass spectrometry, atomic force microscopy, electron microscopy, and molecular modeling, we propose a model of the P. aeruginosa needle that exposes the N-terminal region of each PscF monomer toward the outside of the filament, while the core of the fiber is formed by the C-terminal helix. Among mutations introduced into the needle protein PscF, D76A, and P47A/Q54A caused a defect in the assembly of the needle on the bacterial surface, although the double mutant was still cytotoxic on macrophages in a T3SS-dependent manner and formed filamentous structures in vitro. These results suggest that the T3SS needle of P. aeruginosa displays an architecture that is similar to that of other bacterial needles studied to date and highlight the fact that small, targeted perturbations in needle assembly can inhibit T3SS function. Therefore, the T3SS needle represents an excellent drug target for small molecules acting as virulence blockers that could disrupt pathogenesis of a broad range of bacteria.

The MurG glycosyltransferase provides an oligomeric scaffold for the cytoplasmic steps of peptidoglycan biosynthesis in the human pathogen Bordetella pertussis.

Peptidoglycan is a major component of the bacterial cell wall and thus a major determinant of cell shape. Its biosynthesis is initiated by several sequential reactions catalyzed by cytoplasmic Mur enzymes. Mur ligases (MurC, -D, -E, and -F) are essential for bacteria, metabolize molecules not present in eukaryotes, and are structurally and biochemically tractable. However, although many Mur inhibitors have been developed, few have shown promising antibacterial activity, prompting the hypothesis that within the cytoplasm, Mur enzymes could exist as a complex whose architecture limits access of small molecules to their active sites. This suggestion is supported by the observation that in many bacteria, mur genes are present in a single operon, and pairs of these genes often are fused to generate a single polypeptide. Here, we explored this genetic arrangement in the human pathogen Bordetella pertussis and show that MurE and MurF are expressed as a single, bifunctional protein. EM, small angle X-ray scattering (SAXS), and analytical centrifugation (AUC) revealed that the MurE-MurF fusion displays an elongated, flexible structure that can dimerize. Moreover, MurE-MurF interacted with the peripheral glycosyltransferase MurG, which formed discrete oligomers resembling 4- or 5-armed stars in EM images. The oligomeric structure of MurG may allow it to play a bona fide scaffolding role for a potential Mur complex, facilitating the efficient conveyance of peptidoglycan-building blocks toward the inner membrane leaflet. Our findings shed light on the structural determinants of a peptidoglycan formation complex involving Mur enzymes in bacterial cell wall formation.

Assembly of an atypical α-macroglobulin complex from Pseudomonas aeruginosa.

Alpha-2-macroglobulins (A2Ms) are large spectrum protease inhibitors that are major components of the eukaryotic immune system. Pathogenic and colonizing bacteria, such as the opportunistic pathogen Pseudomonas aeruginosa, also carry structural homologs of eukaryotic A2Ms. Two types of bacterial A2Ms have been identified: Type I, much like the eukaryotic form, displays a conserved thioester that is essential for protease targeting, and Type II, which lacks the thioester and to date has been poorly studied despite its ubiquitous presence in Gram-negatives. Here we show that MagD, the Type II A2M from P. aeruginosa that is expressed within the six-gene mag operon, specifically traps a target protease despite the absence of the thioester motif, comforting its role in protease inhibition. In addition, analytical ultracentrifugation and small angle scattering show that MagD forms higher order complexes with proteins expressed in the same operon (MagA, MagB, and MagF), with MagB playing the key stabilization role. A P. aeruginosa strain lacking magB cannot stably maintain MagD in the bacterial periplasm, engendering complex disruption. This suggests a regulated mechanism of Mag complex formation and stabilization that is potentially common to numerous Gram-negative organisms, and that plays a role in periplasm protection from proteases during infection or colonization.

Structural similarity of secretins from type II and type III secretion systems.

Secretins, the outer membrane components of several secretion systems in Gram-negative bacteria, assemble into channels that allow exoproteins to traverse the membrane. The membrane-inserted, multimeric regions of PscC, the Pseudomonas aeruginosa type III secretion system secretin, and PulD, the Klebsiella oxytoca type II secretion system secretin, were purified after cell-free synthesis and their structures analyzed by single particle cryoelectron microscopy. Both homomultimeric, barrel-like structures display a "cup and saucer" architecture. The "saucer" region of both secretins is composed of two distinct rings, with that of PulD being less segmented than that of PscC. Both secretins have a central chamber that is occluded by a plug linked to the chamber walls through hairpin-like structures. Comparisons with published structures from other bacterial systems reveal that secretins have regions of local structural flexibility, probably reflecting their evolved functions in protein secretion and needle assembly.

Structural basis for the inhibition of the chromatin repressor BAHD1 by the bacterial nucleomodulin LntA.

UNLABELLED: The nucleus has emerged as a key target for nucleomodulins, a family of effectors produced by bacterial pathogens to control host transcription or other nuclear processes. The virulence factor LntA from Listeria monocytogenes stimulates interferon responses during infection by inhibiting BAHD1, a nuclear protein involved in gene silencing by promoting heterochromatin formation. So far, whether the interaction between LntA and BAHD1 is direct and sufficient for inhibiting BAHD1 activity is unknown. Here, we functionally characterized the molecular interface between the two proteins in vitro and in transfected or infected human cells. Based on the known tridimensional structure of LntA, we identified a dilysine motif (K180/K181) in the elbow region of LntA and a central proline-rich region in BAHD1 as crucial for the direct LntA-BAHD1 interaction. To better understand the role played by the dilysine motif in the functionality of LntA, we solved the crystal structure of a K180D/K181D mutant to a 2.2-Å resolution. This mutant highlights a drastic redistribution of surface charges in the vicinity of a groove, which likely plays a role in nucleomodulin target recognition. Mutation of the strategic dilysine motif also abolished the recruitment of LntA to BAHD1-associated nuclear foci and impaired the LntA-mediated stimulation of interferon responses upon infection. Last, the strict conservation of residues K180 and K181 in LntA sequences from 188 L. monocytogenes strains of different serotypes and origins further supports their functional importance. Together, these results provide structural and functional details about the mechanism of inhibition of an epigenetic factor by a bacterial nucleomodulin. IMPORTANCE: Pathogens have evolved various strategies to deregulate the expression of host defense genes during infection, such as targeting nuclear proteins. LntA, a secreted virulence factor from the bacterium Listeria monocytogenes, stimulates innate immune responses by inhibiting a chromatin-associated repressor, BAHD1. This study reveals the structural features of LntA required for BAHD1 inhibition. LntA interacts directly with a central domain of BAHD1 via a surface patch of conserved positive charges, located nearby a groove on the elbow region of LntA. By demonstrating that this patch is required for LntA function, we provide a better understanding of the molecular mechanism allowing a bacterial pathogen to control host chromatin compaction and gene expression.

Membrane and chaperone recognition by the major translocator protein PopB of the type III secretion system of Pseudomonas aeruginosa.

The type III secretion system is a widespread apparatus used by pathogenic bacteria to inject effectors directly into the cytoplasm of eukaryotic cells. A key component of this highly conserved system is the translocon, a pore formed in the host membrane that is essential for toxins to bypass this last physical barrier. In Pseudomonas aeruginosa the translocon is composed of PopB and PopD, both of which before secretion are stabilized within the bacterial cytoplasm by a common chaperone, PcrH. In this work we characterize PopB, the major translocator, in both membrane-associated and PcrH-bound forms. By combining sucrose gradient centrifugation experiments, limited proteolysis, one-dimensional NMR, and ß-lactamase reporter assays on eukaryotic cells, we show that PopB is stably inserted into bilayers with its flexible N-terminal domain and C-terminal tail exposed to the outside. In addition, we also report the crystal structure of the complex between PcrH and an N-terminal region of PopB (residues 51-59), which reveals that PopB lies within the concave face of PcrH, employing mostly backbone residues for contact. PcrH is thus the first chaperone whose structure has been solved in complex with both type III secretion systems translocators, revealing that both molecules employ the same surface for binding and excluding the possibility of formation of a ternary complex. The characterization of the major type III secretion system translocon component in both membrane-bound and chaperone-bound forms is a key step for the eventual development of antibacterials that block translocon assembly.

Structure of internalin InlK from the human pathogen Listeria monocytogenes.

Listeria monocytogenes is a human pathogen that employs a wide variety of virulence factors in order to adhere to, invade, and replicate within target cells. Internalins play key roles in processes ranging from adhesion to receptor recognition and are thus essential for infection. Recently, InlK, a surface-associated internalin, was shown to be involved in Listeria's ability to escape from autophagy by recruitment of the major vault protein (MVP) to the bacterial surface. Here, we report the structure of InlK, which harbors four domains arranged in the shape of a "bent arm". The structure supports a role for the "elbow" of InlK in partner recognition, as well as of two Ig-like pedestals intercalated by hinge regions in the projection of InlK away from the surface of the bacterium. The unusual fold and flexibility of InlK could be essential for MVP binding and concealment from recognition by molecules involved in the autophagic process.

Conformational states of a bacterial α2-macroglobulin resemble those of human complement C3.

α(2) macroglobulins (α(2)Ms) are broad-spectrum protease inhibitors that play essential roles in the innate immune system of eukaryotic species. These large, multi-domain proteins are characterized by a broad-spectrum bait region and an internal thioester, which, upon cleavage, becomes covalently associated to the target protease, allowing its entrapment by a large conformational modification. Notably, α(2)Ms are part of a larger protein superfamily that includes proteins of the complement system, such as C3, a multi-domain macromolecule which is also characterized by an internal thioester-carrying domain and whose activation represents the pivotal step in the complement cascade. Recently, α(2)M/C3-like genes were identified in a large number of bacterial genomes, and the Escherichia coli α(2)M homolog (ECAM) was shown to be activated by proteases. In this work, we have structurally characterized ECAM by electron microscopy and small angle scattering (SAXS) techniques. ECAM is an elongated, flexible molecule with overall similarities to C3 in its inactive form; activation by methylamine, chymotrypsin, or elastase induces a conformational modification reminiscent of the one undergone by the transformation of C3 into its active form, C3b. In addition, the proposed C-terminus of ECAM displays high flexibility and different conformations, and could be the recognition site for partner macromolecules. This work sheds light on a potential bacterial defense mechanism that mimics structural rearrangements essential for activation of the complement cascade in eukaryotes, and represents a possible novel target for the development of antibacterials.

Calcium-dependent complex formation between PBP2 and lytic transglycosylase SltB1 of Pseudomonas aeruginosa.

In Gram-negative bacteria, the bacterial cell wall biosynthetic mechanism requires the coordinated action of enzymes and structural proteins located in the cytoplasm, within the membrane, and in the periplasm of the cell. Its main component, peptidoglycan (PG), is essential for cell division and wall elongation. Penicillin-binding proteins (PBPs) catalyze the last steps of PG biosynthesis, namely the polymerization of glycan chains and the cross-linking of stem peptides, and can be either monofunctional or bifunctional. Their action is coordinated with that of other enzymes essential for cell-wall biosynthesis, such as lytic transglycosylases (LT). Here, we have studied SltB1, an LT from Pseudomonas aeruginosa, and identified that it forms a complex with PBP2, a monofunctional enzyme, which requires the presence of Ca(2+). In addition, we have solved the structure of SltB1 to a high resolution, and identified that it harbors an EF-hand like motif containing a Ca(2+) ion displaying bipyramidal coordination. These studies provide initial structural details that shed light on the interactions between the PG biosynthesis enzymes in P. aeruginosa.

Structural characterization and membrane localization of ExsB from the type III secretion system (T3SS) of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen that employs a finely tuned type III secretion system (T3SS) to inject toxins directly into the cytoplasm of target cells. ExsB is a 15.6-kDa protein encoded in a T3SS transcription regulation operon that displays high sequence similarity to YscW, a lipoprotein from Yersinia spp. whose genetic neighborhood also involves a transcriptional regulator, and has been shown to play a role in the stabilization of the outer membrane ring of the T3SS. Here, we show that ExsB is expressed in P. aeruginosa upon induction of the T3SS, and subcellular fractionation studies reveal that it is associated with the outer membrane. The high-resolution crystal structure of ExsB shows that it displays a compact ß-sandwich fold with interdependent ß-sheets. ExsB possesses a large patch of basic residues that could play a role in membrane recognition, and its structure is distinct from that of MxiM, a lipoprotein involved in secretin stabilization in Shigella, as well as from those of Pil lipoproteins involved in pilus biogenesis. These results reveal that small lipoproteins involved in formation of the outer membrane secretin ring display clear structural differences that may be related to the different functions they play in these systems.