Differential processing of VesB by two rhomboid proteases in .

Unlabelled: Rhomboid proteases are universally conserved and facilitate the proteolysis of peptide bonds within or adjacent to cell membranes. While eukaryotic rhomboid proteases have been demonstrated to harbor unique cellular roles, prokaryotic members have been far less characterized. For the first time, we demonstrate that expresses two active rhomboid proteases that cleave a shared substrate at distinct sites, resulting in differential localization of the processed protein.

Suppressor Mutations in Type II Secretion Mutants of Vibrio cholerae: Inactivation of the VesC Protease.

The type II secretion system (T2SS) is a conserved transport pathway responsible for the secretion of a range of virulence factors by many pathogens, including Disruption of the T2SS genes in results in loss of secretion, changes in cell envelope function, and growth defects. While T2SS mutants are viable, high-throughput genomic analyses have listed these genes among essential genes. To investigate whether secondary mutations arise as a consequence of T2SS inactivation, we sequenced the genomes of six T2SS mutants with deletions or insertions in either the , , or genes and identified secondary mutations in all mutants.

Architecture, Function, and Substrates of the Type II Secretion System.

The type II secretion system (T2SS) delivers toxins and a range of hydrolytic enzymes, including proteases, lipases, and carbohydrate-active enzymes, to the cell surface or extracellular space of Gram-negative bacteria. Its contribution to survival of both extracellular and intracellular pathogens as well as environmental species of proteobacteria is evident. This dynamic, multicomponent machinery spans the entire cell envelope and consists of a cytoplasmic ATPase, several inner membrane proteins, a periplasmic pseudopilus, and a secretin pore embedded in the outer membrane.

CpaA Is a Glycan-Specific Adamalysin-like Protease Secreted by Acinetobacter baumannii That Inactivates Coagulation Factor XII.

Antibiotic-resistant is increasingly recognized as a cause of difficult-to-treat nosocomial infections, including pneumonia, wound infections, and bacteremia. Previous studies have demonstrated that the metalloprotease CpaA contributes to virulence and prolongs clotting time when added to human plasma as measured by the activated partial thromboplastin time (aPTT) assay. Here, we show that CpaA interferes with the intrinsic coagulation pathway, also called the contact activation system, in human as well as murine plasma, but has no discernible effect on the extrinsic pathway.

C-terminal processing of GlyGly-CTERM containing proteins by rhombosortase in Vibrio cholerae.

Vibrio cholerae and a subset of other Gram-negative bacteria, including Acinetobacter baumannii, express proteins with a C-terminal tripartite domain called GlyGly-CTERM, which consists of a motif rich in glycines and serines, followed by a hydrophobic region and positively charged residues. Here we show that VesB, a V. cholerae serine protease, requires the GlyGly-CTERM domain, the intramembrane rhomboid-like protease rhombosortase, and the type II secretion system (T2SS) for localization at the cell surface.

Targeting the Type II Secretion System: Development, Optimization, and Validation of a High-Throughput Screen for the Identification of Small Molecule Inhibitors.

Nosocomial pathogens that develop multidrug resistance present an increasing problem for healthcare facilities. Due to its rapid rise in antibiotic resistance, is one of the most concerning gram-negative species. typically infects immune compromised individuals resulting in a variety of outcomes, including pneumonia and bacteremia.

Measuring In Vitro ATPase Activity for Enzymatic Characterization.

Adenosine triphosphate-hydrolyzing enzymes, or ATPases, play a critical role in a diverse array of cellular functions. These dynamic proteins can generate energy for mechanical work, such as protein trafficking and degradation, solute transport, and cellular movements. The protocol described here is a basic assay for measuring the in vitro activity of purified ATPases for functional characterization.

Zinc coordination is essential for the function and activity of the type II secretion ATPase EpsE.

The type II secretion system Eps in Vibrio cholerae promotes the extracellular transport of cholera toxin and several hydrolytic enzymes and is a major virulence system in many Gram-negative pathogens which is structurally related to the type IV pilus system. The cytoplasmic ATPase EpsE provides the energy for exoprotein secretion through ATP hydrolysis. EpsE contains a unique metal-binding domain that coordinates zinc through a tetracysteine motif (CXXCX CXXC), which is also present in type IV pilus assembly but not retraction ATPases.

Acinetobacter baumannii Is Dependent on the Type II Secretion System and Its Substrate LipA for Lipid Utilization and In Vivo Fitness.

Unlabelled: Gram-negative bacteria express a number of sophisticated secretion systems to transport virulence factors across the cell envelope, including the type II secretion (T2S) system. Genes for the T2S components GspC through GspN and PilD are conserved among isolates of Acinetobacter baumannii, an increasingly common nosocomial pathogen that is developing multidrug resistance at an alarming rate. In contrast to most species, however, the T2S genes are dispersed throughout the genome rather than linked into one or two operons.

Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae.

Background: Outer membrane vesicles (OMVs) are known to release from almost all Gram-negative bacteria during normal growth. OMVs carry different biologically active toxins and enzymes into the surrounding environment. We suggest that OMVs may therefore be able to transport bacterial proteases into the target host cells.

The Type II secretion system delivers matrix proteins for biofilm formation by Vibrio cholerae.

Gram-negative bacteria have evolved several highly dedicated pathways for extracellular protein secretion, including the type II secretion (T2S) system. Since substrates secreted via the T2S system include both virulence factors and degradative enzymes, this secretion system is considered a major survival mechanism for pathogenic and environmental species. Previous analyses revealed that the T2S system mediates the export of ≥ 20 proteins in Vibrio cholerae, a human pathogen that is indigenous to the marine environment.

Functional and structural characterization of Vibrio cholerae extracellular serine protease B, VesB.

The chymotrypsin subfamily A of serine proteases consists primarily of eukaryotic proteases, including only a few proteases of bacterial origin. VesB, a newly identified serine protease that is secreted by the type II secretion system in Vibrio cholerae, belongs to this subfamily. VesB is likely produced as a zymogen because sequence alignment with trypsinogen identified a putative cleavage site for activation and a catalytic triad, His-Asp-Ser.

Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity.

The type II secretion system (T2SS), a multiprotein machinery spanning two membranes in Gram-negative bacteria, is responsible for the secretion of folded proteins from the periplasm across the outer membrane. The critical multidomain T2SS assembly ATPase GspE(EpsE) had not been structurally characterized as a hexamer. Here, four hexamers of Vibrio cholerae GspE(EpsE) are obtained when fused to Hcp1 as an assistant hexamer, as shown with native mass spectrometry.

Fluorescence microscopy and proteomics to investigate subcellular localization, assembly, and function of the type II secretion system.

Investigation of secretion systems is often critical to understanding the virulence mechanisms of bacterial pathogens. With estimates as high as 30-40% of proteins secreted or localized to the cell envelope, information about the subcellular localization and organization of secretion complexes and identification and functional characterization of their substrates are key steps toward understanding these intricate systems. Here we describe a protocol using fluorescent live-cell imaging of fusion proteins that can provide a powerful tool to potentially examine the localization, assembly, and role of each component in the secretion complex.

The type II secretion system: biogenesis, molecular architecture and mechanism.

Many gram-negative bacteria use the sophisticated type II secretion system (T2SS) to translocate a wide range of proteins from the periplasm across the outer membrane. The inner-membrane platform of the T2SS is the nexus of the system and orchestrates the secretion process through its interactions with the periplasmic filamentous pseudopilus, the dodecameric outer-membrane complex and a cytoplasmic secretion ATPase. Here, recent structural and biochemical information is reviewed to describe our current knowledge of the biogenesis and architecture of the T2SS and its mechanism of action.

Structural and functional studies on the interaction of GspC and GspD in the type II secretion system.

Type II secretion systems (T2SSs) are critical for secretion of many proteins from Gram-negative bacteria. In the T2SS, the outer membrane secretin GspD forms a multimeric pore for translocation of secreted proteins. GspD and the inner membrane protein GspC interact with each other via periplasmic domains.

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria.

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (> 80 nm) helical filaments exist near or along either surface of the inner membrane.

Proteomic analysis of the Vibrio cholerae type II secretome reveals new proteins, including three related serine proteases.

The type II secretion (T2S) system is responsible for extracellular secretion of a broad range of proteins, including toxins and degradative enzymes that play important roles in the pathogenesis and life cycle of many gram-negative bacteria. In Vibrio cholerae, the etiological agent of cholera, the T2S machinery transports cholera toxin, which induces profuse watery diarrhea, a hallmark of this life-threatening disease. Besides cholera toxin, four other proteins have been shown to be transported by the T2S machinery, including hemagglutinin protease, chitinase, GbpA, and lipase.

Involvement of the GspAB complex in assembly of the type II secretion system secretin of Aeromonas and Vibrio species.

The type II secretion system (T2SS) functions as a transport mechanism to translocate proteins from the periplasm to the extracellular environment. The ExeA homologue in Aeromonas hydrophila, GspA(Ah), is an ATPase that interacts with peptidoglycan and forms an inner membrane complex with the ExeB homologue (GspB(Ah)). The complex may be required to generate space in the peptidoglycan mesh that is necessary for the transport and assembly of the megadalton-sized ExeD homologue (GspD(Ah)) secretin multimer in the outer membrane.

In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae.

The type II secretion system is a multi-protein complex that spans the cell envelope of Gram-negative bacteria and promotes the secretion of proteins, including several virulence factors. This system is homologous to the type IV pilus biogenesis machinery and contains five proteins, EpsG-K, termed the pseudopilins that are structurally homologous to the type IV pilins. The major pseudopilin EpsG has been proposed to form a pilus-like structure in an energy-dependent process that requires the ATPase, EpsE.

Oligomerization of EpsE coordinates residues from multiple subunits to facilitate ATPase activity.

EpsE is an ATPase that powers transport of cholera toxin and hydrolytic enzymes through the Type II secretion (T2S) apparatus in the gram-negative bacterium, Vibrio cholerae. On the basis of structures of homologous Type II/IV secretion ATPases and our biochemical data, we believe that EpsE is active as an oligomer, likely a hexamer, and the binding, hydrolysis, and release of nucleotide cause EpsE to undergo dynamic structural changes, thus converting chemical energy to mechanical work, ultimately resulting in extracellular secretion. The conformational changes that occur as a consequence of nucleotide binding would realign conserved arginines (Arg(210), Arg(225), Arg(320), Arg(324), Arg(336), and Arg(369)) from adjoining domains and subunits to complete the active site around the bound nucleotide.

Type II Secretion in Escherichia coli.

The type II secretion system (T2SS) is used by Escherichia coli and other gram-negative bacteria to translocate many proteins, including toxins and proteases, across the outer membrane of the cell and into the extracellular space. Depending on the bacterial species, between 12 and 15 genes have been identified that make up a T2SS operon. T2SSs are widespread among gram-negative bacteria, and most E.

Calcium is essential for the major pseudopilin in the type 2 secretion system.

The pseudopilus is a key feature of the type 2 secretion system (T2SS) and is made up of multiple pseudopilins that are similar in fold to the type 4 pilins. However, pilins have disulfide bridges, whereas the major pseudopilins of T2SS do not. A key question is therefore how the pseudopilins, and in particular, the most abundant major pseudopilin, GspG, obtain sufficient stability to perform their function.

Cell envelope perturbation induces oxidative stress and changes in iron homeostasis in Vibrio cholerae.

The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A.

The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae.

The type 2 secretion system (T2SS), a multi-protein machinery that spans both the inner and the outer membranes of Gram-negative bacteria, is used for the secretion of several critically important proteins across the outer membrane. Here we report the crystal structure of the N-terminal cytoplasmic domain of EpsF, an inner membrane spanning T2SS protein from Vibrio cholerae. This domain consists of a bundle of six anti-parallel helices and adopts a fold that has not been described before.