The GspLM inner membrane complex from the bacterial type II secretion system is a dimer of dimers and interacts with the system ATPase with high affinity (original) (raw)
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The Journal of biological chemistry, 2014
The type VI secretion system (T6SS) is a bacterial nanomachine for the transport of effector molecules into prokaryotic and eukaryotic cells. It involves the assembly of a tubular structure composed of TssB and TssC that is similar to the tail sheath of bacteriophages. The sheath contracts to provide the energy needed for effector delivery. The AAA(+) ATPase ClpV disassembles the contracted sheath, which resets the systems for reassembly of an extended sheath that is ready to fire again. This mechanism is crucial for T6SS function. In Vibrio cholerae, ClpV binds the N terminus of TssC within a hydrophobic groove. In this study, we resolved the crystal structure of the N-terminal domain of Pseudomonas aeruginosa ClpV1 and observed structural alterations in the hydrophobic groove. The modification in the ClpV1 groove is matched by a change in the N terminus of TssC, suggesting the existence of distinct T6SS classes. An accessory T6SS component, TagJ/HsiE, exists predominantly in one o...
Biogenesis and structure of a type VI secretion membrane core complex
Nature, 2015
Bacteria share their ecological niches with other microbes. The bacterial Type VI secretion system is one of the key players for microbial competition, as well as an important virulence determinant during bacterial infections. It assembles a nanocrossbow-like structure that propels an arrow made of Hcp tube and VgrG spike into the cytoplasm of the attacker cell and punctures the prey's cell wall. The nano-crossbow is stably anchored to the cell envelope of the attacker by a membrane core complex. Here, we show that this complex is assembled by the sequential addition of three proteins-TssJ, TssM and TssL-and present a 11.6 Å resolution structure of the fully assembled complex, determined by negative stain electron microscopy. With overall C5 symmetry, this 1.7-megadalton complex comprises a large base in the cytoplasm. It extends in the periplasm via 10 arches to form a double-ring structure containing the Cterminal domain of TssM (TssM ct) and TssJ that is anchored in the outer membrane. The crystal structure of the TssM ct-TssJ complex coupled to whole-cell accessibility studies suggest that large conformational changes induce transient pore formation in the outer membrane allowing passage of the attacking Hcp tube/VgrG spike.
PLOS Pathogens, 2011
Type VI secretion systems (T6SS) are trans-envelope machines dedicated to the secretion of virulence factors into eukaryotic or prokaryotic cells, therefore required for pathogenesis and/or for competition towards neighboring bacteria. The T6SS apparatus resembles the injection device of bacteriophage T4, and is anchored to the cell envelope through a membrane complex. This membrane complex is composed of the TssL, TssM and TagL inner membrane anchored proteins and of the TssJ outer membrane lipoprotein. Here, we report the crystal structure of the enteroaggregative Escherichia coli Sci1 TssJ lipoprotein, a two four-stranded b-sheets protein that exhibits a transthyretin fold with an additional a-helical domain and a protruding loop. We showed that TssJ contacts TssM through this loop since a loop depleted mutant failed to interact with TssM in vitro or in vivo. Biophysical analysis of TssM and TssJ-TssM interaction suggest a structural model of the membraneanchored outer shell of T6SS. Collectively, our results provide an improved understanding of T6SS assembly and encourage structure-aided drug design of novel antimicrobials targeting T6SS.
Structure, 2009
Secretins are among the largest bacterial outer membrane proteins known. Here we report the crystal structure of the periplasmic N-terminal domain of GspD (peri-GspD) from the type 2 secretion system (T2SS) secretin in complex with a nanobody, the VHH domain of a heavy-chain camelid antibody. Two different crystal forms contained the same compact peri-GspD:nanobody heterotetramer. The nanobody contacts peri-GspD mainly via CDR3 and framework residues. The peri-GspD structure reveals three subdomains, with the second and third subdomains exhibiting the KH fold which also occurs in ring-forming proteins of the type 3 secretion system. The first subdomain of GspD is related to domains in phage tail proteins and outer membrane TonBdependent receptors. A dodecameric peri-GspD model is proposed in which a solvent-accessible b strand of the first subdomain interacts with secreted proteins and/or T2SS partner proteins by b strand complementation.
Structural analysis of a prototypical ATPase from the type III secretion system
Nature Structural & Molecular Biology, 2007
The type III secretion system (T3SS) ATPase is the conserved and essential inner-membrane component involved in the initial stages of selective secretion of specialized T3SS virulence effector proteins from the bacterial cytoplasm through to the infected host cell, a process crucial to subsequent pathogenicity. Here we present the 1.8-Å -resolution crystal structure of the catalytic domain of the prototypical T3SS ATPase EscN from enteropathogenic Escherichia coli (EPEC). Along with in vitro and in vivo mutational analysis, our data show that the T3SS ATPases share similarity with the F1 ATPases but have important structural and sequence differences that dictate their unique secretory role. We also show that T3SS ATPase activity is dependent on EscN oligomerization and describe the molecular features and possible functional implications of a hexameric ring model.
PLOS Pathogens, 2011
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. Three different crystal structures of the homology region domain of GspC (GspC HR ) in complex with either two or three domains of the N-terminal region of GspD from enterotoxigenic Escherichia coli show that GspC HR adopts an all-b topology. N-terminal b-strands of GspC and the N0 domain of GspD are major components of the interface between these inner and outer membrane proteins from the T2SS. The biological relevance of the observed GspC-GspD interface is shown by analysis of variant proteins in two-hybrid studies and by the effect of mutations in homologous genes on extracellular secretion and subcellular distribution of GspC in Vibrio cholerae. Substitutions of interface residues of GspD have a dramatic effect on the focal distribution of GspC in V. cholerae. These studies indicate that the GspC HR -GspD N0 interactions observed in the crystal structure are essential for T2SS function. Possible implications of our structures for the stoichiometry of the T2SS and exoprotein secretion are discussed.
Structural analysis of EscN, the ATPase from the type III secretion system
The type III secretion system (T3SS) ATPase is the conserved and essential inner-membrane component involved in the initial stages of selective secretion of specialized T3SS virulence effector proteins from the bacterial cytoplasm through to the infected host cell, a process crucial to subsequent pathogenicity. Here we present the 1.8-Å -resolution crystal structure of the catalytic domain of the prototypical T3SS ATPase EscN from enteropathogenic Escherichia coli (EPEC). Along with in vitro and in vivo mutational analysis, our data show that the T3SS ATPases share similarity with the F1 ATPases but have important structural and sequence differences that dictate their unique secretory role. We also show that T3SS ATPase activity is dependent on EscN oligomerization and describe the molecular features and possible functional implications of a hexameric ring model.
The EMBO Journal, 2007
The secretion superfamily ATPases are conserved motors in key microbial membrane transport and filament assembly machineries, including bacterial type II and IV secretion, type IV pilus assembly, natural competence, and archaeal flagellae assembly. We report here crystal structures and small angle X-ray scattering (SAXS) solution analyses of the Archaeoglobus fulgidus secretion superfamily ATPase, afGspE. AfGspE structures in complex with ATP analogue AMP-PNP and Mg 2 þ reveal for the first time, alternating open and closed subunit conformations within a hexameric ring. The closed-form active site with bound Mg 2 þ evidently reveals the catalytically active conformation. Furthermore, nucleotide binding results and SAXS analyses of ADP, ATPcS, ADP-Vi, and AMP-PNP-bound states in solution showed that asymmetric assembly involves ADP binding, but clamped closed conformations depend on both ATP c-phosphate and Mg 2 þ plus the conserved motifs, arginine fingers, and subdomains of the secretion ATPase superfamily. Moreover, protruding N-terminal domain shifts caused by the closed conformation suggest a unified piston-like, push-pull mechanism for ATP hydrolysis-dependent conformational changes, suitable to drive diverse microbial secretion and assembly processes by a universal mechanism.
Targeting Type IV Secretion System Proteins to Combat Multidrug-Resistant Gram-positive Pathogens
The Journal of Infectious Diseases
For many gram-positive pathogens, conjugative plasmid transfer is an important means of spreading antibiotic resistance. Therefore, the search for alternative treatments to fight and prevent infections caused by these bacteria has become of major interest. In the present study, we evaluated the protein TraM, from the conjugative plasmid pIP501, as a potential vaccine candidate. Anti-TraM antiserum mediated in vitro opsonophagocytic killing of the strain harboring the pIP501 plasmid and also proved to be cross-reactive against other clinically relevant enterococcal and staphylococcal strains. Specificity of antibodies toward TraM was confirmed by results of an opsonophagocytic inhibition assay and Western blot. In addition, conjugative transfer experiments proved that TraM is essential for the transfer of pIP501. Finally, immunization with either TraM or anti-TraM antiserum reduced significantly the colony counts in mice livers, demonstrating that TraM is a promising vaccine candidate against enterococci and other gram-positive pathogens. Keywords. vaccine; type IV secretion system (T4SS); gram-positive pathogens; conjugative transfer; mouse sepsis model. Enterococci are among the most common pathogens associated with healthcare infections [1]. These bacteria have become a serious concern because of their ability to acquire antibiotic resistance and virulence determinants [2], demonstrating an urgent need to improve our understanding of enterococcal virulence mechanisms and the necessity to develop new alternative treatments [3, 4]. The most important mechanism for the spread of resistance and virulence factors is conjugative plasmid transfer [5]. Most gram-positive bacteria seem to conjugate through type IV secretion systems (T4SS) [6, 7]. Plasmid pIP501, which confers resistance to multiple antibiotics and was originally isolated from Streptococcus agalactiae [8], can be transferred to a variety of gram-positive bacteria, including streptococci, lactobacilli, lactococci, Listeria species, staphylococci, and enterococci [9]. Plasmids such as pIP501 may contribute to the spread of antibiotic-resistant bacteria among humans and may represent an attractive target for the prevention of infections caused by multidrug-resistant gram-positive bacteria. In the present study, we evaluate a previously characterized component of a T4SS (ie, TraM, a VirB8-like protein) for vaccine development against infections caused by enterococci and other gram-positive pathogens. MATERIALS AND METHODS Production of Recombinant Proteins Purification of proteins was performed as described elsewhere [7, 10]. Briefly, traM was cloned into 7xHis-tag expression vector pQTEV [5]. The recombinant construct was transformed into competent Escherichia coli BL21-CodonPlus (DE3)-RIL (Stratagene, Amsterdam, the Netherlands). E. coli BL21-CodonPlus (DE3)-RIL pQTEV::traM was grown in Luria Bertani (LB) medium with 100 µg/mL of ampicillin; at an OD 600 of 0.6, expression of the protein was induced by addition of 1 mM isopropyl-β-d-thiogalactoside, and incubation was continued for 3 hours at 37°C. Cells were harvested and lysed in 25 mM Hepes (pH 7.6), 75 mM Na 2 SO 4 , 2 U of DNAse (Sigma-Aldrich, St. Louis, MO), 1 mM phenylmethylsulfonyl fluoride, and 2 mM benzamidine. The cell suspension was mixed, kept on ice for 30 minutes, and sonicated continuously for 1 minute at an amplitude of approximately 80% (Sonopuls HD2070, Bandelin). The supernatant obtained after centrifugation for 30 minutes at 8°C and 15 000 × g was subjected to fractionation on a HisTrapFF column (GE Healthcare, Chalfont St. Giles, United Kingdom). Purity was assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein was purified and concentrated using a 3-kDa ultrafiltration/diafiltration system (Amicon tubes, Merck, Darmstadt, Germany).
Proteins:Structure Function & Bioinformatics, 2019
Type Three Secretion Systems (T3SS) from many gram-negative bacteria utilize ATPases for the translocation of effector proteins into the eukaryotic host cells through injectisome. Cytosolic regulators effectively control the action of these ATPases. PscN from Pseudomonas aeruginosa was an ATPase which was regulated by an uncharacterized PscL. Here we have bioinformatically, biochemically , and biophysically characterized PscN as a T3SS ATPase and PscL as its regulator. In solution, PscN exists predominantly as oligomer and hydrolyzes ATP with V max of 3.9 ± 0.2 μmol/ min/mg and K m 0.93 ± 0.06 mM. Hexameric structure of PscN was observed under AFM and TEM in the presence of ATP. PscL was dimeric in solution and interacted with PscN strongly in Ni-NTA pull-down assay and SPR analysis. PscL was shown to downregulate PscN ATPase activity up to 80% when mixed with PscN in 1:2 ratio (PscN:PscL). SEC data reconfirm the PscN-PscL interaction stoichiometry (ie, 1:2 ratio) which can also be visualized under AFM. In the present study, we have also found out the existence of an oligomeric form of the PscN-PscL heterotri-meric complex. PscL being the regulator of PscN and interacts to form this conformation, which may play an important role too in the regulation of T3SS utilized by Pseudomonas aeruginosa. For structural aspect, three dimensional in silico models of PscN, PscL, and PscN-PscL were generated. So, in short, present study tried to enlighten both the structural, functional and mechanistic insights into the action of PscN-PscL complex in T3SS mediated pathogenic pathway. K E Y W O R D S atomic force microscopy, ATPase-regulator complex, hexamer, molecular modeling, transmission electron microscopy