Secretin Complex Research Articles

Functional dissection of structural regions of the Thermus thermophilus competence protein PilW: Implication in secretin complex stability, natural transformation and pilus functions

Uptake of DNA from the environment into the bacterial cytoplasm is mediated by a macromolecular transport machinery that is similar in structure and function to type IV pili (T4P) and, indeed, DNA translocator and T4P share common components. One is the secretin PilQ which is assembled into homopolymeric complexes forming highly dynamic outer membrane (OM) channels mediating pilus extrusion and DNA uptake. How PilQ interacts with the motor is still enigmatic. Here, we have used biochemical and genetic techniques to study the interaction of PilQ with PilW, a unique protein which is essential for natural transformation and T4P extrusion of T. thermophilus. PilQ and PilW form high molecular mass complexes in the OM of T. thermophilus. When pilW was deleted, PilQ complexes were no longer observed but only PilQ monomers, accompanied by a loss of DNA uptake as well as a loss of T4P and twitching motility. Piliation of a ΔpilT2/ΔpilW double mutant suggests that PilW is important for stable assembly of PilQ complexes. To analyze the role of different regions of PilW, partial deletions (pilW∆2–40, pilW∆50–150, pilW∆163–216 and pilW∆216–292) were generated and the effect on DNA uptake, PilQ complex formation and T4P functions such as twitching motility, biofilm formation and cell-cell interaction was studied. These studies revealed that a central disordered region in PilW is required for pilus dynamics. We propose that PilW is part of a protein network that connects the transport ATPase to drive different functions of the DNA translocator and T4P.

Bacterial secretins: Mechanisms of assembly and membrane targeting.

Secretion systems are employed by bacteria to transport macromolecules across membranes without compromising their integrities. Processes including virulence, colonization, and motility are highly dependent on the secretion of effector molecules toward the immediate cellular environment, and in some cases, into the host cytoplasm. In Type II and Type III secretion systems, as well as in Type IV pili, homomultimeric complexes known as secretins form large pores in the outer bacterial membrane, and the localization and assembly of such 1 MDa molecules often relies on pilotins or accessory proteins. Significant progress has been made toward understanding details of interactions between secretins and their partner proteins using approaches ranging from bacterial genetics to cryo electron microscopy. This review provides an overview of the mode of action of pilotins and accessory proteins for T2SS, T3SS, and T4PS secretins, highlighting recent near-atomic resolution cryo-EM secretin complex structures and underlining the importance of these interactions for secretin functionality.

Structural insight into the assembly of the type II secretion system pilotin-secretin complex from enterotoxigenic Escherichia coli.

Secretin is a large outer-membrane channel found in secretion systems of Gram-negative bacteria, facilitating the last step for transfer of proteins into the extracellular environment. In the type II secretion system, a lipoprotein called pilotin is essential to bind and target its corresponding secretin to the outer membrane. However, there is only limited structural information available about the interaction and assembly of the pilotin-secretin complex. Here we report the first near-atomic-resolution structure of a full-length Vibrio-type pilotin-secretin (AspS-GspD) complex from enterotoxigenic Escherichia coli by cryo-electron microscopy, which reveals the detailed assembly mode of the full-length pilotin-secretin complex. The AspS subunits attach to the secretin channel surface with a 15:15 stoichiometric ratio to GspD subunits, and insert their amino terminus into the outer membrane. The AspS subunits interact with all three secondary structural elements of the S domain of GspD, including strong interaction with the carboxy-terminal α-helix and weak interactions with another two elements, an α-helix and a loop. These structural and biochemical details provide a deeper insight to pilotin-secretin interaction and their assembly mode.

Structure and Membrane Topography of the Vibrio-Type Secretin Complex from the Type 2 Secretion System of Enteropathogenic Escherichia coli.

The β-barrel assembly machinery (BAM) complex is the core machinery for the assembly of β-barrel membrane proteins, and inhibition of BAM complex activity is lethal to bacteria. Discovery of integral membrane proteins that are key to pathogenesis and yet do not require assistance from the BAM complex raises the question of how these proteins assemble into bacterial outer membranes. Here, we address this question through a structural analysis of the type 2 secretion system (T2SS) secretin from enteropathogenic Escherichia coli O127:H6 strain E2348/69. Long β-strands assemble into a barrel extending 17 Å through and beyond the outer membrane, adding insight to how these extensive β-strands are assembled into the E. coli outer membrane. The substrate docking chamber of this secretin is shown to be sufficient to accommodate the substrate mucinase SteC.IMPORTANCE In order to cause disease, bacterial pathogens inhibit immune responses and induce pathology that will favor their replication and dissemination. In Gram-negative bacteria, these key attributes of pathogenesis depend on structures assembled into or onto the outer membrane. One of these is the T2SS. The Vibrio-type T2SS mediates cholera toxin secretion in Vibrio cholerae, and in Escherichia coli O127:H6 strain E2348/69, the same machinery mediates secretion of the mucinases that enable the pathogen to penetrate intestinal mucus and thereby establish deadly infections.

Cryo-EM structure of the bifunctional secretin complex of Thermus thermophilus.

Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.

Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes.

ABSTRACTSophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner membrane and a secretin complex in the outer membrane. The engagement of these two megadalton-sized complexes is required in order to secrete toxins, effectors, and hydrolytic enzymes. Pseudomonas aeruginosa has at least two T2SSs, with the ancestral nanomachine having a secretin complex composed of XcpQ. Until now, no high-resolution structural information was available to distinguish the features of this Pseudomonas-type secretin, which varies greatly in sequence from the well-characterized Klebsiella-type and Vibrio-type secretins. We have purified the ~1-MDa secretin complex and analyzed it by cryo-electron microscopy. Structural comparisons with the Klebsiella-type secretin complex revealed a striking structural homology despite the differences in their sequence characteristics. At 3.6-Å resolution, the secretin complex was found to have 15-fold symmetry throughout the membrane-embedded region and through most of the domains in the periplasm. However, the N1 domain and N0 domain were not well ordered into this 15-fold symmetry. We suggest a model wherein this disordering of the subunit symmetry for the periplasmic N domains provides a means to engage with the 6-fold symmetry in the inner membrane platform, with a metastable engagement that can be disrupted by substrate proteins binding to the region between XcpP, in the assembly platform, and the XcpQ secretin.

Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus

Secretins are versatile outer membrane pores used by many bacteria to secrete proteins, toxins, or filamentous phages; extrude type IV pili (T4P); or take up DNA. Extrusion of T4P and natural transformation of DNA in the thermophilic bacterium Thermus thermophilus requires a unique secretin complex comprising six stacked rings, a membrane-embedded cone structure, and two gates that open and close a central channel. To investigate the role of distinct domains in ring and gate formation, we examined a set of deletion derivatives by cryomicroscopy techniques. Here we report that maintaining the N0 ring in the deletion derivatives led to stable PilQ complexes. Analyses of the variants unraveled that an N-terminal domain comprising a unique βββαβ fold is essential for the formation of gate 2. Furthermore, we identified four βαββα domains essential for the formation of the N2 to N5 rings. Mutant studies revealed that deletion of individual ring domains significantly reduces piliation. The N1, N2, N4, and N5 deletion mutants were significantly impaired in T4P-mediated twitching motility, whereas the motility of the N3 mutant was comparable with that of wild-type cells. This indicates that the deletion of the N3 ring leads to increased pilus dynamics, thereby compensating for the reduced number of pili of the N3 mutant. All mutants exhibit a wild-type natural transformation phenotype, leading to the conclusion that DNA uptake is independent of functional T4P.

Type IV pilus secretins have extracellular C termini.

ABSTRACTType IV pili (T4Ps) are surface appendages used by Gram-negative and Gram-positive pathogens for motility and attachment to epithelial surfaces. In Gram-negative bacteria, such as the important pediatric pathogen enteropathogenic Escherichia coli (EPEC), during extension and retraction, the pilus passes through an outer membrane (OM) pore formed by the multimeric secretin complex. The secretin is common to Gram-negative assemblies, including the related type 2 secretion (T2S) system and the type 3 secretion (T3S) system. The N termini of the secretin monomers are periplasmic and in some systems have been shown to mediate substrate specificity. In this study, we mapped the topology of BfpB, the T4P secretin from EPEC, using a combination of biochemical and biophysical techniques that allowed selective identification of periplasmic and extracellular residues. We applied rules based on solved atomic structures of outer membrane proteins (OMPs) to generate our topology model, combining the experimental results with secondary structure prediction algorithms and direct inspection of the primary sequence. Surprisingly, the C terminus of BfpB is extracellular, a result confirmed by flow cytometry for BfpB and a distantly related T4P secretin, PilQ, from Pseudomonas aeruginosa. Keeping with prior evidence, the C termini of two T2S secretins and one T3S secretin were not detected on the extracellular surface. On the basis of our data and structural constraints, we propose that BfpB forms a beta barrel with 16 transmembrane beta strands. We propose that the T4P secretins have a C-terminal segment that passes through the center of each monomer.

The absence of protein Y4yS affects negatively the abundance of T3SS Mesorhizobium loti secretin, RhcC2, in bacterial membranes.

Mesorhizobium loti MAFF303099 has a functional type III secretion system (T3SS) that is involved in the determination of nodulation competitiveness on Lotus. The M. loti T3SS cluster contains gene y4yS (mlr8765) that codes for a protein of unknown function (Y4yS). A mutation in the y4yS gene favors the M. loti symbiotic competitive ability on Lotus tenuis cv. Esmeralda and affects negatively the secretion of proteins through T3SS. Here we localize Y4yS in the bacterial membrane using a translational reporter peptide fusion. In silico analysis indicated that this protein presents a tetratricopeptide repeat (TPR) domain, a signal peptide and a canonical lipobox LGCC in the N-terminal sequence. These features that are shared with proteins required for the formation of the secretin complex in type IV secretion systems and in the Tad system, together with its localization, suggest that the y4yS-encoded protein is required for the formation of the M. loti T3SS secretin (RhcC2) complex. Remarkably, analysis of RhcC2 in the wild-type and M. loti y4yS mutant strains indicated that the absence of Y4yS affects negatively the accumulation of normal levels of RhcC2 in the membrane.

Peptidoglycan-binding protein TsaP functions in surface assembly of type IV pili

Type IV pili (T4P) are ubiquitous and versatile bacterial cell surface structures involved in adhesion to host cells, biofilm formation, motility, and DNA uptake. In Gram-negative bacteria, T4P pass the outer membrane (OM) through the large, oligomeric, ring-shaped secretin complex. In the β-proteobacterium Neisseria gonorrhoeae, the native PilQ secretin ring embedded in OM sheets is surrounded by an additional peripheral structure, consisting of a peripheral ring and seven extending spikes. To unravel proteins important for formation of this additional structure, we identified proteins that are present with PilQ in the OM. One such protein, which we name T4P secretin-associated protein (TsaP), was identified as a phylogenetically widely conserved component of the secretin complex that co-occurs with genes for T4P in Gram-negative bacteria. TsaP contains an N-terminal carbohydrate-binding lysin motif (LysM) domain and a C-terminal domain of unknown function. In N. gonorrhoeae, lack of TsaP results in the formation of membrane protrusions containing multiple T4P, concomitant with reduced formation of surface-exposed T4P. Lack of TsaP did not affect the oligomeric state of PilQ, but resulted in loss of the peripheral structure around the PilQ secretin. TsaP binds peptidoglycan and associates strongly with the OM in a PilQ-dependent manner. In the δ-proteobacterium Myxococcus xanthus, TsaP is also important for surface assembly of T4P, and it accumulates and localizes in a PilQ-dependent manner to the cell poles. Our results show that TsaP is a novel protein associated with T4P function and suggest that TsaP functions to anchor the secretin complex to the peptidoglycan.

Unusual N-terminal ααβαββα Fold of PilQ from Thermus thermophilus Mediates Ring Formation and Is Essential for Piliation

DNA translocators of natural transformation systems are complex systems critical for the uptake of free DNA and provide a powerful mechanism for adaptation to changing environmental conditions. In natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external DNA. Recently, we reported on a novel structure of the DNA translocator secretin complex, PilQ, in Thermus thermophilus HB27 comprising a stable cone and cup structure and six ring structures with a large central channel. Here, we report on structural and functional analyses of a set of N-terminal PilQ deletion derivatives in T. thermophilus HB27. We identified 136 N-terminal residues exhibiting an unusual ααβαββα fold as a ring-building domain. Deletion of this domain had a dramatic effect on twitching motility, adhesion, and piliation but did not abolish natural transformation. These findings provide clear evidence that the pilus structures of T. thermophilus are not essential for natural transformation. The truncated complex was not affected in inner and outer membrane association, indicating that the 136 N-terminal residues are not essential for membrane targeting. Analyses of complex formation of the truncated PilQ monomers revealed that the region downstream of residue 136 is required for multimerization, and the region downstream of residue 207 is essential for monomer stability. Possible implications of our findings for the mechanism of DNA uptake are discussed.

A C-Terminal Region of Yersinia pestis YscD Binds the Outer Membrane Secretin YscC

YscD is an essential component of the plasmid pCD1-encoded type III secretion system (T3SS) of Yersinia pestis. YscD has a single transmembrane (TM) domain that connects a small N-terminal cytoplasmic region (residues 1 to 121) to a larger periplasmic region (residues 143 to 419). Deletion analyses established that both the N-terminal cytoplasmic region and the C-terminal periplasmic region are required for YscD function. Smaller targeted deletions demonstrated that a predicted cytoplasmic forkhead-associated (FHA) domain is also required to assemble a functional T3SS; in contrast, a predicted periplasmic phospholipid binding (BON) domain and a putative periplasmic "ring-building motif" domain of YscD could be deleted with no significant effect on the T3S process. Although deletion of the putative "ring-building motif" domain did not disrupt T3S activity per se, the calcium-dependent regulation of the T3S apparatus was affected. The extreme C-terminal region of YscD (residues 354 to 419) was essential for secretion activity and had a strong dominant-negative effect on the T3S process when exported to the periplasm of the wild-type parent strain. Coimmunoprecipitation studies demonstrated that this region of YscD mediates the interaction of YscD with the outer membrane YscC secretin complex. Finally, replacement of the YscD TM domain with a TM domain of dissimilar sequence had no effect on the T3S process, indicating that the TM domain has no sequence-specific function in the assembly or function of the T3SS.

Multimerization-defective variants of dodecameric secretin PulD

The C-terminal core domain of the secretin PulD from Klebsiella oxytoca forms heat-resistant dodecameric complexes within less than 10 min in an Escherichia coli in vitro transcription-translation system containing liposomes, and is toxic when made in the cytoplasm without a signal peptide. Random mutagenesis of DNA encoding this region of PulD revealed that amino acid changes throughout almost its entire length abolished toxicity. Most of the amino acid substitutions engendered by the mutations retarded or abolished assembly of the dodecameric secretin complex in vitro and/or in the periplasm. Only one of the tested multimerization-defective variants could be rescued by co-production and mixed multimer formation with wild-type secretin in vitro. A three amino acid insertion specifically generated in a region of PulD that was not affected by the spontaneous mutations formed functional multimers that, unlike the wild-type protein, were dissociated by heating in SDS.

Secretins take shape

Secretins are a unique class of bacterial multimeric outer membrane proteins that probably differ considerably from other, less complex outer membrane proteins in their overall structure and organization, and in their requirements for outer membrane targeting and assembly factors. In this MicroCommentary, we discuss these differences with respect to the role of a specific class of lipoproteins, often referred to as pilotins, in secretin complex assembly. We compare them with other lipoproteins that play a role in Omp85/YaeT-mediated assembly of more classical outer membrane proteins. One of the examples we have chosen is the Myxococcus Xanthus lipoprotein Tgl. Coculture of cells with and without Tgl allows secretin assembly (and, hence, type IV pilus assembly) in the cells without Tgl, indicating that it can act by cell-to-cell contact or can transfer between cells.

The penC mutation conferring antibiotic resistance in Neisseria gonorrhoeae arises from a mutation in the PilQ secretin that interferes with multimer stability

The penC resistance gene was previously characterized in an FA19 penA mtrR penB gonococcal strain (PR100) as a spontaneous mutation that increased resistance to penicillin and tetracycline. We show here that antibiotic resistance mediated by penC is the result of a Glu-666 to Lys missense mutation in the pilQ gene that interferes with the formation of the SDS-resistant high-molecular-mass PilQ secretin complex, disrupts piliation and decreases transformation frequency by 50-fold. Deletion of pilQ in PR100 confers the same level of antibiotic resistance as the penC mutation, but increased resistance was observed only in strains containing the mtrR and penB resistance determinants. Site-saturation mutagenesis of Glu-666 revealed that only acidic or amidated amino acids at this position preserved PilQ function. Consistent with early studies suggesting the importance of cysteine residues for stability of the PilQ multimer, mutation of either of the two cysteine residues in FA19 PilQ led to a similar phenotype as penC: increased antibiotic resistance, loss of piliation, intermediate levels of transformation competence and absence of SDS-resistant PilQ oligomers. These data show that a functional secretin complex can enhance the entry of antibiotics into the cell and suggest that the PilQ oligomer forms a pore in the outer membrane through which antibiotics diffuse into the periplasm.