Identification of the gate regions in the primary structure of the secretin pIV

Spagnuolo, Julian; Opalka, Natacha; Wen, Wesley X.; Gagić, Dragana; Chabaud, Elodie; Bellini, Pierdomenico; Bennett, M.D.; Norris, Gillian E.; Darst, Seth A.; Russel, Marjorie; Rakonjac, Jasna

doi:10.1111/j.1365-2958.2010.07085.x

Cited by 41 publications

(61 citation statements)

References 71 publications

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“…This model is supported by the observation that the total amount of PilQ is decreased in the frameshift mutants, meaning that fewer pores that can allow the antibiotics to permeate the outer membrane exist. However, the SDS-resistant oligomer was the form of PilQ least affected by the frameshift mutations, and recent evidence suggests that similar secretins have mechanisms that allow them to close in vivo (28,40,41). In addition, deletion of pilW, the protein required for production of the stable SDS-resistant PilQ multimer (37,39), did not increase penicillin resistance.…”

Section: Discussionmentioning

confidence: 99%

Diffusion of Antibiotics through the PilQ Secretin in Neisseria gonorrhoeae Occurs through the Immature, Sodium Dodecyl Sulfate-Labile Form

et al. 2015

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In strains of Neisseria gonorrhoeae harboring the mtr and penB determinants that decrease permeation of antibiotics into the periplasm, mutation or deletion of the PilQ secretin of type IV pili increases resistance to penicillin by ϳ3-fold, indicating a role for PilQ in antibiotic permeation. In this study, we examined spontaneously arising mutants with decreased susceptibility to penicillin. One class of mutants had a phenotype indistinguishable from that of a previously characterized pilQ2 mutation that interfered with the formation of SDS-resistant PilQ multimers. A second class of mutants contained frameshift mutations in genes upstream of pilQ in the pilMNOPQ operon that increased resistance to levels similar to those of the pilQ2 mutation. Inframe deletions of these genes were constructed, but only the frameshift mutations increased antibiotic resistance, suggesting that the mutations had polar effects on PilQ. Consistent with this result, titration of wild-type PilQ levels revealed a direct correlation between resistance and expression levels of PilQ. To determine which form of PilQ, the monomer or the multimer, was responsible for antibiotic permeation, we manipulated and quantified these forms in different mutants. Deletion of PilW, which is responsible for the maturation of PilQ into SDS-resistant multimers, had no effect on resistance. Moreover, Western blot analysis revealed that while SDS-resistant multimer levels were decreased by 26% in frameshift mutants, the levels of PilQ monomers were decreased by 48%. These data suggest that immature, SDS-labile complexes, not mature, SDS-resistant PilQ complexes, serve as the route of entry of antibiotics into the periplasm. IMPORTANCEThe capacity of antibiotics to reach their target is crucial for their activity. In Neisseria gonorrhoeae, the PilQ secretin of type IV pili plays an important role in antibiotic influx when diffusion of antibiotics through porins is limited (e.g., in most resistant strains). On Western blots, PilQ exists both as a mature higher-order multimer and an immature, SDS-labile monomer. In this study, we examined spontaneously arising mutations in PilQ and in the genes upstream of PilQ in the pilMNOPQ operon that increase resistance to penicillin. We provide evidence that PilQ monomers associate by mass action to form immature multimers and that these complexes likely mediate the diffusion of antibiotics across the outer membrane. G onorrhea, the sexually transmitted infection (STI) caused byNeisseria gonorrhoeae, is the most prevalent STI worldwide, with an estimated 106 million infections per year (1). There is no vaccine against N. gonorrhoeae, and because patients do not develop immunological memory, there is a high rate of reinfection (2). Because of the absence of an adequate immune response, antibiotic therapy is the standard of care for curing gonorrheal infections, but antibiotic resistance has rendered many of the available therapeutics ineffective. Historically, gonorrhea has been treated with penicillin, tetracycli...

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Section: Discussionmentioning

confidence: 99%

Diffusion of Antibiotics through the PilQ Secretin in Neisseria gonorrhoeae Occurs through the Immature, Sodium Dodecyl Sulfate-Labile Form

et al. 2015

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“…Single-residue substitutions in gates 1 and 2 of the C domain of pIV were reported to change the permeability properties of this secretin in the E. coli OM (21). The PulD peptides whose sequences correspond to gates 1 and 2 in pIV (Fig.…”

Section: Secretins Other Than Puld Form Solute-permeable Pores In Lipmentioning

confidence: 99%

“…Electrophysiology studies also suggested that the pIV secretin channel is tightly closed in vitro, although it allows an Escherichia coli strain lacking maltoporin (LamB) to grow on small maltodextrins (20). The permeability of the pIV channel was increased by changes in the pIV sequence that allowed a LamB-deficient strain to regain its ability to import maltopentose (21). These sequence changes clustered mainly in two regions that were named gates 1 and 2 because of their proposed role in channel gating (21).…”

mentioning

confidence: 99%

“…The permeability of the pIV channel was increased by changes in the pIV sequence that allowed a LamB-deficient strain to regain its ability to import maltopentose (21). These sequence changes clustered mainly in two regions that were named gates 1 and 2 because of their proposed role in channel gating (21). To simplify the terminology, we will use the term secretin channel to define the wide-open conduit that permits protein or phage egress from the cell and the term secretin pore to define the (hypothetical) partially closed conduit.…”

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confidence: 99%

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Bacterial Secretins Form Constitutively Open Pores Akin to General Porins

Disconzi¹,

Guilvout²,

Chami³

et al. 2013

Journal of Bacteriology

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e Proteins called secretins form large multimeric complexes that are essential for macromolecular transit across the outer membrane of Gram-negative bacteria. Evidence suggests that the channels formed by some secretin complexes are not tightly closed, but their permeability properties have not been well characterized. Here, we used cell-free synthesis coupled with spontaneous insertion into liposomes to investigate the permeability of the secretin PulD. Leakage assays using preloaded liposomes indicated that PulD allows the efflux of small fluorescent molecules with a permeation cutoff similar to that of general porins. Other secretins were also found to form similar pores. To define the polypeptide region involved in determining the pore size, we analyzed a collection of PulD variants and studied the roles of gates 1 and 2, which were previously reported to affect the pore size of filamentous phage f1 secretin pIV, in assembly and pore formation. Liposome leakage and a novel in vivo assay showed that replacement of the conserved proline residue at position 443 in PulD by leucine increased the apparent size of the pore. The in vitro approach described here could be used to study the pore properties of membrane proteins whose production in vivo is toxic.

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“…35 It is highly conserved and predicted to contain several -strands 36 embedded in the outer membrane to form the actual pore through which transport occurs. 21 The N-terminal periplasmic region displays conservation only in secretins from related secretion pathways 29 and may be involved in substrate recognition, 28,37 gating of the proposed channel 35,38 and DNA binding 39 as well as contributing to subunit oligomerization. 35,40 The atomic structures of periplasmic fragments from two secretins, EscC from enteropathogenic E. coli and GspD from enterotoxigenic E. coli, have previously been determined.…”

Section: (31)mentioning

confidence: 99%

The Extra-Membranous Domains of the Competence Protein HofQ Show DNA Binding, Flexibility and a Shared Fold with Type I KH Domains

Tarry

Jääskeläinen

Paino

et al. 2011

Journal of Molecular Biology

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Secretins form large oligomeric assemblies in the membrane that control both macromolecular secretion and uptake. Several Pasteurellaceae are naturally competent for transformation but the mechanism for DNA assimilation is largely unknown. In Haemophilus influenzae, the secretin ComE has been demonstrated to be essential for the DNA uptake. In closely related Aggregatibacter actinomycetemcomitans, an opportunistic pathogen in periodontitis, the ComE homolog HofQ is believed to be the outer membrane DNA translocase. Here we report the structure of the extra-membranous domains of HofQ at 2.3 Å resolution by X-ray crystallography. We also show that the extra-membranous domains of HofQ are capable of DNA binding. The structure reveals two secretin-like folds, the first of which is formed via means of a domain swap. The second domain displays extensive structural similarity to KH-domains, including the presence of a GxxG motif, which is essential for the nucleotide binding function of KH-domains, suggesting a possible mechanism for DNA binding by HofQ. The data indicate a direct involvement in DNA acquisition and provides insight into the molecular basis for natural competence.

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Identification of the gate regions in the primary structure of the secretin pIV

Cited by 41 publications

References 71 publications

Diffusion of Antibiotics through the PilQ Secretin in Neisseria gonorrhoeae Occurs through the Immature, Sodium Dodecyl Sulfate-Labile Form

Diffusion of Antibiotics through the PilQ Secretin in Neisseria gonorrhoeae Occurs through the Immature, Sodium Dodecyl Sulfate-Labile Form

Bacterial Secretins Form Constitutively Open Pores Akin to General Porins

The Extra-Membranous Domains of the Competence Protein HofQ Show DNA Binding, Flexibility and a Shared Fold with Type I KH Domains

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