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

Abendroth, Jan; Mitchell, Danielle; Korotkov, Konstantin V.; Johnson, Tanya; Kreger, Allison; Sandkvist, Maria; Hól, Wim G. J.

doi:10.1016/j.jsb.2009.03.009

Cited by 48 publications

(65 citation statements)

References 82 publications

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“…Structural and functional similarities between the T2S and T4P systems have long been recognized (51). Despite minimal sequence identity between orthologous components of the alignment subcomplexes in the two systems, atomic resolution structures revealed a high degree of structural similarity, suggesting conservation of function (33,34,46,(52)(53)(54). Previous studies suggested that multiple segments of GspL and GspM, the T2S equivalents of PilMN and PilO, respectively, participate in their interaction (47,48).…”

Section: Discussionmentioning

confidence: 99%

Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations

et al. 2015

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Type IV pili (T4P) are dynamic protein filaments that mediate bacterial adhesion, biofilm formation, and twitching motility. The highly conserved PilMNOP proteins form an inner membrane alignment subcomplex required for function of the T4P system, though their exact roles are unclear. Three potential interaction interfaces for PilNO were identified: core-core, coiled coils (CC), and the transmembrane segments (TMSs). A high-confidence PilNO heterodimer model was used to select key residues for mutation, and the resulting effects on protein-protein interactions were examined both in a bacterial two-hybrid (BTH) system and in their native Pseudomonas aeruginosa context. Mutations in the oppositely charged CC regions or the TMS disrupted PilNO heterodimer formation in the BTH assay, while up to six combined mutations in the core failed to disrupt the interaction. When the mutations were introduced into the P. aeruginosa chromosome at the pilN or pilO locus, specific changes at each of the three interfaces-including core mutations that failed to disrupt interactions in the BTH system-abrogated surface piliation and/or impaired twitching motility. Unexpectedly, specific CC mutants were hyperpiliated but nonmotile, a hallmark of pilus retraction defects. These data suggest that PilNO participate in both the extension and retraction of T4P. Our findings support a model of multiple, precise interaction interfaces between PilNO; emphasize the importance of studying protein function in a minimally perturbed context and stoichiometry; and highlight potential target sites for development of small-molecule inhibitors of the T4P system. IMPORTANCEPseudomonas aeruginosa is an opportunistic pathogen that uses type IV pili (T4P) for host attachment. The T4P machinery is composed of four cell envelope-spanning subcomplexes. PilN and PilO heterodimers are part of the alignment subcomplex and essential for T4P function. Three potential PilNO interaction interfaces (the core-core, coiled-coil, and transmembrane segment interfaces) were probed using site-directed mutagenesis followed by functional assays in an Escherichia coli two-hybrid system and in P. aeruginosa. Several mutations blocked T4P assembly and/or motility, including two that revealed a novel role for PilNO in pilus retraction, while other mutations affected extension dynamics. These critical PilNO interaction interfaces represent novel targets for small-molecule inhibitors with the potential to disrupt T4P function.A larming increases in antibiotic resistance have prompted the search for alternative strategies to combat bacterial infection (1). Genomic strategies designed to identify essential bacterial targets have been informative but less successful for drug development than originally predicted due to high mutation rates and resistance (2). Alternative strategies-such as phage therapy and the use of "antivirulence" drugs that target bacterial factors involved in pathogenesis-show promise since they can effectively disarm bacteria, which are then more easily...

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Section: Discussionmentioning

confidence: 99%

Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations

et al. 2015

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“…1a). GspC, GspL and GspM are bitopic proteins, whereas GspF is a polytopic membrane protein [28][29][30][31] . GspC binds to the periplasmic domains of GspD, thereby connecting the IM platform to the OM complex 32,33 (FIG.…”

Section: Polytopicmentioning

confidence: 99%

Secretion systems in Gram-negative bacteria: structural and mechanistic insights

et al. 2015

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Bacteria have evolved a remarkable array of sophisticated nanomachines to export various virulence factors across the bacterial cell envelope. In recent years, considerable progress has been made towards elucidating the structural and molecular mechanisms of the six secretion systems (types I-VI) of Gram-negative bacteria, the unique mycobacterial type VII secretion system, the chaperone-usher pathway and the curli secretion machinery. These advances have greatly enhanced our understanding of the complex mechanisms that these macromolecular structures use to deliver proteins and DNA into the extracellular environment or into target cells. In this Review, we explore the structural and mechanistic relationships between these single- and double-membrane-embedded systems, and we briefly discuss how this knowledge can be exploited for the development of new antimicrobial strategies.

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“…Abendroth et al (42) proposed previously that based on such similarities, the cytoplasmic domains of the PilC ortholog EpsF and its relatives were likely to have similar folds. We hypothesized that although the NTD of PilC likely interacts with PilB, its CTD may interact with PilT.…”

Section: Pilc Is Essential and Pilmnop Is Dispensable For Pilusmentioning

confidence: 99%

The Platform Protein Is Essential for Type IV Pilus Biogenesis

Takhar

Kemp

Kim

et al. 2013

Journal of Biological Chemistry

109

118

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Background:The platform protein PilC and/or the alignment subcomplex PilMNOP may coordinate type IV pilus (T4P) extension/retraction ATPases. Results: Loss of PilC in a retraction-deficient background abolished assembly. C-terminal mutations were permissive for assembly but not twitching motility. Conclusion: PilC is essential for pilus biogenesis and twitching motility. Significance: Platform proteins are conserved essential components of T4P and type II secretion machines.

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

Cited by 48 publications

References 82 publications

Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations

Novel Role for PilNO in Type IV Pilus Retraction Revealed by Alignment Subcomplex Mutations

Secretion systems in Gram-negative bacteria: structural and mechanistic insights

The Platform Protein Is Essential for Type IV Pilus Biogenesis

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