PilMNOPQ from the Pseudomonas aeruginosa Type IV Pilus System Form a Transenvelope Protein Interaction Network That Interacts with PilA

Tammam, Stephanie; Sampaleanu, L.M.; Koo, Jarley; Manoharan, Kumararaaj; Daubaras, Mark; Burrows, Lori L.; Howell, P. Lynne

doi:10.1128/jb.00032-13

Cited by 100 publications

(110 citation statements)

References 47 publications

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“…1), we suggest that the function of PilO lies in modifying the complex by splitting the PilN dimer and setting up the pilin binding site in the correct orientation to capture pilin subunits from the inner membrane. Our observation that TtPilA4 binds to the TtPilMNO complex is in agreement with other evidence for association of T4P and T2SS pilins with their respective inner membrane complexes (28)(29)(30). Clearly, recruitment of the major pilin subunit could be the first "priming" step in T4P assembly.…”

Section: Discussionsupporting

confidence: 79%

Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis

Karuppiah

Collins

Thistlethwaite

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Significance Type IV pili are long, thin fibers, formed mainly of polymers of a single pilin protein, which are displayed on the surfaces of many bacteria, including several human pathogens. Here, we report three-dimensional reconstructions of the PilMNO inner membrane complex, alone and in complex with pilin protein, through a combination of X-ray crystallography and electron microscopy. PilMNO forms a dimeric T-shaped structure, binding two copies of the pilin protein at its extremities. The results provide a structural model for the way in which pilin is harvested from the inner membrane and made available to other components of the type IV pilus biogenesis machinery.

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Section: Discussionsupporting

confidence: 79%

Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis

Karuppiah

Collins

Thistlethwaite

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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“…Figure 2b shows a hypothetical architectural model built by fitting existing structures into the densities observed by the cryo-ET studies of the M. xanthus system 84 . The ring-forming components of the T4P machinery were modeled in a 12-fold stoichiometry as this provided the best fit with cryo-ET density observed 84 , although other stoichiometries have also been proposed 64,75 . Changes that were observed in the piliated state of both systems include the pilus traversing the periplasm and extending into the extracellular space, the opening of the PilQ gate (there is an additional gate present in the longer T. thermophilus PilQ protein), and the presence of additional cytoplasmic density attributed to the elongating ATPase in both studies 83,84 (FIG.…”

Section: Structure Of T4p Machinerymentioning

confidence: 99%

A comprehensive guide to pilus biogenesis in Gram-negative bacteria

2017

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Pili are critical virulence factors of many Gram-negative pathogens. These surface structures provide bacteria with a link to their outside environments, allowing bacteria to interact with their hosts, other surfaces or with each other. They can even provide a conduit for the exchange of information. The surface chemistries and other biophysical properties of pili are uniquely adapted to the environmental challenges faced by bacteria. The assembly of these surface structures presents a molecular engineering challenge, which bacteria have solved in a variety of unique ways. In this review, we examine recent advances in our structural understanding of various Gram-negative pilus systems and discuss their functional implications.

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“…The motor subcomplex is composed of a platform protein, PilC, and associated cytoplasmic ATPases (PilBTU in P. aeruginosa) thought to provide the mechanical forces that lead to pilus extension/retraction (22,23). The secretin and motor subcomplexes are linked by the alignment subcomplex (PilMNOP), which may also have a role in secretin gating (24,25). The final subcomplex is the pilus, a helical filament of major and minor pilin subunits plus the PilY1 adhesin (13,18,(26)(27)(28).…”

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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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PilMNOPQ from the Pseudomonas aeruginosa Type IV Pilus System Form a Transenvelope Protein Interaction Network That Interacts with PilA

Cited by 100 publications

References 47 publications

Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis

Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis

A comprehensive guide to pilus biogenesis in Gram-negative bacteria

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

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