Interaction with Type IV Pili Induces Structural Changes in the Bacterial Outer Membrane Secretin PilQ

Collins, Richard F.; Frye, Stephan A.; Balasingham, Seetha V.; Ford, Robert C.; Tønjum, Tone; Derrick, Jeremy P.

doi:10.1074/jbc.m411603200

Cited by 62 publications

(71 citation statements)

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“…The structure seen showed a large central cavity, which was closed at the poles by 'plug' and 'cap' features, and four 'arm' features at the sides. The results suggested that the PilQ complex is the channel through which the moving pilus fibre (polymerized PilE) is extruded and retracted from the bacterial surface (Collins et al, 2005). PilQ subunits form dodecameric complex structures that are extremely stable to heat and SDS.…”

Section: Introductionmentioning

confidence: 93%

“…The findings in the non-piliated Mc-656, which produced minute amounts of PilQ multimer, most probably because it is not properly folded (Fig. 2b), might also be complicated by the fact that the conformation of the PilQ complex changes with its reaction state with its pilus substrate (Collins et al, 2005). Our findings that the mutations introduced into the Mc-656 and Mc-678 mutants compromised PilQ multimer formation and/or stability complicated the interpretation of the data obtained from these variants.…”

Section: Antibody Accessibility Of Pilq In Cellsmentioning

confidence: 99%

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Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

et al. 2006

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Neisseria meningitidis is the causative agent of epidemic meningococcal meningitis and septicaemia. Type IV pili are surface organelles that mediate a variety of functions, including adhesion, twitching motility, and competence for DNA binding and uptake in transformation. The secretin PilQ is required for type IV pilus expression at the cell surface, and forms a dodecameric cage-like macromolecular complex in the meningococcal outer membrane. PilQ-null mutants are devoid of surface pili, and prevailing evidence suggests that the PilQ complex facilitates extrusion and retraction of type IV pili across the outer membrane. Defining the orientation of the meningococcal PilQ complex in the membrane is a prerequisite for understanding the structure-function relationships of this important protein in pilus biology. In order to begin to define the topology of the PilQ complex in the outer membrane, polyhistidine insertions in N-and C-terminal regions of PilQ were constructed, and their subcellular locations examined. Notably, the insertion epitopes at residues 205 and 678 were located within the periplasm, whereas residue 656 was exposed at the outer surface of the outer membrane. Using electron microscopy with Ni-NTA gold labelling, it was demonstrated that the insertion at residue 205 within the N-terminus mapped to a site on the arm-like features of the 3D structure of the PilQ multimer. Interestingly, mutation of the same region gave rise to an increase in vancomycin permeability through the PilQ complex. The results yield novel information on the PilQ N-terminal location and function in the periplasm, and reveal a complex organization of the membrane-spanning secretin in vivo.

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Section: Introductionmentioning

confidence: 93%

Section: Antibody Accessibility Of Pilq In Cellsmentioning

confidence: 99%

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

et al. 2006

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“…In Neisseria meningitidis, the ATPase activity of PilF is necessary to drive polymerization of the pilin subunits within the periplasm. PilF associates with PilG in a complex at the inner membrane, and the growing polymerized pilin subunits are extruded across the outer membrane through the central cavity in PilQ (Balasingham et al, 2007;Carbonnelle et al, 2006;Collins et al, 2001Collins et al, , 2005Wall et al, 1999). It is not clear how the Tfp are assembled and cross the outer membrane of F. tularensis in the absence of PilG and PilQ; no additional homologues of these proteins were found by a BLAST search.…”

Section: Discussionmentioning

confidence: 99%

Characterization of the Francisella tularensis subsp. novicida type IV pilus

et al. 2008

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Francisella tularensis causes the disease tularaemia. Type IV pili (Tfp) genes are present in the genomes of all F. tularensis subspecies. We show that the wild-type F. tularensis subsp. novicida expresses pilus fibres on its surface, and mutations in the Tfp genes pilF and pilT disrupt pilus biogenesis. Mutations in other Tfp genes (pilQ and pilG) do not eliminate pilus expression. A mutation in pilE4 eliminates pilus expression, whereas mutations in the other pilin subunits pilE1-3 and pilE5 do not, suggesting that pilE4 is the major pilus structural subunit. The virulence regulator MglA is required for pilus expression, and it regulates the transcription of a putative Tfp glycosylation gene (FTN0431). However, MglA does not regulate transcription of pilF, pilT or pilE4, and a strain lacking FTN0431 still expresses pili; thus, it is unclear how MglA regulates pilus expression. Only pilF was also required for protein secretion, while pilE4 and pilT were not, indicating that there is very little overlap of the protein secretion/Tfp functions of the pil genes. The protein secretion component pilE1 was more important for in vitro intramacrophage growth and mouse virulence than the Tfp component pilE4. Our results provide the first genetic characterization of the novel Tfp system of F. tularensis. INTRODUCTIONFrancisella tularensis is a highly infectious bacterium that causes tularaemia. F. tularensis is found in many different animal hosts and can be transmitted by arthropod vectors (Ellis et al., 2002). Humans can acquire the bacteria by a number of different routes, but inhalation of low doses of the organism can lead to a serious pneumonic form of disease that has a high mortality rate, and this has led to the classification of this bacterium as a category A biothreat agent (Dennis et al., 2001). F. tularensis is further divided into different subspecies (Oyston et al., 2004). F. tularensis subsp. tularensis has historically been associated with bioweapons development, and is reported to have the highest virulence for humans (McLendon et al., 2006). F. tularensis subsp. holarctica is also infectious in humans but typically with a less severe outcome (Sjostedt, 2003). An attenuated 'live vaccine strain' (LVS) was derived by repeated passage of subsp. holarctica in the laboratory; despite the unclear nature of the attenuating mutation(s) in this strain, it is frequently used as a laboratory model for tularaemia (Anthony & Kongshavn, 1987;Eigelsbach et al., 1951). F. tularensis subsp. novicida has low virulence for humans, but maintains high virulence in mice (Kieffer et al., 2003;Lauriano et al., 2004). Whole-genome sequencing has revealed that all three subspecies are closely related, with the less virulent (for humans) subsp. novicida showing less genomic decay than the more virulent subsp. holarctica and subsp. tularensis (Larsson et al., 2005;Petrosino et al., 2006;Rohmer et al., 2006).The ability of F. tularensis to survive and replicate within macrophages has been linked to its virulence (Anthony et al., 19...

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“…In vitro assays demonstrated a direct interaction between PilQ complexes and one end of purified Type IV pili [41]. A negative-stain reconstruction of the pilus-bound PilQ complex differs from the non-bound complex in that its central cavity was filled and the arms are splayed, thus dissociating the cap.…”

Section: The Outer Membrane Secretinmentioning

confidence: 99%

Type IV pili: paradoxes in form and function

Craig

2008

Current Opinion in Structural Biology

254

263

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Type IV pili are filaments on the surfaces of many Gram-negative bacteria that mediate an extraordinary array of functions, including adhesion, motility, microcolony formation and secretion of proteases and colonization factors. Their prominent display on the surfaces of many bacterial pathogens, their vital role in virulence, and their ability to elicit an immune response make Type IV pilus structures particularly relevant for study as targets for component vaccines and therapies. Structural studies of the pili and components of the pilus assembly apparatus have proven extremely challenging, but new approaches and methods have produced important breakthroughs that are advancing our understanding of pilus functions and their complex assembly mechanism. These structures provide insights into the biology of Type IV pili as well as that of the related bacterial secretion and archaeal flagellar systems. This review will summarize the most recent structural advances on Type IV pili and their assembly components and highlight their significance.Type IV pili are homopolymers of a 15-20 kDa pilin subunit that emanate from the surfaces of many Gram-negative bacteria and at least one Gram-positive organism. These filaments, which appear smooth and featureless by electron microscopy (EM), are 6-9 nm in diameter and several microns in length (Fig. 1). Beneath their plain façade lies an exquisite helical architecture that provides for strength, flexibility and a multitude of functions, including twitching and gliding motility, adhesion, immune escape, DNA uptake, biofilm formation, microcolony formation, secretion, phage transduction and signal transduction. Unlike other bacterial pili, which use as few as two proteins for assembly [1,2], Type IV pilus biogenesis requires a dozen or more proteins, many of which share sequence conservation among divergent species. Pili are assembled, and in some cases disassembled, rapidly using powerful molecular motors that hydrolyze ATP. The proteins involved in pilus biogenesis form a dynamic but poorly-defined complex that spans both bacterial membranes and the intervening periplasm. Our knowledge of Type IV pili presents several paradoxes: What type of molecular architecture yields such thin flexible filaments that can withstand stresses greater than 100 pN? How does a single filament design provide for such functional diversity? How does the assembly apparatus allow for rapid polymerization and depolymerization at a rate of more than 1000 subunits/second? This review will focus on the latest structural findings, which help to explain these paradoxes and advance our understanding of this remarkable biological machine.Type IV pilus assembly involves 12 or more proteins that in many cases are encoded within the same operon. Several key components are utilized in all Type IV pilus systems and are also found in Type II secretion and archaeal flagellar systems [3][4][5]. These are: the pilin subunit; an inner membrane prepilin peptidase that cleaves the N-terminal leader peptide; an ...

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Interaction with Type IV Pili Induces Structural Changes in the Bacterial Outer Membrane Secretin PilQ

Cited by 62 publications

References 60 publications

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

Topology of the outer-membrane secretin PilQ from Neisseria meningitidis

Characterization of the Francisella tularensis subsp. novicida type IV pilus

Type IV pili: paradoxes in form and function

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