scholarly journals Interactions between the Lipoprotein PilP and the Secretin PilQ in Neisseria meningitidis

2007 ◽  
Vol 189 (15) ◽  
pp. 5716-5727 ◽  
Author(s):  
Seetha V. Balasingham ◽  
Richard F. Collins ◽  
Reza Assalkhou ◽  
Håvard Homberset ◽  
Stephan A. Frye ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Specific Interaction ◽  
Three Dimensional ◽  
Middle Part ◽  
Dna Uptake ◽  
Type Iv ◽  
Pilus Biogenesis ◽  
Inner Membrane Protein ◽  
Mutant Phenotypes

ABSTRACT Neisseria meningitidis can be the causative agent of meningitis or septicemia. This bacterium expresses type IV pili, which mediate a variety of functions, including autoagglutination, twitching motility, biofilm formation, adherence, and DNA uptake during transformation. The secretin PilQ supports type IV pilus extrusion and retraction, but it also requires auxiliary proteins for its assembly and localization in the outer membrane. Here we have studied the physical properties of the lipoprotein PilP and examined its interaction with PilQ. We found that PilP was an inner membrane protein required for pilus expression and transformation, since pilP mutants were nonpiliated and noncompetent. These mutant phenotypes were restored by the expression of PilP in trans. The pilP gene is located upstream of pilQ, and analysis of their transcripts indicated that pilP and pilQ were cotranscribed. Furthermore, analysis of the level of PilQ expression in pilP mutants revealed greatly reduced amounts of PilQ only in the deletion mutant, exhibiting a polar effect on pilQ transcription. In vitro experiments using recombinant fragments of PilP and PilQ showed that the N-terminal region of PilP interacted with the middle part of the PilQ polypeptide. A three-dimensional reconstruction of the PilQ-PilP interacting complex was obtained at low resolution by transmission electron microscopy, and PilP was shown to localize around the cap region of the PilQ oligomer. These findings suggest a role for PilP in pilus biogenesis. Although PilQ does not need PilP for its stabilization or membrane localization, the specific interaction between these two proteins suggests that they might have another coordinated activity in pilus extrusion/retraction or related functions.

scholarly journals Purification and Three-Dimensional Electron Microscopy Structure of the Neisseria meningitidis Type IV Pilus Biogenesis Protein PilG

2007 ◽  
Vol 189 (17) ◽  
pp. 6389-6396 ◽  
Author(s):  
Richard F. Collins ◽  
Muhammad Saleem ◽  
Jeremy P. Derrick
Keyword(s):  
Electron Microscopy ◽  
Neisseria Meningitidis ◽  
Metal Ion ◽  
Polyacrylamide Gel Electrophoresis ◽  
Three Dimensional ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Iv Pilus ◽  
Type Iv ◽  
Pilus Biogenesis

ABSTRACT Type IV pili are surface-exposed retractable fibers which play a key role in the pathogenesis of Neisseria meningitidis and other gram-negative pathogens. PilG is an integral inner membrane protein and a component of the type IV pilus biogenesis system. It is related by sequence to the extensive GspF family of secretory proteins, which are involved in type II secretion processes. PilG was overexpressed and purified from Escherichia coli membranes by detergent extraction and metal ion affinity chromatography. Analysis of the purified protein by perfluoro-octanoic acid polyacrylamide gel electrophoresis showed that PilG formed dimers and tetramers. A three-dimensional (3-D) electron microscopy structure of the PilG multimer was determined using single-particle averaging applied to samples visualized by negative staining. Symmetry analysis of the unsymmetrized 3-D volume provided further evidence that the PilG multimer is a tetramer. The reconstruction also revealed an asymmetric bilobed structure approximately 125 Å in length and 80 Å in width. The larger lobe within the structure was identified as the N terminus by location of Ni-nitrilotriacetic acid nanogold particles to the N-terminal polyhistidine tag. We propose that the smaller lobe corresponds to the periplasmic domain of the protein, with the narrower “waist” region being the transmembrane section. This constitutes the first report of a 3-D structure of a member of the GspF family and suggests a physical basis for the role of the protein in linking cytoplasmic and periplasmic protein components of the type II secretion and type IV pilus biogenesis systems.

scholarly journals Inhibition of laminin self-assembly and interaction with type IV collagen by antibodies to the terminal domain of the long arm.

1986 ◽  
Vol 103 (5) ◽  
pp. 1689-1697 ◽  
Author(s):  
A S Charonis ◽  
E C Tsilibary ◽  
T Saku ◽  
H Furthmayr
Keyword(s):  
Self Assembly ◽  
Binding Sites ◽  
Type Iv Collagen ◽  
Specific Interaction ◽  
Basement Membranes ◽  
Complex Domain ◽  
Peptide Fragment ◽  
Type Iv ◽  
Collagen Molecules

Laminin is a major glycoprotein of the basement membrane. Although its precise localization and orientation within this structure is unknown, it is presumably anchored to other macromolecules such as type IV collagen or proteoheparan sulfate. In vitro, laminin has the ability to self-assemble and to bind to type IV collagen molecules at distinct sites. To identify more precisely the domains of the complex, cross-shaped laminin molecule that are involved in these interactions, images of laminin-laminin dimers and laminin-type IV collagen complexes obtained by the rotary shadowing method were analyzed. We observed that the complex domain at the end of the long arm of laminin is predominantly involved in these interactions. By using Fab fragments of antibodies specific for a peptide fragment derived from this complex domain, it is shown that laminin self-assembly is inhibited in their presence, as measured by turbidity and by electron microscopy. In addition, these antibodies inhibit the specific interaction of laminin with type IV collagen. These data suggest that the complex domain at the end of the long arm of laminin contains binding sites of potential importance for the assembly of basement membranes.

scholarly journals Three-dimensional observations of an aperiodic oscillatory gliding motility behaviour in Myxococcus xanthus using confocal interference reflection microscopy

2019 ◽  
Author(s):  
Liam M. Rooney ◽  
Lisa S. Kölln ◽  
Ross Scrimgeour ◽  
William B. Amos ◽  
Paul A. Hoskisson ◽  
...  
Keyword(s):  
Myxococcus Xanthus ◽  
Three Dimensional ◽  
Basal Membrane ◽  
Gliding Motility ◽  
The Novel ◽  
Force Transmission ◽  
Topological Information ◽  
Type Iv ◽  
Microscopy Techniques

The Delta-proteobacterium, Myxococcus xanthus, has been used as a model for bacterial motility and to provide insights of bacterial swarming behaviours. Fluorescence microscopy techniques have shown that various mechanisms are involved in gliding motility, but these have almost entirely been limited to 2D studies and there is currently no understanding of gliding motility in a 3D context. We present here the first use of confocal interference reflection microscopy (IRM) to study gliding bacteria, and we reveal aperiodic oscillatory behaviour with changes in the position of the basal membrane relative to the coverglass on the order of 90 nm in vitro. Firstly, we use a model plano-convex lens specimen to show how topological information can be obtained from the wavelength-dependent interference pattern in IRM. We then use IRM to observe gliding M. xanthus and show that cells undergo previously unobserved changes in their height as they glide. We compare the wild-type with mutants of reduced motility, which also exhibit the same changes in adhesion profile during gliding. We find that the general gliding behaviour is independent of the proton motive force-generating complex, AglRQS, and suggest that the novel behaviour we present here may be a result of recoil and force transmission along the length of the cell body following firing of the Type IV pili.

scholarly journals Three-Dimensional Structure of the Neisseria meningitidis Secretin PilQ Determined from Negative-Stain Transmission Electron Microscopy

2003 ◽  
Vol 185 (8) ◽  
pp. 2611-2617 ◽  
Author(s):  
Richard F. Collins ◽  
Robert C. Ford ◽  
Ashraf Kitmitto ◽  
Ranveig O. Olsen ◽  
Tone Tønjum ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Protein Complex ◽  
Rotational Symmetry ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Type Iv ◽  
Negative Stain ◽  
Dominant Feature

ABSTRACT The PilQ secretin from the pathogenic bacterium Neisseria meningitidis is an integral outer membrane protein complex which plays a crucial role in the biogenesis of type IV pili. We present here the first three-dimensional structure of this type of secretin at 2.5-nm resolution, obtained by single-particle averaging methods applied to the purified protein complex visualized in a negative stain. In projection, the PilQ complex is circular, with a donut-like appearance. When viewed from the side it has a rounded, conical profile. The complex was demonstrated to have 12-fold rotational symmetry, and this property was used to improve the quality of the density map by symmetry averaging. The dominant feature of the structure is a cavity, 10 nm deep, within the center of the molecule. The cavity is funnel-shaped in cross section, measures 6.5 nm in diameter at the top of the complex, and tapers to a closed point, effectively blocking formation of a continuous pore through the PilQ complex. These results suggest that the complex would have to undergo a conformational change in order to accommodate an assembled pilus fiber of diameter 6.5 nm running through the outer membrane.

scholarly journals Disease-Associated Neisseria meningitidis Isolates Inhibit Wound Repair in Respiratory Epithelial Cells in a Type IV Pilus-Independent Manner

2014 ◽  
Vol 82 (12) ◽  
pp. 5023-5034 ◽  
Author(s):  
Xiaoyun Ren ◽  
Joanna K. MacKichan
Keyword(s):  
Cell Migration ◽  
Neisseria Meningitidis ◽  
Meningococcal Disease ◽  
Wound Repair ◽  
Disease Onset ◽  
Independent Manner ◽  
Content Type ◽  
Type Iv ◽  
Respiratory Epithelial

ABSTRACTNeisseria meningitidisis the causative agent of meningococcal disease. Onset of meningococcal disease can be extremely rapid and can kill within a matter of hours. However, although a much-feared pathogen,Neisseria meningitidisis frequently found in the nasopharyngeal mucosae of healthy carriers. The bacterial factors that distinguish disease- from carriage-associated meningococci are incompletely understood. Evidence suggesting that disruptions to the nasopharynx may increase the risk of acquiring meningococcal disease led us to evaluate the ability of disease- and carriage-associated meningococcal isolates to inhibit cell migration, using anin vitroassay for wound repair. We found that disease-associated isolates in our collection inhibited wound closure, while carriage-associated isolates were more variable, with many isolates not inhibiting wound repair at all. For isolates selected for further study, we found that actin morphology, such as presence of lamellipodia, correlated with cell migration. We demonstrated that multiple meningococcal virulence factors, including the type IV pili, are dispensable for inhibition of wound repair. Inhibition of wound repair was also shown to be an active process, i.e., requiring live bacteria undergoing active protein synthesis.

scholarly journals Structure of the PilM-PilN Inner Membrane Type IV Pilus Biogenesis Complex from Thermus thermophilus

2011 ◽  
Vol 286 (27) ◽  
pp. 24434-24442 ◽  
Author(s):  
Vijaykumar Karuppiah ◽  
Jeremy P. Derrick
Keyword(s):  
Binding Site ◽  
Atp Hydrolysis ◽  
Thermus Thermophilus ◽  
Narrow Channel ◽  
Dna Uptake ◽  
Type Iv Pilus ◽  
Inner Membrane ◽  
Atp Binding Site ◽  
Type Iv ◽  
Pilus Biogenesis

Type IV pili are surface-exposed filaments, which extend from a variety of bacterial pathogens and play a major role in pathogenesis, motility, and DNA uptake. Here, we present the crystal structure of a complex between a cytoplasmic component of the type IV pilus biogenesis system from Thermus thermophilus, PilM, in complex with a peptide derived from the cytoplasmic portion of the inner membrane protein PilN. PilM also binds ATP, and its structure is most similar to the actin-like protein FtsA. PilN binds in a narrow channel between the 1A and 1C subdomains in PilM; the binding site is well conserved in other Gram-negative bacteria, notably Neisseria meningitidis, Pseudomonas aeruginosa, and Vibrio cholerae. We find no evidence for the catalysis of ATP hydrolysis by PilM; fluorescence data indicate that the protein is likely to be saturated by ATP at physiological concentrations. In addition, binding of the PilN peptide appears to influence the environment of the ATP binding site. This is the first reported structure of a complex between two type IV pilus biogenesis proteins. We propose a model in which PilM binds ATP and then PilN as one of the first steps in the formation of the inner membrane platform of the type IV pilus biogenesis complex.

scholarly journals Neisseria meningitidis Induces Pathology-Associated Cellular and Molecular Changes in Trigeminal Schwann Cells

2020 ◽  
Vol 88 (4) ◽  
Author(s):  
Ali Delbaz ◽  
Mo Chen ◽  
Freda E.-C. Jen ◽  
Benjamin L. Schulz ◽  
Alain-Dominique Gorse ◽  
...  
Keyword(s):  
Neisseria Meningitidis ◽  
Schwann Cells ◽  
Glial Cells ◽  
Trigeminal Nerve ◽  
Three Dimensional ◽  
Cns Infection ◽  
Intercellular Signaling ◽  
Cellular Mechanisms ◽  
Content Type

ABSTRACT Neisseria meningitidis, a common cause of sepsis and bacterial meningitis, infects the meninges and central nervous system (CNS), primarily via paracellular traversal across the blood-brain barrier (BBB) or blood-cerebrospinal fluid barrier. N. meningitidis is often present asymptomatically in the nasopharynx, and the nerves extending between the nasal cavity and the brain constitute an alternative route by which the meningococci may reach the CNS. To date, the cellular mechanisms involved in nerve infection are not fully understood. Peripheral nerve glial cells are phagocytic and are capable of eliminating microorganisms, but some pathogens may be able to overcome this protection mechanism and instead infect the glia, causing cell death or pathology. Here, we show that N. meningitidis readily infects trigeminal Schwann cells (the glial cells of the trigeminal nerve) in vitro in both two-dimensional and three-dimensional cell cultures. Infection of trigeminal Schwann cells may be one mechanism by which N. meningitidis is able to invade the CNS. Infection of the cells led to multinucleation and the appearance of atypical nuclei, with the presence of horseshoe nuclei and the budding of nuclei increasing over time. Using sequential window acquisition of all theoretical mass spectra (SWATH-MS) proteomics followed by bioinformatics pathway analysis, we showed that N. meningitidis induced protein alterations in the glia that were associated with altered intercellular signaling, cell-cell interactions, and cellular movement. The analysis also suggested that the alterations in protein levels were consistent with changes occurring in cancer. Thus, infection of the trigeminal nerve by N. meningitidis may have ongoing adverse effects on the biology of Schwann cells, which may lead to pathology.

scholarly journals Characterization of Three New Competence-Regulated Operons in Haemophilus influenzae

2004 ◽  
Vol 186 (19) ◽  
pp. 6409-6421 ◽  
Author(s):  
Timothy M. VanWagoner ◽  
Paul W. Whitby ◽  
Daniel J. Morton ◽  
Thomas W. Seale ◽  
Terrence L. Stull
Keyword(s):  
Haemophilus Influenzae ◽  
Regulatory Element ◽  
Klebsiella Oxytoca ◽  
Transcriptional Analysis ◽  
Type Ii Secretion ◽  
Dna Uptake ◽  
Exogenous Dna ◽  
Type Iv ◽  
Genes Encoding ◽  
Pilus Biogenesis

ABSTRACT Haemophilus influenzae is one of a growing number of bacteria in which the natural ability to uptake exogenous DNA for potential genomic transformation has been recognized. To date, several operons involved in transformation in this organism have been described. These operons are characterized by a conserved 22-bp regulatory element upstream of the first gene and are induced coincident with transfer from rich to nutrient-depleted media. The previously identified operons comprised genes encoding proteins that include members of the type II secretion system and type IV pili, shown to be essential for transformation in other bacteria, and other proteins previously identified as required for transformation in H. influenzae. In the present study, three novel competence operons were identified by comparative genomics and transcriptional analysis. These operons have been further characterized by construction of null mutants and examination of the resulting transformation phenotypes. The putative protein encoded by the HI0366 gene was shown to be essential for DNA uptake, but not binding, and is homologous to a protein shown to be required for pilus biogenesis and twitching motility in Pseudomonas aeruginosa. An insertion in HI0939 abolished both DNA binding and uptake. The predicted product of this gene shares characteristics with PulJ, a pseudopilin involved in pullulanase export in Klebsiella oxytoca.

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