Attachment role of gonococcal pili. Optimum conditions and quantitation of adherence of isolated pili to human cells in vitro.

Pearce, William A.; Buchanan, Thomas M.

doi:10.1172/jci109018

Cited by 87 publications

(35 citation statements)

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“…In gram-negative pathogens, fimbriae are surface organelles which mediate host cell binding (21) and are important virulence factors in uropathogenic strains of E. coli, Proteus mirabilis, Salmonella, Serratia, Neisseria, etc. (15,29,38,43).…”

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Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

et al. 2004

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Xenorhabdus nematophila is an insect pathogen and produces protein toxins which kill the larval host. Previously, we characterized an orally toxic, large, outer membrane-associated protein complex from the culture medium of X. nematophila. Here, we describe the cloning, expression, and characterization of a 17-kDa pilin subunit of X. nematophila isolated from that protein complex. The gene was amplified by PCR, cloned, and expressed in Escherichia coli. The recombinant protein was refolded in vitro in the absence of its cognate chaperone by using a urea gradient. The protein oligomerized during in vitro refolding, forming multimers. Point mutations in the conserved N-terminal residues of the pilin protein greatly destabilized its oligomeric organization, demonstrating the importance of the N terminus in refolding and oligomerization of the pilin subunit by donor strand complementation. The recombinant protein was cytotoxic to cultured Helicoverpa armigera larval hemocytes, causing agglutination and subsequent release of the cytoplasmic enzyme lactate dehydrogenase. The agglutination of larval cells by the 17-kDa protein was inhibited by several sugar derivatives. The biological activity of the purified recombinant protein indicated that it has a conformation similar to that of the native protein. The 17-kDa pilin subunit was found to be orally toxic to fourth-or fifth-instar larvae of an important crop pest, H. armigera, causing extensive damage to the midgut epithelial membrane. To our knowledge, this is first report describing an insecticidal pilin subunit of a bacterium.

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Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

et al. 2004

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“…One of these is the enzyme prepilin peptidase, which cleaves and then N methylates the precursor pilin subunits prior to their assembly into pili. We have used a PCR amplification strategy to clone the N. Pili are important virulence factors for certain types of human bacterial pathogens, such as Pseudomonas aeruginosa (9, 67), Vibrio cholerae (64), and Neisseria gonorrhoeae (43, 61). These three species all produce type IV pili composed of a single major protein subunit (pilin).…”

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“…Pili are important virulence factors for certain types of human bacterial pathogens, such as Pseudomonas aeruginosa (9,67), Vibrio cholerae (64), and Neisseria gonorrhoeae (43,61). These three species all produce type IV pili composed of a single major protein subunit (pilin).…”

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Type IV prepilin peptidase gene of Neisseria gonorrhoeae MS11: presence of a related gene in other piliated and nonpiliated Neisseria strains

Dupuy

Pugsley

1994

J Bacteriol

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The assembly of type IV pili in Neisseria gonorrhoeae is a complex process likely to require the products of many genes. One of these is the enzyme prepilin peptidase, which cleaves and then N methylates the precursor pilin subunits prior to their assembly into pili. We have used a PCR amplification strategy to clone the N. Pili are important virulence factors for certain types of human bacterial pathogens, such as Pseudomonas aeruginosa (9, 67), Vibrio cholerae (64), and Neisseria gonorrhoeae (43, 61). These three species all produce type IV pili composed of a single major protein subunit (pilin). These are produced as precursors which are processed at a highly conserved consensus cleavage site [G l F(M)TLXE] located close to the amino terminus (12,55, 58). Characteristically, the mature pilins have an N-methylated amino-terminal residue (usually phenylalanine or methionine) and retain a long continuous segment of hydrophobic amino acids close to their N terminus which is essential for their polymerization (19,53, 58).The assembly of type IV pili has been most extensively studied in P. aeruginosa, in which four assembly proteins (PilB, PilC, and PilD [37] and PilQ [29]) have been identified, and in V cholerae, in which at least seven assembly proteins (TcpB,41,44]) have been identified. Although the roles of most of these proteins remain obscure, PilD from P. aeruginosa (PilDpa) and TcpJ are known to correspond to the prepilin peptidase, which cleaves the pilin precursors (22,38), and PilD has additionally been shown to N methylate the processed product (60). Intriguingly, several bacteria which are not known to have type IV pili also produce a protein with significant sequence homology to known prepilin peptidases and which is required for extracellular secretion (45). One of these proteins, PulO of Klebsiella oxytoca, can correctly cleave and N methylate the precursor of gonococcal type IV pilin (11) as well as the precursor of a type IV pilin-like protein (PulG) of K oxytoca (46,47 Relatively little is known about the assembly of type IV pili in N. gonorrhoeae. Expression of the N. gonorrhoeae, Dichelobacter nodosus, or Moraxella bovis type IV pilin genes in P. aeruginosa results in their assembly into pili (13,20,31), suggesting that the mechanism of type IV pilus assembly is basically the same in all of these bacteria. One of the major difficulties encountered in attempts to use genetic techniques to identify factors necessary for pilus formation in N. gonorrhoeae is the high frequency of pilus phase variation caused by genome rearrangements (33). However, Jonsson et al. (21) have identified a gene (N. gonorrhoeae pilC [PlCNg]) which seems to be required for piliation, although this view has recently been challenged by Rudel et al. (51), who demonstrated that PilCNg might be required for pilus-mediated adherence rather than for pilus assembly.One of the aspects of N. gonorrhoeae piliation which seems to be readily amenable to genetic analysis is the processing of the pilin precursor. All type IV pilin...

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“…(19) have demonstrated an intriguing correlation among the properties ofhemolysin production, human erythrocyte agglutination, and virulence for chick embryos in E. coli isolated from extraintestinal infections in humans. Adherence to epithelial cells is a critical requirement for virulence of a number of bacteria, including Enterobacteriaceae (20)(21)(22), gonococci (23,24), and streptococci (25). Alternatively, other investigators have suggested that bacterial adherence may in some circumstances subserve host defenses by the elimination of attached bacteria on sloughing epithelial cells (26,27).…”

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confidence: 99%

Complement-independent Adherence of Escherichia coli to Complement Receptors In Vitro

Fine¹,

Harper²,

Carpenter³

et al. 1980

J. Clin. Invest.

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A B S T R A C T We studied adherence to human cells by a strain of Escherichia coli. Adherence to erythrocytes was assessed directly by phase-contrast microscopy and indirectly by hemagglutination; adherence to peripheral blood leukocytes, using radiolabeled bacteria and subsequent determination of leukocyteassociated radioactivity; and adherence to renal glomeruli, by microscopy of fluoresceinated bacteria and of Gram-stained nonfluoresceinated bacteria.In serum-free systems, E. coli of this strain adhered to human erythrocytes, which have surface receptors for the third component of complement (C3), but not to erythrocytes from species lacking this receptor. 1 mM trypan blue, a reagent that inhibits complement receptor function, inhibited adherence to human erythrocytes, as well as adherence to leukocytes and glomeruli. Preincubation of erythrocytes and leukocytes with complement-coated zymosan particles partially blocked subsequent bacterial adherence. Incubation of human erythrocytes with aging human serum, with trypsin-cleaved C3, or with C3 cleaved by the classical pathway convertase (EAC142)-all of which treatments deposited C3 on the erythrocyte surface, presumably at C3 receptors-inhibited subsequent E. coli adherence. Finally, incubation of E. coli with rabbit antiserum to human C3 blocked adherence to erythrocytes.Bacterial hemagglutination and erythrocyte adherence were not inhibited by mannose in concentrations up to 2.5%. And this strain of E. coli did not adhere to or agglutinate guinea pig erythrocytes, the usual test particle used for demonstration of common pili. Finally, electron microscopy of adherent bacteria showed only rare surface pili. In METHODSBacteria. Laboratory strains of various bacteria were lyophilized or stored at -70°C. After reconstitution, cultures were maintained on trypticase soy (TS) agar containing 5% sheep blood, on Mueller-Hinton (M-H) agar, or in brain-heart infusion (BHI) broth. For individual experiments, organisms were grown 18-24 h on TS agar, M-H agar, or blood agar, or in TS, M-H, or BHI broth; scraped from the agar or centrifuged from the broth; and washed once in Hanks' balanced salt solution (BHS) without phenol red. Organisms were suspended to -1 x 109 organisms/ml as determined by absoYrption at 541 nm (absorbance = 1.5) and confirmed by colony counts.Fluorescent bacteria were prepared according to the method of Gelfand et al. (1). Radiolabeled bacteria were prepared by suspending a loopful of bacteria grown on M-H agar in 0.5 ml BHS containing 50 ItCi of "4C-labeled amino acids (New England Nuclear, Boston, Mass.) and mixing vigorously. The suspension was spread evenly over the surface of M-H agar and incubated 18 h at 37°C in 5% CO2. Bacteria were harvested from the plate, washed twice in BHS by centrifugation at 1,500 g, and finally resuspended in BHS to a concentration of 1 x 109 bacteria/ml and 78,980+4,300 cpm/ml (Beckman liquid scintillation spectrometer, Beckman Instruments Inc., Fullerton, Calif.).A serum-resistant strain of E. coli, designated E...

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Attachment role of gonococcal pili. Optimum conditions and quantitation of adherence of isolated pili to human cells in vitro.

Cited by 87 publications

References 50 publications

Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

Insecticidal Pilin Subunit from the Insect Pathogen Xenorhabdus nematophila

Type IV prepilin peptidase gene of Neisseria gonorrhoeae MS11: presence of a related gene in other piliated and nonpiliated Neisseria strains

Complement-independent Adherence of Escherichia coli to Complement Receptors In Vitro

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