Cryoelectron-Microscopic Structure of the pKpQIL Conjugative Pili from Carbapenem-Resistant Klebsiella pneumoniae.

Conjugative pili are important in mediating bacterial conjugation and horizontal gene transfer. Since plasmid transfer can include antibiotic-resistance genes, conjugation is an important mechanism in the spread of antibiotic resistance. Filamentous bacteriophages have been shown to exist in two different structural classes: those with a 5-fold rotational symmetry and those with a one-start helix with approximately 5 subunits per turn. Structures for the F and the F-like pED208 conjugation pilus have shown that they have 5-fold rotational symmetry. Here, we report the cryoelectron-microscopic structure of conjugative pili from carbapenem-resistant Klebsiella pneumoniae, encoded on the IncFIIK pKpQIL plasmid, at 3.9 Å resolution and show that it has a one-start helix. These results establish that conjugation pili can exist in at least two structural classes, consistent with other results showing that relatively small perturbations are needed to change the helical symmetry of polymers.

Processing and maturation of the pilin of the type IV secretion system encoded within the gonococcal genetic island.

The type IV secretion system (T4SS) encoded within the gonococcal genetic island (GGI) of Neisseria gonorrhoeae has homology to the T4SS encoded on the F plasmid. The GGI encodes the putative pilin protein TraA and a serine protease TrbI, which is homologous to the TraF protein of the RP4 plasmid involved in circularization of pilin subunits of P-type pili. TraA was processed to a 68-amino acid long circular peptide by leader peptidase and TrbI. Processing occurred after co-translational membrane insertion and was independent of other proteins. Circularization occurred after removal of three C-terminal amino acids. Mutational analysis of TraA revealed limited flexibility at the cleavage and joining sites. Mutagenesis of TrbI showed that the conserved Lys-93 and Asp-155 are essential, whereas mutagenesis of Ser-52, the putative catalytic serine did not influence circularization. Further mutagenesis of other serine residues did not identify a catalytic serine, indicating that TrbI either contains redundant catalytic serine residues or does not function via a serine-lysine dyad mechanism. In vitro studies revealed that circularization occurs via a covalent intermediate between the C terminus of TraA and TrbI. The intermediate is processed to the circular form after cleavage of the N-terminal signal sequence. This is the first demonstration of a covalent intermediate in the circularization mechanism of conjugative pili.

A soluble form of the pilus protein FimA targets the VDAC-hexokinase complex at mitochondria to suppress host cell apoptosis.

Inhibition of apoptotic response of host cells during an early phase of infection is a strategy used by many enteroinvasive bacterial pathogens to enhance their survival. Here, we report the identification of a soluble form of the pilus protein FimA from the culture supernatants of E. coli K1, Salmonella, and Shigella that can potently inhibit Bax-mediated release of cytochrome c from isolated mitochondria. Similar to the infected cells, HCT116 cells stably expressing FimA display a delay in the integration of Bax into outer mitochondrial membrane induced by apoptotic stimuli. FimA targets to mitochondria through binding to VDAC1, which is a prerequisite step for E. coli K1 to render the short-term blockade of apoptotic death in the host cells. Interestingly, FimA strengthens the VDAC1-hexokinase interaction and prevents dissociation of hexokinase from VDAC1 triggered by apoptotic stimuli. Together, these data thus reveal a paradigm of antiapoptosis mechanism undertaken by the enteroinvasive bacteria.

Surface components of Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: Research on Porphyromonas gingivalis, a periodontopathogen, has provided a tremendous amount of information over the last 20 years, which may exceed in part than that on other closely related members in terms of phylogenetic as well as proteomic criteria, including Bacteroides fragilis and B. thetaiotaomicron as major anaerobic, opportunistic pathogens in the medical field. In this minireview, we focused on recent research findings concerning surface components such as outer membrane proteins and fimbriae, of P. gingivalis. MATERIAL AND METHODS: Elucidation of the surface components in P. gingivalis was especially difficult because outer membrane proteins are tightly bound to lipopolysaccharide and they are resistant to dissociation and separation from each other, even during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, unless samples are appropriately heated. In addition, P. gingivalis is asaccharolytic and therefore a potent proteolytic bacterium, another factor causing difficulty in research. The study of the surface components was carefully carried out considering these unique features in P. gingivalis when compared with other gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. RESULTS: Separation of outer membrane proteins, and characterization of OmpA-like proteins and RagAB as major proteins, is described herein. Our recent findings on FimA and Mfa1 fimbriae, two unique appendages in this organism, and on their regulation of expression are also described briefly. CONCLUSION: Surface components of P. gingivalis somehow have contact with host tissues and cells because of the outermost cell elements. Therefore, such bacterial components are potentially important in the occurrence of periodontal diseases.

Protein circlets as sex pilus subunits.

The largest circular protein structures discovered define a class of transfer proteins acting in bacterial conjugation and type IV secretion. Proteins ranging from 73 to 78 residues with head-to-tail peptide bonds constitute the major subunit of conjugative pili of some type IV secretion systems. Their plasmid-encoded precursors are enzymatically processed and cyclized before being assembled into pili. These extra-cellular surface filaments mediate physical contact between donor and recipient cell or pathogen and host cell. Pili are essential prerequisites for DNA and protein transfer. A membrane-bound signal peptidase-like enzyme is responsible for the circularization reaction. Site-directed mutagenesis and mass spectrometry has been used extensively to unravel the mechanism of the enzyme-substrate interaction of the pilin maturation process.

Crystallographic analysis of the Pseudomonas aeruginosa strain K122-4 monomeric pilin reveals a conserved receptor-binding architecture.

Adherence of pathogens to host cells is critical for the initiation of infection and is thus an attractive target for anti-infective therapeutics and vaccines. In the opportunistic human pathogen Pseudomonas aeruginosa, host-cell adherence is achieved predominantly by type IV pili. Analysis of several clinical strains of P. aeruginosa reveals poor sequence conservation between pilin genes, including the residues in the receptor-binding site. Interestingly, the receptor-binding sites appear to retain a conserved surface epitope because all Pseudomonas type IV pili recognize the same receptor on the host cell and cross-reactive antibodies specific for the receptor-binding site exist. Here, we present the crystallographic analysis of two crystal forms of truncated pilin from P. aeruginosa strain K122-4 (DeltaK122-4) at 1.54 and 1.8 A resolution, respectively. The DeltaK122-4 structure is compared to other crystallographically determined type IV pilin structures and an NMR structure of DeltaK122-4 pilin. A comparison with the structure of the highly divergent P. aeruginosa strain K (DeltaPAK) pilin indicates that the receptor-binding loop in both pilins forms a shallow depression with a surface that is formed by main-chain atoms. Conservation of this putative binding site is independent of the sequence as long as the main-chain conformation is conserved and could therefore explain the shared receptor specificity and antibody cross reactivity of highly divergent Pseudomonas type IV pilins.

The Agrobacterium tumefaciens T pilus composed of cyclic T pilin is highly resilient to extreme environments.

Agrobacterium tumefaciens T pili are long semi-rigid, flexuous filaments of 10 nm diameter that are primarily composed of T pilin cyclized protein subunits. The cyclic character of T pilin apparently confers a high level of structural stability on the T pilus. Purified T pili subjected to extreme environmental conditions such as acid and alkali, including glycerol remained relatively unaffected morphologically. T pili lost their semi-rigidity when subjected to high temperatures and high pH, and dissociated into donut shaped subunits when exposed to Triton X-100. Sodium dodecyl sulfate increased the uptake of uranyl acetate exposing a 2 nm wide lumen running the length of the T pilus filament.

Conjugative pili of IncP plasmids, and the Ti plasmid T pilus are composed of cyclic subunits.

TrbC propilin is the precursor of the pilin subunit TrbC of IncP conjugative pili in Escherichia coli. Likewise, its homologue, VirB2 propilin, is processed into T pilin of the Ti plasmid T pilus in Agrobacterium tumefaciens. TrbC and VirB2 propilin are truncated post-translationally at the N terminus by the removal of a 36/47-residue leader peptide, respectively. TrbC propilin undergoes a second processing step by the removal of 27 residues at the C terminus by host-encoded functions followed by the excision of four additional C-terminal residues by a plasmid-borne serine protease. The final product TrbC of 78 residues is cyclized via an intramolecular covalent head-to-tail peptide bond. The T pilin does not undergo additional truncation but is likewise cyclized. The circular structures of these pilins, as verified by mass spectrometry, represent novel primary configurations that conform and assemble into the conjugative apparatus.

Periplasmic and fimbrial SefA from Salmonella enteritidis.

Salmonella enteritidis produces thin, filamentous fimbriae composed of the fimbrin subunit SefA. Although insoluble in most detergents and chaotropic agents, these fimbriae were soluble at pH 10.5. Furthermore, in sodium dodecyl sulfate, these fibers depolymerized into monomers, dimers and other multimers of SefA, which precipitated on removal of the detergent. In contrast, unassembled periplasmic SefA fimbrins purified from Escherichia coli expressing cloned sefA and sefB were readily soluble in aqueous solution. Fimbrial and periplasmic SefA also differed in their reaction with an anti-SEF14 monoclonal antibody and in their surface hydrophobicity, indicating that the two forms had different properties. Precise mass measurements of periplasmic and fimbrial SefA by mass spectroscopy showed that these variations were not due to post-translational modifications. Periplasmic SefA consisted primarily of intact as well as some N-terminally truncated forms. The main 24 amino acid, N-terminally truncated form of periplasmic SefA was present as a 12.2 kDa monomer which had a low tendency to dimerize whereas intact periplasmic SefA was present as a 34.1 kDa homodimer. Intact periplasmic SefA also formed stable multimers at low concentrations of chemical cross-linker but multimerization of the truncated form required high concentrations of protein or cross-linker. Thus, SefA fimbrins appear to multimerize through their N-termini and undergo a conformational change prior to assembly into fibers. Within these fibers, subunit-subunit contact is maintained through strong hydrophobic interactions.

Bacterial adhesion pili are heterologous assemblies of similar subunits.

P-pili on uropathogenic bacteria are 68-A-diameter rods typically 1 microm in length. These structures project from the outer membrane of Escherichia coli, and contain on their distal tip a thin fibrillum, 25 A in diameter and 150 A long, displaying an adhesin protein responsible for the binding of the bacterium to the surface of epithelial cells lining the urinary tract. Operationally, it is possible to identify three morphologically distinct states of the 68-A-diameter P-pili rods, based on the degree of curvature each can adopt. These states are designated "straight," "curved," and "highly curved." The rods can also be unwound to form thin "threads" that are very similar to the tip fibrillae. Electron microscope data are used to distinguish among these four morphological states and to define limits on the shapes of the pilus proteins. The mechanical properties of the PapA polymers are assessed, and implications of rod polymorphism for pilus function are discussed. A wide variety of data are considered in light of the possibility that all pilins are similar in molecular architecture, with specific differences designed to optimize their specialized functions in the pilus assembly.

Diagnostic potential of sefA DNA probes to Salmonella enteritidis and certain other O-serogroup D1 Salmonella serovars.

Salmonella enteritidis thin fimbriae, SEF14, were found to be restricted to S. dublin and the predominantly poultry-associated members of the Salmonella O-serogroup D1, S. enteritidis, S. berta, S. gallinarum and S. pullorum, when tested by Western and ELISA analysis from among 90 Salmonella isolates of 42 serovars, as well as from members of several related genera of the Enterobacteriaceae. These five serovars and a single isolate of S. typhi (D1) were also detected by hybridization of genomic DNA from 732 Salmonella isolates of 117 serogroups to gene probes derived from the S. enteritidis sefA (fimbrin gene), sefB (chaperone) or sefC (outer membrane protein) genes encoding proteins involved in SEF14 biosynthesis. None of 250 Enterobacteriaceae or 27 other eubacterial isolates tested hybridized to the sef probes. The sefA, sefB and sefC genes were amplified from these six Salmonella serovars by PCR using primer pairs designed from sefA, sefB or sefC of S. enteritidis. DNA sequencing of sefA genes from these five serovars indicated limited sequence variability among sefA genes and recognition of individual base pairs which could potentially differentiate certain strains of S. enteritidis, S. dublin and S. gallinarum.

Discovery of a novel protein modification: alpha-glycerophosphate is a substituent of meningococcal pilin.

Pili, which are filamentous protein structures on the surface of the meningitis-causing organism Neisseria meningitidis, are known to be post-translationally modified with substituents that affect their mobility in SDS/PAGE and which might play a crucial role in adherence and bloodstream invasion. Tryptic digests of pili were analysed by fast atom bombardment and electrospray MS to identify putative modifications. Serine-93 was found to carry a novel modification of alpha-glycerophosphate. This is the first time that alpha-glycerophosphate has been observed as a substituent of a prokaryotic or eukaryotic protein.

Adaptation of a conjugal transfer system for the export of pathogenic macromolecules.

Conjugal transfer of bacterial plasmids requires a pore through which DNA can traverse the envelopes of the donor and recipient cells. Recent studies indicate that these pores, which are composed of approximately ten proteins, are evolutionarily related to the transport systems required for the transfer of oncogenic T-DNA from Agrobacterium tumefaciens to plant cells and for toxin secretion from Bordetella pertussis.

Production and characterisation of monoclonal antibodies against pyelonephritis-associated P-pili of Escherichia coli.

Pyelonephritis-associated P-pili (PAP) of Escherichia coli O6,H(-),K1(-),F12,haemolysin(-) were purified by salt precipitation and affinity chromatography using Synsorb P1. Purified PAP showed a single band with a molecular weight of 18 kDa by electrophoretic analysis. A monoclonal antibody (mAb) was produced by fusion of the PAI myeloma cell line with splenic lymphocytes from BALB/c mice immunised with the purified PAP. The mAb was of IgM class with kappa light chains and reacted with a 18-kDa moeity of the salt precipitate; the epitope was present near the apical part of the pilus filaments. The mAb reacted with PAP in both immunofluorescence and haemagglutination tests when 108 strains isolated from urine samples were tested; the two tests were in agreement for 202 of 204 strains isolated from faecal samples.