Expression of multiple types of N-methyl Phe pili in Pseudomonas aeruginosa

The nature of pili synthesized by Pseudomonas aeruginosa when plasmid-borne genes of homologous pilins from Bacteroides nodosus are introduced as thermoregulated expression systems has been ascertained. Expression of B. nodosus pili inhibited the production of indigenous P. aeruginosa pili, and an organism harbouring pilin genes from two strains of B. nodosus produced two serologically distinct populations of pili on each cell. Simultaneous production of both indigenous and foreign pili was achieved by partial induction of expression. Homogeneity in pilus structure suggests either that there is an exclusive specificity of interaction between identical pilin subunits in pilus assembly, or that each pilus is produced from the translation products of a single messenger RNA molecule, with translation and pilus assembly closely coupled.     

Role of pili in the pathogenesis of Pseudomonas aeruginosa burn infection

The present study using three isogenic mutants (F+P-, F-P+, F-P-) of Pseudomonas aeruginosa indicates that the presence of pili enhances the virulence of the organisms in experimental P. aeruginosa burn infection of mice. The 50% lethal dose (LD50) value for burned mice inoculated with non-piliated (P-) mutant was at least ten times higher than those inoculated with piliated (P+) bacteria. Meanwhile the LD50 value for burned mice inoculated with non-flagellated (F-) mutant was at least 10(5) times higher than those inoculated with flagellated (F+) bacteria. At 24 hr after inoculation, the bacterial counts in burned skin of mice inoculated with P+ bacteria were ten times higher than those inoculated with P- bacteria; and at 48 hr the bacterial counts became a hundred times higher in the former mice than the latter. At 24 hr after inoculation, P+ bacteria were isolated from blood, liver (F+P+), lung (F+P+), and kidney, while P- bacteria were not present in these tissues. And at 48 hr after inoculation, P+ bacteria were isolated from all tissues, while P- bacteria were isolated from some sites only. These results suggested that pili and flagella each play an important role as virulence factors independently, and that pili-mediated enhancement of virulence of P. aeruginosa was attributed to pili-mediated enhanced colonization of the organisms at the burned skin surfaces.     

Production of Neisseria gonorrhoeae pili (fimbriae) in Pseudomonas aeruginosa.

Pseudomonas aeruginosa K/2PfS, when transformed with an expression plasmid harboring the pilin gene (pilE1) of Neisseria gonorrhoeae MS11, was able to express and assemble gonococcal pilin monomers into surface-associated pili, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and immunoelectron microscopy. Concomitant with the expression of gonococcal pili in P. aeruginosa was the virtual loss of production of P. aeruginosa K/2PfS pili normally associated with the host cell.     

Structure of polar pili from Pseudomonas aeruginosa strains K and O

The polar pili of Pseudomonas aeruginosa strains K and O are hollow cylinders with 52 Å outer diameter and 12 Å inner diameter. There is a girdle of low electron density (interpreted as due to a local concentration of hydrophobic amino acid side-chains) centred at 31 Å diameter. Similar X-ray diffraction patterns are obtained from oriented fibres of the two types of pili, to a resolution of 7 Å in the equatorial direction and 4 Å in the meridional direction. The two types of pilin protein subunits have a similar molecular weight, and their sequences contain a number of homologous regions. They form a helical array with 4.06 to 4.08 units per turn of a basic helix that has a pitch of 40.8 Å for strain K pili and 41.3 Å for strain O pili at 75% relative humidity. A method is described for distinguishing between very similar diffraction patterns.There is strong intensity at 10 Å near the equator and at 5 Å near the meridian on the diffraction patterns. This intensity distribution is characteristic of α-helical rods running roughly in the direction of the fibre axis. The orientation of these rods was established by the fit between the transform of an α-helical polyalanine model and the strong near-equatorial layer-line.     

A function of Pseudomonas aeruginosa PAO polar pili: twitching motility

Twitching motility is a mode of flagella-independent surface translocation exhibited by Pseudomonas aeruginosa and other bacteria on solid media. All species exhibiting it carry thin pili, usually polar. This work shows that only PAO and K strains of P. aeruginosa with retractile (PSA) pili were able to move in this way, those with no pili or non-retractile pili remaining stationary. Specific agents such as anti-pilus serum, which prevents otherwise functional pili from retracting, also prevented twitching motility.     

Biochemical studies on pili isolated from Pseudomonas aeruginosa strain PAO.

Pseudomonas aeruginosa strains PAO and PAK bear polar pili which are flexible filaments having a diameter of 6 nm and an average length of 2500 nm. Both types of pili are retractile and promote infection by a number of bacteriophages. The present communication describes the partial biochemical characterization of PAO pili isolated from a multipiliated nonretractile mutant of PAO. The observed properties are compared to those of PAK pili which were characterized previously. PAO pili were found to contain a single polypeptide subunit of 18 700 daltons. This is similar to PAK pili which contain a single polypeptide of 18 100 daltons. The amino acid composition of PAO pilin was also similar to that of PAK pilin. Neither protein contained phosphate or carbohydrate residues and both were found to contain N-methylphenylalanine at the amino terminus. Sequencing of 20 amino acid residues at the amino terminal end of PAO pilin revealed the sequence to be identical with that of PAK pilin, while tryptic peptide analyses of PAO and PAK pilin indicated that the two proteins probably contain a number of homologous regions within the polypeptide. It was concluded that PAO and PAK pili were closely related structures.     

Morphogenetic expression of Moraxella bovis fimbriae (pili) in Pseudomonas aeruginosa.

Type 4 fimbriae (pili) are found in a wide variety of gram-negative bacteria and are composed of small structural subunits which share significant sequence homology among different species, especially at their amino-terminal ends. Previous studies demonstrating morphogenetic expression of Bacteroides nodosus fimbriae from cloned subunit genes in Pseudomonas aeruginosa suggested that there is a common mechanism for type 4 fimbriae assembly and that the structural subunits are interchangeable (J. S. Mattick et al., J. Bacteriol. 169:33-41, 1987). Here we have examined the expression of Moraxella bovis fimbrial subunits in P. aeruginosa. M. bovis subunits were assembled into extracellular fimbriae in this host, in some cases as a homopolymer but in others as a mosaic with the indigenous subunit, indicating structural equivalence. This result contrasts with other studies in which recombinant P. aeruginosa expressing different subunits produced fimbriae composed almost exclusively of one subunit or the other (T. C. Elleman and J. E. Peterson, Mol. Microbiol. 1:377-380, 1987). Both observations can be explained by reversibility of subunit-subunit interactions at the site of assembly, with the forward equilibrium favoring chain extension between compatible subunits.     

DNA binding: a novel function of Pseudomonas aeruginosa type IV pili

The opportunistic pathogen Pseudomonas aeruginosa produces multifunctional, polar, filamentous appendages termed type IV pili. Type IV pili are involved in colonization during infection, twitching motility, biofilm formation, bacteriophage infection, and natural transformation. Electrostatic surface analysis of modeled pilus fibers generated from P. aeruginosa strain PAK, K122-4, and KB-7 pilin monomers suggested that a solvent-exposed band of positive charge may be a common feature of all type IV pili. Several functions of type IV pili, including natural transformation and biofilm formation, involve DNA. We investigated the ability of P. aeruginosa type IV pili to bind DNA. Purified PAK, K122-4, and KB-7 pili were observed to bind both bacterial plasmid and salmon sperm DNA in a concentration-dependent and saturable manner. PAK pili had the highest affinity for DNA, followed by K122-4 and KB-7 pili. DNA binding involved backbone interactions and preferential binding to pyrimidine residues even though there was no evidence of sequence-specific binding. Pilus-mediated DNA binding was a function of the intact pilus and thus required elements present in the quaternary structure. However, binding also involved the pilus tip as tip-specific, but not base-specific, antibodies inhibited DNA binding. The conservation of a Thr residue in all type IV pilin monomers examined to date, along with the electrostatic data, implies that DNA binding is a conserved function of type IV pili. Pilus-mediated DNA binding could be important for biofilm formation both in vivo during an infection and ex vivo on abiotic surfaces.     

Composition and molecular weight of pili purified from Pseudomonas aeruginosa K.

Pseudomonas aeruginosa strain K (PAK) bears polar pili that promote infection by at least six bacteriophages. Moreover, a recently isolated mutant of strain K (PAK/2PfS) is many times more piliated than the wild-type strain and facilitates the preparation of large amounts of pure pili for biochemical studies. The present investigation was carried out to establish the structural relatedness of PAK and PAK/2PfS pili and to determine their biochemical composition. A purfication procedure is described for PAK and PAK/2PfS pili that yields about 8 mg of pure pili per 100 g (wet weight) of PAK/2PfS cells and 0.8 mg of pure pili per 100 g (wet weight) of PAK cells. PAK and PAK/2PfS pili were found to be free from phosphate, carbohydrate, and lipid and to contain a single polypeptide subunit of 17,800 daltons. Isopycnic centrifugation studies revealed that PAK and PAK(2PfS pili have the same buoyant density in sucrose (1.221) and CsC1 (1.295). Both types of pili banded at pH 3.9 when subjected to isoelectric focusing. Amino acid analyses showed that PAK and PAK/2PfS pili have identical amino acid compositions, whereas microimmunodiffusion studies revealed that the two types of pili are immunologically indistinguishable. It was concluded that PAK and PAK/2PfS pili are identical and that the mutation responsible for producing the multipiliated state in PAK/2PfS is probably located outside the structural gene for PAK pili.     

Pseudomonas aeruginosa exhibits sliding motility in the absence of type IV pili and flagella

Pseudomonas aeruginosa exhibits swarming motility on 0.5 to 1% agar plates in the presence of specific carbon and nitrogen sources. We have found that PAO1 double mutants expressing neither flagella nor type IV pili (fliC pilA) display sliding motility under the same conditions. Sliding motility was inhibited when type IV pilus expression was restored; like swarming motility, it also decreased in the absence of rhamnolipid surfactant production. Transposon insertions in gacA and gacS increased sliding motility and restored tendril formation to spreading colonies, while transposon insertions in retS abolished motility. These changes in motility were not accompanied by detectable changes in rhamnolipid surfactant production or by the appearance of bacterial surface structures that might power sliding motility. We propose that P. aeruginosa requires flagella during swarming to overcome adhesive interactions mediated by type IV pili. The apparent dependence of sliding motility on environmental cues and regulatory pathways that also affect swarming motility suggests that both forms of motility are influenced by similar cohesive factors that restrict translocation, as well as by dispersive factors that facilitate spreading. Studies of sliding motility may be particularly well-suited for identifying factors other than pili and flagella that affect community behaviors of P. aeruginosa.     

Expression of the argF gene of Pseudomonas aeruginosa in Pseudomonas aeruginosa, Pseudomonas putida, and Escherichia coli

R' plasmids carrying argF genes from Pseudomonas aeruginosa strains PAO and PAC were transferred to Pseudomonas putida argF and Escherichia coli argF strains. Expression in P. putida was similar to that in P. aeruginosa and was repressed by exogenous arginine. Expression in E. coli was 2 to 4% of that in P. aeruginosa. Exogenous arginine had no effect, and there were no significant differences between argR' and argR strains of E. coli in this respect.     

铜绿假单胞杆菌QS相关基因差异表达的研究

选取100个与铜绿假单胞杆菌（Pseudomonas aeruginosa）群感效应（quorum-sensing,QS）相关的基因,克隆这些基因片段于pMD-18T载体,测序鉴定,点样制备cDNA基因芯片。制备cy3-dCTP／cy5-dCTP标记的探针,与芯片杂交。初步研究了处于不同生长期的铜绿假单胞杆菌基因的表达差异。指数中期和平台初期相比,有9个QS基因表达量最著增加,有6个基因表达量显著下降。利用芯片做针对铜绿菌假单胞杆菌药物的筛选：妥布霉素（Tobramycin）给药后细菌基因发生差异表达。证明了该cDNA芯片用于药物筛选的可行性。在国内首次研制开发了QS相关基因的cDNA芯片。应用基因芯片技术建立的铜绿假单胞杆菌QS相关基因研究平台,为找到能较好抑制铜绿假单胞杆菌正常生长的药物研究提出新的解决方法。     

FppA, a novel Pseudomonas aeruginosa prepilin peptidase involved in assembly of type IVb pili

Several subclasses of type IV pili have been described according to the characteristics of the structural prepilin subunit. Whereas molecular mechanisms of type IVa pilus assembly have been well documented for Pseudomonas aeruginosa and involve the PilD prepilin peptidase, no type IVb pili have been described in this microorganism. One subclass of type IVb prepilins has been identified as the Flp prepilin subfamily. Long and bundled Flp pili involved in tight adherence have been identified in Actinobacillus actinomycetemcomitans, for which assembly was due to a dedicated machinery encoded by the tad-rcp locus. A similar flp-tad-rcp locus containing flp, tad, and rcp gene homologues was identified in the P. aeruginosa genome. The function of these genes has been investigated, which revealed their involvement in the formation of extracellular Flp appendages. We also identified a gene (designated by open reading frame PA4295) outside the flp-tad-rcp locus, that we named fppA, encoding a novel prepilin peptidase. This is the second enzyme of this kind found in P. aeruginosa; however, it appears to be truncated and is similar to the C-terminal domain of the previously characterized PilD peptidase. In this study, we show that FppA is responsible for the maturation of the Flp prepilin and belongs to the aspartic acid protease family. We also demonstrate that FppA is required for the assembly of cell surface appendages that we called Flp pili. Finally, we observed an Flp-dependent bacterial aggregation process on the epithelial cell surface and an increased biofilm phenotype linked to Flp pilus assembly.     

The cost of multiple drug resistance in Pseudomonas aeruginosa

The spread of bacterial antibiotic resistance mutations is thought to be constrained by their pleiotropic fitness costs. Here we investigate the fitness costs of resistance in the context of the evolution of multiple drug resistance (MDR), by measuring the cost of acquiring streptomycin resistance mutations (StrepR) in independent strains of the bacterium Pseudomonas aeruginosa carrying different rifampicin resistance (RifR) mutations. In the absence of antibiotics, StrepR mutations are associated with similar fitness costs in different RifR genetic backgrounds. The cost of StrepR mutations is greater in a rifampicin‐sensitive (RifS) background, directly demonstrating antagonistic epistasis between resistance mutations. In the presence of rifampicin, StrepR mutations have contrasting effects in different RifR backgrounds: StrepR mutations have no detectable costs in some RifR backgrounds and massive fitness costs in others. Our results clearly demonstrate the importance of epistasis and genotype‐by‐environment interactions for the evolution of MDR.     

Swarming of Pseudomonas aeruginosa is dependent on cell-to-cell signaling and requires flagella and pili

We describe swarming in Pseudomonas aeruginosa as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in P. aeruginosa is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a fliC mutant of P. aeruginosa displayed slightly diminished swarming, a pilR and a pilA mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the las cell-to-cell signaling system showed diminished swarming behavior, while rhl mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of P. aeruginosa in this type of surface motility.     

Intratracheal immunization with pili protein protects against mortality associated with Pseudomonas aeruginosa pneumonia in mice

We examined the protective effect of intratracheal immunization with Pseudomonas aeruginosa pili protein against respiratory infection caused by P. aeruginosa. Mice were immunized intratracheally or subcutaneously with purified pili protein or bovine serum albumin as a control. Intratracheally but not subcutaneously pili protein-immunized mice showed significant improvement of survival after intratracheal challenge with the PAO1 strain. Furthermore, bacterial cell counts in pili protein-immunized murine lungs were significantly decreased compared to controls at 18 h after the challenge. Antipili protein antibody titers in bronchoalveolar lavage fluid of intratracheally pili protein-immunized mice were higher than in bovine serum albumin immunized mice. However, antipili antibody titers were not increased in bronchoalveolar lavage fluid of subcutaneously pili protein-immunized mice, despite the high serum antipili antibody titers. Inoculation of P. aeruginosa induced immediate increases in interleukin-12 and interferon-gamma in bronchoalveolar lavage fluid of pili protein-immunized mice, reflecting an adequate and rapid immune response against P. aeruginosa respiratory tract infection. Our findings suggest that intratracheal pili protein immunization is effective against respiratory tract infection caused by P. aeruginosa in mice.