Pseudomonas aeruginosa pilin activates the inflammasome

IL-1β is produced from inactive pro-IL-1β by activation of caspase-1 brought about by a multi-subunit protein platform called the inflammasome. Many bacteria can trigger inflammasome activity through flagellin activation of the host protein NLRC4. However, strains of the common human pathogen Pseudomonas aeruginosa lacking flagellin can still activate the inflammasome. We set out to identify what non-flagellin components could produce this activation. Using mass spectroscopy, we identified an inflammasome-activating factor from P. aeruginosa as pilin, the major component of the type IV bacterial pilus. Purified pilin introduced into mouse macrophages by liposomal delivery activated caspase-1 and led to secretion of mature IL-1β, as did recombinant pilin purified from Escherichia coli. This was dependent on caspase-1 but not on the host inflammasome proteins NLRC4, NLRP3 or ASC. Mutants of P. aeruginosa strain PA103 lacking pilin did not activate the inflammasome following infection of macrophages with live bacteria. Type III secretion remained intact in the absence of pili, showing this was not due to a lack of effector delivery. Our observations show pilin is a novel activator of the inflammasome in addition to flagellin and the recently described PrgJ protein family, the basal body rod component of the type III apparatus.     

Structural characterization of the Pseudomonas aeruginosa 1244 pilin glycan

An antigenic similarity between lipopolysaccharide (LPS) and glycosylated pilin of Pseudomonas aeruginosa 1244 was noted. We purified a glycan-containing molecule from proteolytically digested pili and showed it to be composed of three sugars and serine. This glycan competed with pure pili and LPS for reaction with an LPS-specific monoclonal antibody, which also inhibited twitching motility by P. aeruginosa bearing glycosylated pili. One-dimensional NMR analysis of the glycan indicated the sugars to be 5N beta OHC(4)7NfmPse, Xyl, and FucNAc. The complete proton assignments of these sugars as well as the serine residue were determined by COSY and TOCSY. Electrospray ionization mass spectrometry (MS) determined the mass of this molecule to be 771.5. The ROESY NMR spectrum, tandem MS/MS analysis, and methylation analysis provided information on linkage and the sequence of oligosaccharide components. These data indicated that the molecule had the following structure: alpha-5N beta OHC(4)7NFmPse-(2-->4)beta-Xyl-(1-->3)-beta-FucNAc-(1-->3)-beta-Ser.     

Glycosylation substrate specificity of Pseudomonas aeruginosa 1244 pilin

The beta-carbon of the Pseudomonas aeruginosa 1244 pilin C-terminal Ser is a site of glycosylation. The present study was conducted to determine the pilin structures necessary for glycosylation. It was found that although Thr could be tolerated at the pilin C terminus, the blocking of the Ser carboxyl group with the addition of an Ala prevented glycosylation. Pilin from strain PA103 was not glycosylated by P. aeruginosa 1244, even when the C-terminal residue was converted to Ser. Substituting the disulfide loop region of strain PA103 pilin with that of strain 1244 allowed glycosylation to take place. Neither conversion of 1244 pilin disulfide loop Cys residues to Ala nor the deletion of segments of this structure prevented glycosylation. It was noted that the PA103 pilin disulfide loop environment was electronegative, whereas that of strain 1244 pilin had an overall positive charge. Insertion of a positive charge into the PA103 pilin disulfide loop of a mutant containing Ser at the C terminus allowed glycosylation to take place. Extending the "tail" region of the PA103 mutant pilin containing Ser at its terminus resulted in robust glycosylation. These results suggest that the terminal Ser is the major pilin glycosylation recognition feature and that this residue cannot be substituted at its carboxyl group. Although no other specific recognition features are present, the pilin surface must be compatible with the reaction apparatus for glycosylation to occur.     

Hyper-recombinogenic RecA protein from Pseudomonas aeruginosa with enhanced activity of its primary DNA binding site

According to one prominent model, each protomer in the activated nucleoprotein filament of homologous recombinase RecA possesses two DNA-binding sites. The primary site binds (1) single-stranded DNA (ssDNA) to form presynaptic complex and (2) the newly formed double-stranded (ds) DNA whereas the secondary site binds (1) dsDNA of a partner to initiate strand exchange and (2) the displaced ssDNA following the strand exchange. RecA protein from Pseudomonas aeruginosa (RecAPa) promotes in Escherichia coli hyper-recombination in an SOS-independent manner. Earlier we revealed that RecAPa rapidly displaces E.coli SSB protein (SSB-Ec) from ssDNA to form presynaptic complex. Here we show that this property (1) is based on increased affinity of ssDNA for the RecAPa primary DNA binding site while the affinity for the secondary site remains similar to that for E.coli RecA, (2) is not specific for SSB-Ec but is also observed for SSB protein from P.aeruginosa that, in turn, predicts a possibility of enhanced recombination repair in this pathogenic bacterium.     

Cloning and sequencing of the Pseudomonas aeruginosa PAK pilin gene

A 1.2-kilobase (kb) HindIII restriction fragment containing the pilin gene from Pseudomonas aeruginosa PAK has been cloned and sequenced. The pilin protein is 144 amino acids in length with a positively charged leader sequence of 6 amino acids. There is probably only one copy of the gene per chromosome.     

The expression of Pseudomonas aeruginosa PAK pilin gene mutants in Escherichia coli

Previous work has demonstrated the expression of the cloned pilin gene of Pseudomonas aeruginosa PAK within Escherichia coli and has pinpointed this protein's localization exclusively to the cytoplasmic membrane (Finlay et al., 1986). To define regions of the pilin subunit necessary for its stability and transport within E. coli, we constructed six mutants of the pilin gene and studied their expression and localization using a T7 promoter system. Two of the mutants have either a 4- or 8-amino-acid deletion at the N-terminus and both were stably expressed and transported primarily to the cytoplasmic membrane of E. coli. The other four mutants are C-terminal truncations having between 36 and 56 amino acids of the N-terminal region of the unprocessed pilin. Studies with these truncated mutants revealed that only the first 36 residues of the unprocessed pilin subunit were required for insertion into the E. coli membrane.     

Identification of pilin pools in the membranes of Pseudomonas aeruginosa.

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.     

铜绿假单胞菌的双组分系统

双组分系统是存在于原核和少部分真核生物细胞中的信号转导系统,主要由组氨酸蛋白激酶和反应调节蛋白组成,通过感应外界环境信号、信号输入、磷酸基团传递、信号输出等环节调节基因表达,使细胞能更加适应环境变化。铜绿假单胞菌为条件致病菌,其双组分系统构成多样、功能复杂且参与介导耐药性产生,因此铜绿假单胞菌的双组分系统日益引起人们关注。本文对铜绿假单胞菌双组分系统的组成、信号转导机制、种类、研究方法及其临床意义进行了综述。     

An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains.

In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two-nucleotide exchange curtailed stimulation of an heterologous promoter. An oligonucleotide known to be recognized with high affinity by Antp-like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.     

Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity

The structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin. Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the O-antigen biosynthetic pathway. In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.     

Expression of the Pseudomonas aeruginosa PAK pilin gene in Escherichia coli.

Pseudomonas aeruginosa is a piliated opportunistic pathogen. We have recently reported the cloning of the structural gene for the pilus protein, pilin, from P. aeruginosa PAK (B. L. Pasloske, B. B. Finlay, and W. Paranchych, FEBS Lett. 183:408-412, 1985), and in this paper we present evidence that this chimera (pBP001) expresses P. aeruginosa PAK pilin in Escherichia coli independent of a vector promoter. The strength of the promoter for the PAK pilin gene was assayed, and the cellular location of the pilin protein within E. coli was examined. This protein was present mainly in the inner membrane fraction both with and without its six-amino-acid leader sequence, but it was not assembled into pili.     

Two unusual pilin sequences from different isolates of Pseudomonas aeruginosa.

The pilin genes of two Pseudomonas aeruginosa strains isolated from two different patients with cystic fibrosis were cloned and sequenced. The predicted protein sequences of these two pilins had several unusual features compared with other published P. aeruginosa pilin sequences.     

The binding of Pseudomonas aeruginosa pili to glycosphingolipids is a tip-associated event involving the C-terminal region of the structural pilin subunit

Pili are one of the adhesins of Pseudomonas aeruginosa that mediate adherence to epithelial cell-surface receptors. The pili of P. aeruginosa strains PAK and PAO were examined and found to bind gangliotetraosyl ceramide (asialo-GM₁) and, to a lesser extend, ll³N-acetylneuraminosylgangliotetraosyl ceramide (GM₁) in solid-phase binding assays. Asialo-GM₁, but not GM₁, inhibited both PAK and PAK pili binding to immobilized asialo-GM₁ on the microtitre plate. PAO pili competitively inhibited PAK pili binding to asialo-GM₁, suggesting the presence of a structurally similar receptor-binding domain in both pilus types. The interaction between asialo-GM₁ and pili occurs at the pilus tip as asialo-GM₁ coated colloidal gold only decorates the tip of purified pili. Three sets of evidence suggest that the C-terminal disulphide-bonded region of the Pseudomonas pilin is exposed at the tip of the pilus: (i) immunocytochemical studies indicate that P. aeruginosa pili have a basal-tip structural differentiation where the monoclonal antibody (mAb) PK3B recognizes an antigenic epitope displayed only on the basal ends of pili (produced by shearing) while the mAb PK99H, whose antigenic epitope resides in residues 134–140 (Wong et al., 1992), binds only to the tip of PAK pili; (ii) synthetic peptides, PAK(128–144)^ox-OH and PAO(128–144)^ox-OH, which correspond to the C-terminal disulphide-bonded region of Pseudomonas pilin are able to bind to asialo-GM₁ and inhibit the binding of pili to the glycolipid; (iii) PK99H was shown to block PAK pilus binding to asialo-GM₁ Monoclonal antibody PK3B had no effect on PAK pili binding to asialo-GM₁ Thus, the adherence of the Pseudomonas pilus to glycosphingolipid receptors is a tip-associated phenomenon Involving a tip-exposed C-terminal region of the pilin structural subunit.