Biomedical subjects

S Lory

Publications and source records attributed to S Lory.

At least 37 records · Page 2Linked to original sources

PubMed1997

Structure-function relationship of type-IV prepilin peptidase of Pseudomonas aeruginosa--a review.

The bifunctional enzyme prepilin peptidase (PilD) from Pseudomonas aeruginosa is a key determinant in both type-IV pilus biogenesis and extracellular protein secretion, in its roles as a leader peptidase and MTase. It is responsible for endopeptidic cleavage of the unique leader peptides that characterize type-IV pilin precursors, as well as proteins with homologous leader sequences that are essential components of the general secretion pathway found in a variety of Gram-negative pathogens. Following removal of the leader peptides, the same enzyme is responsible for the second posttranslational modification that characterizes the type-IV pilins and their homologues, namely N-methylation of the newly exposed N-terminal amino acid residue. This review discusses some of the work begun in order to answer questions regarding the structure-function relationships of the active sites of this unique enzyme.

Amino Acid Sequence↗

PubMed1997

Interactions of the components of the general secretion pathway: role of Pseudomonas aeruginosa type IV pilin subunits in complex formation and extracellular protein secretion.

The general secretion pathway (GSP), found in a wide range of bacteria, is responsible for extracellular targeting of a subset of proteins from the periplasm. In Pseudomonas aeruginosa, the GSP requires the participation of 12 proteins, of which XcpT, XcpU, XcpV, XcpW are homologues of PilA, the major subunit of type IV pili. The interaction between the pilin-like Xcp proteins was investigated using bifunctional crosslinking reagents. Cross-linking analysis of whole cells of wild-type P. aeruginosa, followed by immunoblot analysis, revealed a 34-kDa XcpT-containing complex. This complex was shown to consist of XcpT/PilA heterodimers. The role of PilA in the GSP was examined, using P. aeruginosa mutants in the pilA gene, or in rpoN, a gene regulating pilA expression. Each mutant showed a significant reduction in the efficiency of extracellular protein secretion, and this defect could be restored by expression of the cloned pilA gene in the mutant cells. The formation of the PilA/XcpT complex did not require XcpR or XcpQ, two other components of the secretion machinery, nor did it require the pilus biogenesis factors PilB and PIlC. The dimeric XcpT/PilA complex was also formed in a pilD mutant, which lacks the leader peptidase enzyme, demonstrating that the leader peptide at the N-terminus or PilA or XcpT did not have to be removed for the dimerization to occur. XcpW and XcpU can also be crosslinked to form dimeric complexes with PilA. When expression of XcpT is increased, its homodimers, as well as XcpT/XcpW heterodimers, can be detected. Finally, an oligohistidine-tagged XcpT was shown to form stoichiometric complexes with PilA, and with XcpT, U, V and W. These dimers were co-purified by nickel-affinity chromatography. The results of this study suggest that XcpT can form heterodimers with PilA, and Xcp U, V and W, which may be assembly intermediates of the secretion apparatus. Alternatively, these may represent dynamic intermediates that facilitate protein secretion by continuous association and dissociation. The requirement for PilA for efficient protein secretion argues for a critical role played by PilA in two related processes during P. aeruginosa infections: formation of an adhesive pilus organelle and secretion of exoenzymes.

Bacterial Outer Membrane Proteins↗

PubMed1997

The XcpR protein of Pseudomonas aeruginosa dimerizes via its N-terminus.

Extracellular protein secretion by the main terminal branch of the general secretory pathway in Pseudomonas aeruginosa requires a secretion machinery comprising the products of at least 12 genes. One of the components of this machinery, the XcpR protein, belongs to a large family of related proteins distinguished by the presence of a highly conserved nucleotide binding domain (Walker box A). The XcpR protein is essential for the process of extracellular secretion and amino acid substitutions within the Walker A sequence result in inactive XcpR. The same mutations exert a dominant negative effect on protein secretion when expressed in wild-type bacteria. Transdominance of XcpR mutants suggests that this protein is involved in interactions with other components of the secretion machinery or that it functions as a multimer. In this study, the amino-terminal portion of the cl repressor protein of phage lambda was used as a reporter of dimerization in Escherichia coli following fusion to full-length as well as a truncated form of XcpR. The cl-XcpR hybrid proteins were able to dimerize, as demonstrated by the immunity of bacteria expressing them to killing by lambda phage. The full-length XcpR as well as several deletion mutants of XcpR were able to disrupt the dimerization of the chimeric cl-XcpR protein. The disruption of cl-XcpR dimers using the deletion mutants of XcpR, combined with the analysis of their dominant negative effects on protein secretion, was used to map the minimal dimerization domain of XcpR, which is located within an 85 amino acid region in its N-terminal domain. Taken together, the data presented in this paper suggest that the XcpR protein dimerizes via its N-terminus and that this dimerization is essential for extracellular protein secretion.

Bacterial Proteins↗

PubMed1997

A transcriptional activator, FleQ, regulates mucin adhesion and flagellar gene expression in Pseudomonas aeruginosa in a cascade manner.

Previous work has demonstrated that fleR, the gene for a transcriptional activator belonging to the NtrC subfamily of response regulators, is involved in the regulation of mucin adhesion and flagellar expression by Pseudomonas aeruginosa. This report describes the identification and characterization of fleQ, the gene for another transcriptional regulator which also regulates mucin adhesion and motility in this organism. The complete nucleotide sequence of the fleQ gene was determined on both DNA strands, and an open reading frame (ORF) consisting of 1,493 nucleotides was identified. This ORF coded for a gene product of predicted molecular weight, as confirmed by the overexpression of the fleQ gene as a fusion protein under an inducible promoter. The fleQ gene is flanked by a flagellar operon, fliDSorf126, at the 5' end and the fleSR operon on the 3' end. FleQ also had striking homology to a number of proteins belonging to the NtrC subfamily of response regulators, which work in concert with the alternate sigma factor RpoN (sigma54) to activate transcription. However, FleQ lacks the residues corresponding to Asp-54 and Lys-104 of the NtrC protein which are conserved in most of the members belonging to this subfamily of regulators. In addition, unlike some of the other transcriptional activators of this group, FleQ does not appear to have a cognate sensor kinase. A chromosomal insertional mutation in the fleQ gene abolished mucin adhesion and motility of P. aeruginosa PAK and PAK-NP. Both of these functions were regained by providing the complete fleQ gene on a multicopy plasmid. The location of fleQ immediately upstream of the fleSR operon, which is also necessary for the same process, suggested that these regulators may interact in some way. We therefore examined the regulation of the fleSR operon by fleQ and vice versa. Promoter fusion experiments showed that the fleSR operon was regulated by RpoN and FleQ. On the other hand, the fleQ promoter was independent of RpoN and FleR. FleQ, thus, adds another level of regulation to motility and adhesion in P. aeruginosa, above that of fleSR. We therefore propose the existence of a regulatory cascade which consists of at least two transcriptional regulators, FleQ and FleR, in the control of motility and adhesion in P. aeruginosa.

Amino Acid Sequence↗

PubMed1996

Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection.

Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised patients and those with cystic fibrosis genetic disease. To identify new virulence genes of P. aeruginosa, a selection system was developed based on the in vivo expression technology (IVET) that was first reported in Salmonella system. An adenine-requiring auxotrophic mutant strain of P. aeruginosa was isolated and found avirulent on neutropenic mice. A DNA fragment that can complement the mutant strain, containing purEK operon that is required for de novo biosynthesis of purine, was sequenced and used in the IVET vector construction. By applying the IVET selection system to a neutropenic mouse infection model, genetic loci that are specifically induced in vivo were identified. Twenty-two such loci were partially sequenced and analyzed. One of them was a well-studied virulence factor, pyochelin receptor (FptA), that is involved in iron acquisition. Fifteen showed significant homology to reported sequences in GenBank, while the remaining six did not. One locus, designated np20, encodes an open reading frame that shares amino acid sequence homology to transcriptional regulators, especially to the ferric uptake regulator (Fur) proteins of other bacteria. An insertional np20 null mutant strain of P. aeruginosa did not show a growth defect on laboratory media; however, its virulence on neutropenic mice was significantly reduced compared with that of a wild-type parent strain, demonstrating the importance of the np20 locus in the bacterial virulence. The successful isolation of genetic loci that affect bacterial virulence demonstrates the utility of the IVET system in identification of new virulence genes of P. aeruginosa.

Amino Acid Sequence↗

PubMed1996

Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein.

Only one of the characterized components of the main terminal branch of the general secretory pathway (GSP) in Gram-negative bacteria, GspD, is an integral outer membrane protein that could conceivably form a channel to permit protein transport across this membrane. PulD, a member of the GspD protein family required for pullulanase secretion by Klebsiella oxytoca, is shown here to form outer membrane-associated complexes which are not readily dissociated by SDS treatment. The outer membrane association of PulD is absolutely dependent on another component of the GSP, the outer membrane-anchored lipoprotein PulS. Furthermore, the absence of PulS resulted in limited proteolysis of PulD and caused induction of the so-called phage shock response, as measured by increased expression of the pspA gene. We propose that PulS may be the first member of a new family of periplasmic chaperones that are specifically required for the insertion of a group of outer membrane proteins into this membrane. PulS is only the second component of the main terminal branch of the GSP for which a precise function can be proposed.

Bacterial Outer Membrane Proteins↗

PubMed1996

A specific targeting domain in mature exotoxin A is required for its extracellular secretion from Pseudomonas aeruginosa.

A number of Gram-negative bacteria, including Pseudomonas aeruginosa, actively secrete a subset of periplasmic proteins into their surrounding medium. The presence of a putative extracellular targeting signal within one such protein, exotoxin A, was investigated. A series of exotoxin A truncates, fused to beta-lactamase, was constructed. Hybrid proteins, which carry at their N- termini 120, 255, 355 or the entire 613 residues of the mature exotoxin A, were stable and were secreted into the extracellular medium. Hybrid proteins which carry residues 1-30 and 1-60 of the mature exotoxin A were unstable; however, they could be detected entirely within the cells after a short labeling period. A hybrid with beta-lactamase was constructed which carried only the N-terminal residues 1-3 and region 60-120 of exotoxin A. It was also secreted into the culture medium, suggesting that a specific 60 amino acid domain contains the necessary targeting information for translocation of exotoxin A across the outer membrane. The secretion of the hybrid proteins is independent of the passenger protein, since a similar exotoxin A-murine interleukin 4 hybrid protein was also secreted. The extracellular targeting signal between amino acids 60 and 120 is rich in anti-parallel beta-sheets. It has been shown previously to be involved in the interaction of the exotoxin A with the receptors of the eukaryotic cells. In the three- dimensional view, the targeting region is on the toxin surface where it is easily accessible to the components of the extracellular secretion machinery.

ADP Ribose Transferases↗

PubMed1996

Isolation and characterization of Pseudomonas aeruginosa genes inducible by respiratory mucus derived from cystic fibrosis patients.

Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised individuals and those with cystic fibrosis (CF). In CF patients, the secretion of abnormally high amounts of mucus into the airways contributes to their susceptibility to infection by P. aeruginosa. To identify virulence genes of P. aeruginosa that are important in infection of CF patients, an in vivo selection system (IVET) was used to identify promoters that are specifically inducible by respiratory mucus derived from CF patients. Three genetic loci that are highly inducible by the mucus were identified. One of them is a well-characterized virulence gene (fptA), encoding the receptor for pyochelin, which is a P. aeruginosa iron siderophore. Induction of the fptA gene by mucus is suppressed by the addition of exogenous iron, demonstrating that the mucus is an iron chelator and generates an iron-deficient environment in CF lungs. Therefore, as a part of the host-defence mechanism, the mucus could also be responsible for induction of iron-regulated virulence factors of bacterial pathogens. The second locus, np20, encodes a peptide that shares sequence homology to a number of transcriptional regulators. An identical locus was previously identified to be inducible in vivo during infection of mice and was shown to be important in bacterial virulence in a neutropenic-mouse infection model. The third locus, designated migA (mucus inducible gene), was sequenced and found to encode a 299-amino-acid peptide which is homologous to glycosyltransferases of other bacteria, and is involved in the biosynthesis of lipopolysaccharides or exopolysaccharides. Inducibilities of the np20 and migA genes are not affected by iron and the exact nature of the inducing signals in the mucus is not known. The possible implications of the migA inducibility by respiratory mucus is discussed in relation to the P. aeruginosa infection in CF.

Amino Acid Sequence↗

PubMed1996

Intestinal mucin distribution in the germ-free rat and in the heteroxenic rat harbouring a human bacterial flora: effect of inulin in the diet.

A colorimetric method was used on water-soluble mucin extracted from mucosal scrapings and contents of the caecum and the colon of five germ-free (GF) rats and five heteroxenic (HE) rats harbouring a human flora (GF rats associated with a human flora). These rats were fed on a diet containing either 100 g sucrose/kg or 100 g inulin/kg. Histological stains, periodic acid-Schiff, alcian blue pH 2.5 and alcian blue pH 0.5 were used to discriminate between neutral, acidic and acidic sulphated mucins respectively. Spectrocolorimetric assays led to a calculated absorbance value for 1 mg of the initial mucin extract. Each mucin type was compared between treatments. The caecal contents of GF rats contained more acidic mucin than sulphomucin, which was present in the same proportion as neutral mucin. Their colonic contents contained more acidic mucins than sulphomucin, which in turn was more abundant than neutral mucin. Their caecal mucosa mucin distribution differed from that of the contents: very little acidic mucin was present and neutral and sulphomucin proportions were of the same order of magnitude. Inulin increased the amount of neutral mucin in the caecal contents and of sulphated mucins in the colonic contents and increased the amounts of neutral and acidic mucins in the caecal mucosa. Mucin distribution in the HE rats was very different from that in the GF rats: the caecal contents contained a high proportion of acidic mucins and very little sulphomucin. The same distribution of mucins was observed in the colonic contents. The caecal mucosa contained less acidic mucin and more sulphomucin than the caecal contents. Inulin decreased acidic mucins and increased sulphated mucins in the caecal contents and increased neutral and sulphated mucins in the colonic contents. Inulin increased sulphomucin in the caecal mucosa and decreased acidic mucin in the caecal and colonic mucosas. The very low amount of mucin that was recovered in the colonic mucosa suggests that, in the presence of the bacterial flora and associated with inulin in the diet, mucin was extensively released from the mucosa to the colonic lumen. This might be related to the bacterial metabolites produced.

Animals↗

PubMed1996

Cloning and characterization of Pseudomonas aeruginosa fliF, necessary for flagellar assembly and bacterial adherence to mucin.

Pseudomonas aeruginosa adheres to the mucosal surfaces of the lungs. This process appears to be mediated by nonpilus adhesins which bind to mucin. To find this nonpilus adhesin(s), mutagenesis of a nonpiliated mutant of P. aeruginosa with transposon Tn5G, followed by a screen for mucin adhesion, was used to isolate a series of mutants unable to adhere to mucin. All of these mutants were also found to be defective in motility. One such mutant, PAK-RR20, is characterized here. The site of the transposon insertion in PAK-RR20 was localized to a gene which is homologous to the fliF gene of other organisms and was flanked by other motility-related genes, fliE and fliG. Both adhesion and motility defects in PAK-RR20 were complemented by providing the fliF gene in trans. Since complementation could have been due to the presence of an internal promoter in the fliF gene or in the Tn5G transposon, which allowed the transcription of the downstream genes, another chromosomal mutant of the fliF gene was constructed by insertional inactivation with an antibiotic resistance cassette. This mutant was also nonmotile and nonadhesive. However, the two defects in this new mutant could not be complemented by the fliF gene in trans, consistent with the interpretation that there is no internal fliF promoter but possibly a functional promoter in the Tn5G transposon. The complete nucleotide sequences of the fliE and fliF genes and a partial nucleotide sequence of the fliG gene of P. aeruginosa were determined. Control of the promoter upstream of the fliE gene was analyzed by construction of a fliE-lacZ fusion and the introduction of this construct into strains of P. aeruginosa with mutations in several regulatory genes. Beta-Galactosidase expression measurements indicated that the fliE promoter does not utilize RpoF (sigma(28)) or RpoN (sigma(54)) sigma factors. The characterization of this gene as being responsible for the loss of adhesion indicates that basal body structures are probably important for localization of the adhesin.

Amino Acid Sequence↗

PubMed1996

Dual function of PilS during transcriptional activation of the Pseudomonas aeruginosa pilin subunit gene.

The polar pili of Pseudomonas aeruginosa are composed of subunits encoded by the pilA gene. Expression of pilA requires the alternative sigma factor RpoN and a pair of regulatory elements, PilS and PilR. These two proteins are members of the two-component regulatory family, in which PilS is the sensory component and PilR is the response regulator. By using expression and localization analyses, in this work we show that PilS is synthesized as a 59-kDa polypeptide located in the P. aeruginosa cytoplasmic membrane. When the pilS gene is expressed in Escherichia coli, aberrant translational initiation results in a smaller, 40-kDa polypeptide. Unexpectedly, overexpression of pilS in P. aeruginosa results in decreased transcription of the pilA gene. Moreover, fully functional PilS was not required for this inhibitory effect. A mutation in the histidine residue essential for kinase activity resulted in a protein unable to activate transcription, yet when overexpressed in the presence of the wild-type PilS protein, this protein still repressed pilin synthesis. A shorter form of PilS, lacking its transmembrane segments, was active and fully capable of stimulating pilA transcription but when overexpressed did not show the inhibitory effect on pilin expression seen with full-length PilS. We also show that overexpression of pilR can activate transcription of pilA even in the absence of PilS. On the basis of our studies, we propose a complex mechanism of regulation of PilS function, involving other cellular factors that control PilS and its activities during the phosphorelay mechanism of signal transduction.

Bacterial Outer Membrane Proteins↗

PubMed1996

Differential gene expression by Pseudomonas aeruginosa during interaction with respiratory mucus.

Pseudomonas aeruginosa is a common respiratory tract pathogen that causes serious infections in patients with cystic fibrosis. A number of putative virulence factors have been characterized in several laboratories, and some have been implicated in human infections, based on criteria such as the phenotype of isolates from infected patients, an immune response to a particular antigenic factor, and the effect of a virulence factor on infectivity in an animal model. We have developed a series of genetic tools to study the selective regulation of expression of P. aeruginosa genes during interactions of the pathogen with host tissues. These tools are based on direct enrichment of bacteria, when a particular promoter is induced or repressed. We have found that interaction of bacteria with mucus from patients with cystic fibrosis results in marked induction of expression of several genes, including one that encodes a lipopolysaccharide biosynthetic enzyme, a gene for a protein responsible for uptake of the ferric pyochelin siderophore, and a new gene homologous with a class of iron-responsive repressors. The tools described here are useful for identification of induced or repressed genes in various animal models of infection or in controlled laboratory conditions that mimic natural infections of humans. Such genes might not be detectable when bacteria are cultured in laboratory conditions, and these tools are therefore useful for general probing of a bacterial genome for genes regulated during different stages of infection.

Animals↗

PubMed1995

Cloning and phenotypic characterization of fleS and fleR, new response regulators of Pseudomonas aeruginosa which regulate motility and adhesion to mucin.

This work has identified two genes (designated fleS and fleR) in Pseudomonas aeruginosa which are highly homologous to members of the subclass of two-component systems involved in transcriptional regulation of a diverse array of genes from sigma 54 promoters. The genes are located upstream from fliE, a flagellar gene of P. aeruginosa, and they are arranged in a putative fleSR operon. FleS has a predicted molecular mass of 43.87 kDa and shows strong homology to histidine kinases which in other two-component systems have been shown to be sensor proteins. FleR has a predicted molecular mass of 51.26 kDa and is homologous to other regulatory proteins that bind to specific upstream activating elements to enhance transcription of genes with sigma 54 promoters. The fleSR system is believed to control both flagellar synthesis and adhesion to mucin. Several lines of evidence are presented. (i) A nonpiliated mutant of P. aeruginosa PAK containing a gentamicin cassette in fleR is nonmotile and nonadhesive. (ii) The fleR mutant regained motility and adhesion when complemented with a wild-type copy of fleR. (iii) A Western blot (immunoblot) of the fleR mutant showed no synthesis of flagellin, and electron microscopy of the fleR mutant confirmed the lack of flagella. Previous work has shown that flagellar mutants with mutations in fliA (sigma 28) or fliC (the structural gene for flagellin) retain adhesion; therefore, these new observations suggest that FleSR regulates both the expression of flagella and the nonpilus adhesin(s) for mucin or that one of the flagellar proteins (other than flagellin) may be responsible for adhesion to mucins.

Amino Acid Sequence↗

PubMed1995

Characterization of Pseudomonas aeruginosa fliO, a gene involved in flagellar biosynthesis and adherence.

Pseudomonas aeruginosa binds to eukaryotic cells via both pilus and nonpilus adhesins, while binding of P. aeruginosa to mucin is pilus independent. To characterize genes involved in non-pilus-mediated adherence, transposon mutants of the nonpiliated strain P. aeruginosa PAK-NP that are unable to bind to cells or mucins were isolated. Two such mutants, P. aeruginosa B164 and P. aeruginosa RR18, were identified previously as deficient in binding to eukaryotic cells or mucins as well as nonmotile. The transposon insertion in each of these strains was mapped to the same gene. Sequence analysis of both DNA flanking the transposons and plasmids that could complement the mutations indicated that this open reading frame encodes a putative protein homolog of both Escherichia coli FliO and Erwinia carotovora subsp. atroseptica MopB. The transposons in both of these mutants are nonpolar, since the addition of the P. aeruginosa fliO gene in trans restored adherence to both cells and mucins to these mutants. The cloned fliO gene also complemented the motility defect of both B164 and RR18. A 1.6-kb KpnI fragment from the PAK chromosome that contained the fliO gene was sequenced. The fliO gene appears to be part of an operon with a complete open reading frame upstream of the FliO homolog encoding a putative protein homolog of FliN of both E. coli and Salmonella typhimurium. The partial open reading frame downstream of fliO encodes a putative homolog of both E. coli and S. typhimurium FliP. The fliN gene is flanked on its 5'-end by the 3'-end of a homolog of a fliM gene. The P. aeruginosa FliN protein was identified with a T7 expression system, while all attempts to identify the P. aeruginosa FliO protein were unsuccessful. Homologs of P. aeruginosa FliO are involved in the biosynthesis of flagella, but the function of FliO in this biosynthetic process remains unknown. Further study should reveal the precise role of P. aeruginosa FliO in non-pilus-mediated adherence, which could include regulation of expression or localization of a nonpilus adhesin.

Amino Acid Sequence↗

PubMed1994

Identification and characterization of PilS, an essential regulator of pilin expression in Pseudomonas aeruginosa.

Expression of the pilin gene, pilA, of Pseudomonas aeruginosa requires the alternative sigma factor, sigma 54, and also two other transcriptional regulators encoded by the pilS and pilR genes. These two linked genes, which have been identified by transposon insertion mutagenesis, share significant amino acid sequence homology with members of the two-component family of regulators. The transcriptional regulator, PilR, has been described previously. PilS, a 37,285 Dalton protein, shares significant homology with the protein kinase sensors of the two-component regulatory family. PilS, however, has no hydrophobic domains which might be membrane-spanning alpha-helices, suggesting that PilS is a cytoplasmic protein. Characterization of the pilS gene revealed that when overexpressed in Escherichia coli by the bacteriophage T7 promoter it specifies a protein of approximately 40,000 daltons, corresponding to the molecular weight of PilS predicted from the deduced amino acid sequence. Deletion analysis of the pilS promoter fused to a promoterless lacZ gene further showed that a significant region upstream of pilS is essential for expression of pilS and pilR, suggesting a need for transcriptional activation. The pilA promoter can be activated in E. coli but only when PilR and sigma 54 are present. This work suggests that the PilS activation signal is received in the bacterial cytoplasm, and that the mechanism of PilS/PilR-mediated signal transduction resulting in activation of the pilin gene promoter is likely to be similar to that of other two-component systems.

Amino Acid Sequence↗

PubMed1994

Differences in eucaryotic cell binding of Pseudomonas.

The lungs of cystic fibrosis (CF) patients are frequently chronically colonized by Pseudomonas aeruginosa. Recently there has been an increase in colonization by another pathogen Pseudomonas cepacia, which can cause a rapid decline in clinical condition or death of the patient. The nature of the factor(s) which predispose CF patients to colonization by one or both of these opportunistic pathogens is unknown. It has been suggested that the genetic defect in CF patients results in an increase in the number of epithelial cell receptors available to P. aeruginosa in the lung, thus rendering CF patients more susceptible to bacterial colonization than non-CF individuals. In this study, we have examined adherence of several strains of P. aeruginosa and P. cepacia to a variety of continuous cell lines, as well as primary cultures of CF and non-CF nasal polyp cells. The results suggested that there may be a decrease in the number of receptors available to both strains of Pseudomonas on cells of canine origin compared to human cells. Both strains appear to use pili as the primary adhesin, but there is also evidence that non-pilus adhesins contribute significantly to eucaryotic cell binding. P. cepacia exhibited microcolony formation on all cell types, which is typical of the localized adherence pattern characteristic of the enteropathogenic Escherichia coli. However, we were unable to demonstrate, with either P. cepacia or P. aeruginosa, a significant increase in adherence to CF compared to non-CF nasal polyp cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Adhesins, Bacterial↗

PubMed1994

Posttranslational processing of type IV prepilin and homologs by PilD of Pseudomonas aeruginosa.

We have described the characterization of a protein initially identified as having an essential function in biogenesis of polar pili of P. aeruginosa by processing precursors of pilin. Other findings have also expanded the range of substrates for PilD to include a set of proteins that are essential components of the extracellular secretion machinery. Direct demonstration of prepilin processing necessitates use of purified substrates and enzymes, and we present general protocols for purification of both enzymes and substrates, as well as an assay for prepilin peptidase activity. For a source of enzyme and substrates, mutants of P. aeruginosa defective in pilin processing as well as clones overexpressing the pilin gene and PilD were developed. These methods are applicable to other bacterial systems that express Type IV pili and/or possess the PilD-dependent machinery of extracellular protein secretion. PilD is a bifunctional enzyme, which carries out not only cleavage but also amino-terminal methylation of the mature pilin. Cleavage and N-methylation of the pilin-like Xcp proteins involved in extracellular protein secretion have also been shown to be dependent on PilD. The leader peptidase activity of PilD is inhibited by sulfhydryl blocking reagents such as NEM and PCMB, whereas the methyltransferase activity of the purified enzyme is dependent on reduction with dithiothreitol. The conserved region containing the cysteine residues lies within the largest hydrophilic domain of the protein as predicted from hydrophobicity analysis, and it is probably exposed to the cytoplasmic side of the cytoplasmic membrane. Identification of the active site residues involved in recognition of the substrates for processing and subsequent methylation is currently underway. Studies on substrate specificities of PilD, with respect to its leader peptidase and methyltransferase activity, may prove to be useful in designing inhibitors which would interfere with maturation of Type IV prepilins and components of the extracellular protein secretion machinery. In light of the fact that an increasing number of both mammalian and plant pathogens are being shown to have extracellular secretion pathways homologous to that seen for P. aeruginosa, such inhibitors may be useful tools in the study of the role these peptidases play in bacterial virulence.

Amino Acid Sequence↗

PubMed1994

PilR, a transcriptional regulator of piliation in Pseudomonas aeruginosa, binds to a cis-acting sequence upstream of the pilin gene promoter.

The PilR protein of Pseudomonas aeruginosa is a transcriptional activator of the pilin gene and belongs to a two-component sensor-regulator family. PilR was overproduced by fusing pilR to the gene for the maltose-binding protein (malE), yielding a MalE-PilR hybrid protein. The plasmid with the malE-pilR fusion, when introduced into a non-piliated pilR mutant strain of P. aeruginosa, restored piliation, indicating that the hybrid protein retains PilR function in vivo. The MalE-PilR protein was purified from Escherichia coli and used in a series of DNA-binding studies. A specific pilin promoter-binding activity of MalE-PilR was observed in a gel retardation assay. Subsequent DNase I footprinting analysis revealed a 40 bp PilR-binding site located at the -120 to -80 region, relative to the transcriptional start site of the pilin gene. This PilR-binding region consists of a nine-base sequence and three consensus sequences of 5'-(N)4-6C/GTGTC-3', in a tandem array in which the first 7-9 bp are bound by the PilR on the non-coding strand, leaving the last two nucleotides (TC) unbound. On the coding strand, PilR binds to sequences complementary to the two middle consensus sequences of the non-coding strand. A sequence similar to the NifA recognition site (5'-TGT-(N)11-ACA-3') is also found within the PilR-binding region. Deletion analysis and disruption of the individual consensus PilR-binding sequences by site-directed mutagenesis revealed that all four PilR-binding sites are absolutely required for the PilS/PilR-mediated pilin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Outer Membrane Proteins↗