Molecular basis of mucin-Pseudomonas interactions.
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Adhesins, Bacterial↗
Biomedical subjects
R Ramphal
Publications and source records attributed to R Ramphal.
Is monotherapy for febrile neutropenia still a viable alternative?
Monotherapy for empirical treatment of febrile neutropenia is effective and often less costly than combination therapy but remains controversial. The controversy results from observations that combination therapy for Pseudomonas aeruginosa improved outcomes, and this approach became a standard. Many subsequent publications, including the Infectious Diseases Society of America guidelines for febrile neutropenia, now support monotherapy. However, changes in the pathogens involved in febrile neutropenia and in their resistance prompt a reevaluation. In the evaluation of new antibiotics, recent trials comparing either cefepime or meropenem with combination therapy or with ceftazidime confirm that monotherapy remains a viable therapeutic approach, with infectious mortality in the 5% range in all arms. The choice of monotherapy should, however, be made on the basis of resistance patterns seen in an institution. The agent selected should be very active against the organisms that are likely to cause rapidly fatal infections, and clinicians must be prepared to modify monotherapy as appropriate.
Anti-Bacterial Agents↗
Pneumonia in the compromised host including cancer patients and transplant patients.
Pneumonia remains a major cause of morbidity and mortality in the immunocompromised host. The type and timing of immunosuppression will predispose the patient to infections with certain pathogens. This article discusses the types of immunosuppression and their infectious and noninfectious implications. Key points of the most commonly involved pathogens are mentioned. Finally, an approach to diagnosis and empiric therapy is discussed.
Bacterial Infections↗
The Pseudomonas aeruginosa flagellar cap protein, FliD, is responsible for mucin adhesion.
Mucin-specific adhesion of Pseudomonas aeruginosa plays an important role in the initial colonization of this organism in the airways of cystic fibrosis patients. We report here that the flagellar cap protein, FliD, participates in this adhesion process. A polar chromosomal insertional mutation in the P. aeruginosa fliD gene made this organism nonadhesive to mucin in an in vitro mucin adhesion assay. The adhesive phenotype was restored by providing the fliD gene alone on a multicopy plasmid, suggesting involvement of this gene in mucin adhesion of P. aeruginosa. Further supporting this observation, the in vitro competition experiments demonstrated that purified FliD protein inhibited the mucin adhesion of nonpiliated P. aeruginosa PAK-NP, while the same concentrations of PilA and FlaG proteins of P. aeruginosa were ineffective in this function. The regulation of the fliD gene was studied and was found to be unique in that the transcription of the fliD gene was independent of the flagellar sigma factor sigma28. Consistent with this finding, no sigma28 binding sequence could be identified in the fliD promoter region. The results of the beta-galactosidase assays suggest that the fliD gene in P. aeruginosa is regulated by the newly described transcriptional regulator FleQ and the alternate sigma factor sigma54 (RpoN).
Amino Acid Sequence↗
Monotherapy versus combination therapy for febrile neutropenia.
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Anti-Bacterial Agents↗
Gelsolin activates DNase I in vitro and cystic fibrosis sputum.
Because actin can form a complex in vitro containing both gelsolin and DNase I, gelsolin and DNase I have been assumed to bind independently to actin. Although this assumption is consistent with the known crystalline structures of gelsolin with one actin and of actin with DNase I, which suggest that the binding sites on actin for both gelsolin and DNase I are distinct and separate, we propose that a second actin binding site on gelsolin competes with DNase I for actin. Since actin is an inhibitor of DNase I, competition at the second binding site results in activation of DNase I by gelsolin. Covalent cross-linking experiments confirm that DNase I prevents dimerization of actin by gelsolin, consistent with displacement of one actin from gelsolin by DNase I. Activation of DNase I by gelsolin is a novel function for a cytoskeletal protein and could have broad implications for biology, such as a role in initiating apoptosis. These results also may explain why both gelsolin and DNase I decrease sputum viscosity in cystic fibrosis (CF). While the activity of DNase I had originally been attributed to fragmentation of DNA, subsequent data suggested that both gelsolin and DNase I may affect viscosity by depolymerizing filamentous actin. The current results alternatively suggest aht dissociation of the actin-DNase I complex by gelsolin in CF sputum results in activation of the nuclease activity of constitutive DNase I. The nuclease activity of DNase I alone is therefore sufficient to explain the effects of both gelsolin and DNase I on CF sputum.
Actins↗
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↗
Clinical experience with single agent and combination regimens in the management of infection in the febrile neutropenic patient.
Choice of antibiotic therapy for the management of infection in the neutropenic patient continues to challenge the clinician. The shift toward gram-positive organisms and the continuing need to provide gram-negative coverage demands the use of an agent or agents that provide coverage for the spectrum of potential infecting organisms. Cefepime is an extended-spectrum fourth-generation cephalosporin that has good activity against gram-positive and gram-negative organisms; in addition, it resists degradation by Bush group 1 beta-lactamases. These properties make this agent a promising candidate for empiric therapy with febrile neutropenic patients. Data presented in this article are from febrile neutropenic cancer patients enrolled into two randomized, prospective, nonblinded comparative U.S. clinical trials. Patients were randomized to receive cefepime (2 g thrice daily) or a comparator regimen of either ceftazidime (2 g thrice daily) or piperacillin + gentamicin (3 g every 4 hours + 1.5 mg/kg every 8 hours). When indicated, vancomycin was added to the regimen. A total of 109 febrile episodes were treated with cefepime and 107 episodes were treated with the comparator regimens. Neutropenia (< or = 500 PMNs/mm3) persisted for > or = 10 days in >40% of episodes and severe neutropenia (< or = 100 PMNs/mm3) in >25%. More than 40% of the total number of episodes were documented bacterial infections. These characteristics did not differ among treatment groups. Duration of therapy was similar in both groups (median: cefepime, 9 days; comparators, 11 days). In >40% of episodes, patients received study therapy without addition of other antibacterials (cefepime, 46%; comparators, 41%). Vancomycin was added in almost half of all the episodes (cefepime, 45%; comparators, 53%). Patients became afebrile by the fourth day of study therapy in approximately 60% of episodes (cefepime, 58%; comparators, 60%). In approximately 75% of the episodes, patients had a satisfactory response at the end of therapy (cefepime, 74%; comparators, 76%); and following approximately 90% of episodes, patients survived for >30 days (cefepime, 90%; comparators, 92%). Eradication rates were similar for all pathogens for cefepime and comparator agents. There were similar numbers of superinfecting organisms in each treatment arm; most involved gram-positive organisms. These multiple measures of efficacy suggest that initial empiric cefepime monotherapy is comparable to the pooled experience with standard therapies and that antibacterial modifications occur with similar frequency for cefepime compared with standard empiric regimens.
Anti-Bacterial Agents↗
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↗
Adhesion of Pseudomonas aeruginosa to respiratory mucins and expression of mucin-binding proteins are increased by limiting iron during growth.
The attachment of Pseudomonas aeruginosa to human respiratory mucus represents an important step in the development of lung infection, especially in cystic fibrosis. Local factors in the respiratory tract, such as osmolarity or iron concentration, might influence this colonization. In the present work, we have observed that overall levels of adhesion of two nonmucoid, nonpiliated strains of P. aeruginosa, 1244-NP and PAK-NP, to human airway mucins were higher when these strains were grown in a minimal medium of low osmolarity (M9) than when they were grown in a rich medium of higher osmolarity (tryptic soy broth [TSB]). However, increasing the NaCl concentration of M9 to increase the osmolarity did not modify the level of binding. In order to find out whether these differences were due to variations in nutrients, the influence of iron concentration was investigated: the levels of binding of the two strains increased after TSB was depleted of iron and decreased after iron was added to M9. Since the outer membranes from the two strains have been shown to contain proteins reacting with human bronchial mucins, we compared the mucin-binding proteins expressed by the two strains grown in different media. When the nonpiliated strains 1244-NP and PAK-NP were grown in the different media, previously observed mucin-binding bands were detected in the 42- to 48-kDa range but additional mucin-binding bands in the 77- to 85-kDa range were detected when these strains were grown in M9 or iron-deprived TSB. These results demonstrate that the adhesion of P. aeruginosa and the expression of mucin-binding proteins in the outer membranes of nonpiliated P. aeruginosa are affected by the iron content of the medium in which the bacteria are grown and not by the osmolarity.
Bacterial Adhesion↗
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↗
Recognition of mucin by the adhesin-flagellar system of Pseudomonas aeruginosa.
Pseudomonas aeruginosa colonizes the mucus of patients with chronic lung diseases by a specific mechanism involving an adhesin-receptor system. Several adhesins have been implicated in the adhesion of P. aeruginosa to cells, but the identity of the principal adhesin(s) involved in adhesion to mucin is unknown. Mutagenesis studies have indicated that P. aeruginosa adhesion is under the control of the rpoN gene, which also regulates pilin synthesis, flagellum formation, and other functions. Mutagenesis of certain flagellar genes that are not controlled by RpoN, e.g., flif, also indicates a close relationship between adhesion and flagellar genes and not necessarily an independent effect of rpoN on adhesion. Mutants of certain early flagellar genes lead to the loss of both adhesion and motility, whereas mutants of certain late genes, e.g., fliC, the gene for flagellin, lose motility but retain adhesion. Recent studies indicate that both motility and adhesion are regulated by a two-component regulatory system called fleS-R, which in turn is controlled by another regulator in a cascade that involves rpoN. A fleR mutant possessing pili adheres poorly to mucins, definitively showing that pili do not play a major role in adhesion to mucin. It is unclear whether the adhesin is a flagellar protein or another protein that uses the flagellar export apparatus for localization or both. Finding the gene under control of rpoN may provide answers to these questions.
Adhesins, Bacterial↗
Use of amphotericin B lipid complex for treatment of disseminated cutaneous fusarium infection in a neutropenic patient.
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Amphotericin B↗
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↗
Adhesion of nontypeable Haemophilus influenzae from blood and sputum to human tracheobronchial mucins and lactoferrin.
Nontypeable Haemophilus influenzae strains are the most common pathogens encountered in patients with chronic bronchitis. These organisms chronically colonize the airways of patients and occasionally cause bacteremia. Nontypeable H. influenzae strains have been demonstrated microscopically to bind to mucus, but quantitative studies of adhesion have not been published to date. We have therefore developed a reproducible microtiter plate assay to study mucin binding and have examined the adhesion of sputum and blood strains of nontypeable H. influenzae. The assay is similar to that described for Pseudomonas aeruginosa (S. Vishwanath and R. Ramphal, Infect. Immun. 45:197-202, 1984), but notably 2% Tween 20 is used to desorb bacteria from the wells to quantitate bacterial binding. Using a standard strain, we have established that 1 h of incubation is optimum with an inoculum of < or = 5 x 10(8) CFU/ml. The standard strain binds to bronchitic and cystic fibrosis mucins equally well but binds less to bronchiectasis mucins. It does not bind to bovine serum albumin or fetuin. We have also examined the levels of adhesion of freshly isolated sputum and bacteremia strains and find very significant differences in adhesion. Blood strains bound six to seven times less than sputum strains ([13.8 +/- 7] x 10(2) per well versus [102 +/- 43] x 10(2); P < 0.001). Studies with adhesion to lactoferrin, another glycosylated protein, revealed variable binding of respiratory strains but marked binding of blood strains compared with mucin. An isogenic pair of respiratory and blood isolates was examined by electron microscopy but did not show surface differences. We speculate that bacteremic strains studied may have masked, lost, or downregulated adhesin production to allow them to escape from mucins or upregulated adhesins for lactoferrin to invade the bloodstream.
Bacterial Adhesion↗
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↗
Bilateral pulmonary infiltrates in association with disseminated actinomycosis.
The most common infectious cause of bilateral upper-lobe pulmonary disease is tuberculosis. However, we recently encountered a patient with bilateral apical infiltrates and multiple soft-tissue abscesses caused by Actinomyces odontolyticus. Other findings included fever, weight loss, and leukocytosis, and the patient's only known source of immunosuppression was a long history of alcoholism. There was no history of diabetes, steroid use, or other chronic underlying disease. The diagnosis was made by culture of drainage fluid from one abscess. Therapy with intravenous penicillin G led to rapid clinical improvement and reduction in the infiltrates. To our knowledge, the presentation of pulmonary infection, with bilateral apical infiltrates due to A. odontolyticus has not been previously reported in the medical literature.
Actinomyces↗
Modulation of Pseudomonas aeruginosa adherence to the corneal surface by mucus.
To gain access to the corneal epithelium and cause infections keratitis, bacterial pathogens must first interact with ocular surface factors that could affect bacterial adherence. In this study, we demonstrated that the mucus layer, and, in particular, the mucin fraction of mucus, modulated adherence to intact corneal epithelium of Pseudomonas aeruginosa but not that of Staphylococcus aureus or Streptococcus pyogenes. Removal of endogenous mucus from rat or rabbit eyes increased the adherence of P. aeruginosa by 3- to 10-fold. Ocular mucus obtained from rat eyes, porcine stomach mucin, or bovine submaxillary gland mucin inhibited adherence of P. aeruginosa to uninjured corneal epithelium. The mucin fraction of ocular mucus, purified by ultracentrifugation, was found to contain the inhibitory activity, and inhibition was demonstrated at concentrations of mucin as low as 35 micrograms/ml. Ocular mucin was the only material tested that inhibited adherence of P. aeruginosa to an injured cornea. However, the binding of P. aeruginosa to immobilized substrates in vitro did not predict which fraction would possess antiadherence activity: bacteria bound well to whole ocular mucus, mucin, the nonmucin fraction of ocular mucus, and dilute human tears as well as to porcine stomach mucin and bovine submaxillary gland mucin. The effectiveness of the mucin fraction of ocular mucus at inhibiting the binding of P. aeruginosa to the cornea implies that this material is a barrier that protects the surface of the eye from P. aeruginosa adherence.
Animals↗