Synthetic peptide vaccine and antibody therapeutic development: prevention and treatment of Pseudomonas aeruginosa

Pseudomonas aeruginosa and Pseudomonas maltophilia account for 80% of opportunistic infections by pseudomonads. Pseudomonas aeruginosa is an opportunistic pathogen that causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, and a variety of systemic infections, particularly in patients with severe burns, and in cancer and AIDS patients who are immunosuppressed. Pseudomonas aeruginosa is notable for its resistance to antibiotics, and is therefore a particularly dangerous pathogen. Only a few antibiotics are effective against Pseudomonas, including fluoroquinolones, gentamicin, and imipenem, and even these antibiotics are not effective against all strains. The difficulty treating Pseudomonas infections with antibiotics is most dramatically illustrated in cystic fibrosis patients, virtually all of whom eventually become infected with a strain that is so resistant that it cannot be treated. Since antibiotic therapy has proved so ineffective as a treatment, we embarked on a research program to investigate the development of a synthetic peptide consensus sequence vaccine for this pathogen. In this review article we will describe our work over the last 15 years to develop a synthetic peptide consensus sequence anti-adhesin vaccine and a related therapeutic monoclonal antibody (cross-reactive to multiple strains) to be used in the prevention and treatment of P. aeruginosa infections. Further, we describe the identification and isolation of a small peptide structural element found in P. aeruginosa strain K (PAK) bacterial pili, which has been proven to function as a host epithelial cell-surface receptor binding domain. Heterologous peptides are found in the pili of all strains of P. aeruginosa that have been sequenced to date. Several of these peptide sequences have been used in the development of an consensus sequence anti-adhesin vaccine targeted at the prevention of host cell attachment and further for the generation of a monoclonal antibody capable of prevention and treatment of existing infections.     

Pyoverdine siderophores: from biogenesis to biosignificance

Pyoverdines are a group of structurally related siderophores produced by fluorescent Pseudomonas species. Recent genomic and biochemical data have shed new light on the complex molecular steps of pyoverdine biogenesis and explained the chemical diversity of these compounds. In the opportunistic pathogen Pseudomonas aeruginosa, pyoverdine is necessary for infection in several different disease models. The occurrence of pyoverdine-defective strains in chronic infections of patients with cystic fibrosis and the extremely high sequence diversity of genes involved in pyoverdine synthesis and uptake indicate that pyoverdine production is subject to high evolutionary pressure. Pyoverdine-dependent iron transport is also crucial for biofilm development, further expanding the importance of these siderophores in Pseudomonas biology.     

Microbial ecology and adaptation in cystic fibrosis airways

Chronic infections in the respiratory tracts of cystic fibrosis (CF) patients are important to investigate, both from medical and from fundamental ecological points of view. Cystic fibrosis respiratory tracts can be described as natural environments harbouring persisting microbial communities with Pseudomonas aeruginosa as a dominant pathogen. Various factors contribute to the complexity of this ecosystem, including community composition, dynamics and interactions, as well as heterogeneous distribution and fluctuation of components of the immune system, antibiotics and nutrients. All these elements constitute the selective forces that drive the evolution of the microbes after they migrate from the outer environment to human airways. Pseudomonas aeruginosa adapts to the new environment through genetic changes and exhibits a special lifestyle in chronic CF airways. Understanding the persistent colonization of microbial pathogens in CF patients in the context of ecology and evolution will expand our knowledge of the pathogenesis of chronic infections and improve therapeutic strategies.     

Cystic fibrosis infections: treatment strategies and prospects

Pseudomonas aeruginosa and Burkholderia cepacia are the two major Gram-negative rods that colonize/infect the lungs of patients with cystic fibrosis (CF). These organisms may cause progressive respiratory failure, although occasionally more rapid infections result in the ' Cepacia ' syndrome. Many antibiotics have been used against Pseudomonas and Burkholderia , but once chronic colonization has been established, eradication of these organisms is rare. Drug therapy for CF patients is compromised by a number of bacterial factors that render the infectious agents resistant to antibiotics, including efflux pumps that remove antibiotics, lack of penetration of antibiotics into bacterial biofilms, and changes in the cell envelope that reduce the permeability of antibiotics. Any combination of these mechanisms increases the likelihood of bacterial survival. Therefore, combinations of antibiotics or of antibiotic and nonantibiotic compounds are currently being tested against Pseudomonas and Burkholderia . However, progress has been slow, with only occasional combinations showing promise for the eradication of persistent Gram-negative rods in the airways of CF patients. This review will summarize the current knowledge of CF infections and speculate on potential future pathways to treat these chronic infections.     

Inactivation of the mismatch repair system in Pseudomonas aeruginosa attenuates virulence but favors persistence of oropharyngeal colonization in cystic fibrosis mice

The inactivation of the mismatch repair (MMR) system of Pseudomonas aeruginosa modestly reduced in vitro fitness, attenuated virulence in murine models of acute systemic and respiratory infections, and decreased the initial oropharyngeal colonization potential. In contrast, the inactivation of the MMR system favored long-term persistence of oropharyngeal colonization in cystic fibrosis mice. These results may help in understanding the reasons for the low and high prevalences, respectively, of hypermutable P. aeruginosa strains in acute and chronic infections.     

Molecular Epidemiology of Chronic Pseudomonas aeruginosa Airway Infections in Cystic Fibrosis

Background/Methods

The molecular epidemiology of the chronic airway infections with Pseudomonas aeruginosa in individuals with cystic fibrosis (CF) was investigated by cross-sectional analysis of bacterial isolates from 51 CF centers and by longitudinal analysis of serial isolates which had been collected at the CF centers Hanover and Copenhagen since the onset of airway colonization over 30 years.

Results

Genotyping revealed that the P. aeruginosa population in CF is dominated by a few ubiquitous clones. The five most common clones retrieved from the CF host also belonged to the twenty most frequent clones in the environment and in other human disease habitats. Turnover of clones in CF airways was rare. At the Hanover clinic more than half of the patient cohort was still harbouring the initially acquired clone after twenty years of airway colonization. At the Copenhagen clinic, however, two rare clones replaced the initially acquired individual clones in all but one patient.

Conclusion

The divergent epidemiology at the two sites is explained by their differential management of hygiene and antipseudomonal chemotherapy. Hygienic measures to prohibit patient-to-patient transmission and the modalities of antipseudomonal chemotherapy modify the epidemiology of the chronic P. aeruginosa infections in CF.     

Central role of quorum sensing in regulating the production of pathogenicity factors in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an important opportunistic human pathogen, causing various infections that are often very persistent. P. aeruginosa infections are the major cause of death in cystic fibrosis patients. Infections are difficult to treat since P. aeruginosa is resistant to most antibiotics and its antibiotic susceptibility is decreased when it is present in biofilms. P. aeruginosa produces many exoproducts (including toxins and hydrolytic enzymes) that are involved in virulence. Recent research has elucidated many mechanisms and pathways that regulate the production of these virulence factors. The regulation is extremely complex and many components are influenced by environmental conditions. Quorum sensing is a key regulatory system, which itself is affected by many other regulators. Targeting the regulation of pathogenicity factors provides a novel strategy for combating P. aeruginosa infections. Degradation of acyl homoserine lactones, the signaling molecules of the quorum-sensing system, is a promising therapeutic treatment option.     

Oxidation of calprotectin by hypochlorous acid prevents chelation of essential metal ions and allows bacterial growth: Relevance to infections in cystic fibrosis

Calprotectin provides nutritional immunity by sequestering manganese and zinc ions. It is abundant in the lungs of patients with cystic fibrosis but fails to prevent their recurrent infections. Calprotectin is a major protein of neutrophils and composed of two monomers, S100A8 and S100A9. We show that the ability of calprotectin to limit growth of Staphylococcus aureus and Pseudomonas aeruginosa is exquisitely sensitive to oxidation by hypochlorous acid. The N-terminal cysteine residue on S100A9 was highly susceptible to oxidation which resulted in cross-linking of the protein monomers. The N-terminal methionine of S100A8 was also readily oxidized by hypochlorous acid, forming both the methionine sulfoxide and the unique product dehydromethionine. Isolated human neutrophils formed these modifications on calprotectin when their myeloperoxidase generated hypochlorous acid. Up to 90% of the N-terminal amine on S100A8 in bronchoalveolar lavage fluid from young children with cystic fibrosis was oxidized. Oxidized calprotectin was higher in children with cystic fibrosis compared to disease controls, and further elevated in those patients with infections. Our data suggest that oxidative stress associated with inflammation in cystic fibrosis will stop metal sequestration by calprotectin. Consequently, strategies aimed at blocking extracellular myeloperoxidase activity should enable calprotectin to provide nutritional immunity within the airways.     

Evaluation of the pulmonary and systemic immunogenicity of Fluidosomes, a fluid liposomal-tobramycin formulation for the treatment of chronic infections in lungs.

In previous studies, we have developed a fluid bactericidal liposomal formulation containing tobramycin, called Fluidosomes, which has been shown to be highly bactericidal both in in vitro and in in vivo studies against Pseudomonas aeruginosa and other related and unrelated bacteria. One foreseeable application of these Fluidosomes is the treatment of chronic pulmonary infections in cystic fibrosis patients colonized with P. aeruginosa and other related bacteria. Considering the capacity of some liposomal preparations to play an adjuvant role in vaccines, the non-immunogenicity of Fluidosomes has to be demonstrated. The systemic and local immunogenicity of Fluidosomes were assessed by effectuating repeated intraperitoneal (i.p.) and intratracheal (i.t. ) immunizations in BALB/c mouse. No significant mucosal and serum immune responses against Fluidosomes and/or tobramycin were detected as compared with preimmune sera. These data suggest that Fluidosomes could be administered repeatedly without adverse immune responses to control chronic pulmonary infections in cystic fibrosis.     

Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis

Microbial lung infections are the major cause of morbidity and mortality in the hereditary metabolic disorder cystic fibrosis, yet the molecular mechanisms leading from the mutation of cystic fibrosis transmembrane conductance regulator (CFTR) to lung infection are still unclear. Here, we show that ceramide age-dependently accumulates in the respiratory tract of uninfected Cftr-deficient mice owing to an alkalinization of intracellular vesicles in Cftr-deficient cells. This change in pH results in an imbalance between acid sphingomyelinase (Asm) cleavage of sphingomyelin to ceramide and acid ceramidase consumption of ceramide, resulting in the higher levels of ceramide. The accumulation of ceramide causes Cftr-deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi and high susceptibility to severe Pseudomonas aeruginosa infections. Partial genetic deficiency of Asm in Cftr(-/-)Smpd1(+/-) mice or pharmacological treatment of Cftr-deficient mice with the Asm blocker amitriptyline normalizes pulmonary ceramide and prevents all pathological findings, including susceptibility to infection. These data suggest inhibition of Asm as a new treatment strategy for cystic fibrosis.     

Chemoenzymatic routes to enantiomerically pure 2-azatyrosine and 2-, 3- and 4-pyridylalanine derivatives

Enantiomerically pure 2-, 3- or 4-pyridylalanine (pya) and 2-azatyrosine (azatyr) are known to present various biological activities. After incorporation into appropriate peptide sequences, these heterocyclic non natural α-amino acids could behave as new substrates or inhibitors of elastase from Pseudomonas aeruginosa. This enzyme is known to be involved in nosocomial infections and infections related to the cystic fibrosis disease. New efficient chemoenzymatic preparations of those compounds using α-chymotrypsin (α-CT) are presented.     

Combined chemotherapy of experimental infection in neutropenia

A significant decrease in resistance to infections caused by gramnegative pathogens was observed in mice with neutropenia induced by cytostatics. Efficacy of schemes for combined chemotherapy with beta-lactams, aminoglycosides and a novel peptide antibiotic was studied on model infections in mice with neutropenia. In the neutropenic mice with sepsis caused by Pseudomonas the peptide antibiotic administered parenterally in a single dose of 50 micrograms/kg provided high therapeutic activity. In combination with azlocillin, cefotaxime and amikacin the peptide antibiotic has a synergistic therapeutic action.     

Cystic fibrosis sputum supports growth and cues key aspects of Pseudomonas aeruginosa physiology

The opportunistic human pathogen Pseudomonas aeruginosa causes persistent airway infections in patients with cystic fibrosis (CF). To establish these chronic infections, P. aeruginosa must grow and proliferate within the highly viscous sputum in the lungs of CF patients. In this study, we used Affymetrix GeneChip microarrays to investigate the physiology of P. aeruginosa grown using CF sputum as the sole source of carbon and energy. Our results indicate that CF sputum readily supports high-density P. aeruginosa growth. Furthermore, multiple signals, which reduce swimming motility and prematurely activate the Pseudomonas quinolone signal cell-to-cell signaling cascade in P. aeruginosa, are present in CF sputum. P. aeruginosa factors critical for lysis of the common CF lung inhabitant Staphylococcus aureus were also induced in CF sputum and increased the competitiveness of P. aeruginosa during polymicrobial growth in CF sputum.     

Pseudomonas aeruginosa flagellin and alginate elicit very distinct gene expression patterns in airway epithelial cells: implications for cystic fibrosis disease

Infection with the opportunistic pathogen Pseudomonas aeruginosa remains a major health concern. Two P. aeruginosa phenotypes relevant in human disease include motility and mucoidy. Motility is characterized by the presence of flagella and is essential in the establishment of acute infections, while mucoidy, defined by the production of the exopolysaccharide alginate, is critical in the development of chronic infections, such as the infections seen in cystic fibrosis patients. Indeed, chronic infection of the lung by mucoid P. aeruginosa is a major cause of morbidity and mortality in cystic fibrosis patients. We have used Calu-3 human airway epithelial cells to investigate global responses to infection with motile and mucoid P. aeruginosa. The response of airway epithelial cells to exposure to P. aeruginosa motile strains is characterized by a specific increase in gene expression in pathways controlling inflammation and host defense. By contrast, the response of airway epithelia to the stimuli presented by mucoid P. aeruginosa is not proinflammatory and, hence, may not be conducive to the effective elimination of the pathogen. The pattern of gene expression directed by flagellin, but not alginate, includes innate host defense genes, proinflammatory cytokines, and chemokines. By contrast, infection with alginate-producing P. aeruginosa results in an overall attenuation of host responses and an antiapoptotic effect.     

Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells

Background  

Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF). To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells.     

The occurrence of denitrification in extremely halophilic bacteria

Abstract A total of 97 aerobic and facultatively anaerobic bacteria, and 3 Candida albicans , were tested for their ability to inhibit the growth of Haemophilus influenzae . Only strains of Pseudomonas aeruginosa showed any inhibitory effect and all 5 strains tested clearly inhibited the growth of all 10 strains of H. influenzae . The inhibition of H. influenzae . by Ps. aeruginosa may play a role in the establishment of chronic Ps. aeruginosa infections in the respiratory tracts of patients with bronchiectasis and cystic fibrosis (CF).     

Polycrylamide gel electrophoresis analysis of lipopolysaccharide from Pseudomonas aeruginosa growing planktonically and as biofilm

Abstract Lipopolysaccharide (LPS, endotoxin) was extracted from biofilm and planktonically grown monoagglutinable (1118) and polyagglutinable (258 and 15703) strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients with chronic pulmonary infections. Analysis by polyacrylamide gel electrophoresis (PAGE) followed by immune-detection of LPS fractions showed an S-form appearance of strain 1118 and 258 with three distinct clusters of high molecular weight bands, whereas 15703 appeared semi-rough. LPS of semi-rough cells grown planktonically and as biofilm showed a very similar PAGE pattern; however, the core/lipid A R-LPS fraction was more prominent in biofilm-LPS than in planktonic-LPS extracted from the S-form bacteria (1118 and 258). The apparent change in LPS sub-unit components of the bacteria when grown as biofilm may reflect changes in the outer membrane structure that contribute to the altered physico-chemical properties of biofilm bacteria in foreign-device associated infections and chronic P. aeruginosa lung infection in cystic fibrosis patients.     

Does Pseudomonas aeruginosa use intercellular signalling to build biofilm communities?

Pseudomonas aeruginosa is a Gram-negative bacterial species that causes several opportunistic human infections. This organism is also found in the environment, where it is renowned (like other Pseudomonads) for its ability to use a wide variety of compounds as carbon and energy sources. It is a model species for studying group-related behaviour in bacteria. Two types of group behaviour it engages in are intercellular signalling, or quorum sensing, and the formation of surface-associated communities called biofilms. Both quorum sensing and biofilm formation are important in the pathogenesis of P. aeruginosa infections. Quorum sensing regulates the expression of several secreted virulence factors and quorum sensing mutant strains are attenuated for virulence in animal models. Biofilms have been implicated in chronic infections. Two examples are the chronic lung infections afflicting people suffering from cystic fibrosis and colonization of indwelling medical devices. This review will discuss quorum sensing and biofilm formation and studies that link these two processes.     

Adaptive alterations in the outer membrane of gram-negative bacteria during human infection

The outer membrane of gram-negative bacteria is a dynamic structure that is capable of altering its ultrastructure and chemistry in order to adapt to changes in its environment. In human infections, outer-membrane alterations are known to play a role in mediating serum resistance, iron uptake, adaptation by Pseudomonas aeruginosa to colonization of the lungs of cystic fibrosis patients, and adaptive resistance to the polymyxin and aminoglycoside antibiotics. This adaptive antibiotic resistance is due to alterations in the cation binding sites within the outer membrane so that these cationic antibiotics can no longer penetrate through the membrane effectively. Adaptive resistance is not stable but is maintained only in the continued presence of the antibiotic. Hence, the role that this type of resistance to cationic antibiotics plays in clinical treatment of human infections remains inadequately assessed.     

The bacterial redox signaller pyocyanin as an antiplasmodial agent: comparisons with its thioanalog methylene blue

The quorum sensor and signalling molecule pyocyanin (PYO) contributes significantly to the pathophysiology of Pseudomonas aeruginosa infections. Comparison to phenothiazine drugs suggests that the antimalarial compound methylene blue (MB) can be regarded as a sulfur analog of PYO. This working hypothesis would explain why the synthetic drug MB behaves as a compound shaped in biological evolution. Here we report on redox-associated biological and biochemical properties of PYO in direct comparison to its synthetic analog MB. We quantitatively describe the reactivity of both compounds toward cellular reductants, the reactivity of their reduced leuco-forms towards O2, and their interactions with FAD-containing disulfide reductases. Furthermore, the interaction of PYO with human glutathione reductase was studied in structural detail by x-ray crystallography, showing that a single PYO molecule binds to the intersubunit cavity of the enzyme. Like MB, also PYO was also found to be active against blood schizonts of the malaria parasite P. falciparum in vitro. Furthermore, both compounds were active against the disease transmitting gametocyte forms of the parasites, which was systematically studied in vitro. As shown for mice, PYO is too toxic to be used as a drug. It may, however, have antimalarial activity in numerous human patients with concomitant Pseudomonas infections. MB, in contrast to PYO, is well tolerated and represents a promising agent for MB-based combination therapies against malaria. Current and future clinical studies can be guided by the comparisons between MB and PYO reported here. Additionally, it is of interest to study if and to what extent the protection from malaria in patients with cystic fibrosis or with severe wound infections is based on PYO produced by Pseudomonas species.