- Email: [email protected]
WS07.5 Novel antimicrobial peptides to fight infections in patients with cystic fibrosis
Workshops / Journal of Cystic Fibrosis 16S1 (2017) S1–S62
Methods: 16 Pa strains from CF patients were selected based on (a) clinical laboratory sensitivity to ceftazidime (Cef ) and tobramycin (Tob); (b) biofilm formation in vitro; (c) sensitivity to the phage cocktail in conventional plaque assay. Bacteria were inoculated into 96 well plates and incubated static for 24 hours to allow adherent biofilms to form. Cef and Tob (both at 2 × MIC) were then applied together, with and without the further addition of a cocktail of four phages for a further 24 hours. A fluorescent resazurin assay was used to estimate cell viability and wells were stained with crystal violet to assess biomass. Results: Treatments were described as percent reduction in cell viability and biomass compared to negative control wells (no treatment). Compared with the dual antibiotics, the phage cocktail resulted in a further mean 1.3fold reduction in cell viability ( p < 0.001) and 1.7-fold reduction in biomass ( p < 0.001). Notably, in 4 biofilms which were largely resistant to the antibiotic mix, the addition of phage conferred a 50 ± 15% reduction in cell viability and 60 ± 12% reduction in biomass (95% CI). Conclusion: Our results show a convincing reduction in pre-formed Pa biofilms when a lytic phage cocktail was added to standard antibiotic therapy. The most striking benefits seen were in the 4 clinical strains with the least response to conventional antibiotics. In an era of increased bacterial resistance, bacteriophage is a promising adjunct to standard clinical care. Acknowledgement: Funded by the CF Trust as part of the Strategic Research Centre for Pseudomonas infection. WS07.5 Novel antimicrobial peptides to fight infections in patients with cystic fibrosis M. van Eijk1, C.K. van der Ent2, H.G. Arets2, M.I. Kristensen2, H. de Cock3, A. van Dijk1, B.J. Keijser4, H.P. Haagsman1. 1Utrecht University, Infectious Diseases and Immunology, Utrecht, Netherlands; 2University Medical Center Utrecht, Cystic Fibrosis Center Utrecht, Utrecht, Netherlands; 3Utrecht University, Biology, Utrecht, Netherlands; 4TNO, Microbiolggy and Systems Biology, Zeist, Netherlands Objectives: In view of the current limitations to treat and reduce pulmonary infections and chronic inflammation in Cystic Fibrosis (CF) patients effectively, we explored the potential of a novel therapeutic approach based upon antimicrobial peptides (AMPs). Novel cathelicidinlike peptides have been designed and were tested for their antimicrobial and cytotoxic properties in vitro and ex vivo. Methods: Several well-established microbiological assays (colony counts, bioscreen kinetics) were applied using different media to test the killing efficacy of the different peptide designs against several isolates of CFrelevant bacterial and fungal species. Different mammalian cell culture systems (bronchial cell lines, primary human nasal cells) were used to screen for toxicity using the WST-1 conversion assay (cell viability). Results: Newly developed AMPs were found to display extraordinary strong antimicrobial activities against several clinical isolates of multiresistant Pseudomonas aeruginosa and Staphylococcus aureus and were shown to be effective against pathogens present in CF-patient-derived pulmonary lavage and sputum. In addition, these peptides possessed strong antifungal properties against Candida albicans and Aspergillus fumigatus, two important fungal pathogens associated with CF. In vitro studies revealed that these AMPs are active under physiological conditions (in the presence of NaCl, CaCl2) and at the lower pH that typically exists in the CF lung. Furthermore, these peptides exhibited low cytotoxicity towards several mammalian cell types (e.g. primary human nasal epithelial cells). Conclusion: Novel cathelicidin-inspired peptides show potential as a new anti-infectious therapy to effectively reduce a broad-range of microbial infections in the upper and lower airways of patients suffering from CF. This novel therapeutic approach may help to limit pulmonary inflammation and tissue damage, and will improve clinical outcome in CF-patients. WS07.6 CAT-5571: an autophagy activator that enhances the clearance of intracellular bacteria F. Liu1, A. Amer2, K. Krause2, T.L. Bonfield3, D. Fletcher3, C. Richards4, J.F. Reilly1, A.J. Nichols1, C.B. Vu1. 1Catabasis, Cambridge, United States; 2Ohio State University, Microbial Infection and Immunity, Columbus, United States;
S13
3
Case Western Reserve University, Pediatrics, Cleveland, United States; 4KWS BioTest, Bristol, United Kingdom In cystic fibrosis (CF) patients, chronic intracellular pathogens such as Pseudomonas aeruginosa and Burkholderia cenocepacia are difficult to eradicate with conventional antibiotics. Autophagy is a process cells use to capture and degrade dysfunctional proteins and organelles and invading pathogens. In CF it is known that autophagy is impaired and this can further compromise patients’ ability to clear chronic lung infections. Autophagy activation enables an alternative bacterial clearance mechanism that could be beneficial in combination with antiinfective agents. CAT-5571 is a novel molecular conjugate of a cysteamine moiety that is covalently linked to docosahexaenoic acid (DHA) that can potently activate autophagy. CAT-5571 synergistically activated autophagy in hBE cells from F508del/F508del CF patients at concentrations as low as 3 µM, whereas cysteamine and DHA alone or in simple combination failed to activate autophagy at concentrations as high as 250 µM. In cftr F508del/F508del mice, treatment with CAT-5571 restored the depressed autophagy markers Beclin-1 and LC3B. In in vitro studies using normal or F508del/F508del hBE cells infected with P. aeruginosa, CAT-5571 treatment caused a significant reduction in the intracellular bacterial load. Similarly, in macrophages isolated from cftr F508del/F508del mice, in vitro treatment with CAT-5571 reduced the intracellular bacterial load of B. cenocepacia. In addition to effects on bacterial clearance, CAT-5571 can significantly enhance the chloride current in combination with ivacaftor/lumacaftor. CAT-5571 represents a potential new therapeutic for improving clinical outcomes in conjunction with current therapies for CF. WS07.7 Response of Pseudomonas aeruginosa to hypochlorous acid (HOCl) stress and the role of defence mechanisms in protecting against neutrophil killing
K.V. Farrant1, J.C. Davies2,3, H.D. Williams1. 1Imperial College London, Department of Life Sciences, London, United Kingdom; 2Imperial College London, National Heart and Lung Institute, London, United Kingdom; 3Royal Brompton Hospital, London, United Kingdom Objectives: The immune response to Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterised by neutrophildominated inflammation, which fails to eradicate infection and causes irreversible lung damage. Neutrophils produce reactive oxygen species to combat infection including the potent oxidant hypochlorous acid (HOCl). Defence mechanisms used by P. aeruginosa in response to HOCl stress remain to be elucidated. We aim to identify mechanisms used by P. aeruginosa to protect against HOCl and determine the role of defence mechanisms during infection. Methods: We screened a library of 712 P. aeruginosa regulatory gene mutants for altered susceptibility to a sublethal concentration of HOCl (300 ppm) over 24 hours of growth. Molecular biology techniques were used to construct in-frame deletion mutants and to perform gene expression assays. Results: We identified 12 HOCl-resistant and 16 HOCl-sensitive mutants that had mutations in genes encoding regulators of antibiotic resistance, catabolite repression, methionine biosynthesis, motility and quorum sensing. One HOCl-sensitive strain was mutated in PA14_07340, which encodes an AraC-type regulator and is adjacent to PA14_07355 a putative peroxiredoxin. In-frame deletion mutants of PA14_07340 and PA14_07355 were confirmed to be HOCl-sensitive. PA14_07340 was not required for protection against the oxidants hydrogen peroxide, methyl-viologen or tert-butyl-hydroperoxide. Gene expression assays revealed that PA14_07340 upregulates expression of PA14_07355 in the presence of HOCl. Experiments that examine the role of PA14_07340 and PA14_07355 in protecting P. aeruginosa from killing by neutrophils from healthy and CF patients will be shown. Conclusions: We have identified a novel regulator PA14_07340 that specifically protects against HOCl, raising the prospect that this regulator plays a critical role in defending P. aeruginosa against neutrophil killing.
Methods: 16 Pa strains from CF patients were selected based on (a) clinical laboratory sensitivity to ceftazidime (Cef ) and tobramycin (Tob); (b) biofilm formation in vitro; (c) sensitivity to the phage cocktail in conventional plaque assay. Bacteria were inoculated into 96 well plates and incubated static for 24 hours to allow adherent biofilms to form. Cef and Tob (both at 2 × MIC) were then applied together, with and without the further addition of a cocktail of four phages for a further 24 hours. A fluorescent resazurin assay was used to estimate cell viability and wells were stained with crystal violet to assess biomass. Results: Treatments were described as percent reduction in cell viability and biomass compared to negative control wells (no treatment). Compared with the dual antibiotics, the phage cocktail resulted in a further mean 1.3fold reduction in cell viability ( p < 0.001) and 1.7-fold reduction in biomass ( p < 0.001). Notably, in 4 biofilms which were largely resistant to the antibiotic mix, the addition of phage conferred a 50 ± 15% reduction in cell viability and 60 ± 12% reduction in biomass (95% CI). Conclusion: Our results show a convincing reduction in pre-formed Pa biofilms when a lytic phage cocktail was added to standard antibiotic therapy. The most striking benefits seen were in the 4 clinical strains with the least response to conventional antibiotics. In an era of increased bacterial resistance, bacteriophage is a promising adjunct to standard clinical care. Acknowledgement: Funded by the CF Trust as part of the Strategic Research Centre for Pseudomonas infection. WS07.5 Novel antimicrobial peptides to fight infections in patients with cystic fibrosis M. van Eijk1, C.K. van der Ent2, H.G. Arets2, M.I. Kristensen2, H. de Cock3, A. van Dijk1, B.J. Keijser4, H.P. Haagsman1. 1Utrecht University, Infectious Diseases and Immunology, Utrecht, Netherlands; 2University Medical Center Utrecht, Cystic Fibrosis Center Utrecht, Utrecht, Netherlands; 3Utrecht University, Biology, Utrecht, Netherlands; 4TNO, Microbiolggy and Systems Biology, Zeist, Netherlands Objectives: In view of the current limitations to treat and reduce pulmonary infections and chronic inflammation in Cystic Fibrosis (CF) patients effectively, we explored the potential of a novel therapeutic approach based upon antimicrobial peptides (AMPs). Novel cathelicidinlike peptides have been designed and were tested for their antimicrobial and cytotoxic properties in vitro and ex vivo. Methods: Several well-established microbiological assays (colony counts, bioscreen kinetics) were applied using different media to test the killing efficacy of the different peptide designs against several isolates of CFrelevant bacterial and fungal species. Different mammalian cell culture systems (bronchial cell lines, primary human nasal cells) were used to screen for toxicity using the WST-1 conversion assay (cell viability). Results: Newly developed AMPs were found to display extraordinary strong antimicrobial activities against several clinical isolates of multiresistant Pseudomonas aeruginosa and Staphylococcus aureus and were shown to be effective against pathogens present in CF-patient-derived pulmonary lavage and sputum. In addition, these peptides possessed strong antifungal properties against Candida albicans and Aspergillus fumigatus, two important fungal pathogens associated with CF. In vitro studies revealed that these AMPs are active under physiological conditions (in the presence of NaCl, CaCl2) and at the lower pH that typically exists in the CF lung. Furthermore, these peptides exhibited low cytotoxicity towards several mammalian cell types (e.g. primary human nasal epithelial cells). Conclusion: Novel cathelicidin-inspired peptides show potential as a new anti-infectious therapy to effectively reduce a broad-range of microbial infections in the upper and lower airways of patients suffering from CF. This novel therapeutic approach may help to limit pulmonary inflammation and tissue damage, and will improve clinical outcome in CF-patients. WS07.6 CAT-5571: an autophagy activator that enhances the clearance of intracellular bacteria F. Liu1, A. Amer2, K. Krause2, T.L. Bonfield3, D. Fletcher3, C. Richards4, J.F. Reilly1, A.J. Nichols1, C.B. Vu1. 1Catabasis, Cambridge, United States; 2Ohio State University, Microbial Infection and Immunity, Columbus, United States;
S13
3
Case Western Reserve University, Pediatrics, Cleveland, United States; 4KWS BioTest, Bristol, United Kingdom In cystic fibrosis (CF) patients, chronic intracellular pathogens such as Pseudomonas aeruginosa and Burkholderia cenocepacia are difficult to eradicate with conventional antibiotics. Autophagy is a process cells use to capture and degrade dysfunctional proteins and organelles and invading pathogens. In CF it is known that autophagy is impaired and this can further compromise patients’ ability to clear chronic lung infections. Autophagy activation enables an alternative bacterial clearance mechanism that could be beneficial in combination with antiinfective agents. CAT-5571 is a novel molecular conjugate of a cysteamine moiety that is covalently linked to docosahexaenoic acid (DHA) that can potently activate autophagy. CAT-5571 synergistically activated autophagy in hBE cells from F508del/F508del CF patients at concentrations as low as 3 µM, whereas cysteamine and DHA alone or in simple combination failed to activate autophagy at concentrations as high as 250 µM. In cftr F508del/F508del mice, treatment with CAT-5571 restored the depressed autophagy markers Beclin-1 and LC3B. In in vitro studies using normal or F508del/F508del hBE cells infected with P. aeruginosa, CAT-5571 treatment caused a significant reduction in the intracellular bacterial load. Similarly, in macrophages isolated from cftr F508del/F508del mice, in vitro treatment with CAT-5571 reduced the intracellular bacterial load of B. cenocepacia. In addition to effects on bacterial clearance, CAT-5571 can significantly enhance the chloride current in combination with ivacaftor/lumacaftor. CAT-5571 represents a potential new therapeutic for improving clinical outcomes in conjunction with current therapies for CF. WS07.7 Response of Pseudomonas aeruginosa to hypochlorous acid (HOCl) stress and the role of defence mechanisms in protecting against neutrophil killing
K.V. Farrant1, J.C. Davies2,3, H.D. Williams1. 1Imperial College London, Department of Life Sciences, London, United Kingdom; 2Imperial College London, National Heart and Lung Institute, London, United Kingdom; 3Royal Brompton Hospital, London, United Kingdom Objectives: The immune response to Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterised by neutrophildominated inflammation, which fails to eradicate infection and causes irreversible lung damage. Neutrophils produce reactive oxygen species to combat infection including the potent oxidant hypochlorous acid (HOCl). Defence mechanisms used by P. aeruginosa in response to HOCl stress remain to be elucidated. We aim to identify mechanisms used by P. aeruginosa to protect against HOCl and determine the role of defence mechanisms during infection. Methods: We screened a library of 712 P. aeruginosa regulatory gene mutants for altered susceptibility to a sublethal concentration of HOCl (300 ppm) over 24 hours of growth. Molecular biology techniques were used to construct in-frame deletion mutants and to perform gene expression assays. Results: We identified 12 HOCl-resistant and 16 HOCl-sensitive mutants that had mutations in genes encoding regulators of antibiotic resistance, catabolite repression, methionine biosynthesis, motility and quorum sensing. One HOCl-sensitive strain was mutated in PA14_07340, which encodes an AraC-type regulator and is adjacent to PA14_07355 a putative peroxiredoxin. In-frame deletion mutants of PA14_07340 and PA14_07355 were confirmed to be HOCl-sensitive. PA14_07340 was not required for protection against the oxidants hydrogen peroxide, methyl-viologen or tert-butyl-hydroperoxide. Gene expression assays revealed that PA14_07340 upregulates expression of PA14_07355 in the presence of HOCl. Experiments that examine the role of PA14_07340 and PA14_07355 in protecting P. aeruginosa from killing by neutrophils from healthy and CF patients will be shown. Conclusions: We have identified a novel regulator PA14_07340 that specifically protects against HOCl, raising the prospect that this regulator plays a critical role in defending P. aeruginosa against neutrophil killing.