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Met CM, Hofstaedter CE, O'Keefe IP, Yang H, Moustafa DA, Sherman ME, Doi Y, Rasko DA, Sweet CR, Goldberg JB, Ernst RK. Characterization of Pseudomonas aeruginosa from subjects with diffuse panbronchiolitis. Microbiol Spectr 2024; 12:e0053024. [PMID: 39377602 PMCID: PMC11537112 DOI: 10.1128/spectrum.00530-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Accepted: 08/26/2024] [Indexed: 10/09/2024] Open
Abstract
Diffuse panbronchiolitis (DPB) is a rare, idiopathic inflammatory disease primarily diagnosed in East Asian populations. DPB is characterized by diffuse pulmonary lesions, inflammation of the respiratory bronchioles, and bacterial infections of the airway. Historically, sputum cultures reveal Pseudomonas aeruginosa in 22% of DPB patients, increasing to 60% after 4 years from disease onset. Although DPB patients have a known susceptibility to respiratory P. aeruginosa infections, as is observed in other chronic lung diseases such as cystic fibrosis (CF), the characterization of DPB P. aeruginosa strains is limited. In this study, we characterized 24 strains obtained from a cohort of DPB patients for traits previously associated with virulence, including growth, motility, antibiotic susceptibility, lipopolysaccharide structure, and genomic diversity. Our cohort of DPB P. aeruginosa strains exhibits considerable genomic variability when compared with isolates from people with cystic fibrosis chronically colonized with P. aeruginosa and acute P. aeruginosa infection isolates. Similar to CF, DPB P. aeruginosa strains produce a diverse array of modified lipid A structures. Antibiotic susceptibility testing revealed increased resistance to erythromycin, a representative agent of the macrolide antibiotics used to manage DPB patients. Differences in the O-antigen type among P. aeruginosa strains collected from these different backgrounds were also observed. Ultimately, the characterization of DPB P. aeruginosa strains highlights several unique qualities of P. aeruginosa strains collected from chronically diseased airways, underscoring the challenges in treating DPB, CF, and other obstructive respiratory disease patients with P. aeruginosa infections. IMPORTANCE Diffuse panbronchiolitis (DPB), a chronic lung disease characterized by persistent P. aeruginosa infection, serves as an informative comparator to more common chronic lung diseases, such as cystic fibrosis (CF). This study aimed to better address the interplay between P. aeruginosa and chronically compromised airway environments through the examination of DPB P. aeruginosa strains, as existing literature regarding DPB is limited to case reports, case series, and clinical treatment guidelines. The evaluation of these features in the context of DPB, in tandem with prevailing knowledge of P. aeruginosa strains collected from more common chronic lung diseases (e.g., CF), can aid in the development of more effective strategies to combat respiratory P. aeruginosa infections in patients with chronic lung diseases.
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Affiliation(s)
- Charles M. Met
- Department of Microbial Pathogenesis, University of Maryland – Baltimore, Baltimore, Maryland, USA
| | - Casey E. Hofstaedter
- Department of Microbial Pathogenesis, University of Maryland – Baltimore, Baltimore, Maryland, USA
- Medical Scientist Training Program, University of Maryland – Baltimore, Baltimore, Maryland, USA
| | - Ian P. O'Keefe
- Department of Microbial Pathogenesis, University of Maryland – Baltimore, Baltimore, Maryland, USA
- Department of Biochemistry and Molecular Biology, University of Maryland – Baltimore, Baltimore, Maryland, USA
| | - Hyojik Yang
- Department of Microbial Pathogenesis, University of Maryland – Baltimore, Baltimore, Maryland, USA
| | - Dina A. Moustafa
- Department of Pediatrics, Division of Pulmonary, Asthma, Cystic Fibrosis, and Sleep, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Matthew E. Sherman
- Department of Microbial Pathogenesis, University of Maryland – Baltimore, Baltimore, Maryland, USA
| | - Yohei Doi
- Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA
| | - David A. Rasko
- Department of Microbial Pathogenesis, University of Maryland – Baltimore, Baltimore, Maryland, USA
- Department of Microbiology and Immunology, Institute for Genome Sciences, University of Maryland - Baltimore, Baltimore, Maryland, USA
| | - Charles R. Sweet
- Chemistry Department, USA Naval Academy, Annapolis, Maryland, USA
| | - Joanna B. Goldberg
- Department of Pediatrics, Division of Pulmonary, Asthma, Cystic Fibrosis, and Sleep, Emory University School of Medicine, Atlanta, Georgia, USA
| | - Robert K. Ernst
- Department of Microbial Pathogenesis, University of Maryland – Baltimore, Baltimore, Maryland, USA
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2
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Ji X, Jin P, Yu P, Wang P. Autophagy ameliorates Pseudomonas aeruginosa-infected diabetic wounds by regulating the toll-like receptor 4/myeloid differentiation factor 88 pathway. Wound Repair Regen 2023; 31:305-320. [PMID: 36879445 DOI: 10.1111/wrr.13074] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2022] [Revised: 02/11/2023] [Accepted: 02/18/2023] [Indexed: 03/08/2023]
Abstract
Diabetic foot ulcers (DFUs) are among the most common complications in patients with diabetes and a leading cause of lower extremity amputation. DFUs are exacerbated by prolonged bacterial infection; therefore, there is an urgent need for effective treatments to alleviate the burden associated with this condition. Although autophagy plays a unique role in pathogen phagocytosis and inflammation, its role in diabetic foot infections (DFIs) remains unclear. Pseudomonas aeruginosa (PA) is the most frequently isolated gram-negative bacterium from DFUs. Here, we evaluated the role of autophagy in ameliorating PA infection in wounds in a diabetic rat model and a bone marrow-derived macrophage (BMDM) hyperglycemia model. Both models were pretreated with or without rapamycin (RAPA) and then infected with or without PA. Pretreatment of rats with RAPA significantly enhanced PA phagocytosis, suppressed wound inflammation, reduced the M1:M2 macrophage ratio, and improved wound healing. In vitro investigation of the underlying mechanisms revealed that enhanced autophagy resulted in decreased macrophage secretion of inflammatory factors such as TNF-α, IL-6, and IL-1β but increased that of IL-10 in response to PA infection. Additionally, RAPA treatment significantly enhanced autophagy in macrophages by increasing LC3 and beclin-1 levels, which led to altered macrophage function. Furthermore, RAPA blocked the PA-induced TLR4/MyD88 pathway to regulate macrophage polarisation and inflammatory cytokine production, which was validated by RNA interference and use of the autophagy inhibitor 3-methyladenine (3-MA). These findings suggest enhancing autophagy as a novel therapeutic strategy against PA infection to ultimately improve diabetic wound healing.
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Affiliation(s)
- Xiaoyan Ji
- NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital &Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, China.,Department of Emergency Ward, First Teaching Hospital of Tianjin University of Traditional Chinese Medicine, National Clinical Research Center for Chinese Medicine Acupuncture and Moxibustion, Tianjin, China
| | - Peng Jin
- Department of Gastric Surgery, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin, China.,Key Laboratory of Cancer Prevention and Therapy, Tianjin, China.,Tianjin's Clinical Research Center for Cancer, Tianjin, China
| | - Pei Yu
- NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital &Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, China
| | - Penghua Wang
- NHC Key Laboratory of Hormones and Development, Tianjin Key Laboratory of Metabolic Diseases, Chu Hsien-I Memorial Hospital &Tianjin Institute of Endocrinology, Tianjin Medical University, Tianjin, China
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3
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Mező E, Hartmann-Balogh F, Madarászné Horváth I, Bufa A, Marosvölgyi T, Kocsis B, Makszin L. Effect of Culture Conditions on Fatty Acid Profiles of Bacteria and Lipopolysaccharides of the Genus Pseudomonas-GC-MS Analysis on Ionic Liquid-Based Column. Molecules 2022; 27:molecules27206930. [PMID: 36296523 PMCID: PMC9610168 DOI: 10.3390/molecules27206930] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2022] [Revised: 10/10/2022] [Accepted: 10/11/2022] [Indexed: 12/02/2022] Open
Abstract
The profiling of bacterial fatty acids is a well-established technique in identifying and classifying bacteria. Cultivation conditions may affect the biosynthesis, thereby, changing the fatty acid profile in bacteria. The effect of the culture conditions on the fatty acid components of Pseudomonas aeruginosa PAO1, Pseudomonas aeruginosa ATCC 27853, Pseudomonas aeruginosa polyresistant and Pseudomonas putida all are aligned to the genus Pseudomonas. The fatty acids in the lipopolysaccharides of Pseudomonas aeruginosa PAO1 were also examined. The effects of the cultivation conditions were followed by using agar and blood agar media at the characteristic temperatures, 25 °C, 37 °C and 42 °C, respectively, and an analysis was made during the 1st, 3rd and 5th day following inoculation. In addition to quantitative differences, we also experienced qualitative differences in the fatty acid profiles which detect newly appearing fatty acids, due to changes in environmental factors. The application of ionic liquid-based column unveils new possibilities for the analyses of fatty acids in GC-MS experiments for bacterial fatty acid profiling. The validation results (response linearity, limit of detection, limit of quantification, system suitability, intraday and interday repeatability and accuracy) show the high separation efficiency of the ionic liquid-based column in the analyses.
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Affiliation(s)
- Emerencia Mező
- Institute of Bioanalysis, Medical School, Szentágothai Research Center, University of Pécs, 7622 Pécs, Hungary
| | - Fruzsina Hartmann-Balogh
- Institute of Bioanalysis, Medical School, Szentágothai Research Center, University of Pécs, 7622 Pécs, Hungary
| | - Ibolya Madarászné Horváth
- Institute of Bioanalysis, Medical School, Szentágothai Research Center, University of Pécs, 7622 Pécs, Hungary
| | - Anita Bufa
- Institute of Bioanalysis, Medical School, Szentágothai Research Center, University of Pécs, 7622 Pécs, Hungary
| | - Tamás Marosvölgyi
- Institute of Bioanalysis, Medical School, Szentágothai Research Center, University of Pécs, 7622 Pécs, Hungary
| | - Béla Kocsis
- Department of Medical Microbiology and Immunology, Medical School, University of Pécs, 7622 Pécs, Hungary
| | - Lilla Makszin
- Institute of Bioanalysis, Medical School, Szentágothai Research Center, University of Pécs, 7622 Pécs, Hungary
- Correspondence:
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Simonin JL, Luscher A, Losa D, Badaoui M, van Delden C, Köhler T, Chanson M. Surface Hydration Protects Cystic Fibrosis Airways from Infection by Restoring Junctional Networks. Cells 2022; 11:cells11091587. [PMID: 35563895 PMCID: PMC9105190 DOI: 10.3390/cells11091587] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2022] [Revised: 04/20/2022] [Accepted: 05/06/2022] [Indexed: 02/01/2023] Open
Abstract
Defective hydration of airway surface mucosa is associated with recurrent lung infection in cystic fibrosis (CF), a disease caused by CF transmembrane conductance regulator (CFTR) gene mutations. Whether the composition and/or presence of an airway surface liquid (ASL) is sufficient to prevent infection remains unclear. The susceptibility to infection of polarized wild type and CFTR knockdown (CFTR-KD) airway epithelial cells was determined in the presence or absence of a healthy ASL or physiological saline. CFTR-KD epithelia exhibited strong ASL volume reduction, enhanced susceptibility to infection, and reduced junctional integrity. Interestingly, the presence of an apical physiological saline alleviated disruption of the airway epithelial barrier by stimulating essential junctional protein expression. Thus, rehydrated CFTR-KD cells were protected from infection despite normally intense bacterial growth. This study indicates that an epithelial integrity gatekeeper is modulated by the presence of an apical liquid volume, irrespective of the liquid's composition and of expression of a functional CFTR.
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Affiliation(s)
- Juliette L. Simonin
- Department of Cell Physiology & Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; (J.L.S.); (D.L.); (M.B.)
| | - Alexandre Luscher
- Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; (A.L.); (C.v.D.); (T.K.)
| | - Davide Losa
- Department of Cell Physiology & Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; (J.L.S.); (D.L.); (M.B.)
| | - Mehdi Badaoui
- Department of Cell Physiology & Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; (J.L.S.); (D.L.); (M.B.)
| | - Christian van Delden
- Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; (A.L.); (C.v.D.); (T.K.)
- Department of Medicine Specialties, Division of Infectious Diseases, Geneva University Hospitals, 1211 Geneva, Switzerland
| | - Thilo Köhler
- Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; (A.L.); (C.v.D.); (T.K.)
| | - Marc Chanson
- Department of Cell Physiology & Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva, Switzerland; (J.L.S.); (D.L.); (M.B.)
- Correspondence: ; Tel./Fax: +41-22-37-95-206
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Jafari L, Safinejad K, Nasiri M, Heidari M, Houshmand M. The prevalence of common CFTR gene mutations and polymorphisms in infertile Iranian men with very severe oligozoospermia. J Med Life 2022; 15:547-556. [PMID: 35646184 PMCID: PMC9126445 DOI: 10.25122/jml-2021-0261] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/08/2021] [Accepted: 12/09/2021] [Indexed: 11/05/2022] Open
Abstract
Due to progress in infertility etiology, several genetic bases of infertility are revealed today. This study aimed to investigate the distribution of mutations in the CFTR gene, M470V polymorphism, and IVS8 poly T. Furthermore, we aimed to examine the hotspot exons (4, 7, 9, 10, 11, 20, and 21 exons) to find a new mutation in cystic fibrosis transmembrane conductance regulator (CFTR) gene among infertile Iranian men very severe oligozoospermia (<1 million sperm/mL ejaculate fluid). In the present case-control study, 200 very severe oligozoospermia (20-60s) and 200 fertile men (18-65s) were registered. Five common CFTR mutations were genotyped using the ARMS-PCR technique. The M470V polymorphism was checked out by real-time PCR, and poly T and exons were sequenced. The F508del was the most common (4.5%) CFTR gene mutation; G542X and W1282X were detected with 1.5% and 1%, respectively. N1303K and R117H were detected in 0.5% of cases. F508del was seen as a heterozygous compound with G542X in one patient and with W1282X in the other patient. Also, in the case of M470V polymorphism, there are differences between the case and control groups (p=0.013). Poly T assay showed statistical differences in some genotypes. The study showed no new mutation in the exons mentioned above. Our results shed light on the genetic basis of men with very severe oligozoospermia in the Iranian population, which will support therapy decisions among infertile men.
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Affiliation(s)
- Leyla Jafari
- Department of Biology, Arsanjan Branch, Islamic Azad University, Arsanjan, Iran
| | - Kyumars Safinejad
- Department of Biology, Borujerd Branch, Islamic Azad University, Borujerd, Iran,Corresponding Author: Kyumars Safinejad, Department of Biology, Borujerd Branch, Islamic Azad University, Borujerd, Iran. E-mail:
| | - Mahboobeh Nasiri
- Department of Biology, Arsanjan Branch, Islamic Azad University, Arsanjan, Iran
| | - Mansour Heidari
- Department of Medical Genetics, Tehran University of Medical Sciences (TUMS), Tehran, Iran
| | - Massoud Houshmand
- Department of Medical Genetics, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran
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6
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Bertelsen A, Elborn JS, Schock BC. Microbial interaction: Prevotella spp. reduce P. aeruginosa induced inflammation in cystic fibrosis bronchial epithelial cells. J Cyst Fibros 2021; 20:682-691. [PMID: 34112603 DOI: 10.1016/j.jcf.2021.04.012] [Citation(s) in RCA: 13] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2020] [Revised: 03/30/2021] [Accepted: 04/26/2021] [Indexed: 10/21/2022]
Abstract
BACKGROUND In Cystic Fibrosis (CF) airways, the dehydrated, thick mucus promotes the establishment of persistent polymicrobial infections and drives chronic airways inflammation. This also predisposes the airways to further infections, the vicious, self-perpetuating cycle causing lung damage and progressive lung function decline. The airways are a poly-microbial environment, containing both aerobic and anaerobic bacterial species. Pseudomonas aeruginosa (P. aeruginosa) infections contribute to the excessive inflammatory response in CF, but the role of anaerobic Prevotella spp., frequently found in CF airways, is not known. MATERIALS We assessed innate immune signalling in CF airway epithelial cells in response to clinical strains of P. histicola, P. nigresens and P. aeruginosa. CFBE41o- cells were infected with P. aeruginosa (MOI 100, 2h) followed by infection with P. histicola or P. nigrescens (MOI 100, 2h). Cells were incubated under anaerobic conditions for the duration of the experiments. RESULTS Our study shows that P. histicola and P. nigresens can reduce the growth of P. aeruginosa and dampen the inflammatory response in airway epithelial cells. We specifically illustrate that the presence of the investigated Prevotella spp. reduces Toll-like-receptor (TLR)-4, MAPK, NF-κB(p65) signalling and cytokine release (Interleukin (IL)-6, IL-8) in mixed infections. CONCLUSION Our work, for the first time, strongly indicates a relationship between P. aeruginosa and anaerobic Prevotella spp.. The observed modified NF-κB and MAPK signalling indicates some mechanisms underlying this interaction that could offer a novel therapeutic approach to combat chronic P. aeruginosa infection in people with CF.
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Affiliation(s)
- Anne Bertelsen
- Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Lisburn Road, Belfast, UK; Department of Medicine, University of Cambridge, Addenbrookes Hospital, Hills Road, Cambridge, UK
| | - J Stuart Elborn
- Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Lisburn Road, Belfast, UK; Imperial College London, London, UK
| | - Bettina C Schock
- Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Lisburn Road, Belfast, UK.
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7
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Bertelsen A, Elborn SJ, Schock BC. Toll like Receptor signalling by Prevotella histicola activates alternative NF-κB signalling in Cystic Fibrosis bronchial epithelial cells compared to P. aeruginosa. PLoS One 2020; 15:e0235803. [PMID: 33031374 PMCID: PMC7544055 DOI: 10.1371/journal.pone.0235803] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2020] [Accepted: 09/17/2020] [Indexed: 12/13/2022] Open
Abstract
Cystic Fibrosis (CF), caused by mutations affecting the CFTR gene, is characterised by viscid secretions in multiple organ systems. CF airways contain thick mucus, creating a gradient of hypoxia, which promotes the establishment of polymicrobial infection. Such inflammation predisposes to further infection, a self-perpetuating cycle in mediated by NF-κB. Anaerobic Gram-negative Prevotella spp. are found in sputum from healthy volunteers and CF patients and in CF lungs correlate with reduced levels of inflammation. Prevotella histicola (P. histicola) can suppress murine lung inflammation, however, no studies have examined the role of P. histicola in modulating infection and inflammation in the CF airways. We investigated innate immune signalling and NF-kB activation in CF epithelial cells CFBE41o- in response to clinical stains of P. histicola and Pseudomonas aeruginosa (P. aeruginosa). Toll-Like Receptor (TLR) expressing HEK-293 cells and siRNA assays for TLRs and IKKα were used to confirm signalling pathways. We show that P. histicola infection activated the alternative NF-kB signalling pathway in CF bronchial epithelial cells inducing HIF-1α protein. TLR5 signalling was responsible for the induction of the alternative NF-kB pathway through phosphorylation of IKKα. The induction of transcription factor HIF-1α was inversely associated with the induction of the alternative NF-kB pathway and knockdown of IKKα partially restored canonical NF-kB activation in response to P. histicola. This study demonstrates that different bacterial species in the respiratory microbiome can contribute differently to inflammation, either by activating inflammatory cascades (P. aeruginosa) or by muting the inflammatory response by modulating similar or related pathways (P. histicola). Further work is required to assess the complex interactions of the lung microbiome in response to mixed bacterial infections and their effects in people with CF.
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Affiliation(s)
- Anne Bertelsen
- Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Belfast, United Kingdom
- Department of Medicine, University of Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom
| | - Stuart J. Elborn
- Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Belfast, United Kingdom
- Imperial College London, London, United Kingdom
| | - Bettina C. Schock
- Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Belfast, United Kingdom
- * E-mail:
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8
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Bertelsen A, Elborn JS, Schock BC. Infection with Prevotella nigrescens induces TLR2 signalling and low levels of p65 mediated inflammation in Cystic Fibrosis bronchial epithelial cells. J Cyst Fibros 2019; 19:211-218. [PMID: 31607634 DOI: 10.1016/j.jcf.2019.09.005] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2019] [Revised: 09/05/2019] [Accepted: 09/07/2019] [Indexed: 12/13/2022]
Abstract
Prevotella spp. are frequently identified in Cystic Fibrosis sputum. This study examined whether infection with Prevotella nigrescens, a frequently identified member of this species, contributes to inflammation in CF bronchial epithelial cells through activation of TLR- and NF-κB signalling pathways. CFBE41o- cells were infected with either P.nigrescens or Pseudomonas aeruginosa and incubated under anaerobic conditions for 4h. P.nigrescens activated TLR2 signalling but not TLR4 signalling while P.aeruginosa activated TLR4 signalling with a lesser effect on TLR2. P.aeruginosa induced significant IκBα phosphorylation 10min post infection with a return to control levels by 30min post infection. A significant induction in nuclear p65 DNA binding was observed at 2h post infection. In contrast, infection with P.nigrescens induced phosphorylation of IκBα 120min post infection, with significant induction in nuclear p65 DNA binding at 4h post infection only. Cytokine gene and protein responses were lower for P.nigrescens compared to P.aeruginosa. This study demonstrates the ability of a clinical P.nigrescens isolate to provoke a delayed NF-κB(p65) driven response through induction in TLR2 signalling and activation of sustained levels of IKKα.
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Affiliation(s)
- A Bertelsen
- Department of Veterinary Medicine, The University of Cambridge, Madingley Road, Cambridge, United Kingdom; Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Belfast, United Kingdom
| | - J S Elborn
- Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Belfast, United Kingdom; National Heart and Lung Institute, Imperial College London, London, United Kingdom
| | - B C Schock
- Wellcome-Wolfson Institute for Experimental Medicine, Queens University Belfast, Belfast, United Kingdom.
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Curutiu C, Iordache F, Lazar V, Pisoschi AM, Pop A, Chifiriuc MC, Hoban AM. Impact of Pseudomonas aeruginosa quorum sensing signaling molecules on adhesion and inflammatory markers in endothelial cells. Beilstein J Org Chem 2018; 14:2580-2588. [PMID: 30410619 PMCID: PMC6204754 DOI: 10.3762/bjoc.14.235] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2018] [Accepted: 09/19/2018] [Indexed: 01/20/2023] Open
Abstract
Pseudomonas aeruginosa relies on the quorum sensing (QS) signaling system as a central regulator mechanism of virulence expression that contributes to the formation and maintenance of biofilms and tolerance to conventional antimicrobials. QS Signaling molecules (QSSMs) may be recognized and may function also within the host cells, being potentially involved in the progression of the infectious process. In this study we evaluate the expression of adhesion and inflammatory molecules in endothelial cells treated with P. aeruginosa QSSMs, in order to bring new insights on the mechanisms involved in the interaction of P. aeruginosa with host cells during the infectious process. Endothelial cells were stimulated with 20 µM of main P. aeruginosa QSSMs (OdDHL = N-(3-oxododecanoyl)-L-homoserine lactone, C4HSL = N-butyryl-L-homoserine lactone, PQS = 2-heptyl-3-hydroxy-4(1H)-quinolone and HHQ = 2-heptyl-4-quinolone). Adherence to endothelial cells, inert substratum and biofilm formation was evaluated. The expression of adhesion molecules (VE-cadherin, PECAM-1, ICAM-1, and P-selectin) and inflammatory response molecules (IL-1β, IL-6, TNFα, TGFβ, and eNOS) was assessed by qRT-PCR and flow cytometry. Our results showed that bacterial adherence to inert substratum and biofilm were decreased in the presence of all tested QSSMs. The adherence index of PAO1 laboratory strain to host cells was decreased between 10-40% in the presence of QSSMs, as compared to untreated control. Expression of eukaryotic cells adhesion molecules ICAM-1 and P-selectin was stimulated by QSSMs, whereas VE-cadherin and PECAM-1 levels were increased only by C4HSL. The inflammatory response of endothelial cells was also modulated, as observed by the modified expression of IL-1β (for C4HSL, PQS and HHQ), IL-6 (for C4HSL and HHQ), TNFα (for C4HSL and HHQ), TGFβ, and eNOS factors. Our results demonstrate that the main pseudomonadal QSSMs differentially modulate endothelial cells adhesion and proinflammatory cytokine expression. These observations provide new insights in the mechanisms by which different QSSMs activate endothelial cells and modulate the infectious process, and support the importance of recent studies aiming to develop anti-QS therapeutic strategies to fight against P. aeruginosa infections.
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Affiliation(s)
- Carmen Curutiu
- University of Bucharest, Faculty of Biology, Department of Microbiology-Immunology, Bucharest, Romania.,Research Institute of the University of Bucharest, Romania
| | - Florin Iordache
- University of Agronomical Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Bucharest, Romania.,Institute of Cellular Biology and Pathology Nicolae Simionescu of Romanian Academy, Romania
| | - Veronica Lazar
- University of Bucharest, Faculty of Biology, Department of Microbiology-Immunology, Bucharest, Romania.,Research Institute of the University of Bucharest, Romania
| | - Aurelia Magdalena Pisoschi
- University of Agronomical Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Bucharest, Romania
| | - Aneta Pop
- University of Agronomical Sciences and Veterinary Medicine, Faculty of Veterinary Medicine, Bucharest, Romania
| | - Mariana Carmen Chifiriuc
- University of Bucharest, Faculty of Biology, Department of Microbiology-Immunology, Bucharest, Romania.,Research Institute of the University of Bucharest, Romania
| | - Alina Maria Hoban
- University of Bucharest, Faculty of Biology, Department of Microbiology-Immunology, Bucharest, Romania.,Research Institute of the University of Bucharest, Romania
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10
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Gamaletsou MN, Hayes G, Harris C, Brock J, Muldoon EG, Denning DW. F508del CFTR gene mutation in patients with allergic bronchopulmonary aspergillosis. J Asthma 2017; 55:837-843. [PMID: 29035608 DOI: 10.1080/02770903.2017.1373808] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
OBJECTIVE The F508del mutation occurs in approximately 3.5% of Caucasian population of Northern Europe. Heterozygotes have increased risk for asthma and reduced pulmonary function. Allergic bronchopulmonary aspergillosis (ABPA) is more common in patients with cystic fibrosis (CF). We aimed to establish the frequency of F508del mutation in adult patients with ABPA. METHODS A retrospective matched case-control study of CF genotyped patients with ABPA seen at the National Aspergillosis Centre was undertaken. Key data were collected retrospectively from medical records, including respiratory comorbidities, total IgE, Aspergillus IgG and IgE, and immunoglobulins. Cystic fibrosis transmembrane regulator (CFTR) gene mutation analysis included multiplex PCR and sequencing. RESULTS From a cohort of 189 ABPA patients, 156 were screened for common mutations and variants in the CFTR gene. Eighteen were heterozygous for at least one CFTR mutation; 12 (7.7%) were heterozygous for the F508del, notably; 3 were heterozygous for the intron 8 5T variant; and 1 for an intronic variant of uncertain significance, c.3139 + 18C>T. Eight (67%) had asthma, 7 (58%) had CT-defined bronchiectasis, 4 (33%) hypergammaglobulinemia (>16 g/L), 3 (25%) sinusitis and 1 (8%) chronic pulmonary aspergillosis. Eight (67%) had elevated Aspergillus IgG antibodies (42-98 mg/L), and 8 (67%) had total IgE above 1,000 KIU/L. Two individuals heterozygous for the F508del mutation and the TG12T5 variant were diagnosed with CF, leading to a de novo CF discovery rate of 1.3%. CONCLUSIONS In our ABPA patient cohort, the presence of the delta F508 mutation was higher than that seen in general population. Genetic counseling for CFTR genotyping might be appropriate for these patients.
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Affiliation(s)
- Maria N Gamaletsou
- a The National Aspergillosis Centre , University Hospital of South Manchester, The University of Manchester and Manchester Academic Health Science Centre , Manchester , UK
| | - Gemma Hayes
- a The National Aspergillosis Centre , University Hospital of South Manchester, The University of Manchester and Manchester Academic Health Science Centre , Manchester , UK
| | - Chris Harris
- a The National Aspergillosis Centre , University Hospital of South Manchester, The University of Manchester and Manchester Academic Health Science Centre , Manchester , UK
| | - Joanna Brock
- b Genomic Diagnostics Laboratory, Manchester Centre for Genomic Medicine , Central Manchester University Hospitals NHS Foundation Trust, St Mary's Hospital , Manchester , UK
| | - Eavan G Muldoon
- a The National Aspergillosis Centre , University Hospital of South Manchester, The University of Manchester and Manchester Academic Health Science Centre , Manchester , UK
| | - David W Denning
- a The National Aspergillosis Centre , University Hospital of South Manchester, The University of Manchester and Manchester Academic Health Science Centre , Manchester , UK
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11
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Vu CB, Bridges RJ, Pena-Rasgado C, Lacerda AE, Bordwell C, Sewell A, Nichols AJ, Chandran S, Lonkar P, Picarella D, Ting A, Wensley A, Yeager M, Liu F. Fatty Acid Cysteamine Conjugates as Novel and Potent Autophagy Activators That Enhance the Correction of Misfolded F508del-Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). J Med Chem 2016; 60:458-473. [PMID: 27976892 DOI: 10.1021/acs.jmedchem.6b01539] [Citation(s) in RCA: 36] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
A depressed autophagy has previously been reported in cystic fibrosis patients with the common F508del-CFTR mutation. This report describes the synthesis and preliminary biological characterization of a novel series of autophagy activators involving fatty acid cysteamine conjugates. These molecular entities were synthesized by first covalently linking cysteamine to docosahexaenoic acid. The resulting conjugate 1 synergistically activated autophagy in primary homozygous F508del-CFTR human bronchial epithelial (hBE) cells at submicromolar concentrations. When conjugate 1 was used in combination with the corrector lumacaftor and the potentiator ivacaftor, it showed an additive effect, as measured by the increase in the chloride current in a functional assay. In order to obtain a more stable form for oral dosing, the sulfhydryl group in conjugate 1 was converted into a functionalized disulfide moiety. The resulting conjugate 5 is orally bioavailable in the mouse, rat, and dog and allows a sustained delivery of the biologically active conjugate 1.
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Affiliation(s)
- Chi B Vu
- Catabasis Pharmaceuticals , One Kendall Square, Suite B14202, Cambridge, Massachusetts 02139, United States
| | - Robert J Bridges
- Chicago Medical School, Rosalind Franklin University of Medicine and Science , 3333 Green Bay Road, North Chicago, Illinois 60064, United States
| | - Cecilia Pena-Rasgado
- Chicago Medical School, Rosalind Franklin University of Medicine and Science , 3333 Green Bay Road, North Chicago, Illinois 60064, United States
| | - Antonio E Lacerda
- Charles River Laboratories , 14656 Neo Parkway, Cleveland, Ohio 44128, United States
| | - Curtis Bordwell
- Charles River Laboratories , 14656 Neo Parkway, Cleveland, Ohio 44128, United States
| | - Abby Sewell
- Charles River Laboratories , 14656 Neo Parkway, Cleveland, Ohio 44128, United States
| | - Andrew J Nichols
- Catabasis Pharmaceuticals , One Kendall Square, Suite B14202, Cambridge, Massachusetts 02139, United States
| | - Sachin Chandran
- Catabasis Pharmaceuticals , One Kendall Square, Suite B14202, Cambridge, Massachusetts 02139, United States
| | - Pallavi Lonkar
- Catabasis Pharmaceuticals , One Kendall Square, Suite B14202, Cambridge, Massachusetts 02139, United States
| | - Dominic Picarella
- Catabasis Pharmaceuticals , One Kendall Square, Suite B14202, Cambridge, Massachusetts 02139, United States
| | - Amal Ting
- Catabasis Pharmaceuticals , One Kendall Square, Suite B14202, Cambridge, Massachusetts 02139, United States
| | - Allison Wensley
- Catabasis Pharmaceuticals , One Kendall Square, Suite B14202, Cambridge, Massachusetts 02139, United States
| | - Maisy Yeager
- Catabasis Pharmaceuticals , One Kendall Square, Suite B14202, Cambridge, Massachusetts 02139, United States
| | - Feng Liu
- Catabasis Pharmaceuticals , One Kendall Square, Suite B14202, Cambridge, Massachusetts 02139, United States
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12
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Li R, Fang L, Tan S, Yu M, Li X, He S, Wei Y, Li G, Jiang J, Wu M. Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity. Cell Res 2016; 26:1273-1287. [PMID: 27857054 DOI: 10.1038/cr.2016.135] [Citation(s) in RCA: 85] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2016] [Revised: 07/29/2016] [Accepted: 08/29/2016] [Indexed: 02/05/2023] Open
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems in bacteria and archaea provide adaptive immunity against invading foreign nucleic acids. Previous studies suggest that certain bacteria employ their Type II CRISPR-Cas systems to target their own genes, thus evading host immunity. However, whether other CRISPR-Cas systems have similar functions during bacterial invasion of host cells remains unknown. Here we identify a novel role for Type I CRISPR-Cas systems in evading host defenses in Pseudomonas aeruginosa strain UCBPP-PA14. The Type I CRISPR-Cas system of PA14 targets the mRNA of the bacterial quorum-sensing regulator LasR to dampen the recognition by toll-like receptor 4, thus diminishing the pro-inflammatory responses of the host in cell and mouse models. Mechanistically, this nuclease-mediated RNA degradation requires a "5'-GGN-3'" recognition motif in the target mRNA, and HD and DExD/H domains in Cas3 of the Type I CRISPR-Cas system. As LasR and Type I CRISPR-Cas systems are ubiquitously present in bacteria, our findings elucidate an important common mechanism underlying bacterial virulence.
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Affiliation(s)
- Rongpeng Li
- Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND 58203-9061, USA
| | - Lizhu Fang
- Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND 58203-9061, USA
| | - Shirui Tan
- Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND 58203-9061, USA
| | - Min Yu
- Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND 58203-9061, USA.,State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041, China
| | - Xuefeng Li
- Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND 58203-9061, USA.,State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041, China
| | - Sisi He
- Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND 58203-9061, USA.,State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041, China
| | - Yuquan Wei
- State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041, China
| | - Guoping Li
- Inflammations & Allergic Diseases Research Unit, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646004, China
| | - Jianxin Jiang
- State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing 400042, China
| | - Min Wu
- Department of Biomedical Sciences, University of North Dakota, Grand Forks, ND 58203-9061, USA.,State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, Sichuan 610041, China
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13
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Abstract
The earliest descriptions of lung disease in people with cystic fibrosis (CF) showed the involvement of 3 interacting pathophysiologic elements in CF airways: mucus obstruction, inflammation, and infection. Over the past 7 decades, our understanding of CF respiratory microbiology and inflammation has evolved with the introduction of new treatments, increased longevity, and increasingly sophisticated laboratory techniques. This article reviews the current understanding of infection and inflammation and their roles in CF lung disease. It also discusses how this constantly evolving information is used to inform current therapeutic strategies, measures and predictors of disease severity, and research priorities.
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Affiliation(s)
- Edith T Zemanick
- Children's Hospital Colorado, University of Colorado School of Medicine, 13123 East 16th Avenue, B-395, Aurora, CO 80045, USA
| | - Lucas R Hoffman
- Departments of Pediatrics and Microbiology, Seattle Children's Hospital and University of Washington, 4800 Sand Point Way Northeast, MS OC.7.720, Seattle, WA 98105, USA.
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14
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Hou Y, Guan X, Yang Z, Li C. Emerging role of cystic fibrosis transmembrane conductance regulator - an epithelial chloride channel in gastrointestinal cancers. World J Gastrointest Oncol 2016; 8:282-288. [PMID: 26989463 PMCID: PMC4789613 DOI: 10.4251/wjgo.v8.i3.282] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/05/2015] [Revised: 10/21/2015] [Accepted: 12/18/2015] [Indexed: 02/05/2023] Open
Abstract
Cystic fibrosis transmembrane conductance regulator (CFTR), a glycoprotein with 1480 amino acids, has been well established as a chloride channel mainly expressed in the epithelial cells of various tissues and organs such as lungs, sweat glands, gastrointestinal system, and reproductive organs. Although defective CFTR leads to cystic fibrosis, a common genetic disorder in the Caucasian population, there is accumulating evidence that suggests a novel role of CFTR in various cancers, especially in gastroenterological cancers, such as pancreatic cancer and colon cancer. In this review, we summarize the emerging findings that link CFTR with various cancers, with focus on the association between CFTR defects and gastrointestinal cancers as well as the underlying mechanisms. Further study of CFTR in cancer biology may help pave a new way for the diagnosis and treatment of gastrointestinal cancers.
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15
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Farinde A. TOBI Podhaler for cystic fibrosis patients with Pseudomonas aeruginosa. Nurse Pract 2015; 40:16-17. [PMID: 26080292 DOI: 10.1097/01.npr.0000459736.07899.73] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/04/2023]
Affiliation(s)
- Abimbola Farinde
- Abimbola Farinde is a clinical supervisor in the Pharmacy Department, at Bayshore Medical Center, Pasadena, Tex
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16
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Jørgensen KM, Wassermann T, Johansen HK, Christiansen LE, Molin S, Høiby N, Ciofu O. Diversity of metabolic profiles of cystic fibrosis Pseudomonas aeruginosa during the early stages of lung infection. MICROBIOLOGY-SGM 2015; 161:1447-62. [PMID: 25873584 DOI: 10.1099/mic.0.000093] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Pseudomonas aeruginosa is the dominant pathogen infecting the airways of cystic fibrosis (CF) patients. During the intermittent colonization phase, P. aeruginosa resembles environmental strains but later evolves to the chronic adapted phenotype characterized by resistance to antibiotics and mutations in the global regulator genes mucA, lasR and rpoN. Our aim was to understand the metabolic changes occurring over time and between niches of the CF airways. By applying Phenotype MicroArrays, we investigated changes in the carbon and nitrogen catabolism of subsequently clonally related mucoid and non-mucoid (NM) lung and sinus P. aeruginosa isolates from 10 CF patients (five intermittently colonized/five chronically infected). We found the most pronounced catabolic changes for the early/late NM isolate comparisons, with respiratory reduction seen for all chronically infecting isolates and two intermittently colonizing isolates. Fewer differences were observed between sinus and lung isolates, showing a higher degree of isolate similarity between these two niches. Modest respiratory changes were seen for the early isolate/PAO1 comparisons, indicating colonization with environmental isolates. Assignment of metabolic pathways via the KEGG database showed a prevalence of substrates involved in the metabolism of Ala, Asp and Glu, d-Ala, and Arg and Pro. In conclusion, extensive heterogeneity in the metabolic profiles of the P. aeruginosa isolates was observed from the initial stages of the infection, showing a rapid diversification of the bacteria in the heterogeneous environment of the lung. Metabolic reduction seems to be a common trait and therefore an adaptive phenotype, though it can be reached via multiple metabolic pathways.
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Affiliation(s)
- Karin Meinike Jørgensen
- 1 Department of Immunology and Microbiology, Costerton Biofilm Center, University of Copenhagen, Denmark
| | - Tina Wassermann
- 2 Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark
| | - Helle Krogh Johansen
- 2 Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark 3 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark
| | - Lasse Engbo Christiansen
- 4 Department of Informatics and Mathematical Modelling, Technical University of Denmark, Denmark
| | - Søren Molin
- 3 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark 5 Center for Systems Biology, Technical University of Denmark, Denmark
| | - Niels Høiby
- 1 Department of Immunology and Microbiology, Costerton Biofilm Center, University of Copenhagen, Denmark 2 Department of Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark
| | - Oana Ciofu
- 1 Department of Immunology and Microbiology, Costerton Biofilm Center, University of Copenhagen, Denmark
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17
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Kamath KS, Kumar SS, Kaur J, Venkatakrishnan V, Paulsen IT, Nevalainen H, Molloy MP. Proteomics of hosts and pathogens in cystic fibrosis. Proteomics Clin Appl 2015; 9:134-46. [DOI: 10.1002/prca.201400122] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2014] [Revised: 10/27/2014] [Accepted: 11/18/2014] [Indexed: 12/13/2022]
Affiliation(s)
| | - Sheemal Shanista Kumar
- Department of Chemistry and Biomolecular Sciences; Macquarie University; Sydney Australia
| | - Jashanpreet Kaur
- Department of Chemistry and Biomolecular Sciences; Macquarie University; Sydney Australia
| | | | - Ian T. Paulsen
- Department of Chemistry and Biomolecular Sciences; Macquarie University; Sydney Australia
| | - Helena Nevalainen
- Department of Chemistry and Biomolecular Sciences; Macquarie University; Sydney Australia
| | - Mark P. Molloy
- Department of Chemistry and Biomolecular Sciences; Macquarie University; Sydney Australia
- Australian Proteome Analysis Facility; Macquarie University; Sydney Australia
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18
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Hussain S, Varelogianni G, Särndahl E, Roomans GM. N-acetylcysteine and azithromycin affect the innate immune response in cystic fibrosis bronchial epithelial cells in vitro. Exp Lung Res 2014; 41:251-60. [DOI: 10.3109/01902148.2014.934411] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
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19
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Bouffartigues E, Duchesne R, Bazire A, Simon M, Maillot O, Dufour A, Feuilloley M, Orange N, Chevalier S. Sucrose favors Pseudomonas aeruginosa pellicle production through the extracytoplasmic function sigma factor SigX. FEMS Microbiol Lett 2014; 356:193-200. [PMID: 24861220 DOI: 10.1111/1574-6968.12482] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2014] [Revised: 05/16/2014] [Accepted: 05/16/2014] [Indexed: 11/28/2022] Open
Abstract
Pseudomonas aeruginosa biofilm formation was increased by addition of sucrose to Luria-Bertani medium, whereas addition of NaCl to a final similar osmolarity and use of maltose instead of sucrose, were ineffective. In a previous study, we showed that the extracytoplasmic sigma factor SigX is activated in the presence of sucrose. The sucrose-mediated pellicle increase was abolished in a sigX mutant strain. Sucrose addition led to an increase in pel expression and cyclic-diguanylate (c-di-GMP) pool level production. Interestingly, these two phenotypes were strongly decreased in a sigX mutant. Since pel is not known as a SigX-target, we suspect SigX to be involved in the c-di-GMP production. We found that expression of the diguanylate cyclase PA4843 gene was increased in the presence of sucrose at least partly through SigX activity. Our study shows that sucrose itself rather than osmolarity favours the biofilm mode of P. aeruginosa through the activation of SigX.
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Affiliation(s)
- Emeline Bouffartigues
- Laboratoire de Microbiologie Signaux et Microenvironnement (LMSM), EA 4312, Normandie Université, Université de Rouen, Rouen, France
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20
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Wu X, Chen J, Li X, Zhao Y, Zughaier SM. Culture-free diagnostics of Pseudomonas aeruginosa infection by silver nanorod array based SERS from clinical sputum samples. NANOMEDICINE-NANOTECHNOLOGY BIOLOGY AND MEDICINE 2014; 10:1863-70. [PMID: 24832961 DOI: 10.1016/j.nano.2014.04.010] [Citation(s) in RCA: 59] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/12/2014] [Revised: 03/19/2014] [Accepted: 04/26/2014] [Indexed: 10/25/2022]
Abstract
UNLABELLED Pseudomonas aeruginosa can cause major infection in immunocompromised patients, and successful antibiotic treatment of the infection relies on accurate and rapid identification of the infectious agents. Here, we reported a culture-free diagnostic method based on the surface-enhanced Raman spectroscopy (SERS) of pyocyanin (PCN), a major biomarker of P. aeruginosa. This platform can detect PCN as low as 5 ppb or 2.38 × 10(-8) mol L(-1) in both aqueous solutions and spiked clinical sputum samples. It has also been used to dynamically monitor the excretion of PCN by P. aeruginosa during its growth. The presence of PCN has been detected by SERS in 15 clinical sputum samples, which indicates P. aeruginosa infection, with 95.6% sensitivity and 93.3% specificity. The system can advantageously process multiple specimens rapidly, overcomes the need for bacterial culture and diagnostic microbiology assays, and have widespread implications in the early detection of P. aeruginosa infection. FROM THE CLINICAL EDITOR A surface enhanced Raman spectroscopy method optimized for the detection of P. aureginosa infections is presented in this paper. The presence of pyocyanin, a marker of this bacterium has been detected in 15 clinical sputum samples utilizing this method. A sensitivity of 95.6% and 93.3% specificity was reported, which suggests that the method may enable culture-free high throughput rapid detection of this infection.
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Affiliation(s)
- Xiaomeng Wu
- Department of Food Science and Technology, University of Georgia, Athens, GA, USA; Nanoscale Science and Engineering Center, University of Georgia, Athens, GA, USA.
| | - Jing Chen
- Department of Food Science and Technology, University of Georgia, Athens, GA, USA; Nanoscale Science and Engineering Center, University of Georgia, Athens, GA, USA
| | - Xibo Li
- Science and Technology on Plasma Physics Laboratory, Research Center of Laser Fusion, China Academy of Engineering Physics, Mianyang, China; Department of Physics and Astronomy, University of Georgia, Athens, GA, USA; Nanoscale Science and Engineering Center, University of Georgia, Athens, GA, USA
| | - Yiping Zhao
- Department of Physics and Astronomy, University of Georgia, Athens, GA, USA; Nanoscale Science and Engineering Center, University of Georgia, Athens, GA, USA
| | - Susu M Zughaier
- Department of Microbiology and Immunology, Emory University School of Medicine, and Veterans Affair Medical Center, Atlanta, GA, USA.
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21
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Alipour M, Omri A, Lui EM, Suntres ZE. Co-administration of aqueous ginseng extract with tobramycin stimulates the pro-inflammatory response and promotes the killing of Pseudomonas aeruginosa in the lungs of infected rats. Can J Physiol Pharmacol 2013; 91:935-40. [DOI: 10.1139/cjpp-2013-0073] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
North American ginseng is known to have immunomodulatory and antipseudomonal properties in vitro. In this study we investigated the effects of aqueous ginseng extract, either alone or in a combination with the antibiotic tobramycin, in an animal model of chronic Pseudomonas aeruginosa lung infection. The lungs of male rats (n = 5) were infected with P. aeruginosa (2 × 108 cfu/mL) in agar-beads by intratracheal instillation. Starting on day 7 post-infection, animals were treated daily for 3 consecutive days with saline, tobramycin (300 μg/kg body mass, intratracheal), and (or) ginseng (100 mg/kg body mass, subcutaneous); animals were sacrificed 24 h after the third drug treatment. Lung bacteria counts, cytokine levels in sera, and lung histopathology were examined. The treatment of infected animals with tobramycin [6.6 × 104 colony forming units (cfu)], ginseng (5.3 × 104 cfu), or tobramycin plus ginseng (2.0 × 103 cfu) lessened the lung infection compared with the control group (saline treated) (6.0 × 106 cfu). The levels of pro-inflammatory cytokines (IL-2, IL-4, IL-6, IL-12p70, IFN-γ, GM-CSF, TNF-α) in infected animals were significantly increased with co-treatment of ginseng plus tobramycin. These data suggest that co-administration of aqueous ginseng extract and tobramycin stimulated the pro-inflammatory response and promoted the killing of P. aeruginosa.
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Affiliation(s)
- Misagh Alipour
- Medical Sciences Division, Northern Ontario School of Medicine, Lakehead University, 955 Oliver Road, Thunder Bay, ON P7B 5E1, Canada
- Biomolecular Sciences, Laurentian University, Sudbury, Ontario, Canada
- Ontario Ginseng Innovation and Research Consortium, London, Ontario, Canada
| | - Abdelwahab Omri
- Biomolecular Sciences, Laurentian University, Sudbury, Ontario, Canada
| | - Edmund M.K. Lui
- Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Western University, London, Ontario, Canada
- Ontario Ginseng Innovation and Research Consortium, London, Ontario, Canada
| | - Zacharias E. Suntres
- Medical Sciences Division, Northern Ontario School of Medicine, Lakehead University, 955 Oliver Road, Thunder Bay, ON P7B 5E1, Canada
- Biomolecular Sciences, Laurentian University, Sudbury, Ontario, Canada
- Ontario Ginseng Innovation and Research Consortium, London, Ontario, Canada
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22
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del Carmen Fernández-Alonso M, Díaz D, Berbis MÁ, Marcelo F, Cañada J, Jiménez-Barbero J. Protein-carbohydrate interactions studied by NMR: from molecular recognition to drug design. Curr Protein Pept Sci 2013; 13:816-30. [PMID: 23305367 PMCID: PMC3706953 DOI: 10.2174/138920312804871175] [Citation(s) in RCA: 86] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/26/2012] [Revised: 07/12/2012] [Accepted: 07/25/2012] [Indexed: 02/04/2023]
Abstract
Diseases that result from infection are, in general, a consequence of specific interactions between a pathogenic organism and the cells. The study of host-pathogen interactions has provided insights for the design of drugs with therapeutic properties. One area that has proved to be promising for such studies is the constituted by carbohydrates which participate in biological processes of paramount importance. On the one hand, carbohydrates have shown to be information carriers with similar, if not higher, importance than traditionally considered carriers as amino acids and nucleic acids. On the other hand, the knowledge on molecular recognition of sugars by lectins and other carbohydrate-binding proteins has been employed for the development of new biomedical strategies. Biophysical techniques such as X-Ray crystallography and NMR spectroscopy lead currently the investigation on this field. In this review, a description of traditional and novel NMR methodologies employed in the study of sugar-protein interactions is briefly presented in combination with a palette of NMR-based studies related to biologically and/or pharmaceutically relevant applications.
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23
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Junkins RD, Shen A, Rosen K, McCormick C, Lin TJ. Autophagy enhances bacterial clearance during P. aeruginosa lung infection. PLoS One 2013; 8:e72263. [PMID: 24015228 PMCID: PMC3756076 DOI: 10.1371/journal.pone.0072263] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2013] [Accepted: 07/10/2013] [Indexed: 12/21/2022] Open
Abstract
Pseudomonas aeruginosa is an opportunistic bacterial pathogen which is the leading cause of morbidity and mortality among cystic fibrosis patients. Although P. aeruginosa is primarily considered an extacellular pathogen, recent reports have demonstrated that throughout the course of infection the bacterium acquires the ability to enter and reside within host cells. Normally intracellular pathogens are cleared through a process called autophagy which sequesters and degrades portions of the cytosol, including invading bacteria. However the role of autophagy in host defense against P. aeruginosa in vivo remains unknown. Understanding the role of autophagy during P. aeruginosa infection is of particular importance as mutations leading to cystic fibrosis have recently been shown to cause a blockade in the autophagy pathway, which could increase susceptibility to infection. Here we demonstrate that P. aeruginosa induces autophagy in mast cells, which have been recognized as sentinels in the host defense against bacterial infection. We further demonstrate that inhibition of autophagy through pharmacological means or protein knockdown inhibits clearance of intracellular P. aeruginosa in vitro, while pharmacologic induction of autophagy significantly increased bacterial clearance. Finally we find that pharmacological manipulation of autophagy in vivo effectively regulates bacterial clearance of P. aeruginosa from the lung. Together our results demonstrate that autophagy is required for an effective immune response against P. aeruginosa infection in vivo, and suggest that pharmacological interventions targeting the autophagy pathway could have considerable therapeutic potential in the treatment of P. aeruginosa lung infection.
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Affiliation(s)
- Robert D. Junkins
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
- Department of Pediatrics, IWK Health Centre, Halifax, Nova Scotia, Canada
- Beatrice Hunter Cancer Research Institute, Halifax, Nova Scotia, Canada
| | - Ann Shen
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
| | - Kirill Rosen
- Department of Biochemistry and Molecular Biology, Halifax, Nova Scotia, Canada
| | - Craig McCormick
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
- Beatrice Hunter Cancer Research Institute, Halifax, Nova Scotia, Canada
| | - Tong-Jun Lin
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
- Department of Pediatrics, IWK Health Centre, Halifax, Nova Scotia, Canada
- Beatrice Hunter Cancer Research Institute, Halifax, Nova Scotia, Canada
- * E-mail:
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24
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Melvin TAN, Lane AP, Nguyen MT, Lin SY. Sinonasal epithelial cell expression of Toll-like receptor 9 is elevated in cystic fibrosis-associated chronic rhinosinusitis. Am J Rhinol Allergy 2013; 27:30-3. [PMID: 23406596 DOI: 10.2500/ajra.2013.27.3834] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
BACKGROUND Cystic fibrosis (CF) patients frequently suffer from chronic rhinosinusitis (CRS). The extent to which alterations in sinonasal innate immunity contribute to this disease process is unknown. Activation of sinonasal epithelial cell (SNEC) Toll-like receptors (TLRs), an important component of the innate immune system, may be associated with the hyperinflammatory state observed in sinonasal mucosa of CF patients with CRS. This study compares expression of Toll-like receptor 9 (TLR9), in SNRCs collected from CF subjects with CRS to that of normal control subjects. METHODS This was a prospective study measuring TLR9 on SNECs collected via endoscopic-guided middle meatal brushings from 8 adult controls and 14 adult subjects with CF-associated CRS. RESULTS TLR9 expression was significantly elevated in CF subjects at 91% ± 6% when compared with 76% ± 10% in normal controls (p = 0.001). CONCLUSION The significantly greater expression of sinonasal epithelial TLR9 in CF likely reflects increased antimicrobial innate immune activity in chronically colonized and frequently infected CF individuals. However, this finding contrasts with previously reported decreased epithelial TLR9 expression in eosinophilic CRS with nasal polyposis and may indicate differential modulation of innate immunity in Th1-predominent CF versus Th2-dominated CRS with nasal polyps, despite both being diseases of sinonasal mucosal inflammation.
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Affiliation(s)
- Thuy-Anh N Melvin
- Department of Otolaryngology-Head and Neck Surgery, Johns Hopkins Hospital, Baltimore, MD 21287, USA
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25
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Buchanan PJ, McNally P, Harvey BJ, Urbach V. Lipoxin A₄-mediated KATP potassium channel activation results in cystic fibrosis airway epithelial repair. Am J Physiol Lung Cell Mol Physiol 2013; 305:L193-201. [PMID: 23686859 DOI: 10.1152/ajplung.00058.2013] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/22/2023] Open
Abstract
The main cause of morbidity and mortality in cystic fibrosis (CF) is progressive lung destruction as a result of persistent bacterial infection and inflammation, coupled with reduced capacity for epithelial repair. Levels of the anti-inflammatory mediator lipoxin A₄ (LXA₄) have been reported to be reduced in bronchoalveolar lavages of patients with CF. We investigated the ability of LXA₄ to trigger epithelial repair through the initiation of proliferation and migration in non-CF (NuLi-1) and CF (CuFi-1) airway epithelia. Spontaneous repair and cell migration were significantly slower in CF epithelial cultures (CuFi-1) compared with controls (NuLi-1). LXA₄ triggered an increase in migration, proliferation, and wound repair of non-CF and CF airway epithelia. These responses to LXA₄ were completely abolished by the ALX/FPR2 receptor antagonist, Boc2 and ALX/FPR2 siRNA. The KATP channel opener pinacidil mimicked the LXA₄ effect on migration, proliferation, and epithelial repair, whereas the KATP channel inhibitor, glibenclamide, blocked the responses to LXA₄. LXA₄ did not affect potassium channel expression but significantly upregulated glibenclamide-sensitive (KATP) currents through the basolateral membrane of NuLi-1 and CuFi-1 cells. MAP kinase (ERK1/2) inhibitor, PD98059, also inhibited the LXA₄-induced proliferation of NuLi-1 and CuFi-1 cells. Finally, both LXA₄ and pinacidil stimulated ERK-MAP kinase phosphorylation, whereas the effect of LXA₄ on ERK phosphorylation was inhibited by glibenclamide. Taken together, our results provided evidence for a role of LXA₄ in triggering epithelial repair through stimulation of the ALX/FPR2 receptor, KATP potassium channel activation, and ERK phosphorylation. This work suggests exogenous delivery of LXA₄, restoring levels in patients with CF, perhaps as a potential therapeutic strategy.
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Affiliation(s)
- Paul J Buchanan
- National Children's Research Center, Our Lady's Children Hospital, Dublin, Ireland
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The upregulation of translocator protein (18 kDa) promotes recovery from neuropathic pain in rats. J Neurosci 2013; 33:1540-51. [PMID: 23345228 DOI: 10.1523/jneurosci.0324-12.2013] [Citation(s) in RCA: 76] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
At present, effective drug for treatment of neuropathic pain is still lacking. Recent studies have shown that the ligands of translocator protein (TSPO, 18 kDa), a peripheral receptor for benzodiazepine, modulate inflammatory pain. Here, we report that TSPO was upregulated in astrocytes and microglia in the ipsilateral spinal dorsal horn of rats following L5 spinal nerve ligation (L5 SNL), lasting until the vanishing of the behavioral signs of neuropathic pain (∼50 d). Importantly, a single intrathecal injection of specific TSPO agonists Ro5-4864 or FGIN-1-27 at 7 and 21 d after L5 SNL depressed the established mechanical allodynia and thermal hyperalgesia dramatically, and the effect was abolished by pretreatment with AMG, a neurosteroid synthesis inhibitor. Mechanically, Ro5-4864 substantially inhibited spinal astrocytes but not microglia, and reduced the production of tumor necrosis factor-α (TNF-α) in vivo and in vitro. The anti-neuroinflammatory effect was also prevented by AMG. Interestingly, TSPO expression returned to control levels or decreased substantially, when neuropathic pain healed naturally or was reversed by Ro5-4864, suggesting that the role of TSPO upregulation might be to promote recovery from the neurological disorder. Finally, the neuropathic pain and the upregulation of TSPO by L5 SNL were prevented by pharmacological blockage of Toll-like receptor 4 (TLR4). These data suggested that TSPO might be a novel therapeutic target for the treatment of neuropathic pain.
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Cystic Fibrosis: Alternative Approaches to the Treatment of a Genetic Disease. Mol Pharmacol 2012. [DOI: 10.1002/9781118451908.ch6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
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Efficacy of liposomal bismuth-ethanedithiol-loaded tobramycin after intratracheal administration in rats with pulmonary Pseudomonas aeruginosa infection. Antimicrob Agents Chemother 2012; 57:569-78. [PMID: 23147741 DOI: 10.1128/aac.01634-12] [Citation(s) in RCA: 41] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
We sought to investigate alterations in quorum-sensing signal molecule N-acyl homoserine lactone secretion and in the release of Pseudomonas aeruginosa virulence factors, as well as the in vivo antimicrobial activity of bismuth-ethanedithiol incorporated into a liposome-loaded tobramycin formulation (LipoBiEDT-TOB) administered to rats chronically infected with P. aeruginosa. The quorum-sensing signal molecule N-acyl homoserine lactone was monitored by using a biosensor organism. P. aeruginosa virulence factors were assessed spectrophotometrically. An agar beads model of chronic Pseudomonas lung infection in rats was used to evaluate the efficacy of the liposomal formulation in the reduction of bacterial count. The levels of active tobramycin in the lungs and the kidneys were evaluated by microbiological assay. LipoBiEDT-TOB was effective in disrupting both quorum-sensing signal molecules N-3-oxo-dodeccanoylhomoserine lactone and N-butanoylhomoserine lactone, as well as significantly (P < 0.05) reducing lipase, chitinase, and protease production. At 24 h after 3 treatments, the CFU counts in lungs of animals treated with LipoBiEDT-TOB were of 3 log(10) CFU/lung, comparated to 7.4 and 4.7 log(10) CFU/lung, respectively, in untreated lungs and in lungs treated with free antibiotic. The antibiotic concentration after the last dose of LipoBiEDT-TOB was 25.1 μg/lung, while no tobramycin was detected in the kidneys. As for the free antibiotic, we found 6.5 μg/kidney but could not detect any tobramycin in the lungs. Taken together, LipoBiEDT-TOB reduced the production of quorum-sensing molecules and virulence factors and could highly improve the management of chronic pulmonary infection in cystic fibrosis patients.
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McIsaac SM, Stadnyk AW, Lin TJ. Toll-like receptors in the host defense against Pseudomonas aeruginosa respiratory infection and cystic fibrosis. J Leukoc Biol 2012; 92:977-85. [PMID: 22892106 DOI: 10.1189/jlb.0811410] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
TLRs function in innate immunity by detecting conserved structures present in bacteria, viruses, and fungi. Although TLRs do not necessarily distinguish pathogenic organisms from commensals, in the context of compromised innate immunity and combined with pathogens' effector molecules, TLRs drive the host response to the organism. This review will discuss the evidence and role(s) of TLRs in the response to the opportunistic bacterial pathogen, Pseudomonas aeruginosa, as it relates to respiratory infection and CF, in which innate immune mechanisms are indeed compromised. Outer membrane lipoproteins, LPS, flagellin, and nucleic acids all serve as ligands for TLR2, -4, -5, and -9, respectively. These TLRs and their respective downstream effector molecules have proven critical to the host response to P. aeruginosa, although the protective effects of TLRs may be impaired and in some cases, enhanced in the CF patient, contributing to the particular susceptibility of individuals with this disease to P. aeruginosa infection.
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Affiliation(s)
- Shayla M McIsaac
- Department of Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada
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Griffin PE, Roddam LF, Belessis YC, Strachan R, Beggs S, Jaffe A, Cooley MA. Expression of PPARγ and paraoxonase 2 correlated with Pseudomonas aeruginosa infection in cystic fibrosis. PLoS One 2012; 7:e42241. [PMID: 22860094 PMCID: PMC3409144 DOI: 10.1371/journal.pone.0042241] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2012] [Accepted: 07/02/2012] [Indexed: 12/19/2022] Open
Abstract
The Pseudomonas aeruginosa quorum sensing signal molecule N-3-oxododecanoyl-l-homoserine lactone (3OC12HSL) can inhibit function of the mammalian anti-inflammatory transcription factor peroxisome proliferator activated receptor (PPAR)γ, and can be degraded by human paraoxonase (PON)2. Because 3OC12HSL is detected in lungs of cystic fibrosis (CF) patients infected with P. aeruginosa, we investigated the relationship between P. aeruginosa infection and gene expression of PPARγ and PON2 in bronchoalveolar lavage fluid (BALF) of children with CF. Total RNA was extracted from cell pellets of BALF from 43 children aged 6 months–5 years and analyzed by reverse transcription–quantitative real time PCR for gene expression of PPARγ, PON2, and P. aeruginosa lasI, the 3OC12HSL synthase. Patients with culture-confirmed P. aeruginosa infection had significantly lower gene expression of PPARγ and PON2 than patients without P. aeruginosa infection. All samples that were culture-positive for P. aeruginosa were also positive for lasI expression. There was no significant difference in PPARγ or PON2 expression between patients without culture-detectable infection and those with non-Pseudomonal bacterial infection, so reduced expression was specifically associated with P. aeruginosa infection. Expression of both PPARγ and PON2 was inversely correlated with neutrophil counts in BALF, but showed no correlation with other variables evaluated. Thus, lower PPARγ and PON2 gene expression in the BALF of children with CF is associated specifically with P. aeruginosa infection and neutrophilia. We cannot differentiate whether this is a cause or the effect of P. aeruginosa infection, but propose that the level of expression of these genes may be a marker for susceptibility to early acquisition of P. aeruginosa in children with CF.
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Affiliation(s)
- Phoebe E. Griffin
- Menzies Research Institute, Hobart, Tasmania, Australia
- School of Medicine, University of Tasmania, Hobart, Tasmania, Australia
| | - Louise F. Roddam
- School of Medicine, University of Tasmania, Hobart, Tasmania, Australia
| | - Yvonne C. Belessis
- Department of Respiratory Medicine, Sydney Children’s Hospital, Randwick, New South Wales, Australia
| | - Roxanne Strachan
- Department of Respiratory Medicine, Sydney Children’s Hospital, Randwick, New South Wales, Australia
| | - Sean Beggs
- School of Medicine, University of Tasmania, Hobart, Tasmania, Australia
- Department of Pediatrics, Royal Hobart Hospital, Hobart, Tasmania, Australia
| | - Adam Jaffe
- Department of Respiratory Medicine, Sydney Children’s Hospital, Randwick, New South Wales, Australia
| | - Margaret A. Cooley
- School of Medicine, University of Tasmania, Hobart, Tasmania, Australia
- * E-mail:
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Ben Mohamed F, Garcia-Verdugo I, Medina M, Balloy V, Chignard M, Ramphal R, Touqui L. A crucial role of Flagellin in the induction of airway mucus production by Pseudomonas aeruginosa. PLoS One 2012; 7:e39888. [PMID: 22768318 PMCID: PMC3388098 DOI: 10.1371/journal.pone.0039888] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2012] [Accepted: 05/28/2012] [Indexed: 01/21/2023] Open
Abstract
Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. Flagellin is a P. aeruginosa virulence factor involved in host response to this pathogen. We examined the role of flagellin in P. aeruginosa-induced mucus secretion. Using a mouse model of pulmonary infection we showed that PAK, a wild type strain of P. aeruginosa, induced airway mucus secretion and mucin muc5ac expression at higher levels than its flagellin-deficient mutant (ΔFliC). PAK induced expression of MUC5AC and MUC2 in both human airway epithelial NCI-H292 cell line and in primary epithelial cells. In contrast, ΔFliC infection had lower to no effect on MUC5AC and MUC2 expressions. A purified P. aeruginosa flagellin induced MUC5AC expression in parallel to IL-8 secretion in NCI-H292 cells. Accordingly, ΔFliC mutant stimulated IL-8 secretion at significantly lower levels compared to PAK. Incubation of NCI-H292 cells with exogenous IL-8 induced MUC5AC expression and pre-incubation of these cells with an anti-IL-8 antibody abrogated flagellin-mediated MUC5AC expression. Silencing of TLR5 and Naip, siRNA inhibited both flagellin-induced MUC5AC expression and IL-8 secretion. Finally, inhibition of ERK abolished the expression of both PAK- and flagellin-induced MUC5AC. We conclude that: (i) flagellin is crucial in P. aeruginosa-induced mucus hyper-secretion through TLR5 and Naip pathways; (ii) this process is mediated by ERK and amplified by IL-8. Our findings help understand the mechanisms involved in mucus secretion during pulmonary infectious disease induced by P. aeruginosa, such as in cystic fibrosis.
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Affiliation(s)
- Fatima Ben Mohamed
- Unité de Défense Innée et Inflammation, Institut Pasteur, Paris, France.
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El Houry Mignan S, Witte G, Naue N, Curth U. Characterization of the χψ subcomplex of Pseudomonas aeruginosa DNA polymerase III. BMC Mol Biol 2011; 12:43. [PMID: 21955458 PMCID: PMC3197488 DOI: 10.1186/1471-2199-12-43] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2011] [Accepted: 09/28/2011] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND DNA polymerase III, the main enzyme responsible for bacterial DNA replication, is composed of three sub-assemblies: the polymerase core, the β-sliding clamp, and the clamp loader. During replication, single-stranded DNA-binding protein (SSB) coats and protects single-stranded DNA (ssDNA) and also interacts with the χψ heterodimer, a sub-complex of the clamp loader. Whereas the χ subunits of Escherichia coli and Pseudomonas aeruginosa are about 40% homologous, P. aeruginosa ψ is twice as large as its E. coli counterpart, and contains additional sequences. It was shown that P. aeruginosa χψ together with SSB increases the activity of its cognate clamp loader 25-fold at low salt. The E. coli clamp loader, however, is insensitive to the addition of its cognate χψ under similar conditions. In order to find out distinguishing properties within P. aeruginosa χψ which account for this higher stimulatory effect, we characterized P. aeruginosa χψ by a detailed structural and functional comparison with its E. coli counterpart. RESULTS Using small-angle X-ray scattering, analytical ultracentrifugation, and homology-based modeling, we found the N-terminus of P. aeruginosa ψ to be unstructured. Under high salt conditions, the affinity of the χψ complexes from both organisms to their cognate SSB was similar. Under low salt conditions, P. aeruginosa χψ, contrary to E. coli χψ, binds to ssDNA via the N-terminus of ψ. Whereas it is also able to bind to double-stranded DNA, the affinity is somewhat reduced. CONCLUSIONS The binding to DNA, otherwise never reported for any other ψ protein, enhances the affinity of P. aeruginosa χψ towards the SSB/ssDNA complex and very likely contributes to the higher stimulatory effect of P. aeruginosa χψ on the clamp loader. We also observed DNA-binding activity for P. putida χψ, making this activity most probably a characteristic of the ψ proteins from the Pseudomonadaceae.
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Affiliation(s)
- Sirine El Houry Mignan
- Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
| | - Gregor Witte
- Department of Biochemistry, Gene Center of the Ludwig-Maximilians-University Munich, Feodor-Lynen-Str. 25, D-81377 Munich, Germany
- Center for Integrated Protein Sciences (CIPSM), Gene Center of the Ludwig-Maximilians-University Munich, Feodor-Lynen-Str. 25, D-81377 Munich, Germany
- Munich Center for Advanced Photonics (MAP), Gene Center of the Ludwig-Maximilians-University Munich, Feodor-Lynen-Str. 25, D-81377 Munich, Germany
| | - Natalie Naue
- Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
| | - Ute Curth
- Institute for Biophysical Chemistry, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany
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Nagant C, Feng Y, Lucas B, Braeckmans K, Savage P, Dehaye JP. Effect of a low concentration of a cationic steroid antibiotic (CSA-13) on the formation of a biofilm by Pseudomonas aeruginosa. J Appl Microbiol 2011; 111:763-72. [PMID: 21699631 DOI: 10.1111/j.1365-2672.2011.05085.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
AIMS Cationic steroids like CSA-13 have been designed by analogy with antimicrobial cationic peptides and have bactericidal properties. The purpose of this work was to evaluate the effect of a low concentration (1 mg l(-1)) of CSA-13 on the formation of a biofilm by eight strains of Pseudomonas aeruginosa (four mucoid and four nonmucoid strains) on an inert surface. METHOD AND RESULTS The biofilm formation was measured with the Crystal Violet method. CSA-13 inhibited the formation of a biofilm by three strains. The zeta potential varied among the strains. The inhibition by the cationic steroid analogue affected the populations of bacteria with the lowest zeta potential. P. aeruginosa bound a fluorescent, more hydrophobic analogue of CSA-13 but there was no correlation between this binding and the inhibition by CSA-13 of biofilm formation. The interaction of CSA-13 with bacteria did not modify their ability to produce rhamnolipids. CONCLUSIONS A low concentration of CSA-13 inhibits the formation of a biofilm by P. aeruginosa through electrostatic interactions and without affecting the production of rhamnolipids. SIGNIFICANCE AND IMPACT OF THE STUDY A low, nontoxic concentration of CSA-13 might be beneficial for the prevention of biofilm formation.
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Affiliation(s)
- C Nagant
- Laboratoire de Chimie biologique et médicale et de Microbiologie pharmaceutique, Faculté de Pharmacie, Université libre de Bruxelles, Brussels, Belgium
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Chaudhary N, Marr KA. Impact of Aspergillus fumigatus in allergic airway diseases. Clin Transl Allergy 2011; 1:4. [PMID: 22410255 PMCID: PMC3294627 DOI: 10.1186/2045-7022-1-4] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2011] [Accepted: 06/10/2011] [Indexed: 02/07/2023] Open
Abstract
For decades, fungi have been recognized as associated with asthma and other reactive airway diseases. In contrast to type I-mediated allergies caused by pollen, fungi cause a large number of allergic diseases such as allergic bronchopulmonary mycoses, rhinitis, allergic sinusitis and hypersensitivity pneumonitis. Amongst the fungi, Aspergillus fumigatus is the most prevalent cause of severe pulmonary allergic disease, including allergic bronchopulmonary aspergillosis (ABPA), known to be associated with chronic lung injury and deterioration in pulmonary function in people with chronic asthma and cystic fibrosis (CF). The goal of this review is to discuss new understandings of host-pathogen interactions in the genesis of allergic airway diseases caused by A. fumigatus. Host and pathogen related factors that participate in triggering the inflammatory cycle leading to pulmonary exacerbations in ABPA are discussed.
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Kaunitz JD, Akiba Y. Purinergic regulation of duodenal surface pH and ATP concentration: implications for mucosal defence, lipid uptake and cystic fibrosis. Acta Physiol (Oxf) 2011; 201:109-16. [PMID: 20560899 DOI: 10.1111/j.1748-1716.2010.02156.x] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The duodenum secretes HCO₃⁻ as part of a multi-layered series of defence mechanisms against damage from luminal acid. In the 1980s, an alkaline surface layer was measured over the mucosa which correlated with the rate of HCO₃⁻ secretion. As all biological processes are regulated, we investigated how the alkaline pH of the surface layer was maintained. As the ecto-phosphorylase alkaline phosphatase (AP) is highly expressed in the duodenal brush border, we hypothesized that its extreme alkaline pH optimum (∼pH 8-9) combined with its ability to hydrolyse regulatory purines such as ATP was part of an ecto-purinergic signalling system, consisting also of brush border P2Y receptors and cystic fibrosis transmembrane regulator-mediated HCO₃⁻ secretion. Extracellular ATP increases the rate of HCO₃⁻ secretion through this purinergic system. At high surface pH (pH(s)), AP activity is increased, which then increases the rate of ATP hydrolysis, decreasing surface ATP concentration ([ATP](s)), with a resultant decrease in the rate of HCO₃⁻ secretion, which subsequently decreases pH(s) . This feedback loop is thus hypothesized to regulate pH(s) over the duodenal mucosa, and in several other HCO₃⁻ secretory organs. As AP activity is directly related to pH(s) , and as AP hydrolyses ATP, [ATP](s) and pH(s) are co-regulated. As many essential tissue functions such as ciliary motility and lipid uptake are dependent on [ATP](s) , dysregulation of pH(s) and [ATP](s) may help explain the tissue dysfunction characteristic of diseases such as cystic fibrosis.
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Affiliation(s)
- J D Kaunitz
- Greater Los Angeles Veterans Affairs Healthcare System, CA, USA.
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Coulon C, Vinogradov E, Filloux A, Sadovskaya I. Chemical analysis of cellular and extracellular carbohydrates of a biofilm-forming strain Pseudomonas aeruginosa PA14. PLoS One 2010; 5:e14220. [PMID: 21151973 PMCID: PMC2997053 DOI: 10.1371/journal.pone.0014220] [Citation(s) in RCA: 48] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/13/2010] [Accepted: 11/08/2010] [Indexed: 11/20/2022] Open
Abstract
Background Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A–L) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the “scaffolding” polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A–L biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated. Principal Findings In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanýi type O:2a,c (Lanýi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-α-L-GalNAcA-(1–3)-α-D-QuiNAc-(1–3)- α-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic β-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ∼20% of dry weight) of LPS-like material. Conclusions We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of P. aeruginosa.
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Affiliation(s)
- Charlène Coulon
- Université Lille Nord de France, Lille, France
- Université du Littoral-Côte d'Opale, LR2B, Bassin Napoléon, Boulogne sur Mer, France
| | - Evgeny Vinogradov
- Institute for Biological Sciences, National Research Council of Canada, Ottawa, Canada
| | - Alain Filloux
- Division of Cell and Molecular Biology, Faculty of Natural Science, Centre for Molecular Microbiology and Infection, Imperial College London, London, United Kingdom
| | - Irina Sadovskaya
- Université Lille Nord de France, Lille, France
- Université du Littoral-Côte d'Opale, UMT 08, Boulogne sur Mer, France
- * E-mail:
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Chao J, Wolfaardt GM, Arts MT. Characterization of Pseudomonas aeruginosa fatty acid profiles in biofilms and batch planktonic cultures. Can J Microbiol 2010; 56:1028-39. [DOI: 10.1139/w10-093] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/12/2023]
Abstract
The fatty acid composition of Pseudomonas aeruginosa PAO1 was compared between biofilm and batch planktonic cultures. Strain PAO1 biofilms were able to maintain a consistent fatty acid profile for up to 6 days, whereas strain PAO1 batch planktonic cultures showed a gradual loss of cis-monounsaturated fatty acids over 4 days. Biofilms exhibited a greater proportion of hydroxy fatty acids but a lower proportion of both cyclopropane fatty acids and saturated fatty acids (SAFAs). SAFAs with ≥16 carbons, in particular, decreased in biofilms when compared with that in batch planktonic cultures. A reduced proportion of SAFAs and a decline in overall fatty acid chain length indicate more fluidic biophysical properties for cell membranes of P. aeruginosa in biofilms. Separating the biofilms into 2 partitions and comparing their fatty acid compositions revealed additional trends that were not observed in the whole biofilm: the shear-nonremovable layer consistently showed greater proportions of hydroxy fatty acid than the bulk liquid + shear-removable portion of the biofilm. The shear-nonremovable portion demonstrated a relatively immediate decline in the proportion of monounsaturated fatty acids between days 2 and 4; which was offset by an increase in the proportion of cyclopropane fatty acids, specifically 19:0cyc(11,12). Simultaneously, the shear-removable portion of the biofilm showed an increase in the proportion of trans-monounsaturated fatty acids and cyclopropane fatty acids.
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Affiliation(s)
- Jerry Chao
- Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada
- Aquatic Ecosystem Management Research Division, National Water Research Institute – Environment Canada, 867 Lakeshore Road, P.O. Box 5050, Burlington, ON L7R 4A6, Canada
| | - Gideon M. Wolfaardt
- Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada
- Aquatic Ecosystem Management Research Division, National Water Research Institute – Environment Canada, 867 Lakeshore Road, P.O. Box 5050, Burlington, ON L7R 4A6, Canada
| | - Michael T. Arts
- Department of Chemistry and Biology, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada
- Aquatic Ecosystem Management Research Division, National Water Research Institute – Environment Canada, 867 Lakeshore Road, P.O. Box 5050, Burlington, ON L7R 4A6, Canada
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Korkmaz B, Horwitz MS, Jenne DE, Gauthier F. Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. Pharmacol Rev 2010; 62:726-59. [PMID: 21079042 PMCID: PMC2993259 DOI: 10.1124/pr.110.002733] [Citation(s) in RCA: 628] [Impact Index Per Article: 41.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/17/2023] Open
Abstract
Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.
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Affiliation(s)
- Brice Korkmaz
- INSERM U-618 Protéases et Vectorisation Pulmonaires, Université François Rabelais, Faculté de médecine, 10 Boulevard Tonnellé, Tours, France.
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Current World Literature. Curr Opin Pulm Med 2010; 16:623-7. [DOI: 10.1097/mcp.0b013e32834006f9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
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Giddings AM, Maitra R. A disease-relevant high-content screening assay to identify anti-inflammatory compounds for use in cystic fibrosis. ACTA ACUST UNITED AC 2010; 15:1204-10. [PMID: 20944057 DOI: 10.1177/1087057110384612] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
Chronic lung inflammation caused by bacterial pathogenesis through activation of nuclear factor kappa B (NFκB)-responsive proinflammatory genes is a major hurdle in the management of lung disease in cystic fibrosis (CF) patients. The authors generated a disease-relevant cell-based high-content screen to identify novel anti-inflammatory compounds for treating lung inflammation in CF. The human bronchial epithelial cell line KKLEB, harboring the most common form of mutation that causes CF, was modified to express an NFκB-responsive green fluorescent protein (GFP) reporter. After creation, the cell line was tested for its ability to respond to disease-relevant inflammatory stimuli elicited by treatment of cells with filtrates of Pseudomonas aeruginosa isolated from the airways of a CF patient. P. aeruginosa filtrates potently activated NFκB-responsive GFP reporter expression in cells. Subsequently, the assay was optimized for high-throughput screening (HTS) through generation of a Z factor (~0.5) and by testing its tolerance to the commonly used solvents ethanol and DMSO. A pilot library of clinically approved compounds was screened for assay validation. Several compounds with known NFκB inhibitory activity were identified, including several steroidal compounds that have been clinically tested in CF. Thus, the assay can be used in a broader HTS campaign to find anti-inflammatory agents for use in CF.
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Affiliation(s)
- Angela M Giddings
- Department of Pharmacology and Toxicology, Discovery Sciences, RTI International, Research Triangle Park, NC, USA
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Dixon AS, Lim CS. The nuclear translocation assay for intracellular protein-protein interactions and its application to the Bcr coiled-coil domain. Biotechniques 2010; 49:519-24. [PMID: 20615205 PMCID: PMC2949290 DOI: 10.2144/000113452] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Protein interactions are critical for normal biological processes and molecular pathogenesis. While it is important to study these interactions, there are limited assays that are performed inside the cell, in the native cell environment, where the majority of protein-protein interactions take place. Here we present a method of studying protein interactions intracellularly using one protein of interest fused to a localization-controllable enhanced GFP (EGFP) construct and the other protein of interest fused to the red fluorescent protein, DsRed. Nuclear translocation of the EGFP construct is induced by addition of a ligand, and the difference in nuclear localization between the induced and noninduced states of the DsRed construct provides an indication of the interaction between the two proteins. This assay, the nuclear translocation assay (NTA), is introduced here as broadly applicable for studying protein interactions in the native environment inside cells and is demonstrated using forms of the coiled-coil domain from the breakpoint cluster region (Bcr) protein.
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Affiliation(s)
- Andrew S Dixon
- Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, 84108, USA
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Sriramulu DD. Amino Acids Enhance Adaptive Behaviour ofPseudomonas Aeruginosain the Cystic Fibrosis Lung Environment. Microbiol Insights 2010. [DOI: 10.4137/mbi.s4694] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022] Open
Abstract
Sputum of cystic fibrosis (CF) patients is a nutrient-rich environment. Higher amino acid content of CF sputum compared to normal sputum plays a major role in the CF-specific phenotype of P. aeruginosa. Presence of amino acids in the sputum-like environment influenced P. aeruginosa quorum-sensing activity and the formation of an unknown exopolysaccharide in the biofilm. Lipopolysaccharides isolated from P. aeruginosa grown in the presence of amino acids enhanced the release of cytokine IL-8 by human kidney and lung epithelial cells. The results of this study provide additional evidence on the role of amino acids towards adaptation of P. aeruginosa to the CF lung environment.
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Affiliation(s)
- Dinesh Diraviam Sriramulu
- Division of Cell and Immune Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany
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Sadovskaya I, Vinogradov E, Li J, Hachani A, Kowalska K, Filloux A. High-level antibiotic resistance in Pseudomonas aeruginosa biofilm: the ndvB gene is involved in the production of highly glycerol-phosphorylated beta-(1->3)-glucans, which bind aminoglycosides. Glycobiology 2010; 20:895-904. [PMID: 20348539 DOI: 10.1093/glycob/cwq047] [Citation(s) in RCA: 87] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/15/2023] Open
Abstract
Pseudomonas aeruginosa is an opportunistic pathogen that affects immunocompromised individuals and causes life-threatening infections in cystic fibrosis (CF) patients. Colonization of CF lung by P. aeruginosa involves a biofilm mode of growth, which is promoted by the production of exopolysaccharides. These polymers are essential components of the extracellular biofilm matrix. P. aeruginosa possesses several clusters contributing to the formation of the matrix, including the pel or psl genes. In the present study, we identified anionic cyclic glucans produced by P. aeruginosa, which are associated with the matrix of strains PAKDeltaretS and PA14. Their structure has been elucidated using chemical analysis, 1- and 2D nuclear magnetic resonance techniques and mass spectrometry. They belong to a family of cyclic beta-(1-->3)-linked glucans of 12-16 glucose residues with 30-50% of glucose units substituted by 1-phosphoglycerol at O-6. These glucans were also recovered in pel mutant strains, which indicated that their biosynthesis was pel independent. In an effort to understand the biogenesis of these glucans, we analyzed the matrix components of a previously characterized P. aeruginosa PA14 mutant, the PA14::ndvB mutant strain. The ndvB gene was predicted to be involved in the synthesis of perisplasmic glucans, capable of physically interacting with aminoglycoside antibiotics. We revealed that the highly glycerol-phosphorylated beta-(1-->3)-glucans are lacking in the ndvB mutant, and we showed that these glucans are capable of direct binding with the aminoglycoside antibiotic kanamycin. This observation fills a gap in our understanding of the relationship between biofilm, cyclic glucans and high-level antibiotic resistance.
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Fausther M, Pelletier J, Ribeiro CM, Sévigny J, Picher M. Cystic fibrosis remodels the regulation of purinergic signaling by NTPDase1 (CD39) and NTPDase3. Am J Physiol Lung Cell Mol Physiol 2010; 298:L804-18. [PMID: 20190036 DOI: 10.1152/ajplung.00019.2010] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022] Open
Abstract
Airway defenses are regulated by a complex purinergic signaling network located on the epithelial surfaces, where ATP stimulates the clearance of mucin and pathogens. The present study shows that the obstructive disease cystic fibrosis (CF) affects the activity, expression, and tissue distribution of two ectonucleotidases found critical for the regulation of ATP on airway surfaces: NTPDase1 and NTPDase3. Functional polarities and mRNA expression levels were determined on primary cultures of human bronchial epithelial (HBE) cells from healthy donors and CF patients. The in vitro model of the disease was completed by exposing CF HBE cultures for 4 days to supernatant of the mucopurulent material (SMM) collected from the airways of CF patients. We report that NTPDase1 and NTPDase3 are coexpressed on HBE cultures, where they regulate physiological and excess nucleotide concentrations, respectively. In aseptic conditions, CF epithelia exhibit >50% lower NTPDase1 activity, protein, and mRNA levels than normal epithelia, whereas these parameters are threefold higher for NTPDase3. Exposure to SMM induced opposite polarity shifts of the two NTPDases on both normal and CF epithelia, apical NTPDase1 being mobilized to basolateral surfaces and bilateral NTPDase3 to the apical surface. Their immunolocalization in human tissue revealed that NTPDase1 is expressed in epithelial, inflammatory, and endothelial cells, whereas NTPDase3 is restricted to epithelial cells. Furthermore, the SMM-exposed CF HBE cultures reproduced the impact of the disease on their in vivo distribution. This study provides evidence that an extensive remodeling of the enzymatic network regulating clearance occurs in the airways of CF patients.
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Affiliation(s)
- Michel Fausther
- Centre de Recherche en Rhumatologie et Immunologie, Université Laval, Ste-Foy, Quebec City, Canada
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Alipour M, Suntres ZE, Lafrenie RM, Omri A. Attenuation of Pseudomonas aeruginosa virulence factors and biofilms by co-encapsulation of bismuth-ethanedithiol with tobramycin in liposomes. J Antimicrob Chemother 2010; 65:684-93. [PMID: 20159770 DOI: 10.1093/jac/dkq036] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022] Open
Abstract
OBJECTIVES This study examined the activities of tobramycin and bismuth against quorum sensing, virulence factors and biofilms of Pseudomonas aeruginosa by co-encapsulating the agents in liposomes in order to achieve greater delivery of the agents. METHODS The inhibitory effects of the agents, in either their conventional (free) or vesicle-entrapped (liposomal) formulations, were assessed by measuring the changes in the quorum-sensing signal molecule N-acyl homoserine lactone, pyoverdine, pyocyanin, elastase, protease, chitinase, bacterial attachment and biofilms in vitro. RESULTS The effectiveness of tobramycin and bismuth was superior when they were co-administered as a liposomal formulation as measured by their ability to attenuate the production of N-acyl homoserine lactone, elastase (P < 0.01), protease (P < 0.05) and chitinase (P < 0.01). In the presence of non-lethal concentrations of free and liposomal tobramycin and bismuth, bacterial attachment was attenuated. Biofilm formation was also attenuated with free tobramycin and bismuth, yet, in the presence of liposomal tobramycin and bismuth, biofilm complexes could form but contained mostly dead bacteria. When established biofilms were treated with higher concentrations, free tobramycin and bismuth killed and detached bacteria, while the liposomal tobramycin and bismuth penetrated and killed bacteria in the cores of the biofilms. CONCLUSIONS These data suggest that treatment of P. aeruginosa with tobramycin and bismuth, as measured by the changes in quorum sensing, virulence factors and biofilms, is most effective when delivered as a liposomal formulation at a lower concentration compared with the free formulation.
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Affiliation(s)
- Misagh Alipour
- The Novel Drug & Vaccine Delivery Systems Facility, Department of Chemistry and Biochemistry, Laurentian University, Sudbury, Ontario, P3E 2C6, Canada
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