RESUMO
Drug-resistant Pseudomonas aeruginosa (PA) poses an emerging threat to human health with urgent need for alternative therapeutic approaches. Here, we deciphered the B cell and antibody response to the virulence-associated type III secretion system (T3SS) in a cohort of patients chronically infected with PA. Single-cell analytics revealed a diverse B cell receptor repertoire directed against the T3SS needle-tip protein PcrV, enabling the production of monoclonal antibodies (mAbs) abrogating T3SS-mediated cytotoxicity. Mechanistic studies involving cryoelectron microscopy identified a surface-exposed C-terminal PcrV epitope as the target of highly neutralizing mAbs with broad activity against drug-resistant PA isolates. These anti-PcrV mAbs were as effective as treatment with conventional antibiotics in vivo. Our study reveals that chronically infected patients represent a source of neutralizing antibodies, which can be exploited as therapeutics against PA.
Assuntos
Anticorpos Antibacterianos , Anticorpos Neutralizantes , Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Anticorpos Antibacterianos/farmacologia , Microscopia Crioeletrônica , Imunoglobulinas/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/fisiologia , Infecções por Pseudomonas/tratamento farmacológicoRESUMO
The Cystic Fibrosis Conductance Transmembrane Regulator gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated CFTR transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. CFTR transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: CFTR mRNA expression could be shown for primary CD4+ T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect CFTR transcripts for CD14+ monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8+ T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Leucócitos Mononucleares , RNA Mensageiro , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Leucócitos Mononucleares/metabolismo , Western Blotting , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fibrose Cística/metabolismo , Fibrose Cística/genética , Células Matadoras Naturais/metabolismo , Citometria de Fluxo/métodos , Linfócitos T CD4-Positivos/metabolismoRESUMO
Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which lead to impaired ion transport in epithelial cells. Although lung failure due to chronic infection is the major comorbidity in individuals with cystic fibrosis, the role of CFTR in non-epithelial cells has not been definitively resolved. Given the important role of host defense cells, we evaluated the Cftr deficiency in pulmonary immune cells by hematopoietic stem cell transplantation in cystic fibrosis mice. We transplanted healthy bone marrow stem cells and could reveal a stable chimerism of wild-type cells in peripheral blood. The outcome of stem cell transplantation and the impact of healthy immune cells were evaluated in acute Pseudomonas aeruginosa airway infection. In this study, mice transplanted with wild-type cells displayed better survival, lower lung bacterial numbers, and a milder disease course. This improved physiology of infected mice correlated with successful intrapulmonary engraftment of graft-derived alveolar macrophages, as seen by immunofluorescence microscopy and flow cytometry of graft-specific leucocyte surface marker CD45 and macrophage marker CD68. Given the beneficial effect of hematopoietic stem cell transplantation and stable engraftment of monocyte-derived CD68-positive macrophages, we conclude that replacement of mutant Cftr macrophages attenuates airway infection in cystic fibrosis mice.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Macrófagos/imunologia , Mutação , Infecções por Pseudomonas/terapia , Pseudomonas aeruginosa/isolamento & purificação , Animais , Fibrose Cística/genética , Fibrose Cística/microbiologia , Células Epiteliais/microbiologia , Humanos , Pulmão/microbiologia , Macrófagos/microbiologia , Camundongos , Infecções por Pseudomonas/complicações , Infecções por Pseudomonas/microbiologiaRESUMO
The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Mycobacterium tuberculosis/imunologia , Pigmentos Biológicos/metabolismo , Pseudomonas aeruginosa/imunologia , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Reconhecimento de Padrão/metabolismo , Animais , Antibacterianos/metabolismo , Células da Medula Óssea/citologia , Citocinas/imunologia , Citocinas/metabolismo , Retroalimentação Fisiológica , Humanos , Ligantes , Ativação de Macrófagos , Camundongos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/metabolismo , Fenazinas/metabolismo , Pigmentos Biológicos/química , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Piocianina/metabolismo , Fatores de Virulência/química , Fatores de Virulência/metabolismoRESUMO
NAD is an essential co-factor of redox reactions and metabolic conversions of NAD-dependent enzymes. NAD biosynthesis in the opportunistic pathogen Pseudomonas aeruginosa has yet not been experimentally explored. The in silico search for orthologs in the P. aeruginosa PAO1 genome identified the operon pncA - pncB1-nadE (PA4918-PA4920) to encode the nicotinamidase, nicotinate phosporibosyltransferase and Nad synthase of salvage pathway I. The functional role of the preceding genes PA4917 and PA4916 was resolved by the characterization of recombinant protein. PA4917 turned out to encode the nicotinate mononucleotide adenylyltransferase NadD2 and PA4916 was determined to encode the transcriptional repressor NrtR that binds to an intergenic sequence between nadD2 and pncA. Complex formation between the catalytically inactive Nudix protein NrtR and its DNA binding site was suppressed by the antirepressor ADP-ribose. NrtR plasposon mutagenesis abrogated virulence of P. aeruginosa TBCF10839 in a murine acute airway infection model and constrained its metabolite profile. When grown together with other isogenic plasposon mutants, the nrtR knock-out was most compromised in competitive fitness to persist in nutrient-rich medium in vitro or murine airways in vivo. This example demonstrates how tightly metabolism and virulence can be intertwined by key elements of metabolic control.
Assuntos
Adenosina Difosfato Ribose/metabolismo , Regulação Bacteriana da Expressão Gênica , NAD/metabolismo , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiologia , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias , Cricetulus , Modelos Animais de Doenças , Feminino , Camundongos Endogâmicos C3H , Óperon , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/metabolismo , Infecções Respiratórias/microbiologia , Infecções Respiratórias/patologia , VirulênciaRESUMO
Via the histamine H4 -receptor (H4 R), histamine promotes the pathogenesis of experimental allergic asthma in mice. Application of H4 R antagonists during sensitization as well as during provocation reduces the severity of the disease. However, the specific cell types functionally expressing H4 R in experimental allergic asthma have not been well characterized in vivo. In this study, we identified the cell type(s) responsible for H4 R activity in experimental asthma and related physiological mechanisms. Using H4 R-deficient mice, we studied the role of H4 R in the sensitization and effector phase. DCs lacking H4 R expression during the in vitro sensitization reaction resulted in effector T cells unable to induce an entire eosinophilic inflammation in the lung upon adoptive transfer in vivo. Recipient mice lacking H4 R expression, which were adoptively transferred with H4 R(+/+) T cells polarized in the presence of H4 R(+/+) DCs, showed reduced signs of inflammation and ameliorated lung function. Here, we provide in vivo evidence that in experimental asthma in mice the H4 R specifically regulates activation of DCs during sensitization, while in the effector phase the H4 R is active in cells involved in the activation of eosinophils, and possibly other cells. A putative therapy targeting the H4 R may be an option for asthma patients developing IL-5-dependent eosinophilia.
Assuntos
Asma/imunologia , Células Dendríticas/imunologia , Eosinófilos/imunologia , Inflamação/imunologia , Receptores Acoplados a Proteínas G/imunologia , Receptores Histamínicos/imunologia , Transferência Adotiva , Alérgenos/imunologia , Animais , Asma/induzido quimicamente , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Antígeno CD11c/metabolismo , Citocinas/imunologia , Modelos Animais de Doenças , Histamina/metabolismo , Interleucina-5/imunologia , Pulmão/imunologia , Mastócitos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Ovalbumina , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Receptores Histamínicos/genética , Receptores Histamínicos H4 , Células Th2/imunologia , Células Th2/transplanteRESUMO
The population genomics of Pseudomonas aeruginosa was analysed by genome sequencing of representative strains of the 15 most frequent clonal complexes in the P. aeruginosa population and of the five most common clones from the environment of which so far no isolate from a human infection has been detected. Gene annotation identified 5892-7187 open reading frame (ORFs; median 6381 ORFs) in the 20 6.4-7.4 Mbp large genomes. The P. aeruginosa pangenome consists of a conserved core of at least 4000 genes, a combinatorial accessory genome of a further 10 000 genes and 30 000 or more rare genes that are present in only a few strains or clonal complexes. Whole genome comparisons of single nucleotide polymorphism synteny indicated unrestricted gene flow between clonal complexes by recombination. Using standardized acute lettuce, Galleria mellonella and murine airway infection models the full spectrum of possible host responses to P. aeruginosa was observed with the 20 strains ranging from unimpaired health following infection to 100% lethality. Genome comparisons indicate that the differential genetic repertoire of clones maintains a habitat-independent gradient of virulence in the P. aeruginosa population.
Assuntos
Genoma Bacteriano , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Microbiologia Ambiental , Feminino , Variação Genética , Humanos , Pneumopatias/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Mariposas/microbiologia , Fases de Leitura Aberta , Doenças das Plantas/microbiologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/metabolismo , Virulência/genéticaRESUMO
Mammalian cells contain the cyclic pyrimidine nucleotides cCMP and cUMP. It is unknown whether these tentative new second messenger molecules occur in vivo. We used high performance liquid chromatography quadrupole tandem mass spectrometry to quantitate nucleoside 3',5'-cyclic monophosphates. cCMP was detected in all organs studied, most notably pancreas, spleen and the female reproductive system. cUMP was not detected in organs, probably due to the intrinsically low sensitivity of mass spectrometry to detect this molecule and organ matrix effects. Intratracheal infection of mice with recombinant Pseudomonas aeruginosa harboring the nucleotidyl cyclase toxin ExoY massively increased cUMP in lung. The identity of cCMP and cUMP in organs was confirmed by high performance liquid chromatography quadrupole time of flight mass spectrometry. cUMP also appeared in serum, urine and faeces following infection. Taken together, this report unequivocally shows for the first time that cCMP and cUMP occur in vivo.
Assuntos
CMP Cíclico/metabolismo , Nucleotídeos Cíclicos/metabolismo , Uridina Monofosfato/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Espectrometria de Massas em TandemRESUMO
Hemeoxygenase-1 (HO-1), an inducible heat shock protein, is upregulated in response to multiple cellular insults via oxidative stress, lipopolysaccharides (LPS), and hypoxia. In this study, we investigated in vitro the role of Toll-like receptor 4 (TLR4), hypoxia-inducible factor 1α (HIF-1α), and iron on HO-1 expression in cystic fibrosis (CF). Immunohistochemical analysis of TLR4, HO-1, ferritin, and HIF-1α were performed on lung sections of CFTR-/- and wild-type mice. CFBE41o- and 16HBE14o- cell lines were employed for in vitro analysis via immunoblotting, immunofluorescence, real-time PCR, luciferase reporter gene analysis, and iron quantification. We observed a reduced TLR4, HIF-1α, HO-1, and ferritin in CFBE41o- cell line and CF mice. Knockdown studies using TLR4-siRNA in 16HBE14o- revealed significant decrease of HO-1, confirming the role of TLR4 in HO-1 downregulation. Inhibition of HO-1 using tin protoporphyrin in 16HBE14o- cells resulted in increased iron levels, suggesting a probable role of HO-1 in iron accumulation. Additionally, sequestration of excess iron using iron chelators resulted in increased hypoxia response element response in CFBE41o- and 16HBE14o-, implicating a role of iron in HIF-1α stabilization and HO-1. To conclude, our in vitro results demonstrate that multiple regulatory factors, such as impaired TLR4 surface expression, increased intracellular iron, and decreased HIF-1α, downregulate HO-1 expression in CFBE41o- cells.
Assuntos
Brônquios/metabolismo , Fibrose Cística/metabolismo , Regulação para Baixo , Células Epiteliais/metabolismo , Heme Oxigenase-1/biossíntese , Homeostase , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Ferro/metabolismo , Proteínas de Membrana/biossíntese , Mucosa Respiratória/metabolismo , Receptor 4 Toll-Like/biossíntese , Animais , Brônquios/patologia , Linhagem Celular , Fibrose Cística/genética , Fibrose Cística/patologia , Estabilidade Enzimática/genética , Células Epiteliais/patologia , Heme Oxigenase-1/genética , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Mucosa Respiratória/patologia , Receptor 4 Toll-Like/genéticaRESUMO
Pseudomonas aeruginosa is a ubiquitous, opportunistic human pathogen. Since it often expresses multidrug resistance, new treatment options are urgently required. Such new treatments are usually assessed with one of the canonical laboratory strains, PAO1 or PA14. However, these two strains are unlikely representative of the strains infecting patients, because they have adapted to laboratory conditions and do not capture the enormous genomic diversity of the species. Here, we characterized the major P. aeruginosa clone type (mPact) panel. This panel consists of 20 strains, which reflect the species' genomic diversity, cover all major clone types, and have both patient and environmental origins. We found significant strain variation in distinct responses toward antibiotics and general growth characteristics. Only few of the measured traits are related, suggesting independent trait optimization across strains. High resistance levels were only identified for clinical mPact isolates and could be linked to known antimicrobial resistance (AMR) genes. One strain, H01, produced highly unstable AMR combined with reduced growth under drug-free conditions, indicating an evolutionary cost to resistance. The expression of microcolonies was common among strains, especially for strain H15, which also showed reduced growth, possibly indicating another type of evolutionary trade-off. By linking isolation source, growth, and virulence to life history traits, we further identified specific adaptive strategies for individual mPact strains toward either host processes or degradation pathways. Overall, the mPact panel provides a reasonably sized set of distinct strains, enabling in-depth analysis of new treatment designs or evolutionary dynamics in consideration of the species' genomic diversity. IMPORTANCE: New treatment strategies are urgently needed for high-risk pathogens such as the opportunistic and often multidrug-resistant pathogen Pseudomonas aeruginosa. Here, we characterize the major P. aeruginosa clone type (mPact) panel. It consists of 20 strains with different origins that cover the major clone types of the species as well as its genomic diversity. This mPact panel shows significant variation in (i) resistance against distinct antibiotics, including several last resort antibiotics; (ii) related traits associated with the response to antibiotics; and (iii) general growth characteristics. We further developed a novel approach that integrates information on resistance, growth, virulence, and life-history characteristics, allowing us to demonstrate the presence of distinct adaptive strategies of the strains that focus either on host interaction or resource processing. In conclusion, the mPact panel provides a manageable number of representative strains for this important pathogen for further in-depth analyses of treatment options and evolutionary dynamics.
Assuntos
Antibacterianos , Farmacorresistência Bacteriana Múltipla , Testes de Sensibilidade Microbiana , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/classificação , Antibacterianos/farmacologia , Humanos , Infecções por Pseudomonas/microbiologia , Farmacorresistência Bacteriana Múltipla/genética , Variação Genética , Virulência/genética , Genoma Bacteriano/genética , Farmacorresistência Bacteriana/genéticaRESUMO
Chronic airway infections with Pseudomonas aeruginosa are the major co-morbidity in most people with cystic fibrosis (CF) sustained by neutrophils as the major drivers of lung inflammation, damage, and remodeling. Phagocytosis assays were performed with clonal consortia of longitudinal P. aeruginosa airway isolates collected from people with CF since the onset of lung colonization until patient's death or replacement by another clone. The extra- and intracellular abundance of individual strains was assessed by deep amplicon sequencing of strain-specific single nucleotide variants in the bacterial genome. The varied microevolution of the accessory genome of the P. aeruginosa clones during mild and severe courses of infection corresponded with a differential persistence of clonal progeny in the neutrophil phagosome. By simultaneously exposing the ancestor and its progeny to the same habitat, the study recapitulated the time lapse of the temporal change of the fitness of the clone to survive in neutrophils.
RESUMO
Pseudomonas aeruginosa TBCF10839 is a highly virulent strain that can persist and replicate in human neutrophils. Screening of a signature-tagged mutagenesis (STM) TBCF10839 transposon library in phagocytosis tests identified a mutant that carried the transposon in the VirB4 homolog 5PG21 of an integrative and conjugative element (ICE)-associated type IV secretion system of the pKLC102 subtype. 5P21 TBCF10839 insertion mutants were deficient in metabolic versatility, secretion, quorum sensing, and virulence. The mutants were efficiently killed in phagocytosis tests in vitro and were avirulent in an acute murine airway infection model in vivo. The inactivation of 5PG21 silenced the rhl, las, and pqs operons and the gene expression for the synthesis of hydrogen cyanide, the antimetabolite l-2-amino-4-methoxy-trans-3-butenoic acid, and the H2- and H3-type VI secretion systems and their associated effectors. The mutants were impaired in the utilization of carbon sources and stored compounds that are not funneled into intermediary metabolism. This showcase demonstrates that a single gene of the mobile accessory genome can become an essential element to operate the core genome-encoded features of metabolism and virulence.
Assuntos
Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Camundongos , Humanos , Virulência/genética , Pseudomonas aeruginosa/metabolismo , Adenosina Trifosfatases , Mutagênese , Elementos de DNA Transponíveis , Percepção de Quorum/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Infecções por Pseudomonas/genéticaRESUMO
Chronic respiratory infections with the gram-negative bacterium Pseudomonas aeruginosa are an important co-morbidity for the quality of life and prognosis of people with cystic fibrosis (CF). Such long-term colonization, sometimes lasting up to several decades, represents a unique opportunity to investigate pathogen adaptation processes to the host. Our studies aimed to resolve if and to what extent the bacterial adaptation to the CF airways influences the fitness of the pathogen to grow and to persist in the lungs. Marker-free competitive fitness experiments of serial P. aeruginosa isolates differentiated by strain-specific SNPs, were performed with murine and human precision cut lung slices (PCLS). Serial P. aeruginosa isolates were selected from six mild and six severe CF patient courses, respectively. MPCLS or hPCLS were inoculated with a mixture of equal numbers of the serial isolates of one course. The temporal change of the composition of the bacterial community during competitive growth was quantified by multi-marker amplicon sequencing. Both ex vivo models displayed a strong separation of fitness traits between mild and severe courses. Whereas the earlier isolates dominated the competition in the severe courses, intermediate and late isolates commonly won the competition in the mild courses. The status of the CF lung disease rather than the bacterial genotype drives the adaptation of P. aeruginosa during chronic CF lung infection. This implies that the disease status of the lung habitat governed the adaptation of P. aeruginosa more strongly than the underlying bacterial clone-type and its genetic repertoire.
Assuntos
Fibrose Cística , Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Humanos , Pulmão/microbiologia , Camundongos , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genéticaRESUMO
Constant exposure of the airways to inhaled pathogens requires efficient early immune responses protecting against infections. How bacteria on the epithelial surface are detected and first-line protective mechanisms are initiated are not well understood. We have recently shown that tracheal brush cells (BCs) express functional taste receptors. Here we report that bitter taste signaling in murine BCs induces neurogenic inflammation. We demonstrate that BC signaling stimulates adjacent sensory nerve endings in the trachea to release the neuropeptides CGRP and substance P that mediate plasma extravasation, neutrophil recruitment, and diapedesis. Moreover, we show that bitter tasting quorum-sensing molecules from Pseudomonas aeruginosa activate tracheal BCs. BC signaling depends on the key taste transduction gene Trpm5, triggers secretion of immune mediators, among them the most abundant member of the complement system, and is needed to combat P. aeruginosa infections. Our data provide functional insight into first-line defense mechanisms against bacterial infections of the lung.
Assuntos
Infecções Bacterianas , Paladar , Animais , Células Epiteliais , Imunidade Inata , Camundongos , Pseudomonas aeruginosa , Transdução de Sinais , Paladar/fisiologia , TraqueiaRESUMO
Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity.
Assuntos
Proteínas de Bactérias/genética , Caenorhabditis elegans/microbiologia , Quimiotaxia/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Técnicas Bacteriológicas , Regulação Bacteriana da Expressão Gênica , Camundongos , Infecções por Pseudomonas/genética , Infecções por Pseudomonas/patologia , VirulênciaRESUMO
BACKGROUND: Since the discovery of the human CFTR gene in 1989 various mouse models for cystic fibrosis (CF) have been generated and used as a very suitable and popular tool to approach research on this life-threatening disease. Age related changes regarding the course of disease and susceptibility towards pulmonary infections have been discussed in numerous studies. METHODS: Here, we investigated CftrTgH(neoim)Hgu and Cftrtm1Unc-Tg(FABPCFTR)1Jaw/J CF mice and their non-CF littermates during an acute lung infection with Pseudomonas aeruginosa for age dependent effects of their lung function and immune response.Mice younger than three or older than six months were intratracheally infected with P. aeruginosa TBCF10839. The infection was monitored by lung function of the animals using non-invasive head-out spirometry and the time course of physiological parameters over 192 hours. Quantitative bacteriology and lung histopathology of a subgroup of animals were used as endpoint parameters. RESULTS: Age-dependent changes in lung function and characteristic features for CF like a shallower, faster breathing pattern were observed in both CF mouse models in uninfected state. In contrast infected CF mice did not significantly differ from their non-CF littermates in susceptibility and severity of lung infection in both mouse models and age groups. The transgenic Cftrtm1Unc-Tg(FABPCFTR)1Jaw/J and their non-CF littermates showed a milder course of infection than the CftrTgH(neoim)Hgu CF and their congenic C57Bl/6J non-CF mice suggesting that the genetic background was more important for outcome than Cftr dysfunction. CONCLUSIONS: Previous investigations of the same mouse lines have shown a higher airway susceptibility of older CF mice to intranasally applied P. aeruginosa. The different outcome of intranasal and intratracheal instillation of bacteria implies that infected CF epithelium is impaired during the initial colonization of upper airways, but not in the subsequent response of host defense.
Assuntos
Envelhecimento/imunologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/microbiologia , Pneumonia Bacteriana/imunologia , Infecções por Pseudomonas/imunologia , Pseudomonas aeruginosa/imunologia , Traqueíte/imunologia , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Camundongos , Camundongos Transgênicos , Pneumonia Bacteriana/microbiologia , Infecções por Pseudomonas/microbiologia , Testes de Função Respiratória , Traqueíte/microbiologiaRESUMO
Primary or secondary immunodeficiencies are characterized by disruption of cellular and humoral immunity. Respiratory infections are a major cause of morbidity and mortality among immunodeficient or immunocompromised patients, with Staphylococcus aureus being a common offending organism. We propose here an adoptive macrophage transfer approach aiming to enhance impaired pulmonary immunity against S aureus. Our studies, using human-induced pluripotent stem cell-derived macrophages (iMφs), demonstrate efficient antimicrobial potential against methicillin-sensitive and methicillin-resistant clinical isolates of S aureus. Using an S aureus airway infection model in immunodeficient mice, we demonstrate that the adoptive transfer of iMφs is able to reduce the bacterial load more than 10-fold within 20 hours. This effect was associated with reduced granulocyte infiltration and less damage in lung tissue of transplanted animals. Whole transcriptome analysis of iMφs compared with monocyte-derived macrophages indicates a more profound upregulation of inflammatory genes early after infection and faster normalization 24 hours postinfection. Our data demonstrate high therapeutic efficacy of iMφ-based immunotherapy against S aureus infections and offer an alternative treatment strategy for immunodeficient or immunocompromised patients.
Assuntos
Células-Tronco Pluripotentes Induzidas , Infecções Respiratórias , Infecções Estafilocócicas , Animais , Humanos , Macrófagos , Camundongos , Infecções Estafilocócicas/terapia , Staphylococcus aureusRESUMO
Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.
Assuntos
Variação Genética , Pseudomonas aeruginosa/genética , Substituição de Aminoácidos , Animais , Inversão Cromossômica , Cromossomos Bacterianos , DNA Bacteriano/química , DNA Bacteriano/genética , Feminino , Duplicação Gênica , Laboratórios , Camundongos , Camundongos Endogâmicos C3H , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo , Mutação Puntual , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/patogenicidade , Infecções Respiratórias/microbiologia , Análise de Sequência de DNA , VirulênciaRESUMO
Polymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non-piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendered the bacterium more resistant against killing by neutrophils than the wild type and any other of the more than 3000 tested mutants. Inactivation of pilY1 led to the loss of twitching motility in twitching-proficient wild-type PA14 and PAO1 strains, predisposed to autolysis and impaired the secretion of quinolones and pyocyanin, but on the other hand promoted growth in stationary phase and bacterial survival in murine airway infection models. The PilY1 population consisted of a major full-length and a minor shorter PilY1* isoform. PilY1* was detectable in small extracellular quinolone-positive aggregates, but not in the pilus. P. aeruginosa PilY1 is not an adhesin on the pilus tip, but assists in pilus biogenesis, twitching motility, secretion of secondary metabolites and in the control of cell density in the bacterial population.