RESUMO
Phage therapy is a therapeutic approach to treat multidrug-resistant (MDR) infections that employs lytic bacteriophages (phages) to eliminate bacteria. Despite the abundant evidence for its success as an antimicrobial in Eastern Europe, there is scarce data regarding its effects on the human host. Here, we aimed to understand how lytic phages interact with cells of the airway epithelium, the tissue site that is colonized by bacterial biofilms in numerous chronic respiratory disorders. Using a panel of Pseudomonas aeruginosa phages and human airway epithelial cells (AECs) derived from a person with cystic fibrosis (CF), we determined that interactions between phages and epithelial cells depend on specific phage properties as well as physiochemical features of the microenvironment. Although poor at internalizing phages, the airway epithelium responds to phage exposure by changing its transcriptional profile and secreting antiviral and proinflammatory cytokines that correlate with specific phage families. Overall, our findings indicate that mammalian responses to phages are heterogenous and could potentially alter the way that respiratory local defenses aid in bacterial clearance during phage therapy. Thus, besides phage receptor specificity in a particular bacterial isolate, the criteria to select lytic phages for therapy should be expanded to include mammalian cell responses.
Assuntos
Fibrose Cística , Citocinas , Células Epiteliais , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/virologia , Células Epiteliais/virologia , Células Epiteliais/metabolismo , Células Epiteliais/imunologia , Citocinas/metabolismo , Fibrose Cística/terapia , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Terapia por Fagos , Bacteriófagos/fisiologia , Bacteriófagos/genética , Mucosa Respiratória/virologia , Mucosa Respiratória/metabolismo , Mucosa Respiratória/imunologia , Infecções por Pseudomonas/terapia , Infecções por Pseudomonas/imunologia , Fagos de Pseudomonas/metabolismo , BiofilmesRESUMO
Pseudomonas aeruginosa is a complex nosocomial infectious agent responsible for numerous illnesses, with its growing resistance variations complicating treatment development. Studies have emphasized the importance of virulence factors OprE and OprF in pathogenesis, highlighting their potential as vaccine candidates. In this study, B-cell, MHC-I, and MHC-II epitopes were identified, and molecular linkers were active to join these epitopes with an appropriate adjuvant to construct a vaccine. Computational tools were employed to forecast the tertiary framework, characteristics, and also to confirm the vaccine's composition. The potency was weighed through population coverage analysis and immune simulation. This project aims to create a multi-epitope vaccine to reduce P. aeruginosa-related illness and mortality using immunoinformatics resources. The ultimate complex has been determined to be stable, soluble, antigenic, and non-allergenic upon inspection of its physicochemical and immunological properties. Additionally, the protein exhibited acidic and hydrophilic characteristics. The Ramachandran plot, ProSA-web, ERRAT, and Verify3D were employed to ensure the final model's authenticity once the protein's three-dimensional structure had been established and refined. The vaccine model showed a significant binding score and stability when interacting with MHC receptors. Population coverage analysis indicated a global coverage rate of 83.40%, with the USA having the highest coverage rate, exceeding 90%. Moreover, the vaccine sequence underwent codon optimization before being cloned into the Escherichia coli plasmid vector pET-28a (+) at the EcoRI and EcoRV restriction sites. Our research has developed a vaccine against P. aeruginosa that has strong binding affinity and worldwide coverage, offering an acceptable way to mitigate nosocomial infections.
Assuntos
Biologia Computacional , Infecções por Pseudomonas , Pseudomonas aeruginosa , Sepse , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/genética , Humanos , Infecções por Pseudomonas/prevenção & controle , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Sepse/prevenção & controle , Sepse/imunologia , Sepse/microbiologia , Biologia Computacional/métodos , Epitopos/imunologia , Epitopos/química , Pneumonia/prevenção & controle , Pneumonia/imunologia , Pneumonia/microbiologia , Vacinas contra Pseudomonas/imunologia , Vacinas Bacterianas/imunologia , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/genéticaRESUMO
Microbial pathogenicity often depends on the route of infection. For instance, P. aeruginosa or S. marcescens cause acute systemic infections when low numbers of bacteria are injected into D. melanogaster flies whereas flies succumb much slower to the continuous ingestion of these pathogens, even though both manage to escape from the gut compartment and reach the hemocoel. Here, we have developed a latent P. aeruginosa infection model by feeding flies on the bacteria for a short period. The bacteria stably colonize internal tissues yet hardly cause any damage since latently-infected flies live almost as long as noninfected control flies. The apparently dormant bacteria display particular characteristics in terms of bacterial colony morphology, composition of the outer cell wall, and motility. The virulence of these bacteria can however be reactivated upon wounding the host. We show that melanization but not the cellular or the systemic humoral response is the predominant host defense that establishes latency and may coerce the bacteria to a dormant state. In addition, the lasting activation of the melanization responses in latently-infected flies provides a degree of protection to the host against a secondary fungal infection. Latent infection by an ingested pathogen protects against a variety of homologous or heterologous systemic secondary infectious challenges, a situation previously described for the endosymbiotic Wolbachia bacteria, a guard against viral infections.
Assuntos
Drosophila melanogaster , Imunidade Inata , Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Drosophila melanogaster/microbiologia , Drosophila melanogaster/imunologia , Pseudomonas aeruginosa/patogenicidade , Pseudomonas aeruginosa/imunologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Virulência , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno/imunologiaRESUMO
Obesity is associated with increased morbidity and mortality during bacterial pneumonia. Cyclooxygenase-2 (COX-2) and PGE2 have been shown to be upregulated in patients who are obese. In this study, we investigated the role of obesity and PGE2 in bacterial pneumonia and how inhibition of PGE2 improves antibacterial functions of macrophages. C57BL/6J male and female mice were fed either a normal diet (ND) or high-fat diet (HFD) for 16 wk. After this time, animals were infected with Pseudomonas aeruginosa in the lung. In uninfected animals, alveolar macrophages were extracted for either RNA analysis or to be cultured ex vivo for functional analysis. HFD resulted in changes in immune cell numbers in both noninfected and infected animals. HFD animals had increased bacterial burden compared with ND animals; however, male HFD animals had higher bacterial burden compared with HFD females. Alveolar macrophages from HFD males had decreased ability to phagocytize and kill bacteria and were shown to have increased cyclooxygenase-2 and PGE2. Treating male, but not female, alveolar macrophages with PGE2 leads to increases in cAMP and decreased bacterial phagocytosis. Treatment with lumiracoxib-conjugated nanocarriers targeting alveolar macrophages improves bacterial phagocytosis and clearance in both ND and HFD male animals. Our study highlights that obesity leads to worse morbidity during bacterial pneumonia in male mice because of elevated PGE2. In addition, we uncover a sex difference in both obesity and infection, because females produce high basal PGE2 but because of a failure to signal via cAMP do not display impaired phagocytosis.
Assuntos
Dinoprostona , Macrófagos Alveolares , Camundongos Endogâmicos C57BL , Obesidade , Pneumonia Bacteriana , Infecções por Pseudomonas , Pseudomonas aeruginosa , Regulação para Cima , Animais , Feminino , Masculino , Macrófagos Alveolares/imunologia , Camundongos , Dinoprostona/metabolismo , Pseudomonas aeruginosa/imunologia , Obesidade/imunologia , Infecções por Pseudomonas/imunologia , Pneumonia Bacteriana/imunologia , Regulação para Cima/imunologia , Dieta Hiperlipídica/efeitos adversos , Ciclo-Oxigenase 2/metabolismo , Fagocitose/imunologia , Fatores SexuaisRESUMO
Rationale: Pseudomonas aeruginosa infection is associated with worse outcomes in bronchiectasis. Impaired neutrophil antimicrobial responses contribute to bacterial persistence. Gremubamab is a bivalent, bispecific monoclonal antibody targeting Psl exopolysaccharide and the type 3 secretion system component PcrV. Objectives: This study evaluated the efficacy of gremubamab to enhance killing of P. aeruginosa by neutrophils from patients with bronchiectasis and to prevent P. aeruginosa-associated cytotoxicity. Methods: P. aeruginosa isolates from a global bronchiectasis cohort (n = 100) underwent whole-genome sequencing to determine target prevalence. Functional activity of gremubamab against selected isolates was tested in vitro and in vivo. Patients with bronchiectasis (n = 11) and control subjects (n = 10) were enrolled, and the effect of gremubamab in peripheral blood neutrophil opsonophagocytic killing (OPK) assays against P. aeruginosa was evaluated. Serum antibody titers to Psl and PcrV were determined (n = 30; 19 chronic P. aeruginosa infection, 11 no known P. aeruginosa infection), as was the effect of gremubamab treatment in OPK and anti-cytotoxic activity assays. Measurements and Main Results: Psl and PcrV were conserved in isolates from chronically infected patients with bronchiectasis. Seventy-three of 100 isolates had a full psl locus, and 99 of 100 contained the pcrV gene, with 20 distinct full-length PcrV protein subtypes identified. PcrV subtypes were successfully bound by gremubamab and the monoclonal antibody-mediated potent protective activity against tested isolates. Gremubamab increased bronchiectasis patient neutrophil-mediated OPK (+34.6 ± 8.1%) and phagocytosis (+70.0 ± 48.8%), similar to effects observed in neutrophils from control subjects (OPK, +30.1 ± 7.6%). No evidence of competition between gremubamab and endogenous antibodies was found, with protection against P. aeruginosa-induced cytotoxicity and enhanced OPK demonstrated with and without addition of patient serum. Conclusions: Gremubamab enhanced bronchiectasis patient neutrophil phagocytosis and killing of P. aeruginosa and reduced virulence.
Assuntos
Anticorpos Biespecíficos , Bronquiectasia , Neutrófilos , Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Bronquiectasia/imunologia , Bronquiectasia/microbiologia , Pseudomonas aeruginosa/imunologia , Neutrófilos/imunologia , Neutrófilos/efeitos dos fármacos , Anticorpos Biespecíficos/uso terapêutico , Anticorpos Biespecíficos/farmacologia , Feminino , Masculino , Infecções por Pseudomonas/imunologia , Pessoa de Meia-Idade , Idoso , Adulto , Antígenos de Bactérias , Toxinas Bacterianas , Proteínas Citotóxicas Formadoras de PorosRESUMO
BACKGROUND: Pseudomonas aeruginosa is a frequent pathogen isolated from bacterial bloodstream infection (BSI) and is associated with high mortality. To survive in the blood, P aeruginosa must resist the bactericidal action of complement (ie, serum killing). Antibodies usually promote serum killing through the classical complement pathway; however, "cloaking antibodies" (cAbs) have been described, which paradoxically protect bacteria from serum killing. The relevance of cAbs in P aeruginosa BSI is unknown. METHODS: Serum and P aeruginosa were collected from a cohort of 100 patients with BSI. Isolates were tested for sensitivity to healthy control serum (HCS). cAb prevalence was determined in sera. Patient sera were mixed with HCS to determine if killing of the matched isolate was inhibited. RESULTS: Overall, 36 patients had elevated titers of cAbs, and 34 isolates were sensitive to HCS killing. Fifteen patients had cAbs and HCS-sensitive isolates; of these patients, 14 had serum that protected their matched bacteria from HCS killing. Patients with cAbs were less likely to be neutropenic or have comorbidities. CONCLUSIONS: cAbs are prevalent in patients with P aeruginosa BSI and allow survival of otherwise serum-sensitive bacteria in the bloodstream. Generation of cAbs may be a risk factor for the development of BSI.
Assuntos
Anticorpos Antibacterianos , Bacteriemia , Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/imunologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/epidemiologia , Masculino , Feminino , Pessoa de Meia-Idade , Bacteriemia/microbiologia , Bacteriemia/imunologia , Idoso , Anticorpos Antibacterianos/sangue , Adulto , Idoso de 80 Anos ou mais , Estudos de CoortesRESUMO
Pseudomonas aeruginosa causes chronic lung infection in cystic fibrosis (CF), resulting in structural lung damage and progressive pulmonary decline. P. aeruginosa in the CF lung undergoes numerous changes, adapting to host-specific airway pressures while establishing chronic infection. P. aeruginosa undergoes lipid A structural modification during CF chronic infection that is not seen in any other disease state. Lipid A, the membrane anchor of LPS (i.e., endotoxin), comprises the majority of the outer membrane of Gram-negative bacteria and is a potent Toll-like receptor 4 (TLR4) agonist. The structure of P. aeruginosa lipid A is intimately linked with its recognition by TLR4 and subsequent immune response. Prior work has identified P. aeruginosa strains with altered lipid A structures that arise during chronic CF lung infection; however, the impact of the P. aeruginosa lipid A structure on airway disease has not been investigated. Here, we show that P. aeruginosa lipid A lacks PagL-mediated deacylation during human airway infection using a direct-from-sample mass spectrometry approach on human BAL fluid. This structure triggers increased proinflammatory cytokine production by primary human macrophages. Furthermore, alterations in lipid A 2-hydroxylation impact cytokine response in a site-specific manner, independent of CF transmembrane conductance regulator function. It is interesting that there is a CF-specific reduction in IL-8 secretion within the epithelial-cell compartment that only occurs in CF bronchial epithelial cells when infected with CF-adapted P. aeruginosa that lacks PagL-mediated lipid A deacylation. Taken together, we show that P. aeruginosa alters its lipid A structure during acute lung infection and that this lipid A structure induces stronger signaling through TLR4.
Assuntos
Fibrose Cística , Lipídeo A , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/imunologia , Humanos , Lipídeo A/metabolismo , Lipídeo A/imunologia , Fibrose Cística/microbiologia , Fibrose Cística/imunologia , Fibrose Cística/metabolismo , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor 4 Toll-Like/imunologia , Citocinas/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Líquido da Lavagem Broncoalveolar/imunologia , Pulmão/microbiologia , Pulmão/imunologia , Pulmão/metabolismoRESUMO
Pseudomonas aeruginosa poses a significant threat to immunocompromised individuals and those with cystic fibrosis. Treatment relies on antibiotics, but persistent infections occur due to intrinsic and acquired resistance of P. aeruginosa towards multiple classes of antibiotics. To date, there are no licensed vaccines for this pathogen, prompting the urgent need for novel treatment approaches to combat P. aeruginosa infection and persistence. Here we validated AAV vectored immunoprophylaxis as a strategy to generate long-term plasma and mucosal expression of highly protective monoclonal antibodies (mAbs) targeting the exopolysaccharide Psl (Cam-003) and the PcrV (V2L2MD) component of the type-III secretion system injectosome either as single mAbs or together as a bispecific mAb (MEDI3902) in a mouse model. When administered intramuscularly, AAV-αPcrV, AAV-αPsl, and AAV-MEDI3902 significantly protected mice challenged intranasally with a lethal dose of P. aeruginosa strains PAO1 and PA14 and reduced bacterial burden and dissemination to other organs. While all AAV-mAbs provided protection, AAV-αPcrV and AAV-MEDI3902 provided 100% and 87.5% protection from a lethal challenge with 4.47 × 107 CFU PAO1 and 87.5% and 75% protection from a lethal challenge with 3 × 107 CFU PA14, respectively. Serum concentrations of MEDI3902 were ~10× lower than that of αPcrV, but mice treated with this vector showed a greater reduction in bacterial dissemination to the liver, lung, spleen, and blood compared to other AAV-mAbs. These results support further investigation into the use of AAV vectored immunoprophylaxis to prevent and treat P. aeruginosa infections and other bacterial pathogens of public health concern for which current treatment strategies are limited.
Assuntos
Anticorpos Monoclonais , Dependovirus , Vetores Genéticos , Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Dependovirus/genética , Dependovirus/imunologia , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/genética , Camundongos , Vetores Genéticos/genética , Vetores Genéticos/administração & dosagem , Infecções por Pseudomonas/prevenção & controle , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/terapia , Anticorpos Monoclonais/imunologia , Anticorpos Biespecíficos , Feminino , Antígenos de Bactérias/imunologia , Antígenos de Bactérias/genética , Pneumonia Bacteriana/prevenção & controle , Pneumonia Bacteriana/terapia , Pneumonia Bacteriana/imunologia , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Humanos , Toxinas Bacterianas , Proteínas Citotóxicas Formadoras de PorosRESUMO
Excessive or persistent inflammation may have detrimental effects on lung structure and function. Currently, our understanding of conserved host mechanisms that control the inflammatory response remains incompletely understood. In this study, we investigated the role of type I interferon signaling in the inflammatory response against diverse clinically relevant stimuli. Using mice deficient in type I interferon signaling (IFNAR1-/-), we demonstrate that the absence of interferon signaling resulted in a robust and persistent inflammatory response against Pseudomonas aeruginosa, lipopolysaccharide, and chemotherapeutic agent bleomycin. The elevated inflammatory response in IFNAR1-/- mice was manifested as elevated myeloid cells, such as macrophages and neutrophils, in the bronchoalveolar lavage. The inflammatory cell response in the IFNAR1-/- mice persisted to 14 days and there is impaired recovery and fibrotic remodeling of the lung in IFNAR1-/- mice after bleomycin injury. In the Pseudomonas infection model, the elevated inflammatory cell response led to improved bacterial clearance in IFNAR1-/- mice, although there was similar lung injury and survival. We performed RNA sequencing of lung tissue in wild-type and IFNAR1-/- mice after LPS and bleomycin injury. Our unbiased analysis identified differentially expressed genes between IFNAR1-/- and wild-type mice, including previously unknown regulation of nucleotide-binding oligomerization domain (NOD)-like receptor signaling, retinoic acid-inducible gene-I (RIG-I) signaling, and necroptosis pathway by type I interferon signaling in both models. These data provide novel insights into the conserved anti-inflammatory mechanisms of the type I interferon signaling.NEW & NOTEWORTHY Type I interferons are known for their antiviral activities. In this study, we demonstrate a conserved anti-inflammatory role of type I interferon signaling against diverse stimuli in the lung. We show that exacerbated inflammatory response in the absence of type I interferon signaling has both acute and chronic consequences in the lung including structural changes.
Assuntos
Interferon Tipo I , Pulmão , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta , Transdução de Sinais , Animais , Interferon Tipo I/metabolismo , Pulmão/metabolismo , Pulmão/imunologia , Pulmão/patologia , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/metabolismo , Camundongos , Bleomicina , Pseudomonas aeruginosa , Lipopolissacarídeos/farmacologia , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/metabolismo , Infecções por Pseudomonas/patologia , Infecções por Pseudomonas/microbiologia , Inflamação/metabolismo , Inflamação/patologia , Inflamação/imunologia , MasculinoRESUMO
BACKGROUND: Donor-specific antibodies (DSAs) are common following lung transplantation (LuTx), yet their role in graft damage is inconclusive. Mean fluorescent intensity (MFI) is the main read-out of DSA diagnostics; however its value is often disregarded when analyzing unwanted post-transplant outcomes such as graft loss or chronic lung allograft dysfunction (CLAD). Here we aim to evaluate an MFI stratification method in these outcomes. METHODS: A cohort of 87 LuTx recipients has been analyzed, in which a cutoff of 8000 MFI has been determined for high MFI based on clinically relevant data. Accordingly, recipients were divided into DSA-negative, DSA-low and DSA-high subgroups. Both graft survival and CLAD-free survival were evaluated. Among factors that may contribute to DSA development we analyzed Pseudomonas aeruginosa (P. aeruginosa) infection in bronchoalveolar lavage (BAL) specimens. RESULTS: High MFI DSAs contributed to clinical antibody-mediated rejection (AMR) and were associated with significantly worse graft (HR: 5.77, p < 0.0001) and CLAD-free survival (HR: 6.47, p = 0.019) compared to low or negative MFI DSA levels. Analysis of BAL specimens revealed a strong correlation between DSA status, P. aeruginosa infection and BAL neutrophilia. DSA-high status and clinical AMR were both independent prognosticators for decreased graft and CLAD-free survival in our multivariate Cox-regression models, whereas BAL neutrophilia was associated with worse graft survival. CONCLUSIONS: P. aeruginosa infection rates are elevated in recipients with a strong DSA response. Our results indicate that the simultaneous interpretation of MFI values and BAL neutrophilia is a feasible approach for risk evaluation and may help clinicians when to initiate DSA desensitization therapy, as early intervention could improve prognosis.
Assuntos
Rejeição de Enxerto , Transplante de Pulmão , Infecções por Pseudomonas , Pseudomonas aeruginosa , Transplante de Pulmão/efeitos adversos , Transplante de Pulmão/mortalidade , Humanos , Feminino , Masculino , Pessoa de Meia-Idade , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/mortalidade , Adulto , Pseudomonas aeruginosa/imunologia , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/diagnóstico , Doadores de Tecidos , Estudos Retrospectivos , Sobrevivência de Enxerto , Estudos de Coortes , Isoanticorpos/sangue , IdosoRESUMO
BACKGROUND: Pseudomonas aeruginosa (PA) is an opportunistic pathogen that can cause sight threatening infections in the eye and fatal infections in the cystic fibrosis airway. Extracellular vesicles (EVs) are released by host cells during infection and by the bacteria themselves; however, there are no studies on the composition and functional role of host-derived EVs during PA infection of the eye or lung. Here we investigated the composition and capacity of EVs released by PA infected epithelial cells to modulate innate immune responses in host cells. METHODS: Human telomerase immortalized corneal epithelial cells (hTCEpi) cells and human telomerase immortalized bronchial epithelial cells (HBECs) were treated with a standard invasive test strain of Pseudomonas aeruginosa, PAO1, for 6 h. Host derived EVs were isolated by qEV size exclusion chromatography. EV proteomic profiles during infection were compared using mass spectrometry and functional studies were carried out using hTCEpi cells, HBECs, differentiated neutrophil-like HL-60 cells, and primary human neutrophils isolated from peripheral blood. RESULTS: EVs released from PA infected corneal epithelial cells increased pro-inflammatory cytokine production in naïve corneal epithelial cells and induced neutrophil chemotaxis independent of cytokine production. The EVs released from PA infected bronchial epithelial cells were also chemotactic although they failed to induce cytokine secretion from naïve HBECs. At the proteomic level, EVs derived from PA infected corneal epithelial cells exhibited lower complexity compared to bronchial epithelial cells, with the latter having reduced protein expression compared to the non-infected control. CONCLUSIONS: This is the first study to comprehensively profile EVs released by corneal and bronchial epithelial cells during Pseudomonas infection. Together, these findings show that EVs released by PA infected corneal and bronchial epithelial cells function as potent mediators of neutrophil migration, contributing to the exuberant neutrophil response that occurs during infection in these tissues.
Assuntos
Células Epiteliais , Vesículas Extracelulares , Neutrófilos , Infecções por Pseudomonas , Pseudomonas aeruginosa , Humanos , Pseudomonas aeruginosa/fisiologia , Vesículas Extracelulares/metabolismo , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/metabolismo , Neutrófilos/metabolismo , Neutrófilos/imunologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Citocinas/metabolismo , Células HL-60RESUMO
OBJECTIVE P. AERUGINOSA: (PA), the major pathogen of lung cystic fibrosis (CF), polarizes macrophages into hyperinflammatory tissue damaging phenotype. The main aim of this study was to verify whether training of macrophages with ß-glucan might improve their response to P. aeruginosa infections. METHODS: To perform this task C57BL/6 mice sensitive to infections with P. aeruginosa were used. Peritoneal macrophages were trained with Saccharomyces cerevisiae ß-glucan and exposed to PA57, the strong biofilm-forming bacterial strain isolated from the patient with severe lung CF. The release of cytokines and the expression of macrophage phenotypic markers were measured. A quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages. The effect of in vivo ß-glucan-trained macrophages in the air pouch model of PA57 infection was investigated. In all experiments the effect of trained and naïve macrophages was compared. RESULTS: Trained macrophages acquired a specific phenotype with mixed pro-inflammatory and pro-resolution characteristics, however they retained anti-bacterial properties. Most importantly, transfer of trained macrophages into infected air pouches markedly ameliorated the course of infection. PA57 bacterial growth and formation of biofilm were significantly suppressed. The level of serum amyloid A (SAA), a systemic inflammation biomarker, was reduced. CONCLUSIONS: Training of murine macrophages with S. cerevisiae ß-glucan improved macrophage defense properties along with inhibition of secretion of some detrimental inflammatory agents. We suggest that training of macrophages with such ß-glucans might be a new therapeutic strategy in P. aeruginosa biofilm infections, including CF, to promote eradication of pathogens and resolution of inflammation.
Assuntos
Biofilmes , Citocinas , Camundongos Endogâmicos C57BL , Infecções por Pseudomonas , Pseudomonas aeruginosa , Saccharomyces cerevisiae , beta-Glucanas , Animais , beta-Glucanas/farmacologia , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/imunologia , Citocinas/metabolismo , Biofilmes/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/imunologia , Feminino , Camundongos , Proteína Amiloide A Sérica , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Células CultivadasRESUMO
This study marks the first utilization of reverse vaccinology to develop recombinant subunit vaccines against Pseudomonas koreensis infection in Empurau (Tor tambroides). The proteome (5538 proteins) was screened against various filters to prioritize proteins based on features that are associated with virulence, subcellular localization, transmembrane helical structure, antigenicity, essentiality, non-homology with the host proteome, molecular weight, and stability, which led to the identification of eight potential vaccine candidates. These potential vaccine candidates were cloned and expressed, with six achieving successful expression and purification. The antigens were formulated into two distinct vaccine mixtures, Vac A and Vac B, and their protective efficacy was assessed through in vivo challenge experiments. Vac A and Vac B demonstrated high protective efficacies of 100 % and 81.2 %, respectively. Histological analyses revealed reduced tissue damage in vaccinated fish after experimental infection, with Vac A showing no adverse effects, whereas Vac B exhibited mild degenerative changes. Quantitative real-time PCR results showed a significant upregulation of TNF-α and downregulation of IL-1ß in the kidneys, spleen, gills, and intestine in both Vac A- and Vac B-immunized fish after challenged with P. koreensis. Additionally, IL-8 exhibits tissue-specific differential expression, with significant upregulation in the kidney, gills, and intestine, and downregulation in the spleen, particularly notable in Vac A-immunized fish. The research underscores the effectiveness of the reverse vaccinology approach in fish and demonstrates the promising potential of Vac A and Vac B as recombinant subunit vaccines.
Assuntos
Doenças dos Peixes , Infecções por Pseudomonas , Pseudomonas , Animais , Doenças dos Peixes/imunologia , Doenças dos Peixes/prevenção & controle , Pseudomonas/imunologia , Infecções por Pseudomonas/veterinária , Infecções por Pseudomonas/prevenção & controle , Infecções por Pseudomonas/imunologia , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas Bacterianas/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinologia , Vacinas Sintéticas/imunologia , Cyprinidae/imunologia , Vacinas contra Pseudomonas/imunologia , Proteoma/imunologiaRESUMO
Pseudomonas plecoglossicida is a vital pathogen that poses a substantial risk to aquaculture. Small RNAs (sRNAs) are non-coding regulatory molecules capable of sensing environmental changes and modulating virulence-associated signaling pathways, such as the assembly of flagella. However, the relevant researches on P. plecoglossicida are an urgent need. Here, we report a novel sRNA, sRNA562, which has potential to regulate the post-transcriptional of fliP, a key component of the lateral flagellar type III secretion system. In this study, the effects of sRNA562 on the virulence of P. plecoglossicida and its role in regulating the pathogenic process were investigated through the use of a constructed sRNA562 deletion strain. The deletion of sRNA562 resulted in an up-regulation of fliP in P. plecoglossicida, and leading to increased swarming motility and enhanced the ability of biofilm formation, adhesion and chemotaxis. Subsequent artificial infection experiment demonstrated that the deletion of sRNA562 increased the virulence of P. plecoglossicida towards hybrid grouper, as evidenced by a reduction in survival rate, elevation of tissue bacterial load, and the exacerbation of histopathological damage. Further studies have found that the deletion of sRNA562 lead to an up-regulation of fliP expression during hybrid grouper infection, thereby enhancing bacterial swarming ability and ultimately heightening pathogenicity, leading to a dysregulated host response to infection, tissue damage and eventually death. Our work revealed a sRNA that exerts negative regulation on the expression of lateral flagella in P. plecoglossicida, thereby impacting its virulence. These findings provide a new perspective on the virulence regulation mechanism of P. plecoglossicida, contributing to a more comprehensive understanding in the field of pathogenicity research.
Assuntos
Doenças dos Peixes , Flagelos , Regulação Bacteriana da Expressão Gênica , Pseudomonas , Pequeno RNA não Traduzido , Pseudomonas/patogenicidade , Pseudomonas/genética , Pseudomonas/fisiologia , Virulência/genética , Animais , Doenças dos Peixes/microbiologia , Pequeno RNA não Traduzido/genética , Flagelos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Bacteriano/genética , Sistemas de Secreção Tipo III/genética , Bass , Infecções por Pseudomonas/imunologiaRESUMO
Pseudomonas plecoglossicida, the causative agent of Visceral White Spot Disease, poses substantial risks to large yellow croaker (Larimichthys crocea) aquaculture. Previous genome-wide association studies (GWAS), directed towards elucidating the resistance mechanisms of large yellow croaker against this affliction, suggested that the transmembrane protein 208 (named Lctmem208) may confer a potential advantage. TMEM proteins, particularly TMEM208 located in the endoplasmic reticulum, plays significant roles in autophagy, ER stress, and dynamics of cancer cell. However, research on TMEM's function in teleost fish immunity remains sparse, highlighting a need for further study. This study embarks on a comprehensive examination of LcTmem208, encompassing cloning, molecular characterization, and its dynamics in immune function in response to Pseudomonas plecoglossicida infection. Our findings reveal that LcTmem208 is highly conserved across teleost species, exhibiting pronounced expression in immune-relevant tissues, which escalates significantly upon pathogenic challenge. Transcriptome analysis subsequent to LcTmem208 overexpression in kidney cells unveiled its pivotal role in modulating immune-responsive processes, notably the p53 signaling pathway and cytokine-mediated interactions. Enhanced phagocytic activity in macrophages overexpressing LcTmem208 underscores its importance in innate immunity. Taken together, this is the first time reported the critical involvement of LcTmem208 in regulating innate immune responses of defensing P. plecoglossicida, thereby offering valuable insights into teleost fish immunity and potential strategies for the selective breeding of disease-resistant strains of large yellow croaker in aquaculture practices.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Perfilação da Expressão Gênica , Imunidade Inata , Perciformes , Infecções por Pseudomonas , Pseudomonas , Animais , Doenças dos Peixes/imunologia , Perciformes/imunologia , Perciformes/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Pseudomonas/fisiologia , Imunidade Inata/genética , Perfilação da Expressão Gênica/veterinária , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/veterinária , Regulação da Expressão Gênica/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Transcriptoma , Filogenia , Alinhamento de Sequência/veterinária , Clonagem MolecularRESUMO
The COMMD (Copper Metabolism gene MURR1 Domain) gene family consists of 10 members, which are involved in various biological processes such as copper and sodium transport, NF-κB activity and cell cycle progression. However, the study of COMMD gene family in large yellow croaker (Larimichthys crocea) is largely unknown. In this study, 10 COMMD gene family members (named LcCOMMDs) were successfully identified from large yellow croaker. The results showed that there were differences in the number of LcCOMMDs exons at the level of gene structure, which reflected that they had adjusted and changed accordingly in the process of evolution to adapt to the environment and achieved functional diversification. Through phylogenetic analysis, we found that the LcCOMMDs was highly conserved, indicating their important functions in organisms. It was worth noting that the expression levels of LcCOMMD1, LcCOMMD2, LcCOMMD3, LcCOMMD5 and LcCOMMD10 in the spleen changed significantly after bacterial stress, which suggested that these genes might be involved in the regulation of innate immune response. In addition, the expression levels of LcCOMMD1, LcCOMMD2, LcCOMMD3, LcCOMMD5, LcCOMMD7, LcCOMMD8, LcCOMMD9 and LcCOMMD10 changed significantly after hypoxia exposure, which further proved the role of LcCOMMDs in immune function. In summary, this study not only revealed the important role of COMMD genes in the innate immune response of large yellow croaker, but also provided valuable information for further understanding the regulatory mechanism of COMMD gene family under different conditions.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Imunidade Inata , Perciformes , Filogenia , Infecções por Pseudomonas , Pseudomonas , Animais , Perciformes/imunologia , Perciformes/genética , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , Doenças dos Peixes/imunologia , Imunidade Inata/genética , Pseudomonas/fisiologia , Infecções por Pseudomonas/veterinária , Infecções por Pseudomonas/imunologia , Regulação da Expressão Gênica/imunologia , Perfilação da Expressão Gênica/veterinária , Alinhamento de Sequência/veterinária , Estresse Fisiológico/imunologia , Sequência de Aminoácidos , Hipóxia/imunologia , Hipóxia/veterinária , Hipóxia/genética , Família MultigênicaRESUMO
The suppressor of cytokine signaling (SOCS) gene family is a group of genes involved in the negative regulation of cytokine signal transduction. The members of this family play a crucial role in regulating immune and inflammatory processes. However, comprehensive investigations of these genes have not yet been conducted in the economically significant fish large yellow croaker (Larimichthys crocea). In this study, a total of 13 SOCS genes (LcSOCS1a, LcSOCS1b, LcSOCS2, LcSOCS3a, LcSOCS3b, LcSOCS4, LcSOCS5a, LcSOCS5b, LcSOCS6, LcSOCS7a, LcSOCS7b, LcCISHa and LcCISHb) were identified and analyzed in L. crocea. The phylogenetic tree revealed a high conservation of SOCS genes in evolution, and the gene structure and motif analysis indicated a high similarity in the structure of LcSOCSs in the same subfamily. In addition, the expression patterns of LcSOCSs showed that LcSOCS1b was significantly down-regulated in all time under acute hypoxia stress, but it was markedly up-regulated throughout the entire process after P. plecoglossicida infection, revealing its different immune effects to two stresses. Besides, LcSOCS2a, LcSOCS6 and LcSOCS7a only participated in acute hypoxic stress, while LcSOCS5a was more sensitive to P. plecoglossicida infection. In summary, these results indicated that SOCS genes were involved in stress responses to both biological and non-biological stimuli, setting the foundation for deeper study on the functions of SOCS genes.
Assuntos
Doenças dos Peixes , Proteínas de Peixes , Regulação da Expressão Gênica , Imunidade Inata , Perciformes , Filogenia , Infecções por Pseudomonas , Pseudomonas , Proteínas Supressoras da Sinalização de Citocina , Animais , Perciformes/imunologia , Perciformes/genética , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Proteínas de Peixes/química , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/imunologia , Proteínas Supressoras da Sinalização de Citocina/química , Imunidade Inata/genética , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/veterinária , Infecções por Pseudomonas/genética , Pseudomonas/fisiologia , Regulação da Expressão Gênica/imunologia , Perfilação da Expressão Gênica/veterinária , Estresse Fisiológico/imunologia , Estresse Fisiológico/genética , Alinhamento de Sequência/veterinária , Hipóxia/genética , Hipóxia/imunologia , Hipóxia/veterináriaRESUMO
Infections, such as that by the multiresistant opportunistic bacterial pathogen Pseudomonas aeruginosa, may pose a serious health risk, especially on vulnerable patient populations. The nematode Caenorhabditis elegans provides a simple organismal model to investigate both pathogenic mechanisms and the emerging role of innate immunity in host protection. Here, we review the virulence and infection strategies of P. aeruginosa and host defenses of C. elegans. We summarize the recognition mechanisms of patterns of pathogenesis, including novel pathogen-associated molecular patterns and surveillance immunity of translation, mitochondria, and lysosome-related organelles. We also review the regulation of antimicrobial and behavioral defenses by the worm's neuroendocrine system. We focus on how discoveries in this rich field align with well-characterized evolutionary conserved protective pathways, as well as on potential crossovers to human pathogenesis and innate immune responses.
Assuntos
Caenorhabditis elegans , Interações Hospedeiro-Patógeno , Imunidade Inata , Infecções por Pseudomonas , Pseudomonas aeruginosa , Animais , Caenorhabditis elegans/microbiologia , Caenorhabditis elegans/imunologia , Pseudomonas aeruginosa/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Infecções por Pseudomonas/microbiologia , Infecções por Pseudomonas/imunologia , Humanos , Modelos Animais de Doenças , VirulênciaAssuntos
Antibacterianos , Anticorpos Monoclonais , Bronquiectasia , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/efeitos dos fármacos , Bronquiectasia/tratamento farmacológico , Bronquiectasia/imunologia , Humanos , Infecções por Pseudomonas/tratamento farmacológico , Infecções por Pseudomonas/imunologia , Anticorpos Monoclonais/uso terapêutico , Antibacterianos/uso terapêutico , VirulênciaRESUMO
Sepsis trains stressed granulocytes to boost nonspecific response and trigger a new wave of inflammation when facing secondary infection. Here, we present a protocol for a murine model of sepsis with secondary infection. We describe steps for cecal ligation and puncture operation and rechallenging with lipopolysaccharide or Pseudomonas aeruginosa during the recovery phase. We also detail steps to characterize the stressed granulocytes by assessing their functional phenotypes and effect on the mortality of rechallenged mice. For complete details on the use and execution of this protocol, please refer to Wang et al.1.