RESUMO
Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that is an important cause of acute exacerbations of chronic obstructive pulmonary disease (AECOPD). COPD is an inflammatory disease of the airways, and exacerbations are acute inflammatory events superimposed on this background of chronic inflammation. Azithromycin (AZM) is a macrolide antibiotic with antibacterial and anti-inflammatory properties and a clinically proven potential for AECOPD prevention and management. Relationships between AZM efficacy and resistance by NTHI and between bactericidal and immunomodulatory effects on NTHI respiratory infection have not been addressed. In this study, we employed two pathogenic NTHI strains with different AZM susceptibilities (NTHI 375 [AZM susceptible] and NTHI 353 [AZM resistant]) to evaluate the prophylactic and therapeutic effects of AZM on the NTHI-host interplay. At the cellular level, AZM was bactericidal toward intracellular NTHI inside alveolar and bronchial epithelia and alveolar macrophages, and it enhanced NTHI phagocytosis by the latter cell type. These effects correlated with the strain MIC of AZM and the antibiotic dose. Additionally, the effect of AZM on NTHI infection was assessed in a mouse model of pulmonary infection. AZM showed both preventive and therapeutic efficacies by lowering NTHI 375 bacterial counts in lungs and bronchoalveolar lavage fluid (BALF) and by reducing histopathological inflammatory lesions in the upper and lower airways of mice. Conversely, AZM did not reduce bacterial loads in animals infected with NTHI 353, in which case a milder anti-inflammatory effect was also observed. Together, the results of this work link the bactericidal and anti-inflammatory effects of AZM and frame the efficacy of this antibiotic against NTHI respiratory infection.
Assuntos
Azitromicina/uso terapêutico , Infecções por Haemophilus/tratamento farmacológico , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/patogenicidade , Infecções Respiratórias/tratamento farmacológico , Animais , Linhagem Celular , Células Epiteliais/virologia , Feminino , Humanos , Macrófagos Alveolares/virologia , CamundongosRESUMO
Therapies that are safe, effective, and not vulnerable to developing resistance are highly desirable to counteract bacterial infections. Host-directed therapeutics is an antimicrobial approach alternative to conventional antibiotics based on perturbing host pathways subverted by pathogens during their life cycle by using host-directed drugs. In this study, we identified and evaluated the efficacy of a panel of host-directed drugs against respiratory infection by nontypeable Haemophilus influenzae (NTHi). NTHi is an opportunistic pathogen that is an important cause of exacerbation of chronic obstructive pulmonary disease (COPD). We screened for host genes differentially expressed upon infection by the clinical isolate NTHi375 by analyzing cell whole-genome expression profiling and identified a repertoire of host target candidates that were pharmacologically modulated. Based on the proposed relationship between NTHi intracellular location and persistence, we hypothesized that drugs perturbing host pathways used by NTHi to enter epithelial cells could have antimicrobial potential against NTHi infection. Interfering drugs were tested for their effects on bacterial and cellular viability, on NTHi-epithelial cell interplay, and on mouse pulmonary infection. Glucocorticoids and statins lacked in vitro and/or in vivo efficacy. Conversely, the sirtuin-1 activator resveratrol showed a bactericidal effect against NTHi, and the PDE4 inhibitor rolipram showed therapeutic efficacy by lowering NTHi375 counts intracellularly and in the lungs of infected mice. PDE4 inhibition is currently prescribed in COPD, and resveratrol is an attractive geroprotector for COPD treatment. Together, these results expand our knowledge of NTHi-triggered host subversion and frame the antimicrobial potential of rolipram and resveratrol against NTHi respiratory infection.
Assuntos
Antibacterianos/farmacologia , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Infecções por Haemophilus/tratamento farmacológico , Inibidores da Fosfodiesterase 4/farmacologia , Rolipram/farmacologia , Sirtuína 1/genética , Estilbenos/farmacologia , Animais , Linhagem Celular Tumoral , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Quimioterapia Combinada , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genoma Humano , Infecções por Haemophilus/genética , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/fisiologia , Interações Hospedeiro-Patógeno , Humanos , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/microbiologia , Mucosa Respiratória/patologia , Resveratrol , Transdução de Sinais , Sirtuína 1/metabolismoRESUMO
Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.
Assuntos
Endotoxinas/metabolismo , Haemophilus influenzae/patogenicidade , Lipopolissacarídeos/metabolismo , Fatores de Virulência/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/metabolismo , Biofilmes/crescimento & desenvolvimento , Broncopneumonia/microbiologia , Broncopneumonia/patologia , Adesão Celular , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Feminino , Infecções por Haemophilus/microbiologia , Infecções por Haemophilus/patologia , Haemophilus influenzae/efeitos dos fármacos , Haemophilus influenzae/fisiologia , Humanos , Redes e Vias Metabólicas/genética , Camundongos , Mutação , VirulênciaRESUMO
Non-typable Haemophilus influenzae (NTHi) is a common commensal of the human nasopharynx, but causes opportunistic infection when the respiratory tract is compromised by infection or disease. The ability of NTHi to invade epithelial cells has been described, but the underlying molecular mechanisms are poorly characterized. We previously determined that NTHi promotes phosphorylation of the serine-threonine kinase Akt in A549 human lung epithelial cells, and that Akt phosphorylation and NTHi cell invasion are prevented by inhibition of phosphoinositide 3-kinase (PI3K). Because PI3K-Akt signalling is associated with several host cell networks, the purpose of the current study was to identify eukaryotic molecules important for NTHi epithelial invasion. We found that inhibition of Akt activity reduced NTHi internalization; differently, bacterial entry was increased by phospholipase Cγ1 inhibition but was not affected by protein kinase inhibition. We also found that α5 and ß1 integrins, and the tyrosine kinases focal adhesion kinase and Src, are important for NTHi A549 cell invasion. NTHi internalization was shown to be favoured by activation of Rac1 guanosine triphosphatase (GTPase), together with the guanine nucleotide exchange factor Vav2 and the effector Pak1. Also, Pak1 might be associated with inactivation of the microtubule destabilizing agent Op18/stathmin, to facilitate microtubule polymerization and NTHi entry. Conversely, inhibition of RhoA GTPase and its effector ROCK increased the number of internalized bacteria. Src and Rac1 were found to be important for NTHi-triggered Akt phosphorylation. An increase in host cyclic AMP reduced bacterial entry, which was linked to protein kinase A. These findings suggest that NTHi finely manipulates host signalling molecules to invade respiratory epithelial cells.
Assuntos
Endocitose , Células Epiteliais/microbiologia , Haemophilus influenzae/patogenicidade , Integrinas/metabolismo , Microtúbulos/metabolismo , Fosfotransferases/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Linhagem Celular , HumanosRESUMO
Yersinia enterocolitica (Ye) is a gram-negative bacterium; Ye serotype O:3 expresses lipopolysaccharide (LPS) with a hexasaccharide branch known as the outer core (OC). The OC is important for the resistance of the bacterium to cationic antimicrobial peptides and also functions as a receptor for bacteriophage phiR1-37 and enterocoliticin. The biosynthesis of the OC hexasaccharide is directed by the OC gene cluster that contains nine genes (wzx, wbcKLMNOPQ, and gne). In this study, we inactivated the six OC genes predicted to encode glycosyltransferases (GTase) one by one by nonpolar mutations to assign functions to their gene products. The mutants expressed no OC or truncated OC oligosaccharides of different lengths. The truncated OC oligosaccharides revealed that the minimum structural requirements for the interactions of OC with bacteriophage phiR1-37, enterocoliticin, and OC-specific monoclonal antibody 2B5 were different. Furthermore, using chemical and structural analyses of the mutant LPSs, we could assign specific functions to all six GTases and also revealed the exact order in which the transferases build the hexasaccharide. Comparative modeling of the catalytic sites of glucosyltransferases WbcK and WbcL followed by site-directed mutagenesis allowed us to identify Asp-182 and Glu-181, respectively, as catalytic base residues of these two GTases. In general, conclusive evidence for specific GTase functions have been rare due to difficulties in accessibility of the appropriate donors and acceptors; however, in this work we were able to utilize the structural analysis of LPS to get direct experimental evidence for five different GTase specificities.
Assuntos
Glicosiltransferases/metabolismo , Lipopolissacarídeos/biossíntese , Lipopolissacarídeos/química , Yersinia enterocolitica/enzimologia , Anticorpos Monoclonais/metabolismo , Bacteriófagos/metabolismo , Domínio Catalítico , Biologia Computacional , Farmacorresistência Bacteriana , Galactose/química , Galactose/metabolismo , Glicosiltransferases/química , Glicosiltransferases/genética , Modelos Moleculares , Família Multigênica , Mutagênese Sítio-Dirigida , Antígenos O/química , Antígenos O/metabolismo , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Polimixina B/farmacologia , Yersinia enterocolitica/efeitos dos fármacos , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismoRESUMO
Colistin resistance is rare in Acinetobacter baumannii, and little is known about its mechanism. We investigated the role of PmrCAB in this trait, using (i) resistant and susceptible clinical strains, (ii) laboratory-selected mutants of the type strain ATCC 19606 and of the clinical isolate ABRIM, and (iii) a susceptible/resistant pair of isogenic clinical isolates, Ab15/133 and Ab15/132, isolated from the same patient. pmrAB sequences in all the colistin-susceptible isolates were identical to reference sequences, whereas resistant clinical isolates harbored one or two amino acid replacements variously located in PmrB. Single substitutions in PmrB were also found in resistant mutants of strains ATCC 19606 and ABRIM and in the resistant clinical isolate Ab15/132. No mutations in PmrA or PmrC were found. Reverse transcriptase (RT)-PCR identified increased expression of pmrA (4- to 13-fold), pmrB (2- to 7-fold), and pmrC (1- to 3-fold) in resistant versus susceptible organisms. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry showed the addition of phosphoethanolamine to the hepta-acylated form of lipid A in the resistant variants and in strain ATCC 19606 grown under low-Mg(2+) induction conditions. pmrB gene knockout mutants of the colistin-resistant ATCC 19606 derivative showed >100-fold increased susceptibility to colistin and 5-fold decreased expression of pmrC; they also lacked the addition of phosphoethanolamine to lipid A. We conclude that the development of a moderate level of colistin resistance in A. baumannii requires distinct genetic events, including (i) at least one point mutation in pmrB, (ii) upregulation of pmrAB, and (iii) expression of pmrC, which lead to addition of phosphoethanolamine to lipid A.
Assuntos
Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Colistina/farmacologia , Etanolaminas/metabolismo , Lipídeo A/metabolismo , Acinetobacter baumannii/genética , Proteínas de Bactérias/genética , Farmacorresistência Bacteriana/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Nontypable Haemophilus influenzae (NTHi) is a Gram-negative, non-capsulated human bacterial pathogen, a major cause of a repertoire of respiratory infections, and intimately associated with persistent lung bacterial colonization in patients suffering from chronic obstructive pulmonary disease (COPD). Despite its medical relevance, relatively little is known about its mechanisms of pathogenicity. In this study, we found that NTHi invades the airway epithelium by a distinct mechanism, requiring microtubule assembly, lipid rafts integrity, and activation of phosphatidylinositol 3-kinase (PI3K) signalling. We found that the majority of intracellular bacteria are located inside an acidic subcellular compartment, in a metabolically active and non-proliferative state. This NTHi-containing vacuole (NTHi-CV) is endowed with late endosome features, co-localizing with LysoTracker, lamp-1, lamp-2, CD63 and Rab7. The NTHi-CV does not acquire Golgi- or autophagy-related markers. These observations were extended to immortalized and primary human airway epithelial cells. By using NTHi clinical isolates expressing different amounts of phosphocholine (PCho), a major modification of NTHi lipooligosaccharide, on their surfaces, and an isogenic lic1BC mutant strain lacking PCho, we showed that PCho is not responsible for NTHi intracellular location. In sum, this study indicates that NTHi can survive inside airway epithelial cells.
Assuntos
Células Epiteliais/microbiologia , Haemophilus influenzae/patogenicidade , Viabilidade Microbiana , Técnicas de Tipagem Bacteriana , Endocitose , Endossomos/química , Endossomos/microbiologia , Haemophilus influenzae/classificação , Haemophilus influenzae/metabolismo , Haemophilus influenzae/fisiologia , Humanos , Microdomínios da Membrana/metabolismo , Microtúbulos/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Transdução de Sinais , VirulênciaRESUMO
Respiratory infections caused by Klebsiella pneumoniae are characterized by high rates of mortality and morbidity. Management of these infections is often difficult, due to the high frequency of strains that are resistant to multiple antimicrobial agents. Multidrug efflux pumps play a major role as a mechanism of antimicrobial resistance in Gram-negative pathogens. In the present study, we investigated the role of the K. pneumoniae AcrRAB operon in antimicrobial resistance and virulence by using isogenic knockouts deficient in the AcrB component and the AcrR repressor, both derived from the virulent strain 52145R. We demonstrated that the AcrB knockout was more susceptible, not only to quinolones, but also to other antimicrobial agents, including beta-lactams, than the wild-type strain and the AcrR knockout. We further showed that the AcrB knockout was more susceptible to antimicrobial agents present in human bronchoalveolar lavage fluid and to human antimicrobial peptides than the wild-type strain and the AcrR knockout. Finally, the AcrB knockout exhibited a reduced capacity to cause pneumonia in a murine model, in contrast to the wild-type strain. The results of this study suggest that, in addition to contributing to the multidrug resistance phenotype, the AcrAB efflux pump may represent a novel virulence factor required for K. pneumoniae to resist innate immune defense mechanisms of the lung, thus facilitating the onset of pneumonia.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/patogenicidade , Animais , Líquido da Lavagem Broncoalveolar/microbiologia , Meios de Cultura , DNA Bacteriano/genética , Defensinas/farmacologia , Genótipo , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/efeitos dos fármacos , Lipopolissacarídeos/biossíntese , Camundongos , Camundongos Endogâmicos ICR , Testes de Sensibilidade Microbiana , Fenótipo , Plasmídeos/genética , Polimixina B/farmacologia , Polissacarídeos/biossíntese , RNA Bacteriano/genética , Proteínas Repressoras/genéticaRESUMO
Nontypeable Haemophilus influenzae (NTHI) is an opportunistic gram-negative pathogen that causes respiratory infections and is associated with progression of respiratory diseases. Cigarette smoke is a main risk factor for development of respiratory infections and chronic respiratory diseases. Glucocorticoids, which are anti-inflammatory drugs, are still the most common therapy for these diseases. Alveolar macrophages are professional phagocytes that reside in the lung and are responsible for clearing infections by the action of their phagolysosomal machinery and promotion of local inflammation. In this study, we dissected the interaction between NTHI and alveolar macrophages and the effect of cigarette smoke on this interaction. We showed that alveolar macrophages clear NTHI infections by adhesion, phagocytosis, and phagolysosomal processing of the pathogen. Bacterial uptake requires host actin polymerization, the integrity of plasma membrane lipid rafts, and activation of the phosphatidylinositol 3-kinase (PI3K) signaling cascade. Parallel to bacterial clearance, macrophages secrete tumor necrosis factor alpha (TNF-alpha) upon NTHI infection. In contrast, exposure to cigarette smoke extract (CSE) impaired alveolar macrophage phagocytosis, although NTHI-induced TNF-alpha secretion was not abrogated. Mechanistically, our data showed that CSE reduced PI3K signaling activation triggered by NTHI. Treatment of CSE-exposed cells with the glucocorticoid dexamethasone reduced the amount of TNF-alpha secreted upon NTHI infection but did not compensate for CSE-dependent phagocytic impairment. The deleterious effect of cigarette smoke was observed in macrophage cell lines and in human alveolar macrophages obtained from smokers and from patients with chronic obstructive pulmonary disease.
Assuntos
Haemophilus influenzae/imunologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/microbiologia , Fagocitose/efeitos dos fármacos , Fumaça/efeitos adversos , Fumar/efeitos adversos , Animais , Aderência Bacteriana , Linhagem Celular , Células Cultivadas , Humanos , Lisossomos/metabolismo , Macrófagos Alveolares/efeitos dos fármacos , Camundongos , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: Klebsiella pneumoniae is a capsulated Gram negative bacterial pathogen and a frequent cause of nosocomial infections. Despite its clinical relevance, little is known about the features of the interaction between K. pneumoniae and lung epithelial cells on a cellular level, neither about the role of capsule polysaccharide, one of its best characterised virulence factors, in this interaction. RESULTS: The interaction between Klebsiella pneumoniae and cultured airway epithelial cells was analysed. K. pneumoniae infection triggered cytotoxicity, evident by cell rounding and detachment from the substrate. This effect required the presence of live bacteria and of capsule polysaccharide, since it was observed with isolates expressing different amounts of capsule and/or different serotypes but not with non-capsulated bacteria. Cytotoxicity was analysed by lactate dehydrogenase and formazan measurements, ethidium bromide uptake and analysis of DNA integrity, obtaining consistent and complementary results. Moreover, cytotoxicity of non-capsulated strains was restored by addition of purified capsule during infection. While a non-capsulated strain was avirulent in a mouse infection model, capsulated K. pneumoniae isolates displayed different degrees of virulence. CONCLUSION: Our observations allocate a novel role to K. pneumoniae capsule in promotion of cytotoxicity. Although this effect is likely to be associated with virulence, strains expressing different capsule levels were not equally virulent. This fact suggests the existence of other bacterial requirements for virulence, together with capsule polysaccharide.
Assuntos
Células Epiteliais/microbiologia , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/patogenicidade , Animais , Cápsulas Bacterianas/fisiologia , Linhagem Celular , Células Epiteliais/citologia , Feminino , Humanos , Pulmão/citologia , Pulmão/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Polissacarídeos Bacterianos/fisiologia , VirulênciaRESUMO
OBJECTIVE: We evaluated the expression of Toll-like receptors 2 and 4 (TLR-2 and TLR-4) in circulating monocytes from peripheral blood of critical care patients treated with and without glutamine. Because no research has been published to date on the effect of glutamine on TLR receptors in critical patients, it was determined in an initial sample of 30 patients. METHODS: This was a prospective, randomized, single-blind study with 15 patients assigned to receive parenteral nutrition with a daily glutamine supplement of 0.35 g/kg. The control group received isocaloric-isonitrogenous parenteral nutrition. Blood samples were extracted before beginning the treatment and at 5 and 14 d. Expressions of CD14, TLR-2, and TLR-4 were determined by flow cytometry. Levels of TLRs were expressed as mean fluorescence intensity (mfi). RESULTS: Basal characteristics were similar in both groups. The expressions of TLR-2 in the treatment group with glutamine were 4.67 +/- 3.82 mfi before treatment, 3.91 +/- 2.04 mfi at 5 d, and 4.28 +/- 2.47 mfi at 14 d. The expressions of TLR-2 in the control group were 5.49 +/- 3.20 mfi before treatment, 4.48 +/- 2.15 mfi at 5 d, and 4.36 +/- 2.36 mfi at 14 d. The expressions of TLR-4 in the treatment group were 1.65 +/- 1.89 mfi before treatment, 1.23 +/- 1.10 mfi at 5 d, and 1.77 +/- 1.97 at 14 d. The expressions of TLR-4 in the control group were 1.51 +/- 1.76 mfi before treatment, 1.36 +/- 0.99 mfi at 5 d, and 1.26 +/- 0.59 mfi at 14 d. Infections were detected in 11 patients who received glutamine and 13 control patients (P = 0.51). CONCLUSION: In critical care patients, parenteral nutrition supplemented with glutamine does not increase the expression of TLR-2 or TLR-4 in peripheral blood monocytes.
Assuntos
Estado Terminal/terapia , Glutamina/farmacologia , Receptores de Lipopolissacarídeos/efeitos dos fármacos , Monócitos/imunologia , Nutrição Parenteral , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/efeitos dos fármacos , Adolescente , Adulto , Idoso , Bacteriemia/epidemiologia , Bacteriemia/prevenção & controle , Cuidados Críticos , Feminino , Citometria de Fluxo , Regulação da Expressão Gênica , Humanos , Receptores de Lipopolissacarídeos/metabolismo , Masculino , Pessoa de Meia-Idade , Pneumonia/epidemiologia , Pneumonia/prevenção & controle , Estudos Prospectivos , Sepse/epidemiologia , Sepse/prevenção & controle , Método Simples-Cego , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
Airway infection by nontypeable Haemophilus influenzae (NTHi) associates to chronic obstructive pulmonary disease (COPD) exacerbation and asthma neutrophilic airway inflammation. Lipids are key inflammatory mediators in these disease conditions and consequently, NTHi may encounter free fatty acids during airway persistence. However, molecular information on the interplay NTHi-free fatty acids is limited, and we lack evidence on the importance of such interaction to infection. Maintenance of the outer membrane lipid asymmetry may play an essential role in NTHi barrier function and interaction with hydrophobic molecules. VacJ/MlaA-MlaBCDEF prevents phospholipid accumulation at the bacterial surface, being the only system involved in maintaining membrane asymmetry identified in NTHi. We assessed the relationship among the NTHi VacJ/MlaA outer membrane lipoprotein, bacterial and exogenous fatty acids, and respiratory infection. The vacJ/mlaA gene inactivation increased NTHi fatty acid and phospholipid global content and fatty acyl specific species, which in turn increased bacterial susceptibility to hydrophobic antimicrobials, decreased NTHi epithelial infection, and increased clearance during pulmonary infection in mice with both normal lung function and emphysema, maybe related to their shared lung fatty acid profiles. Altogether, we provide evidence for VacJ/MlaA as a key bacterial factor modulating NTHi survival at the human airway upon exposure to hydrophobic molecules.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Infecções por Haemophilus/metabolismo , Haemophilus influenzae/patogenicidade , Lipoproteínas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Mucosa Respiratória/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Feminino , Infecções por Haemophilus/microbiologia , Humanos , Camundongos , Mucosa Respiratória/microbiologiaRESUMO
Posttranslational processing of proadrenomedullin generates two biologically active peptides, adrenomedullin (AM) and proadrenomedullin N-terminal 20 peptide (PAMP). Sequence comparison of homologous proadrenomedullin genes in vertebrate evolution shows a high degree of stability in the reading frame for AM, whereas PAMP sequence changes rapidly. Here we investigate the functional significance of PAMP phylogenetic variation studying two of PAMP's better characterized physiological activities, angiogenic potential and antimicrobial capability, with synthetic peptides carrying the predicted sequence for human, mouse, chicken, and fish PAMP. All tested peptides induced angiogenesis when compared with untreated controls, but chicken and fish PAMP, which lack terminal amidation, were apparently less angiogenic than their human and mouse homologs. Confirming the role of amidation in angiogenesis, Gly-extended and free acid variants of human PAMP produced responses similar to the natural nonamidated peptides. In contrast, antimicrobial activity was restricted to human PAMP, indicating that this function may have been acquired at a late time during the evolution of PAMP. Interestingly, free acid human PAMP retained antimicrobial activity whereas the Gly-extended form did not. This fact may reflect the need for maintaining a tightly defined structural conformation in the pore-forming mechanism proposed for these antimicrobial agents. The evolution of PAMP provides an example of an angiogenic peptide that developed antimicrobial capabilities without losing its original function.
Assuntos
Evolução Molecular , Peptídeos/genética , Peptídeos/metabolismo , 1-Naftilamina/análogos & derivados , 1-Naftilamina/metabolismo , Adrenomedulina , Animais , Galinhas , Humanos , Camundongos , Modelos Genéticos , Neovascularização Patológica , Fases de Leitura Aberta , Peptídeos/química , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismoRESUMO
An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.
Assuntos
Antígenos O/genética , Yersiniose/imunologia , Yersiniose/microbiologia , Yersinia enterocolitica/genética , Animais , Anticorpos Antibacterianos/sangue , Atividade Bactericida do Sangue , Via Alternativa do Complemento/imunologia , Via Clássica do Complemento/imunologia , Modelos Animais de Doenças , Peróxido de Hidrogênio/metabolismo , Imunoglobulinas/sangue , Macrófagos Peritoneais/metabolismo , Boca/microbiologia , Mutação , Neutrófilos/imunologia , Suínos , Vísceras/microbiologia , Yersinia enterocolitica/crescimento & desenvolvimento , Yersinia enterocolitica/imunologia , Yersinia enterocolitica/patogenicidadeRESUMO
Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/fisiologia , Interações Hospedeiro-Patógeno , Sistema Respiratório/microbiologia , Infecções Respiratórias/microbiologia , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Aderência Bacteriana , Carga Bacteriana , Proteínas da Membrana Bacteriana Externa/química , Proteínas de Bactérias/química , Técnicas de Tipagem Bacteriana , Biofilmes/crescimento & desenvolvimento , Moléculas de Adesão Celular/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Feminino , Genes Bacterianos , Glicosilação , Infecções por Haemophilus/patologia , Haemophilus influenzae/genética , Humanos , Integrina alfa5/metabolismo , Macrófagos Alveolares/microbiologia , Macrófagos Alveolares/patologia , Camundongos , Dados de Sequência Molecular , Mutação/genética , Sistema Respiratório/patologia , Infecções Respiratórias/patologia , Serina Endopeptidases/químicaRESUMO
Non-typeable Haemophilus influenzae (NTHi) is an opportunist pathogen well adapted to the human upper respiratory tract and responsible for many respiratory diseases. In the human airway, NTHi is exposed to pollutants, such as alkylating agents, that damage its DNA. In this study, we examined the significance of genes involved in the repair of DNA alkylation damage in NTHi virulence. Two knockout mutants, tagI and mfd, encoding N³-methyladenine-DNA glycosylase I and the key protein involved in transcription-coupled repair, respectively, were constructed and their virulence in a BALB/c mice model was examined. This work shows that N³-methyladenine-DNA glycosylase I is constitutively expressed in NTHi and that it is relevant for its virulence.
Assuntos
Proteínas de Bactérias/metabolismo , DNA Glicosilases/metabolismo , Infecções por Haemophilus/microbiologia , Haemophilus influenzae/metabolismo , Haemophilus influenzae/patogenicidade , Fatores de Transcrição/metabolismo , Animais , Proteínas de Bactérias/genética , DNA Glicosilases/genética , Feminino , Haemophilus influenzae/enzimologia , Haemophilus influenzae/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Transcrição/genética , VirulênciaRESUMO
Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.
Assuntos
Haemophilus influenzae/citologia , Haemophilus influenzae/genética , Fenótipo , Doença Pulmonar Obstrutiva Crônica/microbiologia , Adulto , Células Epiteliais Alveolares/microbiologia , Análise de Variância , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Aderência Bacteriana/fisiologia , Sequência de Bases , Biofilmes/crescimento & desenvolvimento , Primers do DNA/genética , Eletroforese em Gel de Campo Pulsado , Genótipo , Haemophilus influenzae/efeitos dos fármacos , Humanos , Camundongos , Dados de Sequência Molecular , Análise de Sequência de DNARESUMO
Non-typable Haemophilus influenzae (NTHi) is a gram negative pathogen that causes acute respiratory infections and is associated with the progression of chronic respiratory diseases. Previous studies have established the existence of a remarkable genetic variability among NTHi strains. In this study we show that, in spite of a high level of genetic heterogeneity, NTHi clinical isolates display a prevalent molecular feature, which could confer fitness during infectious processes. A total of 111 non-isogenic NTHi strains from an identical number of patients, isolated in two distinct geographical locations in the same period of time, were used to analyse nine genes encoding bacterial surface molecules, and revealed the existence of one highly prevalent molecular pattern (lgtF+, lic2A+, lic1D+, lic3A+, lic3B+, siaA-, lic2C+, ompP5+, oapA+) displayed by 94.6% of isolates. Such a genetic profile was associated with a higher bacterial resistance to serum mediated killing and enhanced adherence to human respiratory epithelial cells.