RESUMEN
The Burkholderia cepacia complex (Bcc) is a group of Gram-negative opportunistic pathogens causing infections in people with cystic fibrosis (CF). Bcc is highly antibiotic resistant, making conventional antibiotic treatment problematic. The identification of novel targets for anti-virulence therapies should improve therapeutic options for infected CF patients. We previously identified that the peptidoglycan-associated lipoprotein (Pal) was immunogenic in Bcc infected CF patients; however, its role in Bcc pathogenesis is unknown. The virulence of a pal deletion mutant (Δpal) in Galleria mellonella was 88-fold reduced (p < .001) compared to wild type. The lipopolysaccharide profiles of wild type and Δpal were identical, indicating no involvement of Pal in O-antigen transport. However, Δpal was more susceptible to polymyxin B. Structural elucidation by X-ray crystallography and calorimetry demonstrated that Pal binds peptidoglycan fragments. Δpal showed a 1.5-fold reduced stimulation of IL-8 in CF epithelial cells relative to wild type (p < .001), demonstrating that Pal is a significant driver of inflammation. The Δpal mutant had reduced binding to CFBE41o- cells, but adhesion of Pal-expressing recombinant E. coli to CFBE41o- cells was enhanced compared to wild-type E. coli (p < .0001), confirming that Pal plays a direct role in host cell attachment. Overall, Bcc Pal mediates host cell attachment and stimulation of cytokine secretion, contributing to Bcc pathogenesis.
Asunto(s)
Proteínas Bacterianas/química , Infecciones por Burkholderia/inmunología , Burkholderia cenocepacia/inmunología , Células Epiteliales/fisiología , Lipoproteínas/química , Animales , Adhesión Bacteriana , Proteínas Bacterianas/fisiología , Sitios de Unión , Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/patogenicidad , Adhesión Celular , Células Cultivadas , Cristalografía por Rayos X , Fibrosis Quística/microbiología , Citocinas/metabolismo , Farmacorresistencia Bacteriana , Células Epiteliales/microbiología , Escherichia coli , Humanos , Larva/microbiología , Lipopolisacáridos/fisiología , Lipoproteínas/fisiología , Modelos Moleculares , Mariposas Nocturnas , Peptidoglicano/química , Polimixinas/farmacología , Unión Proteica , Dominios ProteicosRESUMEN
Members of the Burkholderia cepacia complex (Bcc) cause chronic opportunistic lung infections in people with cystic fibrosis (CF), resulting in a gradual lung function decline and, ultimately, patient death. The Bcc is a complex of 20 species and is rarely eradicated once a patient is colonized; therefore, vaccination may represent a better therapeutic option. We developed a new proteomics approach to identify bacterial proteins that are involved in the attachment of Bcc bacteria to lung epithelial cells. Fourteen proteins were reproducibly identified by two-dimensional gel electrophoresis from four Bcc strains representative of two Bcc species: Burkholderia cenocepacia, the most virulent, and B. multivorans, the most frequently acquired. Seven proteins were identified in both species, but only two were common to all four strains, linocin and OmpW. Both proteins were selected based on previously reported data on these proteins in other species. Escherichia coli strains expressing recombinant linocin and OmpW showed enhanced attachment (4.2- and 3.9-fold) to lung cells compared to the control, confirming that both proteins are involved in host cell attachment. Immunoproteomic analysis using serum from Bcc-colonized CF patients confirmed that both proteins elicit potent humoral responses in vivo Mice immunized with either recombinant linocin or OmpW were protected from B. cenocepacia and B. multivorans challenge. Both antigens induced potent antigen-specific antibody responses and stimulated strong cytokine responses. In conclusion, our approach identified adhesins that induced excellent protection against two Bcc species and are promising vaccine candidates for a multisubunit vaccine. Furthermore, this study highlights the potential of our proteomics approach to identify potent antigens against other difficult pathogens.
Asunto(s)
Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana , Proteínas de la Membrana Bacteriana Externa/metabolismo , Bacteriocinas/metabolismo , Infecciones por Burkholderia/prevención & control , Complejo Burkholderia cepacia/fisiología , Células Epiteliales/microbiología , Adhesinas Bacterianas/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/inmunología , Bacteriocinas/inmunología , Infecciones por Burkholderia/inmunología , Fibrosis Quística/inmunología , Fibrosis Quística/microbiología , Modelos Animales de Enfermedad , Escherichia coli/genética , Escherichia coli/fisiología , Femenino , Expresión Génica , Humanos , Ratones Endogámicos BALB C , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Resultado del TratamientoRESUMEN
Iron acquisition is vital to microbial survival and is implicated in the virulence of many of the pathogens that reside in the cystic fibrosis (CF) lung. The multifaceted nature of iron acquisition by both bacterial and fungal pathogens encompasses a range of conserved and species-specific mechanisms, including secretion of iron-binding siderophores, utilization of siderophores from other species, release of iron from host iron-binding proteins and haemoproteins, and ferrous iron uptake. Pathogens adapt and deploy specific systems depending on iron availability, bioavailability of the iron pool, stage of infection and presence of competing pathogens. Understanding the dynamics of pathogen iron acquisition has the potential to unveil new avenues for therapeutic intervention to treat both acute and chronic CF infections. Here, we examine the range of strategies utilized by the primary CF pathogens to acquire iron and discuss the different approaches to targeting iron acquisition systems as an antimicrobial strategy.
Asunto(s)
Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Fibrosis Quística/microbiología , Proteínas de Unión a Hierro/metabolismo , Hierro/metabolismo , Depuración Mucociliar/fisiología , Infecciones por Pseudomonas/patología , Infecciones Estafilocócicas/patología , Humanos , Pulmón/microbiología , Microbiota , Depuración Mucociliar/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Sideróforos/metabolismo , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidadRESUMEN
Bacterial tyrosine kinases and their cognate protein tyrosine phosphatases are best known for regulating the biosynthesis of polysaccharides. Moreover, their roles in the stress response, DNA metabolism, cell division, and virulence have also been documented. The aim of this study was to investigate the pathogenicity and potential mechanisms of virulence dependent on the tyrosine kinase BceF and phosphotyrosine phosphatase BceD of the cystic fibrosis opportunistic pathogen Burkholderia contaminans IST408. The insertion mutants bceD::Tp and bceF::Tp showed similar attenuation of adhesion and invasion of the cystic fibrosis lung epithelial cell line CFBE41o- compared to the parental strain B. contaminans IST408. In the absence of bceD or bceF genes, B. contaminans also showed a reduction in the ability to translocate across polarized epithelial cell monolayers, demonstrated by a higher transepithelial electrical resistance, reduced flux of fluorescein isothiocyanate-labeled bovine serum albumin, and higher levels of tight junction proteins ZO-1, occludin, and claudin-1 present in monolayers exposed to these bacterial mutants. Furthermore, bceD::Tp and bceF::Tp mutants induced lower levels of interleukin-6 (IL-6) and IL-8 release than the parental strain. In conclusion, although the mechanisms of pathogenicity dependent on BceD and BceF are not understood, these proteins contribute to the virulence of Burkholderia by enhancement of cell attachment and invasion, disruption of epithelial integrity, and modulation of the proinflammatory response.
Asunto(s)
Complejo Burkholderia cepacia/patogenicidad , Fibrosis Quística/microbiología , Pulmón/microbiología , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Tirosina Quinasas/fisiología , Mucosa Respiratoria/microbiología , Factores de Virulencia/genética , Albúminas/metabolismo , Antibacterianos/farmacología , Adhesión Bacteriana , Infecciones por Burkholderia/microbiología , Infecciones por Burkholderia/patología , Complejo Burkholderia cepacia/enzimología , Complejo Burkholderia cepacia/genética , Línea Celular , Ciprofloxacina/farmacología , Claudina-1/biosíntesis , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Impedancia Eléctrica , Humanos , Inflamación/inmunología , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Interleucina-8/biosíntesis , Interleucina-8/metabolismo , Potenciales de la Membrana , Mutación , Ocludina/biosíntesis , Transporte de Proteínas , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Quinasas/genética , Uniones Estrechas/patología , Proteína de la Zonula Occludens-1/biosíntesisRESUMEN
The cystic fibrosis (CF) lung is a niche colonized by a diverse group of organisms, with a more limited number of species including Pseudomonas aeruginosa dominating in adult patients. Whether all members of this microbial community play a direct or indirect role in pulmonary decline has yet to be fully elucidated, but investigations of their interactions with both co-colonizing species and with host cells are beginning to shed light on their virulence potential. It is also emerging that some microbial species within this community adapt as chronic infection is established to survive the hostile environment of the lung, to minimize host clearance and to resist therapeutic intervention. This review highlights the recent developments in CF microbiology focusing on the cooperative, competitive and adaptive interactions of established and emerging pathogens in the lung microbiome.
Asunto(s)
Fibrosis Quística/microbiología , Pulmón/microbiología , Adaptación Fisiológica , Adulto , Infecciones Bacterianas/complicaciones , Infecciones Bacterianas/microbiología , Fenómenos Fisiológicos Bacterianos , Enfermedad Crónica , Humanos , Interacciones Microbianas , Microbiota , Pseudomonas aeruginosa/fisiología , Percepción de QuorumRESUMEN
In many Gram-negative bacteria, lipopolysaccharide (LPS) and its lipidâ A moiety are pivotal for bacterial survival. Depending on its structure, lipidâ A carries the toxic properties of the LPS and acts as a potent elicitor of the host innate immune system via the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) receptor complex. It often causes a wide variety of biological effects ranging from a remarkable enhancement of the resistance to the infection to an uncontrolled and massive immune response resulting in sepsis and septic shock. Since the bioactivity of lipidâ A is strongly influenced by its primary structure, a broad range of chemical syntheses of lipidâ A derivatives have made an enormous contribution to the characterization of lipidâ A bioactivity, providing novel pharmacological targets for the development of new biomedical therapies. Here, we describe and discuss the chemical aspects regarding lipidâ A and its role in innate immunity, from the (bio)synthesis, isolation and characterization to the molecular recognition at the atomic level.
Asunto(s)
Bacterias Gramnegativas/química , Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/inmunología , Inmunidad Innata , Lípido A/química , Lípido A/inmunología , Animales , Infecciones por Bacterias Gramnegativas/microbiología , Humanos , Lípido A/análogos & derivados , Antígeno 96 de los Linfocitos/inmunología , Modelos Moleculares , Receptor Toll-Like 4/inmunologíaRESUMEN
Burkholderia cenocepacia is a bacterial pathogen which causes severe respiratory infections in cystic fibrosis (CF). These studies were aimed at gaining an insight into the iron acquisition strategies of B. cenocepacia. In iron restricted conditions, genes associated with the synthesis and utilisation of ornibactin (pvdA, orbA, orb F) were significantly upregulated compared to the expression of pyochelin associated genes (pchD, fptA). In the absence of alternative iron sources, B. cenocepacia J2315 and 715j utilised ferritin and haemin, but not transferrin or lactoferrin for growth. Significantly, mutants unable to produce ornibactin, (715j-orbI) or ornibactin and pyochelin, (715j-pobA), utilised haemin and ferritin more efficiently than the wild-type. Moreover, both mutants were also able to utilise lactoferrin for growth (P ≤ 0.01) and additionally 715j-pobA utilised transferrin (P ≤ 0.01), potentially facilitating adaptation to the host environment. Furthermore, B. cenocepacia increased ornibactin gene expression in response to pyoverdine from Pseudomonas aeruginosa (P ≤ 0.01), demonstrating the capacity to compete for iron in co-colonised niches. Pyoverdine also significantly diminished the growth of B. cenocepacia (P < 0.001) which was related to its iron chelating activity. In a study of three B. cenocepacia sequential clonal isolates obtained from a CF patient over a 3.5 year period, ornibactin upregulation in response to pyoverdine was less pronounced in the last isolate compared to the earlier isolates, as was growth in the presence of haemin and ferritin, indicating alternative iron acquisition mechanism(s) may dominate as chronic infection progresses. These data demonstrate the multifaceted iron acquisition strategies of B. cenocepacia and their capacity to be differentially activated in the presence of P. aeruginosa and during chronic infection.
Asunto(s)
Burkholderia cenocepacia/metabolismo , Hierro/metabolismo , Sideróforos/genética , Adaptación Fisiológica , Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/genética , Fibrosis Quística/microbiología , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Hemo/metabolismo , Humanos , Neumonía Bacteriana/microbiología , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/fisiología , Sideróforos/biosíntesis , Activación TranscripcionalRESUMEN
Cystic fibrosis (CF) is a recessive genetic disease characterized by chronic respiratory infections and inflammation causing permanent lung damage. Recurrent infections are caused by Gram-negative antibiotic-resistant bacterial pathogens such as Pseudomonas aeruginosa, Burkholderia cepacia complex (Bcc) and the emerging pathogen genus Pandoraea. In this study, the interactions between co-colonizing CF pathogens were investigated. Both Pandoraea and Bcc elicited potent pro-inflammatory responses that were significantly greater than Ps. aeruginosa. The original aim was to examine whether combinations of pro-inflammatory pathogens would further exacerbate inflammation. In contrast, when these pathogens were colonized in the presence of Ps. aeruginosa the pro-inflammatory response was significantly decreased. Real-time PCR quantification of bacterial DNA from mixed cultures indicated that Ps. aeruginosa significantly inhibited the growth of Burkholderia multivorans, Burkholderia cenocepacia, Pandoraea pulmonicola and Pandoraea apista, which may be a factor in its dominance as a colonizer of CF patients. Ps. aeruginosa cell-free supernatant also suppressed growth of these pathogens, indicating that inhibition was innate rather than a response to the presence of a competitor. Screening of a Ps. aeruginosa mutant library highlighted a role for quorum sensing and pyoverdine biosynthesis genes in the inhibition of B. cenocepacia. Pyoverdine was confirmed to contribute to the inhibition of B. cenocepacia strain J2315. B. multivorans was the only species that could significantly inhibit Ps. aeruginosa growth. B. multivorans also inhibited B. cenocepacia and Pa. apista. In conclusion, both Ps. aeruginosa and B. multivorans are capable of suppressing growth and virulence of co-colonizing CF pathogens.
Asunto(s)
Infecciones por Burkholderia/prevención & control , Burkholderia/fisiología , Burkholderiaceae/crecimiento & desarrollo , Fibrosis Quística/prevención & control , Infecciones por Pseudomonas/prevención & control , Pseudomonas aeruginosa/fisiología , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/crecimiento & desarrollo , Línea Celular , Fibrosis Quística/microbiología , Citocinas/metabolismo , Células Epiteliales , Humanos , Modelos Biológicos , Infecciones por Pseudomonas/microbiología , Percepción de Quorum , VirulenciaRESUMEN
Burkholderia cenocepacia is an important human pathogen in patients with cystic fibrosis (CF). Non-clinical reservoirs may play a role in the acquisition of infection, so it is important to evaluate the pathogenic potential of environmental B. cenocepacia isolates. In this study, we investigated the interactions of two environmental B. cenocepacia strains (Mex1 and MCII-168) with two bronchial epithelial cell lines, 16HBE14o(-) and CFBE41o(-), which have a non-CF and a CF phenotype, respectively. The environmental strains showed a significantly lower level of invasion into both CF and non-CF cells in comparison with the clinical B. cenocepacia LMG16656(T) strain. Exposure of polarized CFBE41o(-) or 16HBE14o(-) cells to the environmental strains resulted in a significant reduction in transepithelial resistance (TER), comparable with that observed following exposure to the clinical strain. A different mechanism of tight junction disruption in CF versus non-CF epithelia was found. In the 16HBE41o(-) cells, the environmental strains resulted in a drop in TER without any apparent effect on tight junction proteins such as zonula occludens-1 (ZO-1). In contrast, in CF cells, the amount of ZO-1 and its localization were clearly altered by the presence of both the environmental strains, comparable with the effect of LMG16656. This study demonstrates that even if the environmental strains are significantly less invasive than the clinical strain, they have an effect on epithelial integrity comparable with that of the clinical strain. Finally, the tight junction regulatory protein ZO-1 appears to be more susceptible to the presence of environmental strains in CF cells than in cells which express a functional cystic fibrosis transmembrane regulator (CFTR).
Asunto(s)
Infecciones por Burkholderia/patología , Burkholderia cenocepacia/patogenicidad , Fibrosis Quística/microbiología , Células Epiteliales/microbiología , Bronquios/citología , Línea Celular , Humanos , Proteínas de la Membrana/metabolismo , Fosfoproteínas/metabolismo , Rizosfera , Uniones Estrechas/microbiología , Zea mays/microbiología , Proteína de la Zonula Occludens-1RESUMEN
Burkholderia cepacia complex (Bcc) is an opportunistic pathogen in cystic fibrosis patients which is inherently resistant to antimicrobial agents. The mechanisms of attachment and pathogenesis of Bcc, a group of 17 species, are poorly understood. The most commonly identified Bcc species in newly colonised patients, Burkholderia multivorans, continues to be acquired from the environment. Development of therapies which can prevent or reduce the risk of colonization on exposure to Bcc in the environment would be a better alternative to antimicrobial agents. Previously, it has been shown that Bcc strains bound to many glycolipid receptors on lung epithelia. Using a real-time PCR method to quantify the levels of binding of B. multivorans to the lung epithelial cells, we have examined glycoconjugate derivatives for their potential to inhibit host cell attachment. Bivalent lactosides previously shown to inhibit galectin binding significantly reduced the attachment of B. multivorans to CF lung epithelial cells at micromolar concentrations. This was in contrast to monosaccharides and lactose, which were only effective in the millimolar range. Development of glycoconjugate therapies such as these, which inhibit attachment to lung epithelial cells, represent an alternative means of preventing infection with inherently antimicrobially resistant pathogens such as B. multivorans.
Asunto(s)
Adhesión Bacteriana/efectos de los fármacos , Burkholderia/efectos de los fármacos , Glicósidos/farmacología , Mucosa Respiratoria/microbiología , Burkholderia/fisiología , Infecciones por Burkholderia/prevención & control , Línea Celular , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Glicósidos/química , Humanos , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/microbiología , Mucosa Respiratoria/efectos de los fármacos , Mucosa Respiratoria/metabolismoRESUMEN
Burkholderia cepacia complex is a group of bacterial pathogens that cause opportunistic infections in cystic fibrosis (CF). The most virulent of these is Burkholderia cenocepacia. Matrix metalloproteinases (MMPs) are upregulated in CF patients. The aim of this work was to examine the role of MMPs in the pathogenesis of B. cepacia complex, which has not been explored to date. Real-time PCR analysis showed that B. cenocepacia infection upregulated MMP-2 and MMP-9 genes in the CF lung cell line CFBE41o- within 1 h, whereas MMP-2, -7, and -9 genes were upregulated in the non-CF lung cell line 16HBE14o-. Conditioned media from both cell lines showed increased MMP-9 activation following B. cenocepacia infection. Conditioned media from B. cenocepacia-infected cells significantly reduced the rate of wound healing in confluent lung epithelia (P < 0.05), in contrast to conditioned media from Pseudomonas aeruginosa-infected cells, which showed predominant MMP-2 activation. Treatment of control conditioned media from both cell lines with the MMP activator 4-aminophenylmercuric acetate (APMA) also resulted in clear activation of MMP-9 and to a much lesser extent MMP-2. APMA treatment of control media also delayed the repair of wound healing in confluent epithelial cells. Furthermore, specific inhibition of MMP-9 in medium from cells exposed to B. cenocepacia completely reversed the delay in wound repair. These data suggest that MMP-9 plays a role in the reduced epithelial repair observed in response to B. cenocepacia infection and that its activation following B. cenocepacia infection contributes to the pathogenesis of this virulent pathogen.
Asunto(s)
Infecciones por Burkholderia/enzimología , Medios de Cultivo Condicionados/farmacología , Fibrosis Quística/enzimología , Células Epiteliales/efectos de los fármacos , Pulmón/enzimología , Metaloproteinasa 9 de la Matriz/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Infecciones por Burkholderia/complicaciones , Infecciones por Burkholderia/microbiología , Infecciones por Burkholderia/patología , Burkholderia cenocepacia/crecimiento & desarrollo , Línea Celular , Medios de Cultivo Condicionados/química , Fibrosis Quística/complicaciones , Fibrosis Quística/microbiología , Fibrosis Quística/patología , ADN Complementario , Células Epiteliales/citología , Expresión Génica , Humanos , Pulmón/microbiología , Pulmón/patología , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Inhibidores de la Metaloproteinasa de la Matriz , Acetato Fenilmercúrico/análogos & derivados , Acetato Fenilmercúrico/farmacología , Reacción en Cadena de la Polimerasa , Inhibidores de Proteasas/farmacología , Infecciones por Pseudomonas/enzimología , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/crecimiento & desarrollo , Regulación hacia ArribaRESUMEN
Burkholderia cepacia complex (Bcc) is a group of Gram-negative pulmonary pathogens associated with life-threatening infections in patients with cystic fibrosis (CF). The airway epithelium plays a crucial role in the initiation and modulation of inflammatory responses to these pathogens. Interleukin (IL)-8 released from epithelial cells is a potent chemoattractant for neutrophils. The aims of this study were to compare the IL-8 response to Bcc infection in different epithelial cell types and to investigate the impact of IL-8 on Bcc growth and intracellular survival. To compare epithelial cell IL-8 responses, 4 human epithelial cell lines were used in the study; A549 cells, an alveolar epithelial cell line, Calu-3 cells, a sub-bronchial epithelial cell line, 16HBE14o- cells, and CFBE41o- cells, which are CFTR-positive and CFTR-negative bronchial epithelial cell lines, respectively. Two B. multivorans and 2 B. cenocepacia strains all induced a significant IL-8 response by 12 h and further increased in all cell lines at 24 h. Furthermore, the levels of IL-8 from Calu-3 and A549 cells were approximately 3 times that of 16HBE14o- or CFBE41o- cells. In 2 of the cell lines examined (16HBE14o- and CFBE41o-), B. cenocepacia LMG 16656 (J2315), an epidemic strain, induced greater levels of IL-8 (P<0.01) compared to other Bcc strains tested. The CFTR-positive and -negative cell lines secreted similar levels of IL-8 indicating a CFTR-independent induction of IL-8. However, the CFTR-negative cells did secrete constitutive levels of IL-8 greater than that of CFTR-positive cells. An investigation of the effect of IL-8 on Bcc extracellular and intracellular growth found that at low concentrations (<10 ng/ml) of recombinant human (rh) IL-8, the growth of B. cenocepacia LMG 16656 and B. multivorans LMG 13010 was enhanced, whereas at higher concentrations (10 ng/ml), growth of both strains was significantly reduced. Growth of both non-CF Bcc strains remained unchanged in the presence of rhIL-8. In contrast to extracellular growth, higher concentrations (10ng/ml) of rhIL-8 enhance the intracellular growth and survival of both LMG 16656 and LMG 13010 in 16HBE14o- and CFBE41o- cell lines. Although LMG 13010 uptake by epithelial cells was higher than LMG 16656 (P<0.01), the intracellular growth of LMG 16656 is greater than LMG 13010 (P<0.05). These studies demonstrated that the type of epithelial cells encountered by Bcc strains determines the extent of the IL-8 responses triggered and that this cytokine in addition to its well-established proinflammatory properties can enhance both the extracellular and intracellular growth of Bcc strains.
Asunto(s)
Complejo Burkholderia cepacia/crecimiento & desarrollo , Complejo Burkholderia cepacia/inmunología , Células Epiteliales/inmunología , Interleucina-8/metabolismo , Viabilidad Microbiana , Línea Celular , Fibrosis Quística/inmunología , Fibrosis Quística/patología , Humanos , Pulmón/inmunología , Pulmón/patología , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/patologíaRESUMEN
Burkholderia cepacia complex (Bcc) is a group of opportunistic cystic fibrosis (CF) pathogens that invade lung epithelial cells. The mechanisms of invasion are poorly understood, in particular, the receptors utilised by this bacterium in the invasion process have not been identified. The aim of this study was to investigate the epithelial receptors involved in the invasion of Bcc isolates. We confirmed that invasion into two lung epithelial cell lines (16HBE14o- and CFBE41o-) which have a non-CF and CF phenotype, respectively, is receptor mediated and showed that pre-treatment of these epithelial cell lines with α- or ß-galactosidase reduced invasion of isolates of two species of Bcc, Burkholderia multivorans and Burkholderia cenocepacia. In contrast, removal of mucin had no significant effect. Biotinylated Bcc strains were shown to bind to purified glycolipids separated by thin layer chromatography, albeit different patterns of binding were associated with different strains. Invasion of CF lung epithelial cells (CFBE41o-) by all three Bcc strains examined was significantly reduced by treatment of cells with inhibitors of glycolipid biosynthesis. Although the specific glycolipid involved in each case has not been elucidated, it is apparent that invasion of lung epithelial cells is mediated via binding to glycosphingolipid receptors.
Asunto(s)
Adhesión Bacteriana , Complejo Burkholderia cepacia/patogenicidad , Células Epiteliales/microbiología , Glicoesfingolípidos/metabolismo , Receptores de Superficie Celular/metabolismo , Línea Celular , Humanos , alfa-Galactosidasa/metabolismo , beta-Galactosidasa/metabolismoRESUMEN
Burkholderia cepacia complex (Bcc) is an important and virulent pathogen in cystic fibrosis patients. The interactions between this pathogen and the host lung epithelium are being widely investigated but remain to be elucidated. The complex is very versatile and its interactions with the lung epithelial cells are many and varied. The first steps in the interaction are penetration of the mucosal blanket and subsequent adherence to the epithelial cell surface. A range of epithelial receptors have been reported to bind to Bcc. The next step in pathogenesis is the invasion of the lung epithelial cell and also translocation across the epithelium to the serosal side. Furthermore, pathogenesis is mediated by a range of virulence factors that elicit their effects on the epithelial cells. This review outlines these interactions and examines the therapeutic implications of understanding the mechanisms of pathogenesis of this difficult, antibiotic-resistant, opportunistic pathogen.
Asunto(s)
Antibacterianos/farmacología , Infecciones por Burkholderia/tratamiento farmacológico , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/fisiología , Células Epiteliales/microbiología , Pulmón/citología , Interacciones Huésped-Patógeno , HumanosRESUMEN
Pandoraea species are emerging opportunistic pathogens capable of causing chronic lung infections in cystic fibrosis patients. This study examined the interactions of 17 Pandoraea isolates from the five identified species (Pandoraea apista, Pandoraea norimbergensis, Pandoraea pulmonicula, Pandoraea sputorum and Pandoraea pnomenusa) plus two Pandoraea genomospecies isolates with lung epithelial cells and their ability to form biofilms in vitro. Only three isolates showed an ability to invade A549 lung epithelial cells, and only one isolate was able to form biofilms. In contrast, all isolates triggered a pronounced pro-inflammatory response, with elevation of both interleukin (IL)-6 (two- to 19-fold) and IL-8 (10- to 50-fold) above that observed for a control strain of Escherichia coli. This property is likely to be a major factor in the pathogenesis of the genus.
Asunto(s)
Betaproteobacteria/patogenicidad , Biopelículas/crecimiento & desarrollo , Células Epiteliales/microbiología , Pulmón/patología , Virulencia/genética , Betaproteobacteria/efectos de los fármacos , Betaproteobacteria/inmunología , Betaproteobacteria/fisiología , Línea CelularRESUMEN
Chronic infection with opportunistic pathogens including Burkholderia cepacia complex (Bcc) is a hallmark of cystic fibrosis (CF). We investigated the adaptive mechanisms facilitating chronic lung infection in sequential Bcc isolates from two siblings with CF (P1 and P2), one of whom also experienced intermittent blood-stream infections (P2). We previously showed increased lung cell attachment with colonisation time in both P1 and P2. WGS analysis confirmed that the isolates are closely related. Twelve genes showed three or more mutations, suggesting these were genes under selection. Single nucleotide polymorphisms (SNVs) in 45 regulatory genes were also observed. Proteomic analysis showed that the abundance of 149 proteins increased over 61-months in sputum isolates, and both time- and source-related alterations in protein abundance between the second patient's isolates. A consistent time-dependent increase in abundance of 19 proteins encoded by a low-oxygen-activated (lxa) locus was observed in both sets of isolates. Attachment was dramatically reduced in a B. cenocepacia K56-2Δlxa-locus deletion mutant, further indicating that it encodes protein(s) involved in host-cell attachment. Time-related changes in virulence in Galleria mellonella or motility were not observed. We conclude that the lxa-locus, associated with anoxic persistence in vitro, plays a role in host-cell attachment and adaptation to chronic colonization in the hypoxic niche of the CF lung.
Asunto(s)
Adaptación Fisiológica , Infecciones por Burkholderia , Burkholderia cenocepacia , Fibrosis Quística , Sitios Genéticos , Oxígeno/metabolismo , Neumonía Bacteriana , Secuencia de Bases , Infecciones por Burkholderia/genética , Infecciones por Burkholderia/metabolismo , Infecciones por Burkholderia/microbiología , Burkholderia cenocepacia/genética , Burkholderia cenocepacia/metabolismo , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Fibrosis Quística/microbiología , Femenino , Humanos , Pulmón/metabolismo , Pulmón/microbiología , Masculino , Neumonía Bacteriana/genética , Neumonía Bacteriana/metabolismo , Neumonía Bacteriana/microbiología , Polimorfismo de Nucleótido Simple , Eliminación de SecuenciaRESUMEN
BACKGROUND: Colonisation with Burkholderia cepacia complex pathogens has been associated with accelerated decline in cystic fibrosis (CF) patients. The two most common species among the CF community are Burkholderia cenocepacia and Burkholderia multivorans. However, Burkholderia dolosa has recently been causing concern due to its transmissibility and virulence in CF patients. METHODS: We have compared the ability of five B. dolosa strains to invade lung epithelial cells in vitro with other members of the Bcc. The bacterial epithelial cell interaction was visualised by transmission electron microscopy. We have also examined the ability of these strains to form biofilms in vitro. RESULTS: We have found that members of this species can invade pulmonary epithelial cells in vitro as readily as those from B. cenocepacia and B. multivorans. Confirmation of intracellular invasion was obtained by transmission electron microscopy. B. dolosa strains were readily observed in membrane bound vesicles inside the lung epithelial cells. In addition, strains from this species were capable of forming strong biofilms at a level comparable to the more clinically relevant species. CONCLUSIONS: B. dolosa shows comparable virulence characteristics in vitro to the two most clinically relevant species indicating precautions should be taken when this species is identified in the CF population.
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Biopelículas/crecimiento & desarrollo , Complejo Burkholderia cepacia/crecimiento & desarrollo , Vesículas Citoplasmáticas/microbiología , Infecciones por Burkholderia , Complejo Burkholderia cepacia/patogenicidad , Línea Celular , Fibrosis Quística/microbiología , Humanos , Microscopía Electrónica de Transmisión , Mucosa Respiratoria/microbiología , VirulenciaRESUMEN
Respiratory infections with Burkholderia cepacia complex (Bcc) bacteria in cystic fibrosis (CF) are associated with a worse prognosis and increased risk of death. In this work, we assessed the virulence potential of three B. cenocepacia clonal isolates obtained from a CF patient between the onset of infection (isolate IST439) and before death with cepacia syndrome 3.5 years later (isolate IST4113 followed by IST4134), based on their ability to invade epithelial cells and compromise epithelial monolayer integrity. The two clonal isolates retrieved during late-stage disease were significantly more virulent than IST439. Proteomic profiling by 2-D DIGE of the last isolate recovered before the patient's death, IST4134, and clonal isolate IST439, was performed and compared with a prior analysis of IST4113 vs. IST439. The cytoplasmic and membrane-associated enriched fractions were examined and 52 proteins were found to be similarly altered in the two last isolates compared with IST439. These proteins are involved in metabolic functions, nucleotide synthesis, translation and protein folding, cell envelope biogenesis and iron homeostasis. Results are suggestive of the important role played by metabolic reprogramming in the virulence potential and persistence of B. cenocepacia, in particular regarding bacterial adaptation to microaerophilic conditions. Also, the content of the virulence determinant AidA was higher in the last 2 isolates. Significant levels of siderophores were found to be secreted by the three clonal isolates in an iron-depleted environment, but the two late isolates were more tolerant to low iron concentrations than IST439, consistent with the relative abundance of proteins involved in iron uptake.
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Proteínas Bacterianas , Infecciones por Burkholderia , Burkholderia cepacia , Neumonía Bacteriana , Proteómica , Factores de Virulencia , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Infecciones por Burkholderia/genética , Infecciones por Burkholderia/metabolismo , Burkholderia cepacia/genética , Burkholderia cepacia/metabolismo , Burkholderia cepacia/patogenicidad , Femenino , Humanos , Masculino , Neumonía Bacteriana/genética , Neumonía Bacteriana/metabolismo , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Burkholderia cepacia complex (Bcc) is an opportunistic bacterial pathogen that causes chronic infections in people with cystic fibrosis (CF). It is a highly antibiotic resistant organism and Bcc infections are rarely cleared from patients, once they are colonized. The two most clinically relevant species within Bcc are Burkholderia cenocepacia and Burkholderia multivorans. The virulence of these pathogens has not been fully elucidated and the virulence proteins expressed during human infection have not been identified to date. Furthermore, given its antibiotic resistance, prevention of infection with a prophylactic vaccine may represent a better alternative than eradication of an existing infection. We have compared the immunoproteome of two strains each from these two species of Bcc, with the aim of identifying immunogenic proteins which are common to both species. Fourteen immunoreactive proteins were exclusive to both B. cenocepacia strains, while 15 were exclusive to B. multivorans. A total of 15 proteins were immunogenic across both species. DNA-directed RNA polymerase, GroEL, 38kDa porin and elongation factor-Tu were immunoreactive proteins expressed by all four strains examined. Many proteins which were immunoreactive in both species, warrant further investigations in order to aid in the elucidation of the mechanisms of pathogenesis of this difficult organism. In addition, identification of some of these could also allow the development of protective vaccines which may prevent colonisation.
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Proteínas Bacterianas/metabolismo , Complejo Burkholderia cepacia/metabolismo , Proteómica , Proteínas Bacterianas/inmunología , Infecciones por Burkholderia/microbiología , Complejo Burkholderia cepacia/inmunología , Fibrosis Quística/inmunología , Fibrosis Quística/microbiología , Humanos , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/metabolismo , Proteómica/métodosRESUMEN
Chronic infection is a hallmark of cystic fibrosis (CF) and the main contributor to morbidity. Microbial infection in CF is complex, due to the number of different species that colonise the CF lung. Their colonisation is facilitated by a host response that is impaired or compromised by highly viscous mucous, zones of hypoxia and the lack of the cystic fibrosis transmembrane regulator (CFTR). Successful dominant CF pathogens combine an effective arsenal to establish infection and counter-attack the host response, together with an ability to adapt readily to an unfavourable environment. Hypermutability is common among CF pathogens facilitating adaptation and as the host response persists, progressive destruction of the normal architecture of lung tissue ensues with catastrophic consequences for the host.