Bacterial pathogenesis and Pathogenomics
Team: Jorge H. Leitão, Leonilde Moreira, Arsénio M. Fialho, Isabel Sá-Correia, Sílvia A. Sousa, Carla Coutinho, Sandra C dos Santos, Dalila Mil-Homens, Inês Silva
The Burkholderia cepacia complex (Bcc) is a group of at least 17 closely related bacterial species recognized as relevant opportunistic pathogens, particularly in patients with cystic fibrosis (CF), the most common life-threatening autosomal recessive disease among Caucasians. Bcc bacteria are a risk factor for increased morbidity and premature mortality among the CF population.
Work carried out by our group envisages the elucidation of the action of Bcc bacteria as relevant opportunistic pathogens, particularly in CF patients. Our research activities involve molecular epidemiology of respiratory infections caused by Bcc among Portuguese CF patients, studies of the molecular mechanisms underlying Bcc adaptation to the CF lung, acquisition of resistance to multiple antimicrobials and virulence mechanisms, using both transcriptomics and proteomics analyses, and identification of virulence factors that might be exploited as potential targets to develop new antimicrobials or therapeutics to fight Bcc infections.
For more than 10 years, our research group has collaborated with the major Portuguese CF Centre and the Bacteriology Laboratory at Santa Maria Hospital, in Lisbon. The first epidemiological study of Bcc infections among CF Portuguese patients was published in 2000 and was followed by others. The role of the exopolysaccharide cepacian produced by clinical isolates of Bcc in biofilm formation and persistence of respiratory infections was also examined.
1. Strategies employed by Bcc bacteria to adapt to the CF lung
CF patients infected by Bcc are subjected to intensive and aggressive antibiotic therapy,
exposing these intrinsically multi-resistant bacteria to prolonged antibiotic selective pressure. A strategy involving phenotypic and molecular biology analyses and expression proteomics and transcriptomics approaches (in the context of the Burkholderiaarray project, funded by CF Foundation Therapeutics) is being used. These studies aim to unveil the molecular mechanisms behind the strategies employed by Bcc bacteria to adapt to the CF lung stressing environment, in particular to high antibiotic concentration, and to be involved either in chronic or in acute infection. Studies involve clonal variants of the Bcc isolates serially isolated from persistently colonized CF patients including those who died with the cepacia syndrome. An Affymetrix dual species Burkholderia custom microarray based on the genomes of B. multivorans ATCC 17616 and B. cenocepacia J2315 was designed and used to assess mucoid phenotype switch mechanisms in clinical isolates.
2. Novel adherence factors of pathogenic Burkholderia cenocepacia: mechanisms of action and their role in virulence
To initiate infection, B. cenocepacia must be able to colonize the respiratory epithelium by binding to extracellular-matrix components. This step, although not fully characterized, is mediated by several proteins collectively termed adhesins, which are surface-exposed proteins. So far, only one B. cenocepacia adhesin has been identified as a factor responsible for cell adhesion in the respiratory epithelium. Investigation of molecular mechanisms of interaction of B. cenocepacia adhesins with host cells will assist in the development of novel tools to control this pathogen. Thus, in our efforts to discover novel surface-exposed proteins to be involved inadherence/invasion to human respiratory epithelium, we have identified three virulence-associated Trimeric Autotransporter Adhesins (TAAs) (BCAM019, BCAM0223 and BCAM0224) in B . cenocepacia, that contain domains homologous to theYersinia enterocolitica adhesin A (YadA). Our goal is to establish the role of these surface exposed proteins in Burkholderiapathogenicity associated with infection of the respiratory tract.
3. Rational design of new therapeutics/antimicrobials against Bcc
An ongoing strategy to develop new therapeutic approaches/molecules to fight Bcc infections is
being pursued, involving the structural characterization of type II PMIs and envisaging the design of specific inhibitors.
- Silva IN, Santos PM, Moreira LM., "Draft Genome Sequences of Two Burkholderia multivorans Sequential Isolates from a Chronic Lung Infection of a Cystic Fibrosis Patient", Genome Announc. 3(1). pii: e01531-14, 2015.
- Ferreira AS, Silva IN, Fernandes F, Pilkington R, Callaghan M, McClean S, Moreira LM. "The tyrosine kinase BceF and the phosphotyrosine phosphatase BceD of Burkholderia contaminans are required for efficient invasion and epithelial disruption of a cystic fibrosis lung epithelial cell line", Infect Immun. 83(2):812-21, 2015.
- Moreira, A.S., Coutinho, C.P., Avezedo, P., Lito, L., Melo-Cristino, J., Sá-Correia, I., "Burkholderia dolosa phenotypic variation during the decline in lung function of a cystic fibrosis patient along 5.5 years of chronic colonization", Journal of Medical Microbiology, 63: 594-601, 2014.
- Ramos, C.G., Grilo, A.M., Sousa, S.A., Feliciano, J.R., da Costa, P.J., Leitão, J.H., "Regulation of Hfq mRNA and protein levels in Escherichia coli and Pseudomonas aeruginosa by the Burkholderia cebocepacia MtvR sRNA", PLoS One, 9(6): e98813, 2014.
- Madeira, A., dos Santos, A.C., Santos, P.M., Coutinho, C.P., Tyrrell, J., McClean, S., Callaghan, M., Sá-Correia, I., Proteomic profiling of Burkholderia cenocepacia clonal isolates with different virulence potential retrieved from a cystic fibrosis patient during chronic lung infection, PLOS ONE 8(12): e83065, 2013.
- Silva, I.N., Ferreira, A.S., Becker, J.D., Zlosnik, J.E., Speert, D.P., He, J., Mil-Homens, D., Moreira, L.M., Mucoid morphotype variation ofBurkholderia multivorans during chronic cystic fibrosis lung infection is correlates with changes in metabolism, motility, biofilm formation and virulence, Microbiology , 157(Pt 11):3124-37, 2011.
- Coutinho, C.P., de Carvalho, C.C.C.R., Madeira, A., Pinto-de-Oliveira, A., Sá-Correia, I., Burkholderia cenocepacia phenotypic clonal variation during three and a half years of residence in the lungs of a cystic fibrosis patient. Infection and Immunity, 79, 2950-2960, 2011.
- Madeira, A., Santos, P.M., Coutinho, C.P., Pinto-de-Oliveira, A., Sá-Correia, I., "Quantitative proteomics (2-D-DIGE) reveals molecular strategies employed by Burkholderia cenocepacia to adapt to the airways of cystic fibrosis patients under antimicrobial therapy",PROTEOMICS , 11: 1313-1328, 2011
- Mil-Homens, D., Rocha, E.P.C., Fialho, A.M. "Genome-wide analysis of DNA repeats in Burkholderia cenocepacia J2315 identifies a novel adhesin-like gene unique to epidemic-associated strains of ET-12 lineage",Microbiology , 156: 1084-1096, 2010.
- Sousa, S.A., Ramos, C.G., Moreira, L.M., Leitão, J.H. "The hfq gene is required for stress resistance and full virulence of Burkholderia cepacia to the nematode Caenorhabditis elegans", Microbiology , 156: 896-908, 2010.
- Ramos, C.G., Sousa, S.A., Grilo, A.M., Eberl, L., Leitão, J.H., "The Burkholderia cenocepacia K56-2 pleiotropic regulator Pbr, is required for stress resistance and virulence", Microbial Pathogenesis , 48: 168-177, 2010.