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Research Project
KINASE- Phosphoproteomics in Burkholderia to assess the role of tyrosine phosphorylation in virulence determinants expression
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Comparative Transcriptomic Analysis of the Burkholderia cepacia Tyrosine Kinase bceF Mutant Reveals a Role in Tolerance to Stress, Biofilm Formation, and Virulence
Publication . Ferreira, A. S.; Silva, I. N.; Oliveira, V. H.; Becker, J. D.; Givskov, M.; Ryan, R. P.; Fernandes, F.; Moreira, L. M.
The bacterial tyrosine-kinase (BY-kinase) family comprises the major group of bacterial enzymes endowed with tyrosine kinase activity. We previously showed that the BceF protein from Burkholderia cepacia IST408 belongs to this BY-kinase family and is involved in the biosynthesis of the exopolysaccharide cepacian. However, little is known about the extent of regulation of this protein kinase activity. In order to examine this regulation, we performed a comparative transcriptome profile between the bceF mutant and wild-type B. cepacia IST408. The analyses led to identification of 630 genes whose expression was significantly changed. Genes with decreased expression in the bceF mutant were related to stress response, motility, cell adhesion, and carbon and energy metabolism. Genes with increased expression were related to intracellular signaling and lipid metabolism. Mutation of bceF led to reduced survival under heat shock and UV light exposure, reduced swimming motility, and alteration in biofilm architecture when grown in vitro. Consistent with some of these phenotypes, the bceF mutant demonstrated elevated levels of cyclic-di-GMP. Furthermore, BceF contributed to the virulence of B. cepacia for larvae of the Greater wax moth, Galleria mellonella. Taken together, BceF appears to play a considerable role in many cellular processes, including biofilm formation and virulence. As homologues of BceF occur in a number of pathogenic and plant-associated Burkholderia strains, the modulation of bacterial behavior through tyrosine kinase activity is most likely a widely occurring phenomenon.
The regulator LdhR and the d-lactate dehydrogenase LdhA of Burkholderia multivorans play a role in carbon overflow and in planktonic cellular aggregates formation
Publication . Silva, Inês N.; Ramires, Marcelo J.; Azevedo, Lisa A.; Guerreiro, Ana R.; Tavares, Andreia C.; Becker, Jörg D.; Moreira, Leonilde M.
LysR-type transcriptional regulators (LTTR) are the most commonly found regulators in Burkholderia cepacia complex, comprising opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. Despite LTTRs being global regulators of pathogenicity in several bacteria, few have been characterized in Burkholderia Here, we showed that gene ldhR of B. multivorans encoding a LTTR is co-transcribed with ldhA encoding a d-lactate dehydrogenase, and evaluate their implication in virulence traits like exopolysaccharide (EPS) synthesis and biofilm formation. Comparison of wild-type (WT) and its isogenic ΔldhR mutant grown in medium with 2% d-glucose revealed a negative impact on EPS biosynthesis and on cells' viability in the presence of LdhR. Loss of viability in WT cells was caused by intracellular acidification as consequence of cumulative organic acids secretion including d-lactate, this last one absent from the ΔldhR mutant supernatant. Furthermore, LdhR is implicated in the formation of planktonic cellular aggregates. WT cell aggregates reached 1000 μm after 24 hours in liquid cultures; in contrast to ΔldhR mutant aggregates that never grew more than 60 μm. Overexpression of d-lactate dehydrogenase LdhA in the ΔldhR mutant partially restored formed aggregates size, suggesting a role for fermentation inside aggregates. Similar results were obtained for surface-attached biofilms, with WT cells producing more biofilm. A systematic evaluation of planktonic aggregates in Burkholderia CF clinical isolates showed aggregates in 40 out of 74. As CF patients' lung environment is microaerophilic and bacteria are found as free aggregates/biofilms, LdhR and LdhA might have central roles in adaptation to this environment.IMPORTANCE Cystic fibrosis patients often suffer from chronic respiratory infections caused by several microorganisms. Among them are the Burkholderia cepacia complex bacteria which cause progressive deterioration of lung function and, in some patients, might develop into fatal necrotizing pneumoniae with bacteremia, known as "cepacia syndrome". Burkholderia pathogenesis is multifactorial since they express several virulence factors, form biofilms, and are highly resistant to antimicrobial compounds, making their eradication from the CF patients' airways very difficult. As Burkholderia is commonly found in the CF lungs in the form of cell aggregates and biofilms, the need to investigate the mechanisms of cellular aggregation is obvious. In this study we demonstrate the importance of a d-lactate dehydrogenase and a regulator, in regulating carbon overflow, cellular aggregates and surface-attached biofilm formation. This not only enhances our understanding of Burkholderia pathogenesis, but can also lead to the development of drugs against these proteins to circumvent biofilm formation.
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Funding agency
Fundação para a Ciência e a Tecnologia
Funding programme
3599-PPCDT
Funding Award Number
PTDC/QUI-BIQ/118260/2010