Regulation of exopolysaccharide biosynthesis and virulence of Burkholderia cepacia by protein-tyrosine phosphorylation

Principal Investigator: Leonilde Morais Moreira
Contract: PTDC/BIA-MIC/65210/2006
Start date: 01/12/2007

Duration: 36 months

Protein tyrosine phosphorylation occurs in all living organisms and plays a central role in eukaryotes by regulating a wide variety of cellular processes, such as proliferation, differentiation and oncogenesis. It has also been demonstrated to occur in a wide array of bacterial species and appears to be ubiquitous among prokaryotes. This covalent modification is catalyzed by autophosphorylating ATP-dependent protein tyrosine kinases (PTKs). The reversibility of the reaction is carried out by protein tyrosine phosphatases (PTPs). In bacterial species, the genes encoding PTKs and PTPs are most often located next to each other and are generally part of large operons that direct the synthesis of proteins involved in the production of polysaccharides and its regulation. Several studies reported a direct relationship between reversible phosphorylation of the PTKs on tyrosine and the production of polysaccharides, which are also known to be important virulence factors. UDP-glucose dehydrogenase, an enzyme catalyzing the formation of nucleotide sugar precursors for polysaccharide synthesis and involved in antibiotic resistance, is another endogenous substrates of PTKs/PTPs. Protein-tyrosine phosphorylation is also involved in the activation or inhibition of transcription factors regulating heat shock responses. A biological system where protein-tyrosine phosphorylation may have an important role in exopolysaccharide (EPS) synthesis and virulence is the Burkholderia cepacia complex (BCC) isolates. These microorganisms are important opportunistic pathogens causing persistent respiratory infections in patients with cystic fibrosis. Most of these bacterial strains produce an EPS, cepacian, hypothesized to be a persistence and virulence determinant. It was recently identified in our laboratory the bce cluster of genes, which directs the synthesis of cepacian. Within this cluster, map the bceD and bceF genes, encoding a phosphotyrosine phosphatase and a tyrosine autokinase, respectively. The disruption of the bceF gene resulted in the abolishment of cepacian accumulation in the culture medium, but 75% of the parental strain EPS production yield was still registered for the bceD mutant. The EPS produced by the bceD mutant exhibited the same sugar composition as the wild-type cepacian but led to less viscous solutions, suggesting a lower molecular mass of this EPS. Due to the importance of bacterial biofilms in pathogenesis, the size of the biofilms produced, in a microtiter plate assay, by bceD and bceF mutants was evaluated and it was observed a smaller biofilm size when compared to the one formed by the parental strain B. cepacia IST408. Virulence tests performed with Caenorhabditis elegans showed a reduced ability of the bceD and bceF mutants to kill the nematode, with the bceF mutant having the more pronounced effect. Whether the bceD and bceF mutants phenotype concerning biofilm formation and virulence in C. elegans is solely due to the EPS or also from other functions of the tyrosine kinase and phosphatase in presently unknown. Therefore, the main goal of this proposal is to understand the role of protein-tyrosine phosphorylation regarding EPS biosynthesis, biofilm formation and virulence in B. cepacia. Specifically we propose to: 1) evaluate the importance on cepacian biosynthesis of the phosphorylation of BceF tyrosine residues by introducing site-specific mutations; 2) determine possible interactions between the proteins involved  in the polymerization/secretion of cepacian (BceA, B, C, D, E, F, I and L) using two different approaches: a) in vivo, by using a bacterial two-hybrid system and b) in vitro, by surface plasmon resonance; 3) assess biofilm development from bceD and bceF mutants and wild-type strain in a continuous flow reactor; 4) evaluate the role of bceD and bceF in the BALB/c mouse model of infection and study their adhesion properties to human neutrophils and macrophages; 5) identify novel substrates for BceD and BceF using two different approaches: a) co-immunoprecipitation of the tyrosine kinase or the phosphatase complexes followed by protein identification, b) comparison the gene expression levels, using B. cenocepacia microarrays, of the wild-type strain, bceD and bceF mutants grown in a medium that mimics the lung environment in a normal atmosphere and under 5% CO2; 6) search for possible inhibitors of PTK activity. With this set of proposed experiments, new insights in the involvement of protein-tyrosine phosphorylation in EPS biosynthesis and in bacterial pathogenicity will be provided. This knowledge could help in the near future in the identification of new targets for the development of antibacterial drugs. This proposal includes researchers from the Center for Biological and Chemical Engineering/IST, from Gulbenkian Institute, and has the collaboration of international laboratories (Univ. of British Columbia, Canada; Technical Univ. of Denmark).