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Identification of TolC-like proteins in the genome of strains from Burkholderia cepacia complex Supervision: Ana Sofia Ferreira ([email protected]) and Leonilde Moreira ([email protected] ) Grupo de Ciências Biológicas, Centro de Engenharia Biológica e Química, Departamento de Engª Química e Biológica, IST The genus Burkholderia is composed of Gram-negative bacteria, which are ubiquitous in the environment and may cause a number of diseases in plants. Human infections can also be caused by Burkholderia, especially in patients with cystic fibrosis (CF), and is often fatal (Cunha et al., 2003). The identification of virulence factors and the understanding of the mechanisms of pathogenesis is crucial to identify new drug targets against these microorganisms. Several species of Burkholderia have the genome fully sequenced and have genetic information for the synthesis of several extracellular proteins like hemolysins, lipases and proteases that are virulence factors in other bacterial systems. Nevertheless, the role of these molecules in human infections caused by this species is still unknown. Experimental data obtained for other Gram-negative bacteria showed that secretion of pathogenesis-related proteins across the bacterial cytoplasmic and outer membranes requires several export systems like type I, type II and type III secretion systems (Sharff et al., 2001). These secretion systems require outer membrane proteins forming channels through which the toxic proteins or antimicrobials are secreted. One of them, TolC, is an important though low-abundance protein in the outer membrane of Gram-negative bacteria. The crystal structure of this protein has recently been determined and it exists functionally as a trimer forming a β-barrel with a large internal diameter, facilitating movement of both large and small molecules through the outer membrane (Koronakis et al., 2000). TolC has been associated with several multidrug resistance (MDR) efflux systems involved in the removal of a broad range of toxic chemicals from the cell (Sulavik et al., 2001) and also implicated in the secretion of virulence-associated proteins like metalloproteases, α-hemolysis, enterotoxins, colicins in E. coli, Vibrio cholerae, Salmonella enterica, Serratia marcescens among others (Stone and Miller, 1995; Yamanaka et al., 1998). Due to the knowledge of the sequence of several Burkholderia cepacia complex strains, it would be interesting to identify and characterize all the TolC-like porins in this strain as well as the associated transporters. Objectives In this project, the whole genome sequences of Burkholderia cepacia complex strains available at www.sanger.ac.uk and http://imgweb.jgi-psf.org/cgi-bin/w/main.cgi will be searched for the presence of genes putatively coding for outer membrane porins homologous to TolC from Enterobacteria. If in the adjacent regions of a porin gene, encoding membrane gene transporters are present, they will be characterized as well. This computational approach will include homology search using known TolC-like proteins. The possible protein candidates obtained will be characterized for their cellular localization and topology, for the presence of conserved motifs, etc using several computational tools available in the Web. The identification of these putative tolC encoding genes in Burkholderia opens the door to experimental analysis aiming their deletion from the genome and phenotypic characterization of the mutants concerning the presence of secreted proteins in the supernatant and the resistance to antimicrobial chemicals as well as pathogenesis in an animal model. References Cunha MV, Leitao JH, Mahenthiralingam E, Vandamme P, Lito L, Barreto C, Salgado MJ, SaCorreia I. Molecular analysis of Burkholderia cepacia complex isolates from a Portuguese cystic fibrosis center: a 7-year study. J Clin Microbiol. 9:4113-20, 2003 Koronakis V, Sharff A, Koronakis E, Luisi B, Hughes C. Crystal structure of the bacterial membrane protein TolC central to multidrug efflux and protein export. Nature. 405:914-9, 2000. Sharff A, Fanutti C, Shi J, Calladine C, Luisi B. The role of the TolC family in protein transport and multidrug efflux. From stereochemical certainty to mechanistic hypothesis. Eur J Biochem. 268:501126, 2001. Sulavik MC, Houseweart C, Cramer C, Jiwani N, Murgolo N, Greene J, DiDomenico B, Shaw KJ, Miller GH, Hare R, Shimer G. Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes. Antimicrob Agents Chemother. 45:1126-36, 2001. Yamanaka H, Nomura T, Fujii Y, Okamoto K. Need for TolC, an Escherichia coli outer membrane protein, in the secretion of heat-stable enterotoxin I across the outer membrane. Microb Pathog. 25:111-20, 1998. Stone BJ, Miller VL. Salmonella enteritidis has a homologue of tolC that is required for virulence in BALB/c mice. Mol Microbiol. 17:701-12, 1995.