Download TolC-like Burkholderia

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.