RESUMO
The flagellar regulon of Brucella melitensis 16M contains 31 genes clustered in three loci on the small chromosome. These genes encode a polar sheathed flagellum that is transiently expressed during vegetative growth and required for persistent infection in a mouse model. By following the expression of three flagellar genes (fliF, flgE, and fliC, corresponding to the MS ring, hook, and filament monomer, respectively), we identified a new regulator gene, ftcR (flagellar two-component regulator). Inactivation of ftcR led to a decrease in flagellar gene expression and to impaired Brucella virulence. FtcR has a two-component response regulator domain as well a DNA binding domain and is encoded in the first flagellar locus of B. melitensis. Both the ftcR sequence and its genomic context are conserved in other related alpha-proteobacteria. During vegetative growth in rich medium, ftcR expression showed a peak during the early exponential phase that paralleled fliF gene expression. VjbR, a quorum-sensing regulator of the LuxR family, was previously found to control fliF and flgE gene expression. Here, we provide some new elements suggesting that the effect of VjbR on these flagellar genes is mediated by FtcR. We found that ftcR expression is partially under the control of VjbR and that the expression in trans of ftcR in a vjbR mutant restored the production of the hook protein (FlgE). Finally, FtcR binds directly to the upstream region of the fliF gene. Therefore, our data support the role of FtcR as a flagellar master regulator in B. melitensis and perhaps in other related alpha-proteobacteria.
Assuntos
Proteínas de Bactérias/genética , Brucella melitensis/genética , Flagelos/genética , Regulon , Rhizobiaceae , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/genética , Brucella melitensis/patogenicidade , Proteínas de Membrana/genética , Dados de Sequência Molecular , Alinhamento de Sequência , VirulênciaRESUMO
Trypanosomatids, unicellular organisms responsible for several global diseases, contain unique organelles called glycosomes in which the first seven glycolytic enzymes are sequestered. We report the crystal structures of glycosomal fructose-1,6-bisphosphate aldolase from two major tropical pathogens, Trypanosoma brucei and Leishmania mexicana, the causative agents of African sleeping sickness and one form of leishmaniasis, respectively. Unlike mammalian aldolases, the T. brucei and L. mexicana aldolases contain nonameric N-terminal type 2 peroxisomal targeting signals (PTS2s) to direct their import into the glycosome. In both tetrameric trypanosomatid aldolases, the PTS2s from two different subunits form two closely intertwined structures. These "PTS2 dimers", which have very similar conformations in the two aldolase structures, are the first reported conformations of a glycosomal or peroxisomal PTS2, and provide opportunities for the design of trypanocidal compounds.
Assuntos
Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/metabolismo , Leishmania mexicana/enzimologia , Peroxissomos/metabolismo , Sinais Direcionadores de Proteínas/química , Trypanosoma brucei brucei/enzimologia , Sequência de Aminoácidos , Animais , Transporte Biológico , Cristalografia por Raios X , Dimerização , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/fisiologia , Estrutura Quaternária de Proteína , Alinhamento de SequênciaRESUMO
Kinetoplastid organisms, such as the protozoan parasite Trypanosoma brucei, compartmentalise several important metabolic pathways in organelles called glycosomes. Glycosomes are related to peroxisomes of yeast and mammalian cells. A subset of glycosomal matrix proteins is routed to the organelles via the peroxisome-targeting signal type 1 (PTS-1). The PEX5 gene homologue has been cloned from T. brucei coding for a protein of the translocation machinery, the PTS-1 receptor. The gene codes for a polypeptide of 654 amino acids with a calculated molecular mass of 70 kDa. Like its homologue in other organisms T. brucei PTS-1 receptor protein (TbPEX5) is a member of the tetratricopeptide repeat (TPR) protein family and contains several copies of the pentapeptide W-X-X-X-F/Y. Northern and Western blot analysis showed that the protein is expressed at different stages of the life cycle of the parasite. The protein has been overproduced in Escherichia coli and purified using immobilized metal affinity chromatography. The purified protein specifically interacts in vitro with glycosomal phosphoglycerate kinase-C (PGK-C) of T. brucei, a PTS-1 containing protein. The equilibrium dissociation constant (Kd) of PGK-C for purified TbPEX5 is 40 nM. Using biochemical and cytochemical techniques a predominantly cytosolic localization was found for TbPEX5. This is consistent with the idea of receptor cycling between the glycosomes and the cytosol.