Bacterial proteins presenting a particular subcellular localization are regularly described in diverse bacteria, and our model of study Brucella abortus, an intracellular pathogen, does not depart from the rule. Indeed, in our laboratory, we recently showed that an essential histidine kinase named PdhS and a fumarate hydratase called FumC localize at the old pole in B. abortus. These data suggest that a set of proteins with various functions could be associated with one of both bacterial poles, old or new. To test this hypothesis, we initiated an exploratory project to identify on one hand, markers of old and new poles and on the other hand, functions specifically associated with the poles. The proposed scientific strategy used an approach on the scale of the Brucella melitensis ORFeome. The detection of the polar localization of proteins was performed by genetic fusion with the fluorescent protein YFP (yellow fluorescent protein). Among seven identified candidate proteins, we were interested in two proteins in particular, a predicted acyl-CoA dehydrogenase, named AidB, given its homology with the Escherichia coli AidB protein, and a hypothetical protein called IfoP (for In Front Of PdhS). The functional characterization of these two candidates, allowed us to demonstrate that the aidB and ifoP genes are not essential for the viability of B. abortus and that AidB and IfoP proteins are distributed asymmetrically in B. abortus. AidB localizes systematically at the new pole and/or at the constriction site in B. abortus. The overexpression of aidB provokes morphological defects, typically the presence of additional poles. The genome of B. melitensis encodes 9 other putative acyl-CoA dehydrogenases and two of them have a diffuse localization in the cytoplasm and do not present particular morphological phenotype when they are overproduced. Thus, the polar localization of AidB and the presence of morphological aberrations for the overexpression mutant seem to be specific characteristics of this acyl-CoA dehydrogenase. These data suggest that AidB is a marker of new poles and constriction sites, that can be considered as sites of preparation of new poles for sibling cells. AidB could have a role in the generation of growing new poles and/or in cell cycle control in B. abortus. Furthermore, AidB remains polar during a cellular infection and in the presence of an alkylating agent (EMS). On the other hand, the aidB mutant is more sensitive than the wild type strain to an EMS treatment, suggesting that the aidB gene would be involved in the repair or the prevention of alkylating damages. IfoP localizes almost always at the opposite pole of PdhS, therefore considering IfoP as a marker of new or “young” pole. Previous studies indicated that B. abortus divides asymmetrically, following the example of Caulobacter crescentus, to produce a small cell and a large cell. The use of IfoP as marker of the small cell suggests that this one would be the privileged state in infection model in epithelial cell, in the non-proliferative phase of the infection, which means at early time of infection.
Identification et caractérisation de deux protéines polaires de Brucella abortus, IfoP et AidB
Dotreppe, D. (Author). 7 Oct 2011
Student thesis: Doc types › Doctor of Sciences