abortusAidB deduced sequence is blasted againstE
abortusAidB deduced sequence is blasted againstE. new poles and at constriction sites seems to be specific for this ACAD homolog since two other ACAD homologs fused to YFP did not show specific localization. The overexpression ofaidB, but not the two other ACAD coding sequences, leads to multiple morphological defects. == Conclusions == Data reported here suggest that AidB is a marker of new poles and constriction sites, that could be considered as sites of preparation of new poles in the sibling cells originating from cell division. The possible role of AidB in the generation or the function of new poles needs further investigation. == Background == Brucellais the etiologic agent of brucellosis, a worldwide zoonosis that affects a broad range of mammals, including humans [1].Brucellais considered as a facultative intracellular pathogen that enters various cell types during the infection process, including macrophages and epithelial cells, and ultimately survives and multiplies inside these cells [2]. After internalization, intracellularBrucellaresides within a vacuole (BCV forBrucella-containing vacuole) that DPCPX interacts with early endosomes [3] and then transiently acquire markers of late endosomes such as LAMP1. In epithelial cells and macrophages, non-opsonized bacteria replicate finally DPCPX in a compartment characterized by the presence of endoplasmic reticulum (ER) markers [4-7]. The mechanisms used byBrucellato sense and respond to changes within the intracellular environment are poorly understood.Brucellaspp. seem well adapted to cope with nutritional [8] and various physicochemical stresses encountered in non-professional and especially professional phagocytes [9]. For example,Brucellaspp. are adapted to oxidative and nitrosative stresses [9] that have been shown to affect genome integrity in other bacterial species. In 2002, Khleret al. identified an attenuated mutant with a mini-transposon in theaidBgene, proposed to encode an acyl-CoA dehydrogenase homolog [10]. InEscherichia coli, AidB protein takes part of the adaptative response to alkylating agents that could damage the genome [11], suggesting that AidB homolog could play a similar role inB. abortus. Moreover, aBrucella melitensismutant in thealkAgene was found to be attenuated in DPCPX mice (Pascal Lestrate, Ph.D. thesis, 2003). ThealkAgene Mouse monoclonal to CD33.CT65 reacts with CD33 andtigen, a 67 kDa type I transmembrane glycoprotein present on myeloid progenitors, monocytes andgranulocytes. CD33 is absent on lymphocytes, platelets, erythrocytes, hematopoietic stem cells and non-hematopoietic cystem. CD33 antigen can function as a sialic acid-dependent cell adhesion molecule and involved in negative selection of human self-regenerating hemetopoietic stem cells. This clone is cross reactive with non-human primate * Diagnosis of acute myelogenousnleukemia. Negative selection for human self-regenerating hematopoietic stem cells is homologous toE. coli alkA, another gene involved in the adaptative response to alkylating stress [12,13]. In summary, these data suggests that DNA alkylation repair systems could play a role in intracellular persistence, possibly by preventing DNA damage that might be induced by alkylating agents, either produced from endogenous sources [14] or induced by the host during the infection process. Here we report that while screeningBrucellaORFeome for polar proteins inBrucella abortus, AidB was found to localize at the new pole, as well as at the constriction site in dividing cells. This pattern of localization is maintained inB. abortusinfecting epithelial cells and macrophages at different times post-infection. Analysis of anaidBmutant revealed on one hand no effect on virulence, and on the other hand that theaidBmutant was more sensitive to the alkylating agent methanesulfonic acid ethyl ester (EMS), suggesting a function of AidB in the defence against DNA methylation damage. While EMS was found to block cell cycle before cell constriction, aB. abortusstrain overexpressingaidBwas found to generate multipolar morphologies, suggesting a link between the response to alkylating agents and cell growth and/or division. == Results == == Screen for polarly localized proteins inBrucella abortus == To identify polar proteins at the genomic scale, we took advantage of theBrucella melitensisORFeome [15], a collection of all predicted coding sequences (pCDSs) fromB. melitensisgenome cloned in a donor vector (pDONR201) allowing the Gateway recombinational cloning. The resulting ~3200 entry clones are physically organized in 96-well plates (34 plates), each well containing one entry clone (one clonedB. melitensispCDS). For some large-scale experiments, theBrucellaORFeome is also organized in 68 pools [16], each pool being a mix of clones from one half-plate of the original ORFeome. Each of the 68 pools was used to transfer the pCDSs in a destination vector allowing pCDS-yfpfusions under the control ofE. coli lacpromoter, on a low copy number plasmid. More than 1000 transformants (> 10 times the diversity of the pool) were generated for each cloned pool of pCDSs. The pools of constructions were transformed intoE. colistrain S17-1 (> 1000 transformants/pool) and were transferred in aBrucella abortusXDB1155 strain [16] by mating. The XDB1155 strain produces the PdhS-CFP (cyan fluorescent protein) fusion protein from.