The second reaction conjugates the cytosolic soluble LC3-I (microtubule-associated protein 1 light chain 3) to a phosphatidylethanolamine (PE) in the presence of Atg4, Atg3 and Atg7 producing the membrane-associated LC3-II form [19–21]. The Atg5-Atg12 conjugates are essential for the maturation of the isolation membrane into autophagosome and targeting of LC3 to the membrane [18]. Recently, using epithelial cells and macrophages deficient in one of the regulatory proteins of the conventional macroautophagic pathway, Starr et al. [12] have found that core selleck proteins of this canonical macroautophagy machinery such as ULK-1, Beclin1, Atg5, Atg7, LC3B were not necessary for the intracellular

trafficking of B. abortus between the endocytic compartments and the ER-derived vesicles and for its replication [12]. Nevertheless, the conversion of rBCV to aBCV at a later stage of infection, i.e. 48 h and 72 h p.i., seems to be dependent on ULK-1, Beclin1, Atg14L and hVps34 but independent on Atg5, Atg7, Atg16L1 and Atg4B [12]. On the other hand, Guo et al. [22] have observed that infection by B. melitensis

induced macroautophagy that in turn favoured its replication in RAW264.7 macrophages [22]. This later study raises the possibility that in contrast to B. abortus, ARRY-438162 research buy B. melitensis could subvert macroautophagy to replicate in host cells. In our present work, we addressed this issue using embryonic fibroblasts from this website wild-type and Atg5-knockout mice infected or not with B. abortus and B. melitensis. Results Relative abundance of LC3-I and LC3-II in infected mouse embryonic fibroblasts As it has been shown that B. melitensis stimulated macroautophagy

in macrophages to favour its replication L-gulonolactone oxidase [22], we sought to determine whether this also occurred in infected MEFs. First, we established clones stably transfected with GFP-LC3 to monitor the formation of autophagic vacuoles by fluorescence microscopy. As expected [19], in basal conditions, the fluorescent staining in GFP-LC3 expressing cells was faint and diffuse while under starvation conditions, it was more punctuate, due to the recruitment of LC3 onto autophagosomal membranes (Additional file 1). In contrast, when the same cells were infected with B. abortus or with B. melitensis, the GFP-LC3 staining remained diffuse and colocalisation between GFP-LC3 and Texas Red-labelled bacteria was only very occasionally detected. Then, we examined the relative abundance of LC3-I and LC3-II by Western blotting. Preliminary experiments showed that in WT MEFs, LC3-II was detected even in basal conditions (Figure 1A). After 2 h of starvation in EBSS, the abundance of both LC3-I and LC3-II decreased, probably due to an acceleration of the autophagic flow since LC3-II is degraded when autophagosomes fuse with lysosomes.