abortus or with B. melitensis when compared to WT MEFs, all time points combined. The counting of fluorescent bacteria per infected cell, which takes into account living bacteria but also dead bacteria and bacteria that are no longer able to replicate, indicates that for B. abortus, there is no difference between the two cell lines even at short times postinfection (Figure 3A) whereas for B. melitensis, there is a significant increase in the Atg5−/− MEFs at 9, 18 h HSP inhibitor and 24 h. p.i.,

as compared to WT MEFs (Figure 3B). Therefore, for B. abortus, the higher CFUs in Atg5−/− MEFs vs WT MEFs could be explained by an increase in the percentage of infected cells among the cell population or by a higher survival rate during the early times after infection rather than by a higher replication rate. In contrast, for B. melitensis, the increase in the log CFU in Atg5-deficient cells could also result from a slight increase in the replication rate. Next, our data

reveal that there is no conversion of LC3-I to LC3-II in WT MEFs upon Brucella invasion and that neither B. abortus nor B. melitensis is detected in autophagic compartments stained with GFP-LC3, even under starvation conditions. This is consistent with the results of Starr et al. [12], which also showed that the siRNA-mediated Entinostat silencing of LC3B in HeLa cells did not impair the maturation of the BCV into a replicative niche in cells infected with B. abortus. In contrast, Guo et al. [22] proposed that B. melitensis infection induced autophagy because they observed an BAY 80-6946 solubility dmso accumulation of GFP-LC3-positive autophagic vacuoles and a conversion of LC3-I to LC3-II in infected

RAW264.7 macrophages, compared to control cells. Moreover, these authors showed that a treatment with the autophagy inhibitor 3MA attenuated the replication Nintedanib (BIBF 1120) efficiency of B. melitensis. It is not clearly indicated how long they incubated cells with this compound but it has been demonstrated that under nutrient-rich conditions, a prolonged treatment (up to 9 h) with 3MA could promote rather than inhibit the autophagy flux [24]. In contrast to Guo et al., [22], we did not observe a significant decrease in the CFU and in the number of Brucella per infected cells (except for B. melitensis at 24 h p.i.) in WT MEFs pretreated with 3MA. This discrepancy could be explained either by the incubation conditions or by a cell-type specificity. The subversion of the autophagic pathway by B. melitensis could occur in RAW264.7 macrophages but not in MEFs. Given the multifactorial effects of 3MA on cell metabolism [25], cells derived from Atg5 KO mice represent a more reliable tool to study the role of autophagy in different biological situations [18]. Based on our results with Atg5−/− MEFs, it is obvious that B. melitensis 16M as well as B. abortus are able to replicate in cells deficient in the canonical macroautophagy pathway.