As expected, in Atg5−/− MEFs, LC3-II was never detected
whatever the cell culture conditions because the presence of Atg5 is absolutely required for the LC3 recruitment onto autophagosome membrane . In WT MEFs infected with B. abortus or with B. melitensis, the relative abundance of LC3-I and LC3-II at 18 h p.i. did not change when compared to non-infected MEFs (Figure 1B). Figure 1 Relative abundance of LC3B-I and LC3B-II in WT MEFs and in Atg5 selleck chemicals −/− MEFs as determined by immunoblotting. A. Cells were maintained in DMEM/FCS (F), starved for 2 h in EBSS (S) or incubated for 5 h in the presence of 100 nM bafilomycin (Baf). B. Cells were infected with B. abortus (BA) or with B. melitensis (BM) for 18 h or left non infected (Ctl). Replication of B. abortus- and B. melitensis-mCherry in Atg5−/− fibroblasts We studied the contribution of the macroautophagic pathway on the replication of Brucellae using Atg5-deficient MEFs. First, we infected cells with B. abortus-mCherry (Figure 2A) or with B. melitensis-mCherry
(Figure 2B) for 1 h at a multiplicity of infection (MOI) of 300. After inoculation, the medium was removed and replaced by a medium containing gentamicin to kill extracellular bacteria. selleck chemicals llc As it can be seen on micrographs taken after increasing times postinfection, B. abortus-mCherry is able to enter, survive and replicate in MEFs, even in Atg5-deficient MEFs. In both cell lines, at 6 h p.i, there are only a few bacteria per infected cell but this number massively increases between 12 and 18 h p.i. and at 24 h p.i., the bacteria are so abundant that it is difficult to Semaxanib enumerate them. B. melitensis-mCherry is also able to replicate in both WT MEFs and Atg5−/− MEFs. However, it is clear that Prostatic acid phosphatase the number of bacteria per infected cell at 24 h p.i. is lower compared to B. abortus-mCherry. Statistical analysis of these observations revealed that there is no significant difference in the number of B. abortus-mCherry per infected cell between the Atg5-deficient MEFs and the WT MEFs whatever the time postinfection (Figure 3A). In contrast, the number of B. melitensis-mCherry
per infected cell significantly increased in Atg5−/− MEFs when compared to WT MEFs at 9 h, 18 h and 24 h p.i. (Figure 3B). These data demonstrate that both Brucella strains can survive and replicate when the conventional Atg5-dependent macroautophagic pathway is impaired. Atg5-deficient cells seem to be even more permissive for B. melitensis replication than WT MEFs. Figure 2 Fluorescence microscopy analysis of WT MEFs and Atg5 −/− MEFs infected with B. abortus -mCherry (A) or with B. melitensis- mCherry (B). MEFs were infected for 1 h with Brucella-mCherry at an MOI of 300 and observed at 6 h, 12 h, 18 h and 24 h p.i. The nuclei were stained with DAPI. Figure 3 Quantification of the infection of WT MEFs and Atg5 −/− MEFs with B. abortus -mCherry (A) or with B. melitensis- mCherry (B). MEFs were infected for 1 h with Brucella-mCherry at an MOI of 300.