The bacterial and cellular determinants controlling the recruitment of mTOR to the Salmonella-containing vacuole

Abstract

Bacterial invasion results in the rapid induction of an acute state of cytosolic amino acid (AA) starvation, provoked by host membrane damage. Bacteria-induced AA starvation, in turn, down-regulates mTOR signaling while triggering autophagy and the integrated stress response pathway dependent on GCN2, eIF2α and ATF3. In Salmonella-infected cells, we now demonstrate that the host AA starvation response program depended on the Salmonella pathogenicity island (SPI)-1, the activity of which was required to damage the Salmonella-containing vacuole (SCV) in the early stage of infection. At a later stage (3-4 hour post-infection), the progressive recruitment of mTOR to the surface of the SCV appeared to be independent of the activity of SPI-2 and of SCV positioning in the cell. Instead, mTOR localization to the SCV required the activity of host AA transporters SLC1A5, SLC3A2 and SLC7A5, resulting in bacterial escape from autophagy. These results expand our understanding of the mechanisms underlying the AA starvation response in Salmonella-infected cells.

Keywords: Amino acid starvation; Salmonella; mTOR.

Fig. 1.. SPI-1-dependent SCV membrane damage causes early host AA starvation responses.

(A,B) HeLa cells were infected with wild type (WT) or ΔSPI-1/Inv Salmonella strain for 2 h or 4 h, analyzed by IF using antibodies against NDP52 and LAMP2 (A) or mTOR and LAMP2 (B). (C) qPCR analysis of AFT3 and IL-8 induction in HeLa cells infected with WT or ΔSPI-1/Inv Salmonella strains. Values are means s.e.m. n = 3.

Fig. 2.. Membrane recruitment of NDP52, p62 and ubiquitin is not influenced by AA starvation.

HeLa cells were infected with Salmonella in either a normal AA-rich DMEM medium or in an AA-depleted medium (KRB) for 1 h, 2 h or 4 h. Next, cells were fixed and analyzed by IF using antibodies against NDP52, p62 and ubiquitin.

Fig. 3.. Enforced peri-nuclear clustering of SCVs does not impact on the recruitment of mTOR.

HeLa cells were transfected with an expression vector encoding for RILP-GFP, infected with Salmonella for 2 h or 4 h, and mTOR sub-cellular localization was identified by IF using an anti-mTOR antibody.

Fig. 4.. AA starvation responses in Salmonella-infected cells are SPI-2-independent.

(A) HeLa cells were infected with wild type (WT) or ΔSPI-2 Salmonella strains for 4 h, analyzed by IF using antibodies against mTOR and LAMP2. (B) HeLa cells were infected with the WT or ΔSPI-2 Salmonella strain for 1 h or 3 h, analyzed by blotting using the antibodies indicated. (C) qPCR analysis of AFT3 and IL-8 induction in Hela cells infected with WT or ΔSPI-2 Salmonella strain. Values are means ± s.e.m.

Fig. 5.. Essential role of LNAAs and L-glutamine in mTOR targeting to the SCV.

(A,B) HeLa cells were placed in AA-depleted medium (KRB) supplemented with the indicated AA, left uninfected (A) or infected with Salmonella for 4 h (B), and analyzed by IF using antibodies against mTOR and LAMP2. (C) Percentage of cells displaying mTOR targeting to the SCV following infection by Salmonella in AA-depleted medium supplemented with various AA or DMEM. Values are means s.e.m. n = 3. *P<0.05, **P<0.01 over infected in KRB (−AA). (D) HeLa cells were placed in various minimal media (−AA, −AA/L-Leu, −AA/L-Gln, −AA/L-Gln+L-Leu) as indicated, and infected with Salmonella for 4 h. At 2 h p.i., medium was removed and replaced for an additional 2 h. Next, cells were fixed and analyzed by IF using antibodies against mTOR and LAMP2. (E) HeLa cells were incubated in various media as indicated, left uninfected or infected with Salmonella for 4 h, analyzed by blotting using the antibodies indicated. In the “DMEM → KRB 2 h” condition, cells were incubated for 2 h in DMEM before medium changed to KRB for an additional 2 h.

Fig. 6.. Essential role of LNAA transporters in mTOR targeting to the SCV.

(A) HeLa cells either uninfected or infected with Salmonella for 4 h, in the presence or absence of the LNAA transporter inhibitor D-Phenylalanine (D-Phe), were fixed and analyzed by IF using antibodies against mTOR and LAMP2. (B) HeLa cells were transduced with lentiviruses targeting a scramble sequence or SLC7A5, left unstimulated (CTR) or infected with Salmonella for 4 h, analyzed by IF using antibodies against mTOR and LAMP2. (C) HeLa cells were transduced with lentiviruses targeting a scramble sequence (Scr), SLC1A5, SLC3A2, or SLC7A5, left unstimulated (CTR) or infected with Salmonella for 4 h, analyzed by blotting using the antibodies indicated.

Fig. 7.. Sub-cellular localization of SLC7A5 in Salmonella-infected cells.

(A) HeLa cells were transfected overnight with an expression vector encoding for Myc-SLC7A5 and analyzed by IF using antibodies against Myc and Golgin-97. (B) HeLa cells were infected with Salmonella in the absence or presence of the Golgi dissassembly promoting drug Brefeldin A, added 30 min after HeLa cells were infected with Salmonella, in order to avoid potential side-effects on bacterial entry. Next, cells were fixed and analyzed by IF using antibodies against mTOR and LAMP2. (C) HeLa cells infected with Salmonella WT for 1 h or 4 h were analyzed by IF using antibodies against the Golgi marker protein Golgin-97 and SLC7A5 (D) HeLa cells were infected with Salmonella in the absence or presence of the inhibitor of clathrin-dependent endocytosis, Dynasore, added 30 min after HeLa cells were infected with Salmonella, in order to avoid potential side-effects on bacterial entry. Next, cells were fixed and analyzed by IF using antibodies against mTOR and LAMP2.

Fig. 8.. Sustained targeting of Salmonella by the autophagic machinery in SLC7A5-silenced cells.

MDAMC cells stably expressing GFP-LC3, transduced with a lentivirus targeting either a scramble sequence or SLC7A5, were infected with Salmonella for 4 h and analyzed by fluorescence microscopy.