Mammalian target of Rapamycin inhibition and mycobacterial survival are uncoupled in murine macrophages

33Citations
Citations of this article
60Readers
Mendeley users who have this article in their library.

This article is free to access.

Abstract

Background: Autophagy is a cellular response to intracellular pathogens including mycobacteria and is induced by the direct inhibitors of mammalian target of Rapamycin (mTOR), a major negative regulator of autophagy. Autophagy induction by mTOR inhibition (mTOR dependent autophagy), through chemical means or starvation, leads to mycobacterial killing in infected cells. However, previous work by our group has shown that mycobacterial infection of macrophages naturally induces both autophagy and mammalian target of Rapamycin (mTOR) activity (mTOR independent autophagy). In the current work, we further explore the relationship between mTOR activity and mycobacterial killing in macrophages. Results: While low concentrations of the mTOR inhibitors, Rapamycin, Torin 1, and Torin 2, can effectively reduce or block mTOR activity in response to lipopolysaccharides (LPS) or mycobacteria, higher concentrations (10 uM) are required to observe Mycobacterium smegmatis killing. The growth of M. smegmatis was also inhibited by high concentrations of Rapamycin in LC3B and ATG5 deficient bone marrow derived macrophages, suggesting that non-autophagic mechanisms might contribute to killing at high doses. Since mycobacterial killing could be observed only at fairly high concentrations of the mTOR inhibitors, exceeding doses necessary to inhibit mTOR, we hypothesized that high doses of Rapamycin, the most commonly utilized mTOR inhibitor for inducing autophagic killing, may exert a direct bactericidal effect on the mycobacteria. Although a short-term treatment of mycobacteria with Rapamycin did not substantially affect mycobacterial growth, a long-term exposure to Rapamycin could impact mycobacterial growth in vitro in select species. Conclusions: This data, coupled with previous work from our laboratory, further indicates that autophagy induction by mTOR inhibition is an artificial means to increase mycobacterial killing and masks more relevant endogenous autophagic biochemistry that needs to be understood. © 2014 Zullo et al.; licensee BioMed Central Ltd.

Figures

  • Figure 1 Low doses of Rapamycin, Torin 1, and Torin 2 inhibit mTOR and induce autophagy. (A) RAW264.7 cells were pretreated with 1 uM or 10 uM of the mTOR inhibitors indicated and then challenged with 1 ug/ml E. coli derived LPS for 3 hours. Protein lysates were prepared and western blots for total ribosomal S6 and phosphorylated ribosomal S6 are shown. Shown are data representative of two independent assays (B) RAW264.7 cells were infected with M. smegmatis (MOI 5) and treated with the mTOR inhibitors shown. Protein lysates were prepared and western blots for Actin and phosphorylated ribosomal S6 were performed. Shown are data representative of two independent assays (C) A549 cells were treated with 10 uM of the indicated inhibitor for 3 hours and then stained for endogenous LC3B, or an isotype control IgG, and imaged by fluorescence microscopy. Shown are data representative of two independent assays. (D) RAW264.7 cells were loaded with DQ-BSA, either left untreated (−DMSO) or treated overnight with the indicated concentrations of the mTOR inhibitors shown, and analyzed by flow cytometry. Shown is the combined percentage of DQ-BSA positive cells (+/− SEM) and the mean fluorescent intensity (and intensity range) derived from two independent assays with 3 replicates per assay. For analysis of the percent DQ-BSA positive cells, asterisks indicated p < 0.05 for drug treated samples versus untreated.
  • Figure 2 Low doses of Rapamycin, Torin 1, and Torin 2 are insufficient to kill M. smegmatis. RAW264.7 cells were infected with M. smegmatis and treated with Rapamycin (A), Torin 1 (B), or Torin 2 (C). Cells were then lysed and plated for CFU determination. Shown are combined results of two independent assays performed with each inhibitor. On the left are the raw CFU values for each replicate. On the right is the percentage change (+/− SEM) from the mean value of cells treated with DMSO that was set at 100%. For the comparison of raw CFU values, asterisks indicate p≤ 0.05 for drug treated groups versus DMSO treated cells.
  • Figure 3 High doses of Rapamycin can kill M. smegmatis in wildtype and autophagy deficient macrophages. (A) Bone marrow derived macrophages from C57BL/6 mice were infected with M. smegmatis and treated with the indicated concentrations of Rapamycin. The cells were lysed and CFU was determined as described in Figure 2. (B-C) Bone marrow derived macrophages from B6.129 LC3KO mice and B6 LysM-ATG5 mice were isolated and treated as described above. For parts (A-C), shown are results representative of two independent assays per mouse strain. (D) Western blot of protein lysates of samples described in (A) and (B) for ribosomal S6 and phosphorylated ribosomal S6. Asterisks indicate p≤ 0.05 drug treated groups versus DMSO treated cells.
  • Figure 4 Rapamycin does not kill M. smegmatis or BCG in the absence of macrophages. M. smegmatis (A) or BCG (B) were cultured in DMEM+DMSO (control) or the indicated doses of Rapamycin. After 3 hours at 37C and 5% CO2, the wells were mixed and the bacteria were plated for CFU determination. On the left are the raw CFU values for each replicate. On the right is the percentage change (+/− SEM) from the mean value of cells treated with DMSO, which was set at 100%. For both (A) and (B), shown are the combined results of two independent assays.
  • Figure 5 Long incubations with Rapamycin can alter the growth of some mycobacterial species. (A) Fast-growing M. smegmatis was grown in 7H9 + OADC in the presence of either DMSO (control) or Rapamycin (10 uM). Shown is the OD600 (+/− SEM) at the time intervals described of 7 replicate cultures from 2 independent experiments. The last time point for each growth curve comparing DMSO treatment versus Rapamycin treatment was compared by Student’s T test and found non significant (NS). (B) Slow-growing BCG and M. kanasii were grown in 7H9 + OADC in the presence of either DMSO (control) or Rapamycin (10 uM). Shown is the OD600 (+/− SEM) at the time intervals described of 10 replicate cultures from 2–3 independent experiments per species. The last time points for each growth curve comparing DMSO treatment versus Rapamycin treatment were compared by Student’s T test. Asterisks indicate p < 0.05. (C) The indicated pathogenic mycobacterial species were grown in 7H9-OADC in the presence of DMSO (control) or Rapamycin (10 ug/ml). A laboratory strain (H37Rv), KZN drug-sensitive strain (V9124 [S]), a multidrug resistant (MDR) strain (V2475 [M]), and an extensively drug resistant (XDR) strain (TF275 [X]) were used. Shown is the OD600 (+/− SEM) at the time intervals described of 7–8 replicates from 2 independent experiments. The last time points for each growth curve comparing DMSO treatment versus Rapamycin treatment were compared by Student’s T test. Asterisks indicate p < 0.05.

References Powered by Scopus

Regulation mechanisms and signaling pathways of autophagy

3120Citations
N/AReaders
Get full text

Autophagy proteins regulate innate immune responses by inhibiting the release of mitochondrial DNA mediated by the NALP3 inflammasome

2520Citations
N/AReaders
Get full text

Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages

1922Citations
N/AReaders
Get full text

Cited by Powered by Scopus

Programmedcell death ligand 1 (PD-L1) signaling regulates macrophage proliferation and activation

253Citations
N/AReaders
Get full text

Rewiring cellular metabolism via the AKT/mTOR pathway contributes to host defence against Mycobacterium tuberculosis in human and murine cells

116Citations
N/AReaders
Get full text

Autophagy induction targeting mTORC1 enhances Mycobacterium tuberculosis replication in HIV co-infected human macrophages

57Citations
N/AReaders
Get full text

Register to see more suggestions

Mendeley helps you to discover research relevant for your work.

Already have an account?

Cite

CITATION STYLE

APA

Zullo, A. J., Jurcic Smith, K. L., & Lee, S. (2014). Mammalian target of Rapamycin inhibition and mycobacterial survival are uncoupled in murine macrophages. BMC Biochemistry, 15(1). https://doi.org/10.1186/1471-2091-15-4

Readers over time

‘14‘15‘16‘17‘18‘19‘20‘21‘22‘23‘24036912

Readers' Seniority

Tooltip

PhD / Post grad / Masters / Doc 22

63%

Researcher 10

29%

Professor / Associate Prof. 3

9%

Readers' Discipline

Tooltip

Agricultural and Biological Sciences 16

38%

Immunology and Microbiology 13

31%

Biochemistry, Genetics and Molecular Bi... 8

19%

Medicine and Dentistry 5

12%

Article Metrics

Tooltip
Social Media
Shares, Likes & Comments: 45

Save time finding and organizing research with Mendeley

Sign up for free
0