All other transporter mRNA was detectable in the tissue and cell samples

This could be accomplished by a number of methodologies including, proteomics ap MAPK リン酸化反応 proaches that identify specific post translational modifica tions induced shortly after mycobacterial infection, the identification of mycobacterial transposon mutants that are susceptible to macrophage autophagy, the continued use of newly created mouse model systems that are more or less susceptible to mycobacterial infection, and the identifica tion of pharmacological agents that induce autophagy and mycobacterial killing without inhibiting the immunologic ally sensitive mTOR pathway. Lastly, while mTOR inhib ition does carry with it substantial global effects on cellular metabolism, it can not be overlooked that finely tuned mTOR inhibition, especially if restricted to macrophages, could provide a valuable means to favor host defense against mycobacterial infection.

Conclusions While low doses of several mTOR inhibitors are sufficient to reduce mTOR signaling as measured by a reduction in phosphorylated purchase MK-1775 ribosomal S6, the same doses of these compounds are incapable of eliciting robust killing of M. smegmatis. In contrast, high doses of Rapamycin, the most common mTOR inhibitor used in autophagy research, in duces substantial M. smegmatis killing in wildtype macro phages and macrophages from autophagy deficient mice. As it does not appear that Rapamycin has a direct effect on mycobacteria in the short time frames of standard au tophagy assays, it suggests that high dose inhibition of mTOR may be acting through an unappreciated cellular mechanism to elicit killing activity.

When combined with our previous studies demonstrating that mycobacterial in fection naturally induces both autophagy and mTOR sig naling, this data reinforces the idea that mTOR inhibition through drugs オーダー MS-275 or starvation is an artificial means of study ing mycobacterial killing. We contend that the use of mTOR inhibition to study the molecular mechanisms of host pathogen interactions is masking the relevant bio chemistry that needs to be understood and exploited to favor host defense. However, additional studies further examining the connection between mycobacteria, the mTOR pathway, and host defense need to be performed, as fine tuning mTOR activity to favor host defense with out additional effects would be advantageous and could be developed as a valuable therapeutic.

Wildtype and LC3B knockout mice were purchased from Jackson laboratories. LysM ATG5 mice were a gener ous gift from Herbert Virgin. All mice were housed in the Duke Human Vaccine Institute Regional Biocontainment laboratory in accordance with in stitutional animal care and use guidelines. mTOR inhibitors Rapamycin, Torin 1, and Torin 2, were dissolved in DMSO to a concentration of 10 mM, ali quoted, and stored at −20 C. Inhibitors were diluted fresh in culture media immediately before use. Lipopoly sacharride was purchased from Sigma, dissolved in DMEM, aliquoted, and stored at −20 C. Mycobacteria Mycobacterium bovis Bacille Calmette Guérin and M. smegmatis have been described previously. This study also uses a KZN drug sensitive strain, a multidrug resistant strain, and an extensively drug resistant strain. All KZN strains were recovered from patients in KwaZulu Natal province, South Africa.