Lysosome repositioning as an autophagy escape mechanism by Mycobacterium tuberculosis Beijing strain.

Induction of host cell autophagy by starvation was shown to enhance lysosomal delivery to mycobacterial phagosomes, resulting in the restriction of Mycobacterium tuberculosis reference strain H37Rv. Our previous study showed that strains belonging to M. tuberculosis Beijing genotype resisted starvation-induced autophagic elimination but the factors involved remained unclear. Here, we conducted RNA-Seq of macrophages infected with the autophagy-resistant Beijing strain (BJN) compared to macrophages infected with H37Rv upon autophagy induction by starvation. Results identified several genes uniquely upregulated in BJN-infected macrophages but not in H37Rv-infected cells, including those encoding Kxd1 and Plekhm2, which function in lysosome positioning towards the cell periphery. Unlike H37Rv, BJN suppressed enhanced lysosome positioning towards the perinuclear region and lysosomal delivery to its phagosome upon autophagy induction by starvation, while depletion of Kxd1 and Plekhm2 reverted such effects, resulting in restriction of BJN intracellular survival upon autophagy induction by starvation. Taken together, these data indicated that Kxd1 and Plekhm2 are important for the BJN strain to suppress lysosome positioning towards the perinuclear region and lysosomal delivery into its phagosome during autophagy induction by starvation to evade starvation-induced autophagic restriction.

A novel potent autophagy inhibitor ECDD-S27 targets vacuolar ATPase and inhibits cancer cell survival.

Autophagy is a conserved lysosomal-dependent cellular degradation process and its dysregulation has been linked to numerous diseases including neurodegeneration, infectious diseases, and cancer. Modulation of autophagy is therefore considered as an attractive target for disease intervention. We carried out a high-content image analysis screen of natural product-derived compounds to discover novel autophagy modulating molecules. Our screen identified ECDD-S27 as the most effective compound for increasing the number of autophagic vacuoles inside cells. The structure of ECDD-S27 revealed that it is a derivative of cleistanthin A, a natural arylnaphthalene lignan glycoside found in plants. ECDD-S27 increases the number of autophagic vacuoles by inhibiting the autophagic flux and is able to restrict the survival of different cancer cells at low nanomolar concentrations. Molecular docking and SERS analysis showed that ECDD-S27 may potentially target the V-ATPase. Upon treatment of various cancer cells with ECDD-S27, the V-ATPase activity is potently inhibited thereby resulting in the loss of lysosomal acidification. Taken together, these data indicated that ECDD-S27 retards the autophagy pathway by targeting the V-ATPase and inhibits cancer cell survival. The observed antitumor activity without cytotoxicity to normal cells suggests the therapeutic potential warranting further studies on lead optimization of the compound for cancer treatment.

Controlled rapid synthesis and in vivo immunomodulatory effects of LM α(1,6)mannan with an amine linker.

The synthetic lipomannan (LM) α(1,6)mannans, already equipped with an amine linker on the reducing end, are rapidly synthesized in a size-, regio-, and stereocontrolled reaction. The size of the mannans is regulated through the concentration of the linker, applied during the controlled ring-opening polymerization reaction. The versatile amine linker enables a variety of glycan conjugations. The synthetic α(1,6)mannans exert adjuvant activities for a real vaccine antigen, tetanus toxoid (TT) in vitro, as demonstrated by the increased secretion of proinflammatory cytokines TNF-α and IL-6 from the treated macrophages. A conjugation of synthetic α(1,6)mannan with TT can also enhance immune response to TT in vivo after immunization as shown by an increase in TNF-α, IFN-γ, and IL-2 production in splenocytes.

Synthesis of synthetic mannan backbone polysaccharides found on the surface of Mycobacterium tuberculosis as a vaccine adjuvant and their immunological properties.

Surface components of Mycobacterium tuberculosis (Mtb) play crucial roles in modulating host immune responses. Thorough understandings of immunological properties of the Mtb's surface components are essential for the development of tuberculosis treatment and prevention. Unfortunately, the accessibility to the molecules on the surface of Mtb is limited by the structural complexity due to their various macromolecular nature and the hazard of culturing Mtb. In this study, we reveal a practical synthesis of lipomannan (LM) backbone polysaccharides - the core glycans found on Mtb's surface. A rapid synthetic approach based on a controlled polymerization was developed for the chemical synthesis of mannopyranans, the core structure of LM. The size of the LM glycans can be controlled by using specific monomer concentrations in addition to stereo- and regioselectivity derived from the versatile tricyclic orthoester mannose monomer. The immunological properties of the synthesized mannopyranans were investigated and their adjuvant potential was revealed. The adjuvanticity mechanism of the synthetic mannopyranans appears to involve the NF-κB and inflammasome pathways.