The SapM phosphatase can arrest phagosome maturation in an ESX-1 independent manner in Mycobacterium tuberculosis and BCG.

Mycobacterium tuberculosis (Mtb) is an intracellular pathogen that survives and grows in macrophages. A mechanism used by Mtb to achieve intracellular survival is to secrete effector molecules that arrest the normal process of phagosome maturation. Through phagosome maturation arrest (PMA), Mtb remains in an early phagosome and avoids delivery to degradative phagolysosomes. One PMA effector of Mtb is the secreted SapM phosphatase. Because the host target of SapM, phosphatidylinositol-3-phosphate (PI3P), is located on the cytosolic face of the phagosome, SapM needs to not only be released by the mycobacteria but also travel out of the phagosome to carry out its function. To date, the only mechanism known for Mtb molecules to leave the phagosome is phagosome permeabilization by the ESX-1 secretion system. To understand this step of SapM function in PMA, we generated identical in-frame sapM mutants in both the attenuated Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine strain, which lacks the ESX-1 system, and Mtb. Characterization of these mutants demonstrated that SapM is required for PMA in BCG and Mtb. Further, by establishing a role for SapM in PMA in BCG, and subsequently in a Mtb mutant lacking the ESX-1 system, we demonstrated that the role of SapM does not require ESX-1. We further determined that ESX-2 or ESX-4 is also not required for SapM to function in PMA. These results indicate that SapM is a secreted effector of PMA in both BCG and Mtb, and that it can function independent of the known mechanism for Mtb molecules to leave the phagosome.

Thiopeptides Induce Proteasome-Independent Activation of Cellular Mitophagy.

Thiopeptide antibiotics are emerging clinical candidates that exhibit potent antibacterial activity against a variety of intracellular pathogens, including Mycobacterium tuberculosis (Mtb). Many thiopeptides directly inhibit bacterial growth by disrupting protein synthesis. However, recent work has shown that one thiopeptide, thiostrepton (TSR), can also induce autophagy in infected macrophages, which has the potential to be exploited for host-directed therapies against intracellular pathogens, such as Mtb. To better define the therapeutic potential of this class of antibiotics, we studied the host-directed effects of a suite of natural thiopeptides that spans five structurally diverse thiopeptide classes, as well as several analogs. We discovered that thiopeptides as a class induce selective autophagic removal of mitochondria, known as mitophagy. This activity is independent of other biological activities, such as proteasome inhibition or antibiotic activity. We also find that many thiopeptides exhibit potent activity against intracellular Mtb in macrophage infection models. However, the thiopeptide-induced mitophagy occurs outside of pathogen-containing autophagosomes and does not appear to contribute to thiopeptide control of intracellular Mtb. These results expand basic understanding of thiopeptide biology and provide key guidance for the development of new thiopeptide antibiotics and host-directed therapeutics.