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In macrophages

Autophagy
Autophagy is an evolutionarily-conserved eukaryotic self-degradation process in which intracellular components of a cell are degraded. The cargo is first enveloped in a double-membraned autophagosome, which is transported to the lysosome, where the two compartment fuses and the cargo is degraded.

There are two modes of autophagy - general and selective. General autophagy randomly take up cargo for degradation; this is to ensure a periodic turnover of macromolecules, as well as for generating substrates for energy metabolism during nutritional deprivation. Selective autophagy have specific receptors which binds to the substrate and initiate autophagy; this is to target protein aggregates and dysfunctional organelles for degradation to prevent cellular damage. Selective autophagy is often mediated via ubiquitin, where the cargo is ubiquinated and then recognized and targeted for autophagy by, for example, recruiting LC3.

LC3 allows other components of the autophagosome machinery, such as the ATG proteins, to form a scaffold and initiate a structure called phagophore, which expands, distorts and fuses to form a double-membraned autophagosome.

Intracellular bacteria are generally degraded by being transported using the process of autophagy, to the lysosome for degradation.

Mycobacterium tuberculosis is a vacuolar pathogen. Autophagy limit replication.

M. tuberculosis have a ESX-1 type VII secretion system which secretes ESAT-6 that permeabilize the phagosome, to allow the bacteria to escape, but this also allow components of the autophagy machinery from accessing the bacteria. This is similar to listeriolysin-O (LLO)'s role in the escape of Listeria

STING-dependent cytosolic pathway recognizes extracellular bacterial DNA marks bacteria with ubiquitin. p62 and NDP52 binds to ubiquitinated substrates

TBK1.

Intracellular bacteria can survive inside non-phagocytic cells by being endocytosed and remain within the cell in specialized compartments. There are intracellular bacteria, such as Shigella flexneri, Burkholderia pseudomallei, Francisella tularensis, Rickettsia spp., Listeria monocytogenes, and Salmonella enterica serovar Typhimurium (S. Typhimurium), which can break free from these compartments and invade the cytosol, where it will gain nutrients and be able to replicate. L. monocytogenes and S. Typhimurium have been confirmed to be ubiquinated and targeted for selective autophagy through the action of p62 and ndp52, although the mechanism that targets their ubiquination in the first place is not known.

M. tuberculosis and autophagy
Mycobacterium bovis Bacille Calmette-Gue´ rin (BCG) was used to model for autophagic response to mycobacteria clearance. In these study, for autophagy to take place, extracellular stimulation is required. The BCG model also lacks virulence factors such as type VII secretion system ESX-1, which means it is limited in replication and spread of infection. BCG model also do not initiate innate immune response or undergo selective autophagy.

ESX-1 is required for vacuolar escape and subsequent localization of ubiquitin

Also DNA sensing

Experiments
Primary murine bone marrow-derived macrophages (BMDMs) and other similar cell lines that LC3 increased its selectively envelopment of M. tuberculosis during infection. Western blot analysis of the infected cells also show a higher proportion of LC3-II to LC3-I.

M. marinum do not recruit p62 or LC3 but M. tuberculosis does. One difference is the lack of ESX-1 in M. marinum, and this is likely to be a target for ubiquination, leading to selective autophagy.

Restoration of ESX-1 in macrophage-like cell line RAW 264.7 increased LC3 recruitment; conversely, prevention of ESX-1 secretion led to failure to recruit LC3 or LC3 processing.

It is thought that the pore formation is required for recognition, to expose the bacteria to the cytoplasm where it is recognized.

p62 and ndp52 are receptors which recognizes M. tuberculosis, and recruits LC3, although there may be others (only 25% of ESX-1+ cells was seen to be bound by both, and only 70% of LC3+ population interacted with both p62 and ndp52)

ATG5 is involved in the recruitment of LC3

ndp52 interacts with NAP1 and SINTBAD to recruit Tank-binding kinase (TBK1), which initiates type I interferon (IFN) transcription.

Most TBK1+ recruited LC3 - may explain for why not all ESX-1+ p62+ ndp52+ recruited LC3; although again other factors are involved as knockout of TBK1 only reduced recruitment of LC3 to M. tuberculosis by 60%.

~30% of WT M. tuberculosis colocalized with ubiquitin after 4 hours, the same proportion has LC3 recruited. 70% of LC3-coated M. t was associated with ndp52 and p62, and 70% of ubiquinated M. t has LC3 recruited.

Ubiquitination preceded LC3 recruitment as ubiquitination was unaffected in Atg5-/- macrophages.

Bacteria often form biofilms - self-produced hydrated polymeric matrix to which the bacteria is enclosed and prevent the action of antibiotics. In the matrix (the matrix holds the biofilm together) of these biofilms, there contains extracellular DNA. These were traditionally thought of as being derived from cell lysis and is simply a byproduct. However, it was observed that P. aeruginosa produce these extracellular DNA through secretion of small vesicles. It has been previously been shown that eDNA activated TBK1 and induced Type I IFN transcription.

dsDNA plasmid invoked an autophagic response characterized by increased LC3 puncta and higher proportion of LC3-II. HSV-1, HCMV can induce LC3 lipidation. LC3 localize at the dsDNA. Co-immunoprecipitation of dsDNA showed LC3. dsDNA appeared in lysosomes (marked by LAMP-1 and ATG12). Specific to dsDNA, as ssDNA, dsRNA, ssRNA did not induce an autophagic response.

dsDNA, STING, ubiquitin, ndp52 and TBK1 - better with live cell imaging. KO out ndp52 led to reduced targeting of dsDNA to LC3 vesicles

TBK1 phosphorylates p62

STING KO led to lack of recruitment of ubiquitin, ndp52 and LC3 to the dsDNA and M. tuberculosis; activation of STING was sufficient to induce autophagosome formation. M. tuberculosis activated STING in macrophages.

dsDNA - ubiquitin - p62/ndp52 - TBK1 - LC3 - Autophagy

It has been shown that the DNases TREX1 and DNASE IIa reduced the innate immune response to cytosolic DNA as well as reduced type I IFN signalling. Overexpression of DNases reduced recruitment of ubiquitin and LC3 to M. tubeculosis. KO of Trex1 led to increase targetting.

Atg5, TBK1, STING, ndp52 KO or KD prevented maturation of M. t to lysosome - cells from these had more viable bacteria (determined by CFU) The recruitment of STING might not be to the bacteria per se, but through membrane damage

Shigella disrupt the membrane and this is recognized by NOD proteins (pattern recognition receptors) and ATG16L1 to the site of bacterial entry and triggers autophagy. Membrane remnants from bacterial escape are also ubiquitinated and are recognized by p62, NBR1 and NDP52 and delivered to autophagosomes. Damaged vesicles may also be marked with galectin 8, which recruits ndp52 and induce autophagy that way. recruitment of diacylglycerol (DAG),(Shahnazari et al., 2010; Cemma and Brumell, 2012).

other
IFN activate macrophages, break down pathogen to present antigens (seen in dendritic cells)

Induction of autophagy are not through other pathways such as inflammation activation or cytokine signalling