Host defenses against invading microorganisms include targeting the pathogens for degradation in acidic lysosomal organelles, and generating adaptive immune responses by orchestrating successful antigen presentation. Infected cells employ evolutionarily-conserved cellular machinery that is normally used in the degradation of intracellular protein aggregates and damaged organelles to accomplish these responses. This self-digestion pathway, known as autophagy, helps cells survive under starvation conditions by restoring nutrient balance. During autophagy, cytoplasmic material is engulfed in de novo double-membrane vesicles (referred to as autophagosomes) and is delivered to lysosomes where the cargo is degraded into its constituent parts for reuse by the cell. This process is controlled by ATG5 and ATG7, which by mechanisms similar to ubiquitin-conjugation are involved in the conjugation of lipid to microtuble-associated protein 1 light chain 3 (LC3, a homolog of yeast ATG8). Conjugation with phosphatidylethnolamine (PE) converts the soluble form of LC3 (LC3-I) to another (LC3-II) that specifically associates with autophagic membrane vesicles and thus causes a shift from a diffuse staining pattern for LC3 to a punctuate pattern, which is often used to monitor autophagosome formation by fluorescence microscopy. Unlike classical autophagy that involves nonselective bulk degradation of cytosolic material, infected cells target intracellular bacteria (or bacteria-containing phagosomes) for sequestration into LC3-positive vacuoles for eventual destruction. This process of selective removal of invading microbes using autophagic machinery, termed xenophagy, plays a key role in the restriction by destruction of several kinds of bacteria, including Escherchia coli, Salmonella enterica, Mycobacterium tuberculosis, Listeria monocytogenes, and Group A Streptococcus as well as parasites such as Toxoplasma gondii. Genetic and pharmacological interference with the autophagic machinery has been shown to increase the number of intracellular bacteria. Xenophagy can also protect against infection by the Sindbis virus in mice and the singlestranded tobacco mosaic virus in plants. In addition to eliminating intracellular microbes, xenophagy elicits adaptive immune responses by contributing to the cross-presentation of microbial peptide antigens on both MHC class I and II molecules. The signaling pathways involved in the targeted elimination of microbes by autophagy are just beginning to be understood. Toll-like receptors (TLRs) on macrophages recognize pathogen-associated molecular patterns (PAMPs) and engage autophagic processes to clear pathogens. Lysosomal maturation of bacteria-containing phagosomes appear to be enhanced by TLR- mediated recruitment of the autophagosomal marker LC3 to phagosomes and their fusion with lysosomes, leading to rapid clearance of invading bacteria. Moreover, engagement of TLR-4 is also known to induce do novo formation of LC3-positive autophagosomes that contain bacteria, a process that requires p38 MAP kinase activity. Engagement of other TLRs, such as TLR-3 with poly I:C or TLR-7 with ssRNA, have also been shown to trigger autophagic responses. While these findings have linked TLRs and autophagy in host defense processes, many questions, such as how TLR-initiated signaling leads to the assembly of LC3-positive vesicles, remain unanswered. Given the importance of autophagy for pathogen clearance, cancer and neurodegenerative diseases we have focused our efforts on identifying molecules that regulate these processes. We found that p62 (also known as SQSTM1), a ubiquitin- and LC3- binding molecule, controls this innate immune autophagic process. Activation of primary macrophages with either E. coli or LPS triggered the formation of p62-associated LC3-positive vesicular compartments. Engagement of TLR-4 resulted in increased protein ubiquitination and accumulation in p62/LC3-positive vesicles. p62 expression was upregulated in response to TLR-4 activation, which required the functions of myeloid differentiation factor 88 (MyD88), Toll-interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF), and p38 kinase, and knockdown of p62 expression in primary macrophages suppressed the assembly of LPS- or microbe-induced LC3-postive autophagosomes. These findings reveal an anti-microbial function for p62 that links the xenophagic process with ubiquitination. We have also found that FYVE motif-containing molecules have similar roles in distinct pathways. We have generated mice that lack the expression of these genes and experiments are underway in the laboratory to validate the physiological function of these molecules in autophagy using these knock out mice.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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National Cancer Institute Division of Basic Sciences
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