Unc-51-like Kinase 1 (ULK1) Regulates Bacterial Ubiquitylation and p62 Recruitment during Xenophagic Clearance of Listeria monocyt ogenes

ABSTRACT Autophagy is an essential cellular homeostatic process, that also serves as an innate immune mechanism against intracellular bacterial pathogens, through a highly selective form of autophagy known as xenophagy. Despite advances in understanding how bacteria are targeted for autophagic degradation, the specific regulatory mechanisms that drive the initial steps and ensure bacterial selection remain incompletely defined. Our study uncovers a pivotal role for Unc-51-like kinase 1 (ULK1) in the xenophagic clearance of the intracellular bacterial pathogen Listeria monocytogenes . We observed that ULK1 is essential for the efficient ubiquitylation of bacteria and subsequent recruitment of the autophagic adaptor protein p62 to the bacterial surface. Furthermore, we show that the impact of ULK1 deficiency in these early events - reduction in bacterial ubiquitylation followed by impaired p62 targeting – later result in diminished formation of bacteria-targeted autophagosomes. Notably, phosphorylation of p62 at the S409 residue, which is known to enhance its affinity for ubiquitin, is necessary for its recruitment and effective bacterial clearance, highlighting the regulatory role of ULK1 in this process. These findings unveil a previously unrecognized function of ULK1 in modulating early xenophagy steps, contributing to the autophagic control of intracellular pathogens. Our findings offer new perspectives into the manipulation of ULK1activity for therapeutic interventions against infectious diseases. IMPORTANCE Autophagy is a vital process in eukaryotic cells that enables them to digest intracellular components, helping them respond to various stresses, including starvation, the accumulation of dysfunctional organelles, and infections. While the autophagic flux has been extensively studied over the past few decades, some key mechanisms remain poorly understood. In our research, we aimed to clarify one such mechanism: the way the autophagic machinery specifically targets intracellular bacteria. We identified a novel role for the protein ULK1 in this process, demonstrating that ULK1 is essential for marking bacteria within the cell and recruiting the protein p62. This marking and recruitment of p62 are critical steps for effective bacterial clearance. This underscores the pivotal role of ULK1 in initiating the cellular defense against bacterial infections. Our findings could pave the way for new therapeutic strategies aimed at enhancing the body’s capacity to combat bacterial infections.