Individual Atg8 paralogs and a bacterial metabolite sequentially promote hierarchical CASM-xenophagy induction and transition

Atg8 paralogs, consisting of LC3A/B/C and GBRP/GBRPL1/GATE16, function in canonical autophagy; however, their function is controversial because of functional redundancy. In innate immunity, xenophagy and non-canonical single membranous autophagy called “conjugation of Atg8s to single membranes” (CASM) eliminate bacteria in various cells. Previously, we reported that intracellular Streptococcus pneumoniae can induce unique hierarchical autophagy comprised of CASM induction, shedding, and subsequent xenophagy. However, the molecular mechanisms underlying these processes and the biological significance of transient CASM induction remain unknown. Herein, we profile the relationship between Atg8s, autophagy receptors, poly-ubiquitin, and Atg4 paralogs during pneumococcal infection to understand the driving principles of hierarchical autophagy and find that GATE16 and GBRP sequentially play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, and LC3A and GBRPL1 are involved in CASM/xenophagy induction. Moreover, we reveal ingenious bacterial tactics to gain intracellular survival niches by manipulating CASM-xenophagy progression by generating intracellular pneumococci-derived H2O2. [Display omitted] •Recruitment of each Atg8 shows a distinctive pattern during pneumococcal infection•Atg8s play separate roles in CASM induction and shedding and xenophagy induction•GATE16 and GBRP are vital for CASM shedding and xenophagy induction, respectively•Bacterial H2O2 manipulates CASM shedding and xenophagy induction via Ca2+ axis Sakuma et al. show that GATE16 and GBRP play a pivotal role in CASM shedding and subsequent xenophagy induction, respectively, by establishing an autophagy matrix based on the relationship between Atg8 paralogs, autophagy adaptors, poly-ubiquitin, and Atg4 paralogs. Bacterial manipulation of CASM shedding and xenophagy induction via H2O2-Ca2+ axis is revealed.