bioRxiv2025
Xenophagy is a crucial innate subcellular defense mechanism, functioning as a selective autophagy pathway that captures intracellular pathogens within xenophagosomes for subsequent lysosomal degradation. However, many pathogens have evolved strategies to subvert this process, ensuring their intracellular replication and survival within the host cell. Despite its significance, the precise mechanisms by which intracellularly motile xenophagy cargo like Listeria monocytogenes (Lm) circumvent this selective autophagy pathway are not yet completely understood. In this study, we identify a previously unrecognized function of the secreted virulence factor listeriolysin (LLO) in orchestrating a multi-layered disruption of host autophagy-lysosomal machinery. While LLO is classically known for its role in mediating vacuolar escape, our findings reveal that it also contains a functional LIR motif which enables LLO to directly bind LC3 in the cytosol, rendering it unavailable for xenophagosome formation. Concurrently, LLO impairs lysosomal function by reducing its hydrolytic capacity and suppresses nuclear translocation of TFEB, a key transcription factor that governs autophagy and lysosome biogenesis, further compromising host degradative pathways. Using various Lm mutant strains, we show that actin-based motility is dispensable for xenophagy evasion. Instead, the intracellular persistence of Lm primarily stems from two factors: LLO-mediated LC3 binding, which diverts LC3 from its xenophagic functions, and the inhibition of TFEB nuclear translocation. Importantly, pharmacological activation of TFEB or genetic disruption of the LLO-LC3 interaction restores xenophagy and substantially reduces bacterial load. Collectively, our findings position LLO as a multifaceted virulence determinant that employs previously uncharacterized mechanisms to subvert host cell-autonomous defense. This study deepens mechanistic insight into the pathogenesis of Lm and highlights LC3 and TFEB as promising therapeutic targets for host-directed therapies against intracellular infections. Highlights{blacksquare} Listeria monocytogenes (Lm) evades xenophagy independently of its host-actin polymerization ability {blacksquare}Listeriolysin O (LLO) impairs lysosomal degradative potential and suppresses TFEB nuclear translocation {blacksquare}LLO binds LC3 through its LC3-interacting region (LIR) motif, effectively limiting LC3 availability for xenophagosome formation {blacksquare}Restoring LC3 and TFEB function enables intracellular clearance of Lm Graphical abstractThe graphical abstract illustrates how vacuolar ({Delta}hlyA), and cytosolic (WT and {Delta}actA) Listeria monocytogenes differentially engage with host-degradative pathways. Listeriolysin O (LLO), a critical virulence factor, is highlighted as the linchpin determining whether these bacteria get targeted for degradation or successfully evade the host response. In {Delta}hlyA strain infected cells, where LLO is absent, (1) the bacteria remain confined within the phagosome. (2 - 4) The phagosomal compartment then fuse with lysosome, to form phagolysosome that is both acidified and hydrolytically active, enabling effective bacterial degradation while sustaining autophagy flux. (5) This process coincides with nuclear translocation of the master transcription regulator, TFEB, which enhances lysosomal biogenesis and function, boosting bacterial clearance. On the other hand, both WT and {Delta}actA strains produce LLO, (1, 2) which allows them to escape from the phagosome into the host cytosol. (3) WT strain induces polymerization of host cell actin, leading to the formation of actin clouds that mature into actin tails, facilitating bacterial mobility and cell to cell spread. (4) The {Delta}actA strain, despite lacking the ability to interact with host actin, still replicates in the cytosol. LLO plays a multifaceted role in evading host defenses. (5 - 6) It limits LC3 availability for nascent xenophagosome formation by binding to LC3, reducing bacterial capture by the xenophagy machinery. (7 - 8) Furthermore, even when a few bacteria are successfully sequestered within phagosome or xenophagosome, LLO-mediated lysosomal dysfunction impair their maturation into degradative phagolysosome and xenolysosome. (9) Compounding this, LLO suppresses nuclear translocation of TFEB, thereby dampening transcriptional activation of autophagy-lysosomal pathways. Collectively, these mechanisms compromise the host degradative capacity, allowing cytosolic bacteria to replicate and evade clearance. Several unanswered questions remain regarding the structural and mechanistic aspects of the interaction of LLO with LC3. It is still unclear whether LC3 conjugation occurs on single or double membrane vesicles, and whether these vesicular structures are fully sealed. Additionally, it is not yet determined if LLO binds LC3 specifically at sites of omegasome or phagophore formation. The aggregation behavior of LLO also raises intriguing possibilities: could these aggregates play a protective role in shielding LLO from its degradation? Furthermore, it remains to be clarified whether LLO exists freely in the cytosol or is associated with specific membrane-bound compartments. Finally, the precise mechanism by which LLO interferes with TFEB nuclear translocation is another critical open question requiring further investigation. O_FIG O_LINKSMALLFIG WIDTH=165 HEIGHT=200 SRC="FIGDIR/small/691302v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@21c235org.highwire.dtl.DTLVardef@553714org.highwire.dtl.DTLVardef@932e53org.highwire.dtl.DTLVardef@c686ef_HPS_FORMAT_FIGEXP M_FIG C_FIG