α-hemolysin targets LGALS3 (galectin 3) to promote intracellular survival of Staphylococcus aureus via lysosomal disruption and autophagy inhibition

Intracellular persistence caused by Staphylococcus aureus (S. aureus) is among the primary reasons for recurrence and difficulty in eradicating S. aureus infections. In this study, we identify the secreted protein Hla (α-hemolysin) by S. aureus as a key factor enabling its intracellular retention. We demonstrate that intracellular Hla secreted by S. aureus inhibits lysosome degradation via disrupting lysosomal function, which sustains the survival and proliferation of S. aureus within autophagosomes. Furthermore, we identify the interaction between Hla and intracellular LGALS3 (galectin 3) as crucial for sustaining intracellular survival of S. aureus, resolve the structure of the Hla-LGALS3 complex, and identify the Loop 68–75 region of Hla as the key binding domain with LGALS3. Moreover, the interaction between Hla and LGALS3 influences the recruitment of PDCD6IP/ALIX (programmed cell death 6 interacting protein) to the damaged lysosomal surface, resulting in disruption of lysosomal degradative function. Our results highlight an unknown role of Hla in the intracellular survival of S. aureus and suggest that interrupting the interaction between Hla and LGALS3 May be a potential therapeutic strategy for treating S. aureus infections.Abbreviations: 3 MA: 3-methyladenine; AECII: alveolar epithelial cells II; Agr: accessory gene regulator; ATG13: autophagy related 13; Baf A₁: bafilomycin A₁; BLI: biolayer interferometry; CFU: colony-forming units; ClfA: clumping factor A; Co-IP: co-immunoprecipitation; CRD: carbohydrate recognition domain; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; FnbA: fibronectin-binding protein A; FnbB: fibronectin-binding protein B; Hla: α-hemolysin; IP-MS: immunoprecipitation-mass spectrometry; LGALS3: galectin 3; LLoMe: L-leucyl-L-leucine methyl ester hydrobromide; LMP: lysosomal-membrane permeabilization; MOI: multiplicity of infection; PDCD6IP/ALIX: programmed cell death 6 interacting protein; S. aureus: Staphylococcus aureus; SPA: staphylococcal protein A; SSPA: staphylococcal surface protein A; TEM: transmission electron microscopy; TRAF3: TNF receptor associated factor 3; ULK1: unc-51 like autophagy activating kinase 1.