Aerolysins are virulence factors belonging to the bacterial β-poreforming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. βγ-CAT, a complex of an aerolysin-like protein subunit (two βγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When βγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1β release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1β was potently triggered by βγ-CAT from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1β maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.innate immunity | infectious disease | interleukin-1beta

Endolysosomes are key players in cell physiology, including molecular exchange, immunity, and environmental adaptation. They are the molecular target of some pore-forming aerolysin-like proteins (ALPs) that are widely distributed in animals and plants and are functionally related to bacterial toxin aerolysins. βγ-CAT is a complex of an ALP (BmALP1) and a trefoil factor (BmTFF3) in the firebelly toad (Bombina maxima). It is the first example for a secreted endogenous pore-forming protein that modulates the biochemical properties of endolysosomes by inducing pore formation in these intracellular vesicles. Here, using a large array of biochemical and cell biology methods, we report the identification of BmALP3, a paralog of BmALP1 that lacks membrane pore- forming capacity. We noted that both BmALP3 and BmALP1 contain a conserved cysteine in their C-terminal regions. BmALP3 was readily oxidized to a disulfide bond-linked homodimer, and this homodimer then oxidized BmALP1 via disulfide bond exchange, resulting in the dissociation of βγ-CAT subunits and elimination of its biological activity. Consistent with its behavior in vitro, BmALP3 sensed environmental oxygen tension in vivo, leading to modulation of βγ-CAT activity. Interestingly, we found that this C-terminal cysteine site is well conserved in numerous vertebrate ALPs. These findings uncover the existence of a regulatory ALP (BmALP3) that modulates the activity on an active ALP (BmALP1) in a redox-dependent manner, a property that differs from those of bacterial toxin aerolysins.

Many intracellular pathogens invade cells via endocytic organelles and have adapted to the drop in pH along the endocytic pathway. However, the strategy by which the host cell counteracts this pathogen adaptation remains unclear. βγ-CAT is an aerolysin-like pore-forming protein and trefoil factor complex in the frog Bombina maxima. We report here that βγ-CAT, as a host-secreted factor with an intrinsic channel-forming property, is the first example of a molecule that actively neutralizes the acidification of endocytic organelles to counteract Listeria monocytogenes infection. Immunodepletion of endogenous βγ-CAT largely impaired the control of L. monocytogenes by frog cells. βγ-CAT elevates the pH of L. monocytogenes-containing vacuoles to limit the vacuole escape of L. monocytogenes to cytosol. Furthermore, βγ-CAT promotes intracellular L. monocytogenes clearance via autophagy and by that the nonlytic expulsion of the bacteria from host cells. Finally, βγ-CAT attenuated the dissemination of L. monocytogenes in vivo. These findings reveal a novel host strategy and effectors that combat pathogen adaptation to acidic conditions along the endocytic pathway.

Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. βγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of βγ-CAT remain elusive. Here, the actions of βγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of βγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of βγ-CAT. Finally, the ability of βγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses.

Bacterial pore‐forming toxin aerolysin‐like proteins are widely distributed in animals and plants. Emerging evidence supports their roles in host innate immunity, but their direct actions in adaptive immunity remain elusive. In this study, we found that βγ‐CAT, an aerolysin‐like protein and trefoil factor complex identified in the frog Bombina maxima, modulated several steps of endocytic pathways during dendritic cell antigen presentation. The protein augmented the antigen uptake of dendritic cells and actively neutralized the acidification of cellular endocytic organelles to favor antigen presentation. In addition, the release of functional exosome‐like extracellular vesicles was largely enhanced in the presence of βγ‐CAT. The cellular action of βγ‐CAT increased the number of major histocompatibility complex (MHC) I‐ovalbumin and MHC II molecules on dendritic cell surfaces and the released exosome‐like extracellular vesicles. An enhanced antigen presentation capacity of dendritic cell for priming of naive T cells was detected in the presence of βγ‐CAT. Collectively, these effects led to strong cytotoxic T lymphocyte responses and antigen‐specific antibody responses. Our findings provide evidence that a vertebrate‐secreted pore‐forming protein can augment antigen presentation by directly modulating cellular endocytic and exocytic pathways, leading to robust activation of adaptive immunity.

It has been established that Adenosine-5'-triphosphate (ATP) can activate the NLRP3 inflammasome. However, the physiological effect of extracellular ATP on NLRP3 inflammasome activation has not yet been investigated. In the present study, we found that ATP was indeed released during bacterial infection. By using a murine peritonitis model, we also found that ATP promotes the fight against bacterial infection in mice. ATP induced the secretion of IL-1β and chemokines by murine bone marrow-derived macrophages in vitro. Furthermore, the intraperitoneal injection of ATP elevated the levels of IL-1β and chemokines in the mouse peritoneal lavage. Neutrophils were rapidly recruited to the peritoneum after ATP injection. In addition, the effects on cytokine and chemokine secretion and neutrophil recruitment were markedly attenuated by the pre-administration of the caspase-1 inhibitor Ac-YVAD-cho. Ac-YVAD-cho also significantly attenuated the protective effect of ATP against bacterial infection. In the present study, we demonstrated a protective role for ATP during bacterial infection and this effect was related to NLRP3 inflammasome activation. Together, these results suggest a role for ATP in initiating the immune response in hosts suffering from infections.

Tissue repair is a highly dynamic process, and the immediate onset of acute inflammation has been considered necessary for repair. Pore-forming proteins are important, both in pathogen invasion and host immunity. However, their roles in wound healing and tissue repair are unclear. βγ-crystallin fused aerolysin-like protein (α-subunit) and trefoil factor (β-subunit) complex (βγ-CAT) is a complex of a bacterial pore-forming toxin aerolysin-like protein and trefoil factor identified in the frog Bombina maxima. In this study, we established mouse cutaneous wound models to explore the effects of βγ-CAT on skin wound healing. βγ-CAT accelerated the healing of full-thickness wounds by improving re-epithelialization. This complex relieved dermal edema and promoted scarless healing. βγ-CAT treatment resulted in a rapid release of IL-1β, which initiated an acute inflammation response in the early stage of healing. Meanwhile, the expression levels of TGF-β1, VEGF, and bFGF and the recruitment of M2 macrophages around the wound significantly increased after βγ-CAT treatment. βγ-CAT protected skin wounds against methicillin-resistant Staphylococcus aureus by improving neutrophil recruitment at the site of the wound. Overall, our results suggest that βγ-CAT can promote tissue repair and protect skin wounds against antibiotic-resistant bacterial infection by triggering the acute inflammatory response. This is the first example that aerolysin-like pore-forming proteins widely existing in plants and animals may act in wound healing and tissue repair.-Gao, Z.-H., Deng, C.-J., Xie, Y.-Y., Guo, X.-L., Wang, Q.-Q., Liu, L.-Z., Lee, W.-H., Li, S.-A., Zhang, Y. Pore-forming toxin-like protein complex expressed by frog promotes tissue repair.

Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that bg-crystallin fused aerolysin-like protein and trefoil factor complex (bg-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. bg-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, bg-CAT formed ring-like oligomers of ~10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the bg-CATtreated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that bg-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the bg-CATtreated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of bg-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that bg-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.