Host-derived, pore-forming toxin–like protein and trefoil factor complex protects the host against microbial infection Yang Xianga,1, Chao Yana,b,1, Xiaolong Guoa,b, Kaifeng Zhoua,b, Sheng’an Lia, Qian Gaoa, Xuan Wanga,b, Feng Zhaoa,b, Jie Liua,b, Wen-Hui Leea, and Yun Zhanga,2 aKey Laboratory of Animal Models and Human Disease Mechanisms, The Chinese Academy of Sciences and Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China; and bKunming College of Life Science, University of Chinese Academy of Sciences, Beijing 100049, China Edited by B. Brett Finlay, University of British Columbia, Vancouver, BC, Canada, and accepted by the Editorial Board March 26, 2014 (received for review November 18, 2013) Aerolysins are virulence factors belonging to the bacterial β-pore– with a noncovalently linked form of αβ2 was recently identified in forming toxin superfamily. Surprisingly, numerous aerolysin-like the skin secretions of this species. The α-subunit is composed proteins exist in vertebrates, but their biological functions are un- of an aerolysin domain fused to two N-terminal βγ-crystallin known. βγ-CAT, a complex of an aerolysin-like protein subunit domains (9–11). The β-subunit is a three-domain TFF (Fig. (two βγ-crystallin domains followed by an aerolysin pore-forming 1A). This protein complex was therefore named βγ-CAT to domain) and two trefoil factor subunits, has been identified in reflect its domain composition. frogs (Bombina maxima) skin secretions. Here, we report the rich For the purpose of invasion, several pathogenic bacteria pro- expression of this protein, in the frog blood and immune-related duce PFTs, such as β-barrel–type aerolysins, which can oligo- tissues, and the induction of its presence in peritoneal lavage by merize and form pores in host-cell membranes (12, 13). The bacterial challenge. This phenomena raises the possibility of its aerolysin domain is defined according to its structural similarity βγ involvement in antimicrobial infection. When -CAT was admin- to the transmembrane domain of aerolysin toxins. Strikingly, a istrated in a peritoneal infection model, it greatly accelerated bac- diverse array of proteins harboring an aerolysin domain fused terial clearance and increased the survival rate of both frogs and β with other domains have been identified in various vertebrate mice. Meanwhile, accelerated Interleukin-1 release and enhanced species (9). Similar to the α-subunit of βγ-CAT, ep37 proteins local leukocyte recruitments were determined, which may partially from the amphibian newt Cynops pyrrhogaster harbor an aerolysin explain the robust and effective antimicrobial responses observed. domain fused to two N-terminal βγ-crystallin domains that share The release of interleukin-1β was potently triggered by βγ-CAT structural similarity with eye lens βγ-crystallins (14). TFFs are from the frog peritoneal cells and murine macrophages in vitro. βγ-CAT was rapidly endocytosed and translocated to lysosomes, characterized by one to four trefoil domains, which are highly where it formed high molecular mass SDS-stable oligomers (>170 conserved among TFF proteins, from frogs to humans. These kDa). Lysosomal destabilization and cathepsin B release were proteins are constitutively released within the mucosal layer that detected, which may explain the activation of caspase-1 inflamma- covers the gastrointestinal, urinary, respiratory, and skin surfaces some and subsequent interleukin-1β maturation and release. To and are believed to play pivotal roles in mucosal barrier function our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter Significance microbial infection by eliciting rapid and effective host innate im- mune responses. The findings will also largely help to elucidate Pore-forming toxins are a common type of bacterial toxins the possible involvement and action mechanisms of aerolysin-like and are important bacterial virulence factors. Aerolysin is proteins and/or trefoil factors widely existing in vertebrates in the produced by Aeromonas species. It is interesting that aero- host defense against pathogens. lysin-like proteins are also found in vertebrates. However, the physiological roles of these proteins are still unknown. innate immunity | infectious disease | interleukin-1beta Previously, a βγ-crystallin fused aerolysin-like protein (α-subunit) and trefoil factor (β-subunit) complex, hence named βγ-CAT, was he eradication of invading microorganisms is essential for the identified in frogs. Here, we found that this complex is inducible Tsurvival of vertebrates, including frogs. Innate antimicrobial by bacterial challenge. The complex was able to protect the host responses play a key role in host defense against many infections from microbial infection. It oligomerized along the endo-lyso- (1). Host innate immune cells, such as neutrophils, monocytes, some pathway to trigger lysosome destabilization and led to and macrophages, can be activated by various cytokines to engulf interleukin-1β maturation and secretion via inflammasome acti- pathogens and release toxic oxygen and nitrogen radicals (2). vation. Our present work provides functional evidence for Interleukin-1β (IL-1β) is one of the key cytokines that initiate uncovering the physiological roles of vertebrate-derived bac- – and amplify a wide variety of effects associated with the host terial pore-forming toxin like proteins. response to microbial invasion (3, 4). The strategies and mo- Author contributions: Y.X. and Y.Z. designed research; Y.X., C.Y., X.G., K.Z., S.L., and J.L. lecular identities of such host endogenous regulators that ensure performed research; Q.G. and X.W. contributed new reagents/analytic tools; Y.X., C.Y., rapid, effective, and controllable antimicrobial responses are X.G., S.L., Q.G., F.Z., W.-H.L., and Y.Z. analyzed data; and Y.X. and Y.Z. wrote the paper. incompletely understood (2, 5–7). The authors declare no conflict of interest. The frog species Bombina maxima lives in very harsh envi- This article is a PNAS Direct Submission. B.B.F. is a guest editor invited by the Editorial ronments, such as pools containing microorganism-rich mud, and Board. its skin is very “toxic.” Our previous study demonstrated that 1Y.X. and C.Y. contributed equally to this work. B. maxima possesses an immune system similar to that of mammals 2To whom correspondence should be addressed. E-mail: [email protected]. (8). A 72-kDa complex of bacterial pore-forming toxin (PFT) aer- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. olysin-like protein (α-subunit) and trefoil factor (TFF) (β-subunit) 1073/pnas.1321317111/-/DCSupplemental. 6702–6707 | PNAS | May 6, 2014 | vol. 111 | no. 18 www.pnas.org/cgi/doi/10.1073/pnas.1321317111 Downloaded by guest on September 29, 2021 Results Frog βγ-CAT α-Subunit Is a Bacterial Pore-Forming Toxin–Like Protein. The domain architecture of βγ-CAT has been previously de- scribed (10). Its α-subunit contains an aerolysin domain at the C terminus (Fig. 1A). Modeling of the aerolysin domain of βγ-CAT in 3D showed that it is similar to the aerolysin domain of the hemolytic lectin from the mushroom Laetiporus sulphureus, with a confidence score (C-score) of −0.74 (Fig. 1B). Indeed, the aerolysin domain-containing proteins are widely distributed, from bacteria to vertebrates (9) (Table S1). Specifically, the aerolysin domain of βγ-CAT showed high similarity to the aer- olysin domains of other vertebrate proteins (Fig. S1A). However, few other vertebrate-derived aerolysin-like proteins have been purified and characterized. A sufficient amount of the βγ-CAT protein could be purified and characterized from B. maxima skin secretions, allowing us to explore its physiological and patho- logical functions without genetic manipulation, which is very hard to perform in B. maxima. βγ-CAT Is Inducible During Bacterial Infection. RT-PCR of the βγ-CAT α-subunit and β-subunit revealed that each subunit was constitutively expressed in the skin, spleen, kidney, and intestine of frogs (Fig. 1C). These organs possess immune functions in amphibians. Skin and blood from frogs fed in bacterium-rich water showed up-regulated mRNA levels of both subunits of Fig. 1. βγ-CAT possesses an aerolysin domain and can be induced by βγ-CAT (Fig. 1D). A peritonitis model was also used to test bacterial challenge. (A) Schematic structure of βγ-CAT. A βγ-CAT molecule whether βγ-CAT was induced during i.p. infection. The presence contains one α-subunit and two β-subunits. One α-subunit contains two of bacteria (Comamonas sp.) significantly up-regulated βγ-CAT IMMUNOLOGY βγ-crystallin domains and one aerolysin domain, and one β-subunit con- at 2, 4, and 8 h postchallenge (Fig. 1E). The amount of βγ-CAT tains three trefoil domains. (B) The model of the βγ-CAT aerolysin domain produced in the peritoneum after bacterial challenge is semi- in 3D based on the crystal structure of the hemolytic lectin from the quantified (Fig. S1B). The βγ-CAT produced at 4 and 8 h after mushroom L. sulphureus (PDB ID code 1w3g), which harbors an aerolysin bacterial challenge was estimated to be 0.33 ± 0.16 μg and 0.40 ± domain at the C terminus. The structure was predicted by the I-TASSER μ βγ server. The predicted aerolysin domain of βγ-CATisshowninred,andthe 0.21 g, which could result in the concentrations of -CAT in hemolytic lectin from the mushroom L. sulphureus is shown in gray. The pic- 100-μL peritoneum fluids to be 46 ± 22 nM and 56 ± 29 nM, ture was generated by the PyMOL program (for Windows). (C) Expression respectively. profile of βγ-CAT in different organs. (D)Theβγ-CAT mRNA level was up- regulated 24 h after being fed in a bacteria-containing environment (Coma- βγ-CAT Protects Frogs and Mice Against Bacterial Infection.