1.C.41 The Tripartite Haemolysin BL (HBL) Family

The HBL family includes a tipartite haemolysin from Bacillus cereus (Beecher and Wong 2000). The three components are homologous but distantly related to each other. They are called HBL component B, HBL component L1 and HBL component L2. The toxin forms pores and has also been called the enterotoxic, necrotizing, vascular, permeability toxin, a likely virulence factor of B. cereus diarrheal food poisoning and necrotic infections (Sastalla et al. 2013). Two distinct sets of all three HBL components have been isolated from a single B. cereus isolate, MGBC145. Both exhibit haemolytic and vascular permeability activities, and the homologues could function interchangeably. In addition to B. cereus, a homologue, called Haemolysin YhlA has been isolated and characterized from Edwardsiella tarda (Chen et al. 1996).

The generalized transport reaction catalyzed by HBL is:

Solutes (in) ⇌ solutes (out)



This family belongs to the Pore-forming Cytotoxin (PfCTx) Superfamily.

 

References:

Beecher, D.J. and A.C. Wong. (2000). Tripartite haemolysin BL: isolation and characterization of two distinct homologous sets of components from a single Bacillus cereus isolate. Microbiology 146(Pt6): 1371-1380.

Bräuning, B., E. Bertosin, F. Praetorius, C. Ihling, A. Schatt, A. Adler, K. Richter, A. Sinz, H. Dietz, and M. Groll. (2018). Structure and mechanism of the two-component α-helical pore-forming toxin YaxAB. Nat Commun 9: 1806.

Chen, J.D., S.Y. Lai, and S.L. Huang. (1996). Molecular cloning, characterization, and sequencing of the hemolysin gene from Edwardsiella tarda. Arch. Microbiol. 165: 9-17.

Dementiev, A., J. Board, A. Sitaram, T. Hey, M.S. Kelker, X. Xu, Y. Hu, C. Vidal-Quist, V. Chikwana, S. Griffin, D. McCaskill, N.X. Wang, S.C. Hung, M.K. Chan, M.M. Lee, J. Hughes, A. Wegener, R.V. Aroian, K.E. Narva, and C. Berry. (2016). The pesticidal Cry6Aa toxin from Bacillus thuringiensis is structurally similar to HlyE-family alpha pore-forming toxins. BMC Biol 14: 71.

Fagerlund, A., T. Lindbäck, A.K. Storset, P.E. Granum, and S.P. Hardy. (2008). Bacillus cereus Nhe is a pore-forming toxin with structural and functional properties similar to the Cl- yA (HlyE, SheA) family of haemolysins, able to induce osmotic lysis in epithelia. Microbiology 154: 693-704.

Fagerlund, A., T. Lindbäck, and P.E. Granum. (2010). Bacillus cereus cytotoxins Hbl, Nhe and CytK are secreted via the Sec translocation pathway. BMC Microbiol 10: 304.

Huang, J., Z. Guan, L. Wan, T. Zou, and M. Sun. (2016). Crystal structure of Cry6Aa: A novel nematicidal ClyA-type α-pore-forming toxin from Bacillus thuringiensis. Biochem. Biophys. Res. Commun. 478: 307-313.

Kopanja, L., Z. Kovacevic, M. Tadic, M.C. Žužek, M. Vrecl, and R. Frangež. (2018). Confocal micrographs: automated segmentation and quantitative shape analysis of neuronal cells treated with ostreolysin A/pleurotolysin B pore-forming complex. Histochem Cell Biol 150: 93-102.

Liu, X., S. Ding, P. Shi, R. Dietrich, E. Märtlbauer, and K. Zhu. (2016). Non-haemolytic enterotoxin (Nhe) of Bacillus cereus induces apoptosis in Vero cells. Cell Microbiol. [Epub: Ahead of Print]

Sastalla, I., R. Fattah, N. Coppage, P. Nandy, D. Crown, A.P. Pomerantsev, and S.H. Leppla. (2013). The Bacillus cereus Hbl and Nhe tripartite enterotoxin components assemble sequentially on the surface of target cells and are not interchangeable. PLoS One 8: e76955.

Schubert, E., I.R. Vetter, D. Prumbaum, P.A. Penczek, and S. Raunser. (2018). Membrane insertion of α-xenorhabdolysin in near-atomic detail. Elife 7:.

Vigneux, F., R. Zumbihl, G. Jubelin, C. Ribeiro, J. Poncet, S. Baghdiguian, A. Givaudan, and M. Brehélin. (2007). The xaxAB genes encoding a new apoptotic toxin from the insect pathogen Xenorhabdus nematophila are present in plant and human pathogens. J. Biol. Chem. 282: 9571-9580.

Wagner, N.J., C.P. Lin, L.B. Borst, and V.L. Miller. (2013). YaxAB, a Yersinia enterocolitica pore-forming toxin regulated by RovA. Infect. Immun. 81: 4208-4219.

Zhu, K., A. Didier, R. Dietrich, U. Heilkenbrinker, E. Waltenberger, N. Jessberger, E. Märtlbauer, and R. Benz. (2015). Formation of small transmembrane pores: An intermediate stage on the way to Bacillus cereus non-hemolytic enterotoxin (Nhe) full pores in the absence of NheA. Biochem. Biophys. Res. Commun. [Epub: Ahead of Print]

Examples:

TC#NameOrganismal TypeExample
1.C.41.1.1

The tripartite haemolysin BL, consisting of HblA, HblC and HblD (Sastalla et al. 2013). These proteins are secreted via the general secretory pathway (Fagerlund et al. 2010).

Bacteria

HBL of Bacillus cereus

 
1.C.41.1.2Haemolysin YhlA Bacteria YhlA of Edwardsiella tarda
 
1.C.41.1.3

The non-hemolytic pore-forming cyto-enterotoxin, Nhe (Fagerlund et al., 2008; Sastalla et al. 2013), a three-partite toxin.  Pore formation and subsequent lysis of target cells caused by Nhe is an orchestrated process comprising three steps: (i) formation of NheB/C oligomers in solution, (ii) attachment of the oligomers to the cell membrane, (iii) binding of NheA to the oligomers. The benefit of these complexes is more stable cell binding as well as stronger and earlier cytotoxic effects. High molecular mass hetero-oligomers (~620 kDa), probably consist of one NheC and up to 15 NheB. NheBC induces membrane permeability. Formation of stable transmembrane channels with a conductance of about 870 pS and a diameter of about 2 nm due to the application of NheBC could be demonstrated in lipid bilayer experiments (Zhu et al. 2015). Thus, the NheBC complex increases the membrane permeability prior to the emergence of full pores containing also NheA.  Can induce apoptosis (Liu et al. 2016).

Firmicutes

Nhe of Baccilus cereus
Nhe-L1 (NheB; 402aas) (Q63CS3)
Nhe-L2 (NheA; 386aas) (Q63CS4)
NheC (359aas) (Q2TM55)

 
Examples:

TC#NameOrganismal TypeExample
1.C.41.2.1

Nematicidal pesticide pore-forming crystal protein α-toxin, Cry6Aa (Cry6A; CryVIa) of 475 aas.  It is structurally similar to HlyE (TC# 1.C.10.1.1) (Dementiev et al. 2016).  The X-ray struction of residues 1 - 396 at 1.9 Å resolution shows a structure similar to to those of Cly toxins (Huang et al. 2016).

Cry6Aa of Bacillus thuringiensis

 
1.C.41.2.10

Binary cytotoxin component of 321 aas.

Binary cytotoxin component of Pseudomonas syringae

 
1.C.41.2.2

Uncharacterized toxin of 407 aas,

Toxin of Pseudoalteromonas piscicida

 
1.C.41.2.3

Uncharacterized toxin of 383 aas.

Toxin of Clostridium kluyveri

 
1.C.41.2.4

Uncharacterized toxin of 389 aas

Toxin of Schizophyllum commune

 
1.C.41.2.5

Uncharacterized toxin of 420 aas

Toxin of Hypocrea virens (Gliocladium virens) (Trichoderma virens)

 
1.C.41.2.6

Putative toxin of 415 aas and 1 TMS

Toxin of Pseudomonas cichorii

 
1.C.41.2.7

The two component pore-forming toxin (PFT), YaxA-YaxB, where YaxA is 410 aas with 1 central TMS, and YaxB is 344 aas with no TMS. X-ray structures are available (Bräuning et al. 2018). While a yaxAB mutant (ΔyaxAB) is capable of colonizing mice at the same level as the wild type, the mutation slightly delays the course of infection and results in differing pathology in the spleen.  Wagner et al. 2013 found that yaxAB encode a cytotoxin capable of lysing mammalian cells, that both YaxA and YaxB are required for cytotoxic activity, and that the two proteins associate. YaxAB-mediated cell death occurs via osmotic lysis through the formation of distinct membrane pores. In silico tertiary structural analysis identified predicted structural homology between YaxA and proteins in pore-forming toxin complexes from Bacillus cereus (HBL-B) and Escherichia coli (HlyE). Thus, it appears that YaxAB function as virulence factors by inducing cell lysis through the formation of pores in the host cell membrane (Wagner et al. 2013). YaxAB represents a family of binary α-PFTs with orthologues in human, insect, and plant pathogens. Bräuning et al. 2018 presented crystal structures of YaxA and YaxB, together with a cryo-electron microscopy map of the YaxAB complex. Their structures revealed a pore predominantly composed of decamers of YaxA-YaxB heterodimers. Both subunits bear membrane-active moieties, but only YaxA is capable of binding to membranes by itself. YaxB can subsequently be recruited to membrane-associated YaxA and induced to present its lytic transmembrane helices. Pore formation can progress by further oligomerization of YaxA-YaxB dimers (Bräuning et al. 2018).

YaxAB of Yersinia enterocolitica

 
1.C.41.2.8

Two component cytotoxin consisting of XaxA of 408 aas and XaxB of 350 aas. Xenorhabdus nematophila, a member of the Enterobacteriaceae, kills many species of insects by strongly depressing the immune system and colonizing the entire body. The peptide cytotoxin, XaxAB, has been purified from X. nematophila broth, and the cytolytic effect on insect immunocytes and the hemolytic effect on mammalian red blood cells have been described (Vigneux et al. 2007). This toxin, Xenorhabdus alpha-xenorhabdolysin (Xax), triggers apoptosis in both insect and mammalian cells. Vigneux et al. 2007 also cloned and sequenced xaxAB, and showed that hemolytic activity was observed only if the two proteins were added in the appropriate order. The membrane inserted complex forms a 1-1.3 MDa large pore complexes to perforate the host cell membrane. Schubert et al. 2018 reported the cryo-EM structure of the XaxAB pore complex and the crystal structures of the soluble monomers of XaxA and XaxB. The structures reveal that XaxA and XaxB are built similarly and appear as heterodimers in the 12-15 subunit containing pore, classifying XaxAB as bi-component α-PFT. Major conformational changes in XaxB, including the swinging out of an amphipathic helix, are responsible for membrane insertion. XaxA acts as an activator and stabilizer for XaxB that forms the actual transmembrane pore. A novel structural model for the mechanism of Xax intoxication was proposed (Schubert et al. 2018). Kopanja et al. 2018 determined the influence of an ostreolysin A/pleurotolysin B complex (OlyA/PlyB) on the morphology of murine neuronal NG108-15 cells.

XaxAB of Xenorhabdus nematophila