Extending the aerolysin family: from bacteria to vertebrates

Abstract

A number of bacterial virulence factors have been observed to adopt structures similar to that of aerolysin, the principal toxin of Aeromonas species. However, a comprehensive description of architecture and structure of the aerolysin-like superfamily has not been determined. In this study, we define a more compact aerolysin-like domain--or aerolysin fold--and show that this domain is far more widely spread than anticipated since it can be found throughout kingdoms. The aerolysin-fold could be found in very diverse domain and functional contexts, although a toxic function could often be assigned. Due to this diversity, the borders of the superfamily could not be set on a sequence level. As a border-defining member, we therefore chose pXO2-60--a protein from the pathogenic pXO2 plasmid of Bacillus anthracis. This fascinating protein, which harbors a unique ubiquitin-like fold domain at the C-terminus of the aerolysin-domain, nicely illustrates the diversity of the superfamily. Its putative role in the virulence of B. anthracis and its three dimensional model are discussed.

Figure 1. A simplified topology of a common core for aerolysin-like β-PFTs.

Five β-strands that span the structure of common core of aerolysin-like toxins have been numbered from 1 to 5. The first β-strand in most cases does not maintain extended secondary structure through, therefore we divided it into two (denoted as β1a and β1b). β-strands in so-called “insertion” loop are not strictly preserved, so we did not number them. The fifth strand (marked in orange) is not present in the alignment. Due to different lenghts of the variable loop connecting β4 and β5, we were unable to precisely align the last strand (see text).

Figure 2. Topology of a common core for aerolysin-like β-PFTs mapped onto structures.

A: hemolytic lectin, PDB code 1w3f; B: parasporin, PDB code 1ztb; C: epsilon-toxin, PDB code 1uyj; D: proaerolysin, PDB code 3c0n. Color scheme as on Fig. 1: blue - core with conserved sequence; red – variable loop; orange – the fifth, weakly conserved β-strand (see text).

Figure 3. Alignment of the conserved common core of aerolysin like β-PFTs.

Beta strands corresponding to the structure of proaerolysin (3c0n) and previously shown topology are marked above the alignment. The B1a is marked in light blue as only part of it seem to hit the alignment. A putative “insertion loop” is marked with red box. This fragment has calculated an average hydrophobicity according to Kyte-Doolittle scale in each column and shown above the alignment. Abbrevations: Banth|pXO2-60 - pXO2-60 protein, gi: 51704196 [Bacillus anthracis]; PDB|3c0n_A - proaerolysin, PDB structure 3c0n, chain A [Aeromonas hydrophila]; Ahydr|aeropre - aerolysin-3 precursor, gi: 2501301 [Aeromonas hydrophila]; Ggall|snatte3 - protein similar to nattering-3, gi: 118105776 [Gallus gallus]; Cpyrr|ep37-L2 - ep37-L2 protein, gi: 2339973, [Cynops pyrrhogaster]; Vvini|hypprot - hypothetical protein, gi: 147838248 [Vitis vinifera]; Nvect|hypprot - hypothetical protein, gi: 156349328 [Nematostella vectenensis]; Fvelu|teerdec - TEER-decreasing protein, gi: 3551186 [Flammulina velutipes]; Bunif|hypprot - hypothetical protein, gi: 160892167 [Bacteroidetes uniformis]; Dreri|hypprot - hypothetical protein, gi: 162139040 [Danio rerio].

Figure 4. Domain organization of the Aerolysin/ETX pore-forming superfamily.

Proteins 1–4 are present in bacteria, 5–11 in Eukaryotes. Domain organization representatives: (1) alpha-toxin, gi:452163 [Clostridium septicum]; (2) Hemolysin-3, gi:2501300 [Aeromonas hydrophila]; (3) hypothetical protein BACUNI_04630, gi:160892167 [Bacteroides uniformis ATCC 8492]; (4) pXO2-60 protein, gi: 10956450 [Bacillus anthracis]; (5) hypothetical protein CAN71829, gi:147838248 [Vitis vinifera]; (6) ep37-L2, gi:2339973 [Cynops pyrrhogaster]; (7) Natterin-3 precursor, gi:75571591 [Thalassophryne nattereri]; (8) hypothetical protein NEMVEDRAFT_v1g221281, gi:156349328 [Nematostella vectensis]; (9) hypothetical protein LOC494812, gi:148223884 [Xenopus laevis]; (10) hypothetical protein LOC613112, gi:73853870 [Xenopus tropicalis]; (11) hypothetical protein LOC568775, gi:162139040 [Danio rerio].

Figure 5. Cluster map of aerolysin superfamily.

All sequenced from nr database similar to aerolysin core profile at E-value of 100 and better were used including false positives. Major groups of proteins are highlighted with ovals and described. Presence of a clear false positive (protective antigen) is highlighted to indicate difficulties in assessing the border of the superfamily.

Figure 6. Structural model of pXO2-60.

Color scheme: blue - core with conserved sequence; red – variable loop; orange – the fifth, weakly conserved β-strand; green – β-grasp domain modeled on ubiquitin protein. Two parts of the conserved core that form b-sandwich structures are marked as domain III and IV (historically). Variable loop that connects β4 and weakly conserved β5 is barely visible because of its short length.

Figure 7. Alignment between ubiquitin fold domain of pXO2-60 and ubiquitin protein 1ubi.

Green segments correspond to beta conformation, while orange to alpha conformation. Model with the same color scheme is shown below alignment.