Secretion and properties of the large and small lobes of the channel‐forming toxin aerolysin

Abstract

SummaryAerolysin is a dimeric protein secreted by Aeromonas spp. that binds to glycosylphosphatidylinositol-anchored receptors on target cells and becomes insertion competent by oligomerizing. The protein comprises two lobes joined by a short arm. The large lobe is thought to be responsible for channel formation, whereas the small lobe is believed to stabilize the dimer, and it may also contain the receptor binding site. We cloned and expressed the DNA for both lobes of the toxin separately and together in A. salmonicida. The large lobe produced alone was secreted, although more poorly than native protein. The small lobe with the arm produced by itself was not secreted. When the large lobe without the arm was co-produced with the small lobe with the arm, both were secreted, and they co-purified as a stoichiometric complex. Analytical ultracentrifugation showed that they form a heterotetramer corresponding to the native dimer. The purified product was nearly as active as aerolysin, but lost activity and became trypsin sensitive above 25ЊC. The large lobe with the arm was also purified. It was shown to be monomeric, confirming that the small lobe is responsible for dimer stabilization. The large lobe had very low channel-forming activity, although it was correctly processed by trypsin, and it could form stable oligomers. Surprisingly, the large lobe was found to bind to several glycosylphosphatidylinositol-anchored proteins, indicating that it contains at least part of the receptor-binding domain.

Functional Properties of the p33 and p55 Domains of the Helicobacter pylori Vacuolating Cytotoxin

“…Two previous studies have investigated putative func- tional domains of the pore-forming toxins aerolysin (58) and listeriolysin (59). When the two domains of aerolysin and listeriolysin were co-expressed in Aeromonas and Listeria respectively, in both cases the two domains were able to assemble into proteins with hemolytic activity (58,59). In contrast, when the two domains of listeriolysin were expressed separately and then mixed together, no hemolytic activity was detected (59).…”

The Identification and Structure of the Membrane-spanning Domain of the Clostridium septicum Alpha Toxin

“…This difference may reflect the fact that aerolysin exists as a head-to-tail dimer in solution (13), whereas AT exists as a monomer, 4 and so the aerolysin dimer must dissociate prior to oligomerization. Aerolysin dimer formation is, in part, mediated via its small lobe (13,37), which is missing in AT. When the small lobe is removed from aerolysin-like AT, it exists as a monomer in solution (37).…”

“…Aerolysin dimer formation is, in part, mediated via its small lobe (13,37), which is missing in AT. When the small lobe is removed from aerolysin-like AT, it exists as a monomer in solution (37). Therefore, it appears that the movement of the D3 loop of aerolysin may also be necessary for the disruption of the aerolysin dimer after receptor binding (38).…”

Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes

“…Fragment complementation has been performed either with fragments produced by limited proteolysis or chemical cleavage, or with incomplete polypeptide chains expressed independently by genetic manipulations. The in vivo assembly of functional proteins from complementing fragments has been demonstrated for several proteins of Gram-negative bacteria, including integral membrane proteins (Bibi & Kaback, 1990) and soluble cytoplasmic, periplasmic or secreted proteins (Betton & Hofnung, 1994 ;Diep et al, 1998 ;Shiba & Schimmel, 1992). Taking advantage of the sequence similarities between LLO and PFO, we elaborated a theoretical 3D model of LLO folding, and engineered truncated and modified LLO proteins and expressed them in L. monocytogenes.…”