Identification and structural analysis of the tripartite α-pore forming toxin of Aeromonas hydrophila

Abstract

The alpha helical CytolysinA family of pore forming toxins (α-PFT) contains single, two, and three component members. Structures of the single component Eschericia coli ClyA and the two component Yersinia enterolytica YaxAB show both undergo conformational changes from soluble to pore forms, and oligomerization to produce the active pore. Here we identify tripartite α-PFTs in pathogenic Gram negative bacteria, including Aeromonas hydrophila (AhlABC). We show that the AhlABC toxin requires all three components for maximal cell lysis. We present structures of pore components which describe a bi-fold hinge mechanism for soluble to pore transition in AhlB and a contrasting tetrameric assembly employed by soluble AhlC to hide their hydrophobic membrane associated residues. We propose a model of pore assembly where the AhlC tetramer dissociates, binds a single membrane leaflet, recruits AhlB promoting soluble to pore transition, prior to AhlA binding to form the active hydrophilic lined pore.

The A component (SmhA) of a tripartite pore-forming toxin from Serratia marcescens: expression, purification and crystallographic analysis

“…Tripartite -pore-forming toxins (-PFTs) are members of the ClyA -pore-forming toxin family (Fagerlund et al, 2008;Wilson et al, 2019); however, unlike ClyA, where the pore is formed from an oligomer of a single protomer, three proteins (A, B and C) are involved in active pore formation (Sastalla et al, 2013;Lindbä ck et al, 2004;Wilson et al, 2019;Beecher & Macmillan, 1991). In the ClyA family the active pore is formed when the soluble protein(s) undergo a large-scale conformational change to expose the membrane-binding regions, with protomers assembling into a hydrophilic-lined pore (Benke et al, 2015;Roderer & Glockshuber, 2017).…”

“…In the ClyA family the active pore is formed when the soluble protein(s) undergo a large-scale conformational change to expose the membrane-binding regions, with protomers assembling into a hydrophilic-lined pore (Benke et al, 2015;Roderer & Glockshuber, 2017). It is proposed that each protein of the tripartite -PFT (A, B and C) fulfils a role in the active pore that is provided by different regions of the ClyA protomer (Wilson et al, 2019). The C component makes the first attachment to the target cell, binding to a single leaflet of the membrane, and is equivalent in function to the -tongue region of soluble ClyA.…”

“…In the tripartite -PFTs the A and B components are functionally equivalent to this region of ClyA. The B component acts as the pore-forming unit, using two hydrophobic helices to span the membrane, with the A component proposed to provide amphipathic helices that produce the hydrophilic interior lining of the oligomeric pore (Wilson et al, 2019;Mueller et al, 2009;Benke et al, 2015).…”

“…Tripartite -PFTs were first identified in the pathogenic Gram-positive bacterium Bacillus cereus, when the Hbl system, and later the NheABC system, were identified as vital toxins in its pathogenicity and the cause of a major foodpoisoning outbreak in Norway (Thompson et al, 1984;Lund & Granum, 1996;Beecher et al, 1995). The tripartite -PFT family has recently been expanded into a large number of clinically and economically important Gram-negative bacteria, including the fish and opportunistic human pathogen Aeromonas hydrophila (Wilson et al, 2019). The -PFT toxin AhlABC from A. hydrophila, like NheABC, has been shown to be lytic to mammalian cells and forms pores in membranes (Wilson et al, 2019;Lindbä ck et al, 2004).…”

“…The tripartite -PFT family has recently been expanded into a large number of clinically and economically important Gram-negative bacteria, including the fish and opportunistic human pathogen Aeromonas hydrophila (Wilson et al, 2019). The -PFT toxin AhlABC from A. hydrophila, like NheABC, has been shown to be lytic to mammalian cells and forms pores in membranes (Wilson et al, 2019;Lindbä ck et al, 2004).…”