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1.
Sci Rep ; 11(1): 6447, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33742033

ABSTRACT

Tripartite members of the ClyA family of α-PFTs have recently been identified in a number of pathogenic Gram-negative bacteria, including the human pathogen Serratia marcescens. Structures of a Gram-negative A component and a tripartite α-PFT complete pore are unknown and a mechanism for pore formation is still uncertain. Here we characterise the tripartite SmhABC toxin from S. marcescens and propose a mechanism of pore assembly. We present the structure of soluble SmhA, as well as the soluble and pore forms of SmhB. We show that the ß-tongue soluble structure is well conserved in the family and propose two conserved latches between the head and tail domains that are broken on the soluble to pore conformational change. Using the structures of individual components, sequence analysis and docking predictions we illustrate how the A, B and C protomers would assemble on the membrane to produce a complete tripartite α-PFT pore.

2.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 12): 577-582, 2020 Dec 01.
Article in English | MEDLINE | ID: mdl-33263568

ABSTRACT

Tripartite α-pore-forming toxins are constructed of three proteins (A, B and C) and are found in many bacterial pathogens. While structures of the B and C components from Gram-negative bacteria have been described, the structure of the A component of a Gram-negative α-pore-forming toxin has so far proved elusive. SmhA, the A component from the opportunistic human pathogen Serratia marcescens, has been cloned, overexpressed and purified. Crystals were grown of selenomethionine-derivatized protein and anomalous data were collected. Phases were calculated and an initial electron-density map was produced.


Subject(s)
Bacterial Toxins/chemistry , Bacterial Toxins/metabolism , Serratia marcescens/genetics , Bacterial Toxins/genetics , Bacterial Toxins/isolation & purification , Cloning, Molecular , Crystallography, X-Ray
3.
Nat Commun ; 10(1): 2900, 2019 07 01.
Article in English | MEDLINE | ID: mdl-31263098

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.


Subject(s)
Aeromonas hydrophila/metabolism , Bacterial Toxins/chemistry , Hemolysin Proteins/chemistry , Pore Forming Cytotoxic Proteins/chemistry , Aeromonas hydrophila/chemistry , Aeromonas hydrophila/genetics , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Crystallography, X-Ray , Hemolysin Proteins/genetics , Hemolysin Proteins/metabolism , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Pore Forming Cytotoxic Proteins/genetics , Pore Forming Cytotoxic Proteins/metabolism
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