Rapid Purification of Endotoxin-Free RTX Toxins.

Ondrej Stanek,Adriana Osickova,Jiri Masin,David Jurnecka,Radim Osicka,Peter Sebo

doi:10.3390/toxins11060336

Ondrej Stanek, Adriana Osickova + Show 4 more

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https://doi.org/10.3390/toxins11060336

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Abstract

Cytolytic leukotoxins of the repeat in toxin (RTX) family are large proteins excreted by gram-negative bacterial pathogens through the type 1 secretion system (T1SS). Due to low yields and poor stability in cultures of the original pathogens, it is useful to purify recombinant fatty-acylated RTX cytolysins from inclusion bodies produced in E. coli. Such preparations are, however, typically contaminated by high amounts of E. coli lipopolysaccharide (LPS or endotoxin). We report a simple procedure for purification of large amounts of biologically active and endotoxin-free RTX toxins. It is based on the common feature of RTX cytolysins that are T1SS-excreted as unfolded polypeptides and fold into a biologically active toxin only upon binding of calcium ions outside of the bacterial cell. Mimicking this process, the RTX proteins are solubilized from inclusion bodies with buffered 8 M urea, bound onto a suitable chromatographic medium under denaturing conditions and the contaminating LPS is removed through extensive on-column washes with buffers containing 6 to 8 M urea and 1% Triton X-100 or Triton X-114. Extensive on-column rinsing with 8 M urea buffer removes residual detergent and the eluted highly active RTX protein preparations then contain only trace amounts of LPS. The procedure is exemplified using four prototypic RTX cytolysins, the Bordetella pertussis CyaA and the hemolysins of Escherichia coli (HlyA), Kingella kingae (RtxA), and Actinobacillus pleuropneumoniae (ApxIA).

Highlights

Repeat in toxin (RTX) leukotoxins/cytolysins are a large family of pore-forming and immunomodulatory toxins of gram-negative pathogens belonging to the genera Bordetella, Escherichia, Kingella, Moraxella, Morganella, Photorhabdus, Proteus, Vibrio, and the Pasteurellaceae family
repeat in toxin (RTX) toxins exert cytotoxic activities on a broad spectrum of eukaryotic host cells and share several characteristic features, such as: (i) the presence of a pore-forming hydrophobic domain; (ii) the requirement for activation by a posttranslational fatty-acyl modification accomplished by a cognate toxin activating acyltransferase (RtxC); (iii) the presence of an extensive C-terminal calcium-binding domain that consists of acidic glycine-rich repeats exhibiting a nonapeptide consensus sequence
We aimed to develop a simplified single step purification procedure (Figure 1a) that would be generally applicable for removal of LPS from urea-solubilized RTX toxin preparations

Summary

Introduction

Repeat in toxin (RTX) leukotoxins/cytolysins are a large family of pore-forming and immunomodulatory toxins of gram-negative pathogens belonging to the genera Bordetella, Escherichia, Kingella, Moraxella, Morganella, Photorhabdus, Proteus, Vibrio, and the Pasteurellaceae family. Enzyme domain of CyaA translocates into cell cytosol and once activated by intracellular calmodulin, it subverts cellular signaling by catalyzing the unregulated conversion of cytosolic ATP to cAMP [4,5]. Another prototypic RTX cytolysin is α-hemolysin (HlyA), which is produced by numerous pathogenic as well as commensal Escherichia coli isolates. Actinobacillus pleuropneumoniae, the etiological agent of swine pleuropneumonia, secretes the ApxIA hemolysin exhibiting strong hemolytic and cytotoxic activities [8,9]

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