Staphylococcus aureus alpha-toxin. Production of functionally intact, site-specifically modifiable protein by introduction of cysteine at positions 69, 130, and 186

M Palmer,R Jursch,U Weller,A Valeva,K Hilgert,M Kehoe,S Bhakdi

doi:10.1016/s0021-9258(19)50293-9

Abstract

Staphylococcal alpha-toxin, the prototype of an oligomerizing, pore-forming cytotoxin, is sensitive to biochemical modifications and cannot be labeled with biotin or fluorescein under preservation of its biological activity. In this study, we have used site-directed mutagenesis to introduce cysteine residues at positions 69, 130, and 186. Each mutant was fully and rapidly reactive with several sulfhydryl-specific reagents, indicating superficial location. Coupling of SH-groups with fluorescein-maleimide or biotin-maleimide was tolerated without loss of hemolytic activity at position 130, and the formed hexamers were visible on target cells by fluorescence microscopy and could be detected on electroblots by reaction with streptavidin-peroxidase. At the two other positions, modification caused significant loss of activity. However, the labeled proteins still bound to red cells, as shown by fluorescence microscopy and electroblotting. Intrinsically labeled alpha-toxin represents a novel tool to study the interaction of this pore-former with target membranes.

Highlights

From the Institute of Medical Microbiology,University of Mainz, Augustwplutz, W-6500Germany and the §Department of Microbiology, MedicalSchool, University of Newcastle upon Tyne, Newcastleupon Tyne NE2 4HH, United Kingdom
We show here that introduction of cysteine chemical modifications and cannot be labweiltehdbio- at residue 130 generates toxin molecules that can be labeled tin or fluorescein under preservation of its biological with biotin or fluorescein at the sulfhydryl group with full activity
Each mutant was fully and rapidly binding to target cells. Use of these labeled probes renders reactive with several sulfhydryl-specificreagents, in- detection of the toxin possible after cell binding without the wtdc1oei3ilclte0lahsr,tabiafntylneugddfosluurtwheopsreiecterhefsfoiocinrcuemi-ntamlcoleelasdoslcemhoaiemftixciohiardnemoem.sCoecorrooslpbyuwyitpoeicltraiainenncg-vtdmiivsaciiotbolfyeluieSamldotHind-pbgetoerasorwidutgiaepeotstsnecttupseousodxelsfishnoiybfrdleaernn.ydtliebgrosrdoiuiedpses.nFitniufirmcthaoetniroomnmoeorrefi,csp,urfporeebreifn,icgainatdlhlyemrleoemaccabttirevadinterye-bsoiofdutunhedes on electroblots by reaction with streptavidin-peroxidase

Summary

MATERIALS AND METHODS

After our attempts to produce active toxin labeled with amino-reactive biotin or fluorescein derivatives uniformly failed, we sought to introduce selectively reactive sulfhydryl (DTT)’ (Serva, Heidelberg, Germany) was added and supernatants harvested by centrifugation at room temperature (Sorvall GS3 rotor, 20 min, 8000 rpm). Labeling of Cells-When cells were labeled for fluorescence microscopy, PBS supplementedwith 30 mM dextran 4 (Pharmacia) was used throughout for incubations and washing steps to prevenotsmotic lysis of cells [12].In the depicted experiments, high concentrations of toxins (0.2 mg/ml) were used in order to obtain documentable pictures. The cells were incubated with fluoresceinated toxins, washed twice, and subjected to fluorescence microscopy. Treatment with mM N-methylmaleimide for 15 min, followed by addition of a molar excess of DTT and buffer exchange on a PD-10 column, removed all DTNB-titratable SH-groups. PAGE yielded fluorescent protein bands with cysteine mutants buftailed to doso with wild type toxin (data nsohtown).

The cysteine mutants alsodiffered in their ability to form

Findings

CONCLUSION