Figure 1.
Strategies for site-specific labeling of proaerolysin.
A Structure of the proaerolysin monomer (PDB: 1PRE). Proaerolysin consists of several different domains, two of which are responsible for receptor binding (domains 1 and 2), one containsing the trans-membrane domain, and the C-terminal peptide (CP), which functions as a chaperone and dissociates from the rest of the complex upon heptamer association and pore formation. B Sortase reaction mechanism. C-terminal sortagging: sortase cleaves after threonine in the context of its recognition motif resulting in the formation of a new covalent bond with the N-terminus of an added oligoglycine or oligoalanine nucleophile coupled to a label of choice. N-terminal sortagging: the N-terminal glycine of proaerolysin is recognized as a nucleophile by sortase and conjugated to an LPXTG/A probe bearing a label. C Structures of probes used in this study. Not depicted is AAA.Alexa Fluor 647, which is similar to GGG.Alexa Fluor 647, but with alanine replacing glycine. PelB: periplasm targeting sequence, cleaved off by the producer bacteria upon export of proaerolysin to the periplasm. H6: hexahistidine handle for affinity purification. Protease cleavage sites are recognized by target cell surface proteases such as furin. CP: C-terminal peptide, serves as a chaperone for proaerolysin. Upon its loss, proaerolysin is converted to mature aerolysin (AeL). D Scheme for wild type (WT) and sortaggable versions of proaerolysin with their designations. The LPXTG/A pentapeptides are sortase recognition motifs.
Figure 2.
Impact of aerolysin modification on toxic activity.
Aerolysin variants were titrated on KBM7 cells. 0.5×105 cells per sample were incubated with toxin for 1 hour at 37°C in a total volume of 100 µL, stained with propidium iodide (PI), and the PI negative percentage determined by flow cytometry. The concentration range for the aerolysin variants ranged from 60 ng/mL to 4 pg/µL. Every condition was tested in triplicate. The percentage of PI negative controls was set to 100%, and the 50% lethal dose (LC50) calculated in R. 0.001 was added to all concentration values to avoid taking a log2 of 0.
Figure 3.
Installation of a single label on proaerolysin.
The fluorophore carboxytetramethylrhodamine (TAMRA) was installed at the N-terminus of aerolysin (NAeL.CP), at the C-terminus of aerolysin upstream of the CP (AeLC) and at the C-terminus of the C-terminal peptide (AeL.CPC) with sortase. A, C, E Schematic representation of the sortagging reactions using of NAeL.CP, AeL.CPC, AeLC respectively. B, D Sortagging of NAeL.CP and AeL.CPC, respectively, with respective control conditions, resolved by SDS PAGE and imaged with a fluorescence scanner. Product is visible by fluorescent signal. SrtAStrep and SrtAStaph recognize and cleave LPXTA and LPXTG motives, respectively. F Purification of labeled AeLTAMRA, gel filtration. The first peak in the A280 elution profile corresponds to aerolysin, the second to sortase, and the third to free nucleophile. G Analysis of the first peak of the gel filtration elution profile with SDS PAGE followed by fluorescence image scan and Coomassie stain. A fraction of AelC is not converted to fluorescent product.
Figure 4.
Double-labeling of proaerolysin.
Double-labeling was achieved with a two-step approach. A Schematic representation of the dual labeling strategy of proaerolysin. B We used SrtAStrep to install an oligoalanine coupled to the fluorophore AF647 at the C-terminus of proaerolysin, followed by a gel filtration purification step. C Elution profiles were analyzed by SDS-PAGE, fluorescence scan and coomassie stain. D The reaction product was subjected to the second round of sortagging with SrtAStaph7M and LPETG-coupled TAMRA fluorophore for N-terminal labeling. SrtAStaph does not recognize or cleave LPXTA, hence the C-terminal label remains intact. A single peak is observed on the elution profile as immobilized sortase was used for the reaction and removed prior to gel filtration. E Elution profiles were analyzed by SDS-PAGE followed by fluorescence scan.
Figure 5.
Aerolysin variants, fluorescently labeled, bind to the cell surface of HeLa cells. Images were acquired by confocal fluorescence microscopy. A Single labeled aerolysin versions. For comparable signal intensity, different aerolysin concentrations were required as indicated. B Double-labeled aerolysin and unlabeled aerolysin control.
Figure 6.
Dissociation of the C-terminal chaperone in the course of intoxication.
HeLa cells were incubated with TAMRAAeL.CPAF647 for 30 minutes at 4°C, washed, and the temperature shifted to 37°C. Images were acquired by confocal microscopy.
Figure 7.
Identification of new aerolysin receptors.
BiotinAeL.CP was used to identify new GPI-anchored proteins that bind Aerolysin. A Biotin.LPETG was attached to the N-terminus of proaerolysin via sortagging. The purified reaction product was analyzed by immunoblot. B HeLa cells were incubated with BiotinAeL.CP for 3 hours at 4°C and subsequently lysed with 0.5% NP-40. After pull-down with neutravidin beads, proteins were eluted, analyzed by SDS-PAGE, and subjected to mass spectrometry. Five GPI-anchored proteins were identified. UniProt accession codes are indicated. Peptides identified by mass spectrometry, lipidated amino acids, signal peptides, as well as peptides cleaved off from the pro-proteins are highlighted. C Binding of BiotinAeL.CP to mesothelin and to CD59 was verified by immunoblot.