Species-specific peptide ligands for the detection of Bacillus anthracis spores

Abstract

Currently available detectors for spores of Bacillus anthracis, the causative agent of anthrax, are inadequate for frontline use and general monitoring. There is a critical need for simple, rugged, and inexpensive detectors capable of accurate and direct identification of B. anthracis spores. Necessary components in such detectors are stable ligands that bind tightly and specifically to target spores. By screening a phage display peptide library, we identified a family of peptides, with the consensus sequence TYPXPXR, that bind selectively to B. anthracis spores. We extended this work by identifying a peptide variant, ATYPLPIR, with enhanced ability to bind to B. anthracis spores and an additional peptide, SLLPGLP, that preferentially binds to spores of species phylogenetically similar to, but distinct from, B. anthracis. These two peptides were used in tandem in simple assays to rapidly and unambiguously identify B. anthracis spores. We envision that these peptides can be used as sensors in economical and portable B. anthracis spore detectors that are essentially free of false-positive signals due to other environmental Bacillus spores.

FIG. 1.

FACS analysis of the binding of the TYPLPIRGGGC-PE conjugate to selected Bacillus spores. The concentrations of the peptide-PE conjugate (TYP peptide-PE) are indicated. The spore species is indicated in each panel, where possible. The data shown in the bottom panel are for spores of B. cereus ATCC 4342 (representative of the other 15 strains), and the minimal binding at a conjugate concentration of 4,000 nM was not peptide specific. Other details are provided in Materials and Methods.

FIG. 2.

FACS analysis comparing the abilities of the TYPLPIRGGGC-PE (TYP peptide-PE) and ATYPLPIRGGGC-PE (ATYP peptide-PE) conjugates to bind to selected Bacillus spores. Also shown are the binding results for a control peptide (HWHHHGHGGGC)-PE conjugate. Spores were mixed with a 40 nM concentration of each peptide-PE conjugate.

FIG. 3.

Fluorescence microscopy showing selective binding of ATYPLPIRGGGC-PE to B. anthracis spores. The indicated spores (B. anthracis Sterne or B. thuringiensis subsp. kurstaki) were mixed with a 40 nM concentration of either ATYPLPIRGGGC-PE (ATYP) or a control peptide (ILPRPYTGGGC)-PE conjugate and examined by either phase-contrast or fluorescence microscopy.

FIG. 4.

FACS analysis contrasting the binding of the ATYPLPIRGGGC-PE (ATYP peptide-PE) and SLLPGLPGGGC-PE (SLLPGL peptide-PE) conjugates to selected Bacillus spores. Also shown are the binding results for a control peptide (HWHHHGHGGGC)-PE conjugate. Spores were mixed with a 400 nM concentration of each peptide-PE conjugate.