Gamma-phage lysin PlyG sequence-based synthetic peptides coupled with Qdot-nanocrystals are useful for developing detection methods for Bacillus anthracis by using its surrogates, B. anthracis-Sterne and B. cereus-4342

Abstract

Background: Previous reports of site-directed deletion analysis on gamma (gamma)-phage lysin protein (PlyG) have demonstrated that removal of a short amino acid sequence in the C-terminal region encompassing a 10-amino acid motif (190LKMTADFILQ199) abrogates its binding activity specific to the cell wall of Bacillus anthracis. Whether short synthetic peptides representing the10-amino acid PlyG putative binding motif flanked by surrounding N- and C-terminal residues also selectively bind to the bacterial cell wall has not been evaluated. If such peptides do demonstrate selective binding to the cell wall, they could serve as bio-probes towards developing detection technologies for B. anthracis. Furthermore, by using B. anthracis (Sterne, 34F2), an animal vaccine and B. cereus-4342, a gamma-phage susceptible rare strain as surrogates of B. anthracis, development of proof-of-concepts for B. anthracis are feasible.

Results: Using four different methods, we evaluated six synthetic peptides representing the putative binding motif including flanking sequences (PlyG-P1 through P6) for the bacterial cell wall binding capacity. Our analysis identified PlyG-P1, PlyG-P3 and PlyG-P5 to have binding capability to both B. anthracis (Sterne, 34F2) and B. cereus-4342. The peptides however did not bind to B. cereus-11778, B. thuringiensis, and B. cereus-10876 suggesting their specificity for B. anthracis-Sterne and B. cereus-4342. PlyG-P3 in combination with fluorescent light microscopy detected even a single bacterium in plasma spiked with the bacteria.

Conclusion: Overall, these studies illustrate that the short 10-amino acid sequence 'LKMTADFILQ' in fact is a stand-alone bacterial cell wall-binding motif of PlyG. In principle, synthetic peptides PlyG-P1, PlyG-P3 and PlyG-P5, especially PlyG-P3 coupled with Qdot-nanocrystals are useful as high-sensitivity bio-probes in developing detection technologies for B. anthracis.

Figure 1

Schematic representation of PlyG peptides indicating amino acid (aa) position 185–204 of PlyG. Top sequence Represents PlyG amino acid sequence from 185–204 within the C-terminal 156–233 region. Residues underlined at position 190 and 199 in peptide 2 and 6 indicate amino acid substitutions at that position.

Figure 2

Dot-blot analysis of peptides binding to B. cereus-4342, B. anthracis-Sterne, B. cereus-11778, B. thuringiensis, and B. cereus-10876. Bacterial suspensions (10³ CFU) were blotted on to the membrane and the membrane was cut in to strips. The membrane strips were incubated individually with the six PlyG peptides. Following streptavidin-HRP incubation, the membranes were incubated with diaminobenzidine (DAB) reagent and peptide binding was detected by development of brown spots.

Figure 3

ELISA-based analysis of PlyG peptide binding to B. cereus-4342, B. anthracis-Sterne, B. cereus-11778, B. thuringiensis, and B. cereus-10876. Individual wells of a 96-well microplate were coated with desired bacterial suspensions (10³ CFU) and incubated with biotinylated PlyG peptides prior to incubation with streptavidin-HRP. Color was developed by adding TMB. Optical density of the reactions were measured at 490 nm using a microplate reader. Error bars indicate the SD from three independent experiments. Statistically significant difference (P <0.05) is represented by (*).

Figure 4

Streptavidin-conjugated Quantum Dot (QD) based analysis of PlyG peptides binding to bacteria. Human plasma was spiked with B. cereus-4342, B. anthracis-Sterne, B. cereus-11778, B. thuringiensis and B. cereus-10876. Each PlyG peptide (P1-P6) were incubated with the bacteria. Following incubation, the samples were centrifuged and resuspended in PBS. Streptavidin-conjugated quantum-dots (QDs) were then added to the sample (bacteria-peptide complex) and analyzed by a fluorescence plate reader (Synergy 4TM, Biotek, USA) with excitation spectrum set at 360–485 nm and emission spectrum at 605 nm. Error bars indicate the SD from 3 independent experiments. Statistically significant difference (P < 0.05) is represented by (*).

Figure 5

Microscopy-based sensitivity analysis of PlyG-P3 peptide binding. Bacterial cultures of Bacillus cereus 4342, B. anthracis-Sterne, B. cereus-11778, B. thuringiensis, and B. cereus-10876 were incubated with PlyG-P3 and the complex was detected by incubating with strepatividin-conjugated quantum dots. Peptide binding was detected by the fluorescing Q dots using appropriate UV filters (605 nm) under a Nikon microscope, either in phase-contrast (panels on left) or fluorescence (panels on right) fields. Note that even a single bacterium is visible in the B. cereus-Sterne field.