Abstract
Summary
The two-step labeling protocol using Lysostaphin and bio-orthogonal click chemistry for staining bacteria is described. The click protocol is efficient in labeling staphylococci and is non-toxic. This protocol promises the efficient of infections that are difficult to assess by conventional imaging.
Introduction
Infection diagnostics in clinics is time consuming, invasive and relays on microbiological cultures. New probes and labeling protocols enabling rapid and specific detection of infection in vivo shall improve the situation. We investigated the potential of a new click labeling protocol to detect staphylococci. Azido (N3) - modified Lysostaphin and DIBO (Di-benzocyclooctyne) - dye were used in the two-step bacteria-labeling protocol. N3 and DIBO were the counterparts of the bioorthogonal “click” reaction. In the first step, Lysostaphin-N3 bound to Staphylococcus aureus. In the second step, N3 clicked to DIBO thus achieving S. aureus selective labeling.
Methods
S. aureus NCTC 10788 and E. coli NCTC 12241 (from National Collection of Type Cultures), primary sheep osteoblasts and C57BL/6 mice were used for this study. DIBO-Alexa488 (Invitrogen ®), DyeLight488 (Thermofisher ®), NHS-N3 (Lumiprobe ®), Lysostaphin (Sigma-Aldrich ®) were purchased. In vitro we used standard microbiological protocols to assess antimicrobial and labeling activity of the “click” probe (Lysostaphin-N3 plus DIBO-dye), one-step-labeled Lysostaphin-Dye and non-labeled Lysostaphin. Flow cytometry, Fluorescence microscopy, and Spectrophotometry were employed to measure binding of the probes to bacteria. The cytotoxicity of the probes on osteoblasts was performed using Presto Blue Cell Viability test (Invitrogen ®). In vivo we used Fluorescence Intravital Microscopy and mice with dorsal skin-fold chambers (approved by the local governmental animal care committee). Subsequently to anesthesia each mouse received S. aureus strain Cowan I intravenously. This was followed by intravenous injections of the test probes.
Results
Lysostaphin-N3 partially lost its antimicrobial property if compare to Lysostaphin alone, but still bound to S. aureus efficiently and clicked DIBO-dye afterwards. There was no significant cytotoxicity of “click” reagents on sheep osteoblasts. In vitro the two-step labeling with Lysostaphin-Click was more efficient than the one-step labeling with Lysostaphin-Dye. In vivo the two-step and one-step labeling differed rather qualitatively. In the two-step protocol the “click” probe labeled bacteria adherent to the blood vessels and bacteria extravasated into the soft tissue around 30 minutes post-injection. In the one-step protocol bacteria were labeled quickly (within minutes) in the blood flow and on the blood vessels but not in the soft tissue.
Discussion/Conclusion
Effective labeling bacteria with the click probe could be useful for a quantitative and selective assessment of infection, in particular for staphylococci infections, which are common in orthopedic implants but often difficult to detect. The one and two-step protocols successfully labeled bacteria in mice but the labeling was different. In the one-step protocol bacteria were labeled in the blood flow quickly; in the two-step protocol bacteria adherent to the blood vessels and extravasated into the soft tissue bacteria were labeled with a delay.
