A labelled-ubiquicidin antimicrobial peptide for immediate in situ optical detection of live bacteria in human alveolar lung tissue

Abstract

The in situ immediate detection of the presence of bacteria in the distal human lung is of significant clinical utility. Herein we describe the development and optimization of a bacterial binding fragment (UBI29-41) of the antimicrobial peptide, ubiquicidin (UBI), conjugated to an environmentally sensitive fluorophore to enable rapid live bacterial imaging within human lung tissue. UBI29-41 was modified for stability in the presence of human lung bronchoalveolar lavage fluid, for affinity to bacterial membranes and functionality in human lung tissue. The optimized cyclic structure yields an optical molecular Smartprobe for bacterial detection in human lung tissue.

Figures

Fig. 1. NBD–UBI enables live confocal imaging of bacteria. UBI-2 demonstrates higher signal to noise for bacterial labelling over UBI-1 when imaged via confocal microscopy. Upper three panels showing UBI-1 (10 μM) with P. aeruginosa; lower panel demonstrates UBI-2 (10 μM) with P. aeruginosa. Panel insets show cross section of a single bacterium with a plot profile (right of main panel) across the FAM/NBD image in green and the counterstain in red, demonstrating fluorescence of UBI-2 but not UBI-1 across the bacteria scale bar = 5 μm.

Fig. 2. UBI-2 is unstable at 37 °C. HPLC chromatograms (gradient acetonitrile + 0.1% formic acid/water + 0.1% formic acid from 5/95 to 95/5 over 10 min, then isocratic for 4 min; detection at 380 nm). UBI-2 (1 mg mL–1 in PBS, upper panels) and UBI-3 (1 mg mL–1 in PBS, lower panels) at (A) day 0 and (B) day 3 after incubation at 37 °C.

Fig. 3. UBI-3 retains specificity and sensitivity for bacterial labelling. (A) Methionine (UBI-2) and norleucine (UBI-3) variants label bacteria with the same fluorescence intensity. Panels show bacteria labelled with either UBI-2 or UBI-3 with quantification of fluorescence (values normalised to UBI-2) (Bars represent mean, error bars represent standard error of mean, n ≥ 3, ns = not significant, scale bar = 10 μm). (B) Quantification from confocal images of UBI-3 (10 μM) imaging a panel of bacteria (statistical analyses shown compared to unlabelled bacteria, n ≥ 3 for each bacteria, *** = p < 0.001). (C) UBI-3; labels bacteria but not isolated human neutrophils (blue arrows). Dotted box enlarged in lower panel and line plot of fluorescence from green channel shown with yellow representing background, red indicating neutrophil area and blue representing single bacteria demonstrating labelling of bacteria but not neutrophils, scale bar = 10 μm. [MRSA: methicillin-resistant Staphylococcus aureus, MSSA: methicillin-sensitive Staphylococcus aureus].

Fig. 4. Reduction of fluorescent intensity of P. aeruginosa labelled with UBI-3 in the presence of human ARDS BALF. Representative images of P. aeruginosa imaged with UBI-3 (10 μM) by confocal microscopy in the presence of phosphate buffered saline (PBS) or ARDS BALF. Quantification of fluorescence intensity demonstrating significantly reduced intensity when compared to PBS (bars represent mean (±SEM), n = 3 for PBS and n = 5 for ARDS, *** = p < 0.001, scale bar = 10 μm).

Fig. 5. Sites of structural modifications undertaken on UBI-3 based on MALDI-TOF analysis of proteolytic degradation.

Fig. 6. Confocal Images of compounds UBI-3 to 9 with quantified fluorescence. Panels show methicillin-sensitive Staphylococcus aureus incubated with UBI-3 to 9 (all 10 μM) with quantification of bacterial fluorescence normalized to UBI-3 (bars represent mean (±SEM), n ≥ 3, ns = not significant, *** = p < 0.001, * = p < 0.05, scale bar = 10 μm).

Fig. 7. UBI-10 remains stable in ARDS BALF at 5 minutes. (A) Structure of UBI-10 with breakdown products in ARDS BALF. The lower panel shows a MALDI-TOF time course of UBI-10 in ARDS BALF, demonstrating initial stability but the formation of degradation products after 10 minutes.

Fig. 8. Confocal images and quantification of cyclic variants when compared to UBI-3. Panels show methicillin-sensitive Staphylococcus aureus incubated with cyclic UBI compounds (all 10 μM) with quantification of bacterial fluorescence normalized to UBI-3 (bars represent mean (±SEM), n ≥ 3, ns = not significant, ** = p < 0.01, scale bar = 10 μm).

Fig. 9. UBI-3, UBI-4 and UBI-10 label bacteria in a concentration dependent manner. (A) UBI-3, UBI-4 and UBI-10 (0.5 μM to 50 μM) co-incubated with methicillin sensitive S. aureus and imaged by confocal microscopy with quantification of fluorescence, demonstrating a concentration dependent increase in bacterial fluorescence. UBI-10 retains a higher fluorescence (per molar equivalents) at all concentrations tested. Right panel shows representative images. (B) Representative images of a co-culture of isolated human neutrophils and methicillin sensitive S. aureus and UBI-10 at 10 μM (upper panels) and at 50 μM (lower panels) demonstrating no labelling of neutrophils at 10 μM, however at 50 μM, occasional neutrophil labelling was observed (blue arrows).

Fig. 10. UBI-10 can be used to image bacteria in ex vivo human lung tissue by fibered confocal fluorescence microscopy (FCFM). FCFM imaging of bacteria in suspension (upper panels) or when co-incubated with ex vivo human lung tissue (lower panels). Left images demonstrate no intrinsic autofluorescence of bacteria, middle panels show UBI-3 can image bacteria in suspension but not in the presence of ex vivo human lung and right panels demonstrate UBI-10 can image bacteria by FCFM in the presence of ex vivo human lung with a characteristic small round, punctate fluorescence. All compounds at 10 μM, n = 3.