Abstract
Rapid in situ detection of pathogens coupled with high resolution imaging in the distal human lung has the potential to provide new insights and diagnostic utility in patients in whom pneumonia is suspected. We have previously described an antimicrobial peptide (AMP) Ubiquicidin (fragment UBI29–41) labelled with an environmentally sensitive fluorophore that optically detected bacteria in vitro but not ex vivo. Here, we describe further chemical development of this compound and demonstrate that altering the secondary structure of the AMP to generate a tri-branched dendrimeric scaffold provides enhanced signal in vitro and ex vivo and consequently allows the rapid detection of pathogens in situ in an explanted human lung. This compound (NBD-UBIdend) demonstrates bacterial labelling specificity for a broad panel of pathogenic bacteria and Aspergillus fumigatus. NBD-UBIdend demonstrated high signal-to-noise fluorescence amplification upon target engagement, did not label host mammalian cells and was non-toxic and chemically robust within the inflamed biological environment. Intrapulmonary delivery of NBD-UBIdend, coupled with optical endomicroscopy demonstrated real-time, in situ detection of bacteria in explanted whole human Cystic Fibrosis lungs.
Highlights
Rapid in situ detection of pathogens coupled with high resolution imaging in the distal human lung has the potential to provide new insights and diagnostic utility in patients in whom pneumonia is suspected
Bacterial labelling was demonstrated in a concentration dependent manner (Fig. 1D) and of a panel of clinically relevant lung pathogenic bacteria, including both Gram-positive and Gram-negative species (Fig. 1E) with varying labelling intensity (Fig. 1F)
To ensure the compound retains specify and does not label the inflammatory cell infiltrate observed with pneumonia, NBD-UBIdend was co-cultured with freshly isolated primary human neutrophils, monocytes, lymphocytes and human alveolar macrophages (Fig. 2A–D) with no labelling seen
Summary
Rapid in situ detection of pathogens coupled with high resolution imaging in the distal human lung has the potential to provide new insights and diagnostic utility in patients in whom pneumonia is suspected. We have previously described an antimicrobial peptide (AMP) Ubiquicidin (fragment UBI29–41) labelled with an environmentally sensitive fluorophore that optically detected bacteria in vitro but not ex vivo. We describe further chemical development of this compound and demonstrate that altering the secondary structure of the AMP to generate a tri-branched dendrimeric scaffold provides enhanced signal in vitro and ex vivo and allows the rapid detection of pathogens in situ in an explanted human lung. This compound (NBD-UBIdend) demonstrates bacterial labelling specificity for a broad panel of pathogenic bacteria and Aspergillus fumigatus. Linear fragment coupled with a fluorophore is insufficient for bacterial imaging in ex vivo lungs due to rapid degradation, despite being utilised as a nuclear medicine imaging agent[15]
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