Vauchelles, R. | bioRxiv Science

bioRxiv2025

Bioimaging with fluorescent nucleic-acid aptamers for the specific detection and quantification of Pseudomonas aeruginosa alone and in heterogeneous bacterial populations

The rising prevalence of bacterial infections, antibiotic resistance, and emerging pathogens underscores the urgent need for innovative diagnostic approaches. Aptamers, short nucleic acid sequences with high specificity and affinity for their targets, are promising candidates for diagnostic applications due to their ability to detect a wide range of pathogens. In this study, we present a fluorescent aptamer-based bioimaging approach for detecting Pseudomonas aeruginosa, a multidrug-resistant pathogen of significant clinical concern. Conjugated with fluorescent dye, the detection efficacy of the F23 aptamer was evaluated on 15 different Gram-negative and Gram-positive bacteria, including fixed and lived cells, as well as homogeneous and heterogeneous population. To quantify microscopy images, we developed an automated, open-access identification software using ImageJ. Its high sensitivity provides a robust platform for accurately quantifying bacteria labeled with aptamers and potentially other fluorescent ligands. For instance, it successfully detected 1122 P. aeruginosa cells labeled with aptamer F23 out of a total of 1123 P. aeruginosa cells in a single image. With almost 200,000 analyzed bacteria and an exceptionally clear signal-to-noise ratio, we demonstrated that the F23 aptamer effectively detects various reference and clinical strains of Pseudomonas aeruginosa, while failing to detect Gram-positive Staphylococcus aureus, Staphylococcus haemolyticus, Staphylococcus epidermidis and Corynebacterium striatum, as well as Gram-negative Klebsiella pneumonia, Acinetobacter baumannii, and Escherichia coli. The F23 aptamer is therefore a promising tool to distinguish Pseudomonas aeruginosa from different strains of the skin microbiota. However, our quantitative method also revealed partial labeling to other bacterial cells, highlighting the issue of refining aptamer selection to improve selectivity.

microbiology↗

bioRxiv2024

New therapeutic combination to enhance endocytosis of antibodies and nucleic-acid aptamers targeting EGFR in glioblastoma cells

Active targeting is based on the binding of ligands to receptors present on the surface of targeted cells, in order to promote the internalization of the drugs conjugated to the ligands. Several conjugates are already in use or under development for active targeting of tumors, the most widely known being antibody-drug conjugates (ADC). They combine the specificity of monoclonal antibodies with the cytotoxicity of chemotherapeutic molecules. Other than antibodies, nucleic-acid aptamers, are promising ligands to deliver conjugated drugs by active targeting in tumor cells. The therapeutic efficacy of conjugates largely depends on their endocytosis and vesicular trafficking. However, so far, no therapeutic approach to enhance endocytosis of conjugates is available. In recent studies, we showed that gefitinib, a tyrosine kinase inhibitor directed against the epidermal growth factor receptor EGFR, induces a massive, non-physiological endocytosis of EGFR, known as gefitinib-mediated endocytosis (GME), in different glioblastoma cell lines. We thus hypothesized that besides promoting endocytosis of EGFR, gefitinib could also promote endocytosis of its ligands. In this study, we proved by quantitative fluorescence bioimaging, that gefitinib is indeed able to strengthen the endocytosis of fluorophore-conjugated EGFR-specific antibodies and aptamers. We also showed that the GME potentiates the toxicity of an antibody-drug conjugate, even at low concentrations. Our results suggest the development of a new therapeutic combination, of ADC and gefitinib, to potentiate the delivery of ADC and likely other conjugates targeting EGFR in glioblastoma, while limiting side effects on non-targeted cells.

cell biology↗