Jianghong Rao, Doctoral Dissertation Advisor (AC)
A Fluorogenic Trehalose Probe for Tracking Phagocytosed Mycobacterium tuberculosis.
Journal of the American Chemical Society
Tuberculosis (TB) disease is a global epidemic caused by the pathogenic Mycobacterium tuberculosis (Mtb). Tools that can track the replication status of viable Mtb cells within macrophages are vital for the elucidation of host-pathogen interactions. Here, we present a cephalosphorinase-dependent green trehalose (CDG-Tre) fluorogenic probe that enables fluorescence labeling of single live Bacille Calmette-Guérin (BCG) cells within macrophages at concentrations as low as 2 µM. CDG-Tre fluoresces upon activation by BlaC, the β-lactamase uniquely expressed by Mtb, and the fluorescent product is subsequently incorporated within the bacterial cell wall via trehalose metabolic pathway. CDG-Tre showed high selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder. The unique labeling strategy of BCG by CDG-Tre provides a versatile tool for tracking Mtb in both pre- and post-phagocytosis and elucidating fundamental physiological and pathological processes related to the mycomembrane.
View details for DOI 10.1021/jacs.0c07700
View details for PubMedID 32813512
Targeting MMP-14 for dual PET and fluorescence imaging of glioma in preclinical models.
European journal of nuclear medicine and molecular imaging
PURPOSE: There is a clinical need for agents that target glioma cells for non-invasive and intraoperative imaging to guide therapeutic intervention and improve the prognosis of glioma. Matrix metalloproteinase (MMP)-14 is overexpressed in glioma with negligible expression in normal brain, presenting MMP-14 as an attractive biomarker for imaging glioma. In this study, we designed a peptide probe containing a near-infrared fluorescence (NIRF) dye/quencher pair, a positron emission tomography (PET) radionuclide, and a moiety with high affinity to MMP-14. This novel substrate-binding peptide allows dual modality imaging of glioma only after cleavage by MMP-14 to activate the quenched NIRF signal, enhancing probe specificity and imaging contrast.METHODS: MMP-14 expression and activity in human glioma tissues and cells were measured in vitro by immunofluorescence and gel zymography. Cleavage of the novel substrate and substrate-binding peptides by glioma cells in vitro and glioma xenograft tumors in vivo was determined by NIRF imaging. Biodistribution of the radiolabeled MMP-14-binding peptide or substrate-binding peptide was determined in mice bearing orthotopic patient-derived xenograft (PDX) glioma tumors by PET imaging.RESULTS: Glioma cells with MMP-14 activity showed activation and retention of NIRF signal from the cleaved peptides. Resected mouse brains with PDX glioma tumors showed tumor-to-background NIRF ratios of 7.6-11.1 at 4 h after i.v. injection of the peptides. PET/CT images showed localization of activity in orthotopic PDX tumors after i.v. injection of 68Ga-binding peptide or 64Cu-substrate-binding peptide; uptake of the radiolabeled peptides in tumors was significantly reduced (p < 0.05) by blocking with the non-labeled-binding peptide. PET and NIRF signals correlated linearly in the orthotopic PDX tumors. Immunohistochemistry showed co-localization of MMP-14 expression and NIRF signal in the resected tumors.CONCLUSIONS: The novel MMP-14 substrate-binding peptide enabled PET/NIRF imaging of glioma models in mice, warranting future image-guided resection studies with the probe in preclinical glioma models.
View details for DOI 10.1007/s00259-019-04607-x
View details for PubMedID 31773232
Rapid and specific labeling of single live Mycobacterium tuberculosis with a dual-targeting fluorogenic probe
SCIENCE TRANSLATIONAL MEDICINE
2018; 10 (454)
Tuberculosis (TB) remains a public health crisis and a leading cause of infection-related death globally. Although in high demand, imaging technologies that enable rapid, specific, and nongenetic labeling of live Mycobacterium tuberculosis (Mtb) remain underdeveloped. We report a dual-targeting strategy to develop a small molecular probe (CDG-DNB3) that can fluorescently label single bacilli within 1 hour. CDG-DNB3 fluoresces upon activation of the β-lactamase BlaC, a hydrolase naturally expressed in Mtb, and the fluorescent product is retained through covalent modification of the Mtb essential enzyme decaprenylphosphoryl-β-d-ribose 2'-epimerase (DprE1). This dual-targeting probe not only discriminates live from dead Bacillus Calmette-Guérin (BCG) but also shows specificity for Mtb over other bacterial species including 43 nontuberculosis mycobacteria (NTM). In addition, CDG-DNB3 can image BCG phagocytosis in real time, as well as Mtb in patients' sputum. Together with a low-cost, self-driven microfluidic chip, we have achieved rapid labeling and automated quantification of live BCG. This labeling approach should find many potential applications for research toward TB pathogenesis, treatment efficacy assessment, and diagnosis.
View details for PubMedID 30111644