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Principal Investigator: GREGORY W FARIS
Organization: NUMENTUS TECHNOLOGIES INC.
Fiscal Year: 2023
Award: $298,794
Funding agency: National Institute of Allergy and Infectious Diseases
PROJECT SUMMARY
There is growing recognition that extracellular vesicles (EVs)–micrometer- or nanometer-sized lipid
particles containing protein and nucleic acid cargoes–are shed by all domains of life. The ubiquity of
prokaryotic EVs suggests that the presence of bacterial EVs in biofluids could be exploited for new
diagnostics/prognostics and even therapies for diverse pathogenic bacteria. There is, however, an unmet need
for methods able to solve a fundamental problem confounding the exploitation of EV information in biology and
medicine--EVs are naturally highly heterogeneous particles and thus those of interest may be present only at
very low abundance in a mixture of other EVs. In this Phase I project, our overall experimental goal is to
address the need for analyzing EV heterogeneity (subpopulations) by focusing on an important class of
bacterial nanoparticles called outer membrane vesicles (OMVs). Specifically, our novel technology platform is
designed to address the problem of resolving OMV subpopulations at the single OMV level by directly
correlating surface protein and nucleic acid cargoes (here small RNAs or sRNAs) of dispersed OMVs, through
highly multiplexed fluorescence imaging analysis coupled with amplified readouts of both cargoes. Our ultimate
goal is to develop a unique imaging platform for the high-content, high-throughput analysis of the protein and
nucleic acid cargoes of single OMVs (and other EVs) obtained from any biological sample. Our platform could
enable novel diagnostic/prognostic “liquid biopsy” tests for bacterial infections by providing a minimally invasive
and more informative alternative (or supplement) to conventional methods, which often rely on lengthy
culturing of target organisms from clinical samples or specimens.
Our innovative platform simultaneously analyzes, in one pass, up to 10 potential OMV biomarkers in as
many as 106 dispersed OMVs obtained from a biofluid such as blood, saliva, or stool. Unlike conventional
methods, our platform can rapidly: 1) analyze diverse EVs; 2) simultaneously read multiple OMV surface
markers and thus detect subpopulations; 3) read single OMV cargoes, greatly raising information yield com-
pared to typical methods producing pooled cargo data; and 4) identify OMV subpopulations based on unique
combinations of biomarkers from points 1-3. We therefore propose three stepwise objectives. First, to optimize
the combined protein/nucleic acid analyses of OMVs shed by representative examples of Gram-negative and
Gram-positive bacteria (E. coli and S.aureus, respectively). Second, to test the capability of our platform for the
analysis in situ of sRNA cargo using dispersed single OMVs representing these major group classifications.
Third, combine our surface molecule and sRNA analysis methods for the correlated direct detection in situ of
both protein and nucleic acid cargoes in dispersed single OMVs representing the two major groups. We
anticipate that our platform could become a new research/diagnostic/prognostic tool for managing pathologies
in which OMV analysis is clinically informative, and for monitoring normal or aberrant microbiome status.
Terms: <Address><Antibodies><Autoregulation><Back><Bacteria><Bacterial Infections><Biological><Biological Markers><Biology><Biotech><Biotechnology><Blood><Blood Plasma><Blood Reticuloendothelial System><Blood Serum><Cell Body><Cell Culture Techniques><Cell to Cell Communication and Signaling><Cell-Cell Signaling><Cells><Centrifugation><Centrifugation Fractionation><Cerebrospinal Fluid><Classification><Clinical><Coated vesicle><Communication><Complex><Cost Analyses><Cost Analysis><Coupled><Data><Detection><Diagnosis><Diagnostic><Dorsum><E coli><E. coli><Equilibrium><Escherichia coli><Eukaryotic Cell><Feces><Goals><Gram-Positive Bacteria><Health><Heterogeneity><Homeostasis><Human><Human Figure><Human Microbiome><Human body><Image><Immune Precipitation><Immunoprecipitation><In Situ><Individual><Infection><Life><Lipid Bilayers><Lipids><Liquid substance><Mammalian Cell><Mediating><Medicine><Membrane><Membrane Protein Gene><Membrane Proteins><Membrane-Associated Proteins><Methods><Microbe><Modern Man><Monitor><Non-Polyadenylated RNA><Normal Cell><Nucleic Acids><Organ><Organism><Organism-Level Process><Organismal Process><Outcome><Pathogenesis><Pathology><Patients><Phase><Physiologic Processes><Physiological Homeostasis><Physiological Processes><Physiology><Plasma><Plasma Serum><Population><Process><Prognosis><Protein Analysis><Proteins><RNA><RNA Gene Products><RT-PCR><RTPCR><Reporting><Research><Research Specimen><Reticuloendothelial System, Serum, Plasma><Reverse Transcriptase Polymerase Chain Reaction><Ribonucleic Acid><S aureus><S. aureus><Saliva><Sampling><Serum><Small RNA><Specimen><Speed><Staph aureus><Staphylococcus aureus><Surface><Surface Proteins><Systematics><Technology><Testing><Time><Urine><Vesicle><amplification and detection><amplification detection><antibody based detection><antibody detection><assess cost><bacteria infection><bacteria pathogen><bacterial disease><bacterial pathogen><balance><balance function><bio-markers><biologic><biologic marker><biomarker><cell culture><cell cultures><cell type><cerebral spinal fluid><cost assessment><cost evaluation><design><designing><detect antibodies><detection method><detection of amplification><detection procedure><detection technique><evaluate cost><examine cost><exosome><experiment><experimental research><experimental study><experiments><extracellular vesicles><fluid><fluorescence imaging><fluorescent imaging><high throughput analysis><host microbiome><human-associated microbiome><imaging><imaging platform><imaging system><improved><innovate><innovation><innovative><intercellular communication><interest><lipid bilayer membrane><liquid><liquid biopsy><living system><membrane structure><meter><microbial><microbiome><minimally invasive><nano meter scale><nano meter sized><nano particle><nano scale><nano-sized particle><nanometer scale><nanometer sized><nanoparticle><nanoscale><nanosized particle><new diagnostics><new technology><next generation diagnostics><novel><novel diagnostics><novel technologies><particle><pathogen><pathogenic bacteria><potential biological marker><potential biomarker><prevent><preventing><prognostic><prognostic tool><reverse transcriptase PCR><spinal fluid><stool><technology platform><technology system><vesicle release><vesicular release>