Lymphadenitis takes place in the pathogenesis of many infectious diseases, including the ones caused by the bioterror agents such as Y. pestis, F. tularenis, and B. anthracis. In the case of B. anthracis, the lymphatic system serves as the conduit by which germinating spores are delivered by macrophages to the sentinel lymph nodes (SLNs) within hours after exposure. The critical role of SLNs in anthrax makes them important targets for proteomic analyses. For this purpose, we will develop a novel nanoparticle-based technique to study the dynamics of SLN proteome during infection. Currently, direct application of the proteomic tools to study HL has a number of limitations relevant to the assay sensitivity, sample size and preparation procedures, especially in small-rodent models often used in anthrax research. To alleviate these limitations we invented the core-shell, affinity bait, hydroge nanoparticles that quickly capture, protect from degradation and separate from the abundant proteins the low-molecular-weight (LMW) fraction of the sample proteins in one step for direct downstream analysis by mass spectrometry (MS). We will identify the host protein targets associated with tissue damage and obtain new information on the hemorrhage-inducing mechanism of pathogenicity operating in the specific environment of lymphatics. At certain time points during the course of infection the SLNs will be visualized with the tracer dye. We will inject SLNs with harvesting nanoparticles to capture biomolecules within the SLN interstitium for deciphering its proteomic composition. Nanoparticles will be readily retrieved from the surgically excised SLNs by laser capture microdissection of the whole lymph node or any portion of its architecture. The nanoparticle-harvested biomolecules can be further analyzed by any analytical platform including MS, immunoassays, or microarrays. Our preliminary data support feasibility of the suggested experimental approach. The core shell nanoparticles containing different baits demonstrate high-affinity capture of host and bacterial proteins that can be reliably identified by MS and further validated using the highly-sensitive, high-throughput proteomics platform, Reverse-Phase Microarray (RPMA). The approach we develop will be broadly applicable to analyses of SLNs during different diseases.
Aim 1. Inject nanoparticles into lymph nodes in a murine model to harvest the in vivo proteome of the LN microenvironment.
Aim 2 : Assess the LN proteome during the time course of anthrax infection with lethal and non-lethal strains.
The objective of this proposal is to develop the procedure for in situ harvesting and analyzing the lymph node content in mice using nanoparticles in order to assess the proteins targeted by B. anthracis during anthrax lymphadenitis. We will use mass spectroscopy of the low-molecular-weight proteome and validate the identified proteins using the reverse-phase microarray technology. In the experiments with mutant B. anthracis strains, we will identify contribution of major virulence factors to lymph node damage during infection.
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