Conventional diagnoses for tuberculosis (TB), the most prevalent and deadliest infectious diseases worldwide, suffer from low specificity in HIV-TB co-infected individuals. Recently extracellular microvesicles, including exosomes, that transport molecular cargo between intracellular compartments have become an attractive marker for pathogen-associated diagnosis. Microvesicles carry not only Mycobacterium tuberculosis (Mtb) secreted antigens but also host associated markers. Although the analysis of microvesicles has great potential to allow development of new diagnostic and therapeutic applications, lack of an efficient and standard separation technique remains a major challenge. We propose to adapt our cutting-edge microfluidic filtration technology to specifically profile biocomponents inside microvesicles secreted by Mtb infected host cells. The microfluidic architecture incorporates an embedded weir-like filtration barrier that is oriented parallel to the flow direction. The transverse centrifugal flow generated within the microchannel's curved path, creates a driving force that transports smaller-sized components across the barrier. This microfluidic design can minimize the problems of pressure drop and clogging usually appearing in conventional filtration assays;hence, the system can be operated with continuous flow at high flow rate so that large volume samples, such as urine, can be easily processed. We propose to design a multi-stage filtration system that consists of a series of microfluidic channels with various barrier gaps to simultaneously isolate different sizes of microvesicles at different stages Simulated biofluids containing synthetic lipid vesicles will be used to evaluate the efficiency of our microfluidic system. We will optimize the design of microfluidic channel based on our theoretical calculations and experimental observations. In addition, the prototype multi-stage microfluidic filtration system will be utilized to recover and analyze microvesicles derived from healthy and Mtb infected macrophages. Because the production of microvesicles is strongly influenced by Mtb infection, we will analyze the physical properties (size distribution, morphology, and surface charges) and proteomics of harvested microvesicles to discriminate between healthy and infected cells. The concentrations of common exosome markers (CD63, CD9, Alix, and TSG101) and Mtb antigens carried by microvesicles (LAM and CFP-10) will also be measured. These preliminary results will allow us to investigate the potential mechanisms of intracellular transport of mycobacterial components. In summary, this Investigator-Initiated Small Research Grant can help us to establish the methodology for microvesicle analysis and to explore the potential role of microvesicles in the host response to Mtb infection. This preliminary study will allow us to understand the mechanism of Mtb infection and further develop new diagnostic and therapeutic tools.
Although extracellular microvesicles that transport Mycobacterium tuberculosis (Mtb) associated components between intracellular compartments have become an attractive marker for Mtb diagnosis, lack of an efficient and standard separation technique remains a major challenge. We propose to establish a multi- stage filtration system consisting of a series of microfluidic channels that can be operated with continuous flow at high flow rate and simultaneously isolate different sizes of intact microvesicles. This multi-stage filtration system, integrated with bioanalytical techniques including mass spectrometry, allows us to analyze the proteomics of microvesicles derived from Mtb infected host cells and discover new Mtb markers for diagnostic applications.