I have been continuing to meet with my faculty mentor, Dr. Marcy Kelly on a weekly basis to discuss the project. I have been conducting literature reviews on my project. I am still currently waiting for the microscope I will need for my experiments to be properly configured to the appropriate computer. The microscope repairperson will be coming to Pace on Tuesday to do this. In addition to literature reviews, I have also been modifying and strengthening my protocol for the experiments. I will be conducting my first trial on Monday, so that I can have slides ready to view on the microscope on Tuesday after the microscope is fixed.
This project has taught me that research will have many setbacks. It is important to keep a positive attitude and look into alternative options to overcome obstacles that will inevitably come up. This experience has helped me to better my literature reviewing skills. It has also helped me to develop my critical thinking skills which are applied to the process of designing experiments and figuring out how to solve obstacles. In conclusion, this experience, though not without difficulties, has been positive and has enabled me to grow as a scientist.
Since the last update, I have been able to design the protocols for my control experiments. This includes analyzing VASP location in wild-type, untreated macrophages, macrophages with small beads, macrophages with the addition of factors needed for activation, and macrophages with beads and the factors needed for activation. Each of these controls will need to be repeated three times to obtain results that can be analyzed using statistics. I will also be working on creating Mycobacterium bovis-BCG that is fluorescently labeled with GFP (green fluorescent protein). Once this is generated and my controls have been completed and analyzed, I will be able to infect the macrophages with the bacteria and look at the location of VASP in the macrophage. These experiments will require the use of primary and secondary antibodies for immunofluorescent examination on the fluorescent microscope.
One challenge I am currently facing is due to the computer connected to the microscope crashing. This has caused me to delay my control experiments because the cells cannot be analyzed without the fluorescent microscope. Successes of this project have included correct culturing of the macrophages and actively growing M. bovis-BCG. So far this project has strengthened my ability to design protocols. It has also helped me to learn more about literature searches and how to solve setbacks. I am looking forward to starting my control experiments as soon as the computer is repaired.
There were a few setbacks for my research this semester. Our incubator malfunctioned and was too hot while I began to grow my mycobacteria. This caused them to go into “heatshock” and cease growth. However, they have just begun to recover and are growing slowly! Their growth is essential to my project so I am also beginning to transform M. bovis-BCG with a plasmid containing GFP, just to ensure that I will have an uncontaminated, growing stock of the bacteria for use on my project. This will be completed in the coming weeks. Additionally, I have successfully grown up murine macrophages that are growing as they should. I froze them today so that I will have a supply of frozen macrophage stocks for my future work. This semester involved a lot of preparation and growth of both types of cells needed for my project. This will allow me to experiment with them over the winter break and next semester.
So far, I have learned that mycobacteria, such as M.bovis-BCG grow very slowly. They can take anywhere from two weeks to a month to grow. My bacteria have been “growing” for about one month, and I am just beginning to see growth. Additionally, I have learned that macrophages are easily contaminated so proper aseptic technique is needed at all times. I am excited to transform M.bovis BCG next week and to start on my project over the winter break. My work this semester has set up all of the components necessary to complete my experimental goals. It will be interesting to see how the macrophage actin filament network responds to infection by M. bovis-BCG. So far, my timeline is on track and I will be able to complete the project on time, even though there were a few setbacks at the beginning!
I am working on a project titled, “The Effects of the Vasodilator-stimulated Phosphoprotein on Mycobacterium bovis-BCG and the Macrophage Actin Filament Network.” It is a continuation of research that I started in September of 2011. The primary aim of the project is to determine if the Vasodilator-stimulated phosphoprotein (VASP) is involved in mediating cytoskeletal rearrangement in cells infected with Mycobacterium bovis-BCG, a model organism for the study of Mycobacterium tuberculosis. M. tuberculosis is the causative agent of tuberculosis. Tuberculosis is responsible for nearly 2 million deaths worldwide every year. There are about 10 million new cases of tuberculosis each year and about one-third of the world’s population is infected with the bacterium.
VASP is a scaffold protein involved in regulation of the host cell cytoskeleton (de Chastellier et al, 2000). VASP-dependent actin polymerization regulates membrane architecture, cell motility, and pathophysiologic processes such as metastatic invasion. Studies on VASP have shown that it is localized to the site of pathogen attachment on the host cell for enteropathogenic Escherichia coli and Cryptosporidium parvum and led to the intriguing possibility that VASP functionally contributes to the attachment and invasion of these pathogens. In addition, Listeria monocytogenes, Shigella flexneri, and Rickettsia rickettsia have all been show to exploit actin polymerization in the host cytoskeleton to aid in cell-to-cell spread of the organisms (Ball et al 2000). It has also been found that actin regulation in host macrophages is disrupted due to infection by some pathogenic species of mycobacteria, such as M. avium and M. marinum (de Chastellier et al, 2000). However, the role of VASP in the disruption of actin filaments has not been described for M. tuberculosis.
In order to understand the potential role that VASP and actin polymerization might have in M. tuberculosis infection, it is important to understand how the infection initiates. The airborne transmission of M. tuberculosis bacilli from an infected individual upon inhalation places the bacteria in the inner lining of the lungs. There, the bacilli are engulfed by alveolar macrophages through phagocytosis. M. tuberculosis is able to survive the innate antimicrobial defenses of the alveolar macrophages. Mycobacteria are distinct in that they are able to live inside host macrophages, rather than as free-living organisms in the host body. It is well established that rearrangement of the actin cytoskeleton is important to the early steps of phagocytosis by host macrophages (de Chastellier et al, 2000). Entry of a particle into a cell by the process of phagocytosis entails the reorganization of the actin cytoskeleton underlying the area of the plasma membrane of the host cell that is in contact with the particle. When a macrophage ingests a pathogenic mycobacterium, it becomes trapped in a phagosome, or a membrane-bound vesicle. This phagosome does not become fused with a lysosome, which occurs in cases with nonpathogenic bacteria, and thus the phagosome does not mature. The inhibition of association with a lysosome helps the pathogenic mycobacterium evade degradation by the potent, cytolytic environment of the phagolysosome. This occurs due to pathogenic disruption of the host macrophage’s actin cytoskeletal network in the phagosome (de Chastellier et al, 2000). This disruption suggests that VASP is being exploited by the pathogenic mycobacteria to evade normal, immune response by the host macrophages.
Based on the mentioned previous studies, and due to the fact that several pathogens have evolved to utilize the actin cytoskeleton of their host, we propose to determine whether M. tuberculosis is able to utilize VASP to alter the actin filament network during macrophage phagocytosis. In this project we will use an INF-gamma stimulated macrophage derived cell line infected with M. bovis-BCG to test for VASP-mediated cytoskeletal rearrangement. Uninfected cells will be used as a control. Immunofluorescent and time lapse confocal microscopy will be performed at selected time points after infection to determine if and how VASP plays a role in the infection process. Changes in macrophage morphology and VASP distribution will be examined and compared to the uninfected cells. Western blots will also be performed to test for concentration levels of VASP in the macrophages.
Successful completion of these experiments will lead to an understanding of the role of VASP in M. tuberculosis infection. It will lead to an increase in knowledge about how pathogenic bacteria infect host tissues as well as increasing knowledge about the regulation of actin filaments and VASP function. The overall goal from this work and the results that will be produced will provide more information to aid in the understanding of the M. tuberculosis infection process and aid in the development of vaccines or treatments to prevent the disease. I hope that my project will provide you with a fun and engaging opportunity to learn about this infectious disease!