Johns Hopkins University Class of 2012
Molecular & Cellular Biology/Psychology Double Major
Malaria kills close to a million people yearly. It is most prevalent in countries in Africa, Latin America and Asia (Breman , 2004). The Plasmodium parasites in mosquitoes that cause malaria can be transmitted to humans by mosquito vectors of the genus Anopheles. A female mosquito bite transfers the parasite from an infected Anopheles mosquito to the human body and the Plasmodium sporozoites eventually make their way to the liver (Sinnis, 2007). The life cycle of sporozoites both in the mosquito as well as the host cell is important to consider because each step determines infection (Mota, 2001). Sporozoites are made and released in the mosquito midgut; they bind to the salivary glands and inhabit their secretory cells. Once they enter the mammalian host cell, they travel to the liver and traverse multiple cells until successfully invading hepatocytes (Coppi, 2007). Without successful invasion of hepatocytes, the infection is not viable.
The life stages in the liver are a valuable area of study because small numbers of parasites make them an attractive target for drugs and vaccines, they can be attacked by CD8+ T cells which cannot, attack red blood cells. To reach the liver, parasites must travel out of the skin into the bloodstream and then cross the liver endothelium in order to access hepatocytes. Parasite motility may affect immune responses as antigens are shed as parasites migrate. Two important components of this process include, gliding motility (movement of a living organism on a substrate) and cell traversal (movement through cells).
The mechanism by which Plasmodium sporozoites migrate has been observed to be movement in spiral motions that allow it to move forward and leave a trail of proteins behind. This type of locomotion has been described as gliding motility. One of the discarded proteins, known as circumsporozoite protein, is an important target of immunity and can be observed as a peptide presented to CD8+ T cells on the cell surface of MHC class-1 molecules. Parasites can also penetrate tissues by traversing cells. The conventional method of hepatocyte invasion by the sporozoite was the folding of the plasma membrane thereby creating a vacuole around the sporozoite. An alternative method of invasion by which sporozoites go through the plasma membrane of cells without the formation of the parasitophorous vacuole and in which the cell membrane is repaired by the cell has been shown to exist. In order to test the importance of parasite motility and cell migration for invasion, the Zavala lab has developed a number of mutant parasites and identified a number of compounds that should interfere with these processes. My project, under the supervision of post-doctoral fellow Ian Cockburn, was to establish assays to observe parasite motility and cell traversal of sporozoites in Hepa 1-6 cells.
METHODS AND RESULTS
To observe sporozoite motility, we established a standard sporozoite gliding motility assay. Plasmodium sporozoites were obtained through dissection of mosquito salivary glands and were incubated after MAb 3D-11 antibody had been used to coat slides. After 1 hour, parasites were fixed at room temperature for 15 minutes. The slides were then washed three times with PBS and blocked with PBS with 3% BSA for 30 minutes. After 30 minutes, FITC-3D11 antibody was introduced to the cells and stained overnight at 4°C to reveal trails. Observation of trails under fluorescence microscope revealed that some parasites underwent significant motility.
The results were characterized into four categories based on the number of loops/circle made by each individual sporozoite. Some sporozoites were observed to have no loops/circle (Figure 1a) while others were observed to have more than 10 loops/circle (Figure 1b). The quantification of the number of loops of motility by 100 different sporozoites can be found in figure 2 below. On the x-axis, 1is for 1 loop, 2 is for 2-10 loops, 3 is for >10 loops and 4 is for the total. It is observed that no sporozoites made only 1 loop. About 20% of the sporozoites had between 2-10 loops and the vast majority of sporozoites, ~82% were observed to have over 10 loops in their gliding motility assays.
To determine the ability of sporozoites to traverse Hepa 1-6 host cells, we used a standard cell traversal assay. Plasmodium sporozoites, obtained through the dissection of mosquito salivary glands were incubated with mouse hepatoma cells, Hepa 1-6 grown on a coverslip, in the presence of rhodamine dextran. After 2 hours, the cells were washed with PBS/1% FCS and fixed. After 10 minutes, they were permeabilized with methanol. The cells were then blocked with 3% BSA and left at 4°C overnight. Following this, FITC-3D11 antibody and DAPI were introduced to the cells to reveal hepatocyte cells and the coverslip was mounted on a microscope slide. We expect to see that cells that are traversed appear red because of the rhodamine dextran present in the media entering the cells. To validate results, cell traversal was compared between normal and heat inactivated sporozoites in Hepa 1-6 cells. Observation of sporozoite gliding motility under fluorescence microscope showed that live parasite indeed traversed Hepa 1-6 cells while the heat inactivated parasite did not traverse any Hepa 1-6 cells.
Plasmodium sporozoites appear to traverse the plasma membrane of the HEPA 1-6 cells. The blue dye represents the cell nuclei and the purple dye represents the cells that have been traversed by the sporozoite. Figure 3a illustrates the traversal of multiple HEPA 1-6 cells by the neighboring sporozoite. Figure 3b shows the traversal of no HEPA 1-6 cells by the neighboring sporozoite. The quantification of the number of cells positively traversed in the normal and heat inactivated cells can be found in figure 4 below.
Since traversal can be observed with liver parasites but not with heat-killed parasites, we have established the assay as a valid representation of cell traversal by sporozoites. With these two assays, we have been able to establish that we able to measure both cell motility and traversal by parasites.
DISCUSSION AND FUTURE DIRECTIONS
We have developed and tested the gliding motility assay in vitro with wild-type sporozoites and it has concluded that sporozoites exhibit gliding motility. In 2006, it was shown that sporozoites injected into the skin by mosquito bite or by intradermal inoculation arrive in the draining lymph node. In 2007, members of the Zavala lab observed that CD8+ T cells that were protective against malaria sporozoites are primed in the draining lymph node. It is important to address the immunological significance of sporozoite migration to the draining lymph node in future experiments. Future directions will involve inhibiting migration with pharmacological agents and using a transgenic parasite with an impaired migration phenotype in vivo. We will use chemicals and/or antibodies as agents to inhibit sporozoite migration. First, we will determine if there is a defect in migration to the draining lymph node in vivo and also a priming defect. We will also evaluate gliding motility impairment in vitro. We also have plans to evaluate the in vitro gliding motility of a transgenic parasite that shows impairment in priming CD8+ T cells in vivo. In essence, by establishing these two assays through this experiment, we have been able to better understand how sporozoites travel through various body organs in mouse which serves as a representation for the human body. The reason for using heat-killed sporzoites in the cell traversal assay was to observe the effect of heat on the ability for sporozoites to infect cells. We have been able to confirm our hypothesis that heat will reduce the viability for this to happen. This discovery opens the door of opportunity for the development of various pharmaceutical agents that are able to kill sporozoites before infecting cells in the body.
BREMAN, JOEL G., ALILIO, MARTIN S., MILLS, ANNE
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