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University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 1 Effe cts of Cytoskeleton inhibiting D rugs on Leishmania amazonensis D evelopment in Macrophages Kelly Johnson Dr. Blaise Ndjamen, and Dr. Peter E. Kima College of Agricultural and Life Sciences University of Florida Leishmaniasis is a disease that affects approximately 2 million people yearly around the world. This disease is caused by a p arasite called Leishmania which has two hosts: mammals and sandflies. In mammals, Leishmania enter s macrophages by phagocytosis and develop in a specific compartment called the parasitophorous vacuole (PV). The main objective of this study was to examine th e impact of vesicular transport in the host cell on Leishmania development within PVs. We hypothe sized that blocking the host vesicular transport will subsequently impair the interaction between PVs and host cell organelles that may provide important sources of nutrients and factors for Leishmania development within the PV. To achieve our objective, R aw264.7 macrophages were first exposed to L amazonensis promastigotes and after 24 hours, some cultures were treated with colchicine, a microtubule inhibiting drug, or cytochalasin D, an actin inhibiting drug, for a 48 hour time course. Samples were proce ssed by immuno fluorecence assays and analyzed by fluorescence microscopy. We found that blocking the host cell cytoskeleton function significantly inhibits th e development of L. amazonensis within the PV s in Raw 264.7 mouse macrophages. INTRODUCTION Leishmania is a protozoan parasite that is transmitted to mammals during a bite from infected sandflies. Leishmania causes a disease called Leishmaniasis, which occurs in at least 8 8 countries worldwide [1] There are three clinical presentations of Leishmaniasis: cutaneous, mucocutaneous, and visceral. Cutaneous Leishmaniasis causes skin lesions on the body. According to the Center for Disease Control, there are an estimated 1.5 million new cases annually of cutaneous Leishmaniasis. Mucocutaneous Leishmaniasis affects the mucous membranes, causing mucosal ulcerations. Visceral Leishmaniasis and kala azar can be lethal if left untreated. In the visceral form, the parasites infect the macrophages of internal organs, like the liver and spleen, and subsequently cause the organ to enlarge [2] Controlling Leishmaniasis remains a serious public health issue for many countries There is no vaccine for Leishmaniasis [3] Additionally, co infection with HIV/AIDS has led to the spread of Leishmaniasis. The immune deficiency from the HIV causes the increased susceptibility of Leishmania infection, and the parasite accelerates the o nset of AIDS in patients [1] Leishmania has two hosts in its life cycle. The vector of the parasite is a blood sucking sandfly, which harbors the promastigote form of the parasite. When the infected sandfly takes a blood meal from a mammal, Leishmania tr ansfers to its mammalian hosts, such as dogs and humans. Within the mammalian host, Leishmania transforms from the promastigote to the amastigote form within the macrophages [4] Macrophages are designed to eliminate foreign material from the body through the process of phagocytosis. Phagocytosis begins by the engulfment of the foreign particle to form a phagosome. Host cell endocytic components, such as early endosomes, late endosomes and lysosomes, which contain digestive enzymes and other microbiocidal f actors, fuse with the phagosome and lead to the destruction of the foreign material [5] However, Leishmania parasites are generally not destroyed by the macrophage. These parasites survive and replicate in macrophages within a parasitophorous vacuole (PV ) [4] There are two different types of PVs. The first is the large communal PV, associated with L. amazonensis and L. Mexicana where many parasites occupy a single vacuole. There is also the tight individual PV, associated with L. major and L. donovani, which has only one parasite per vacuole [6] Mechanisms by which Leishmania for their development within PVs in macrophages remain to be established. Recent research has suggested that endoplasmic reticulum components may be recruited to Leishmania PVs through the process of vesicular transport [7] Intracellular transport of vesicles occurs by using mi crotubules from ER to cis Golgi and post Golgi trafficking [8, 9] The cell cytoskeleton is made of microtubules and actin filaments. Microtubules and actin filaments are both polar structures with associated motor proteins that can transport material along their tubules or filaments. Microtubules are composed of alpha and beta tubulin subunits that form a tubulin heterodimer or a microtubule subunit [10] There are two types of actin subunits. G actin is the globular subunits of actin that

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KELLY J OHNSON DR BLAISE NDJAMEN & DR PETER E K IMA University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 2 assemble into filaments, and F actin is the filament form of actin. Like microtu bules, actin has a plus and minus end of the filament, where the plus end grows faster than the minus end. Myosins are the motor protein associated with actin. Microtubules in the mammalian cell enable long distance transport of vesicles and organelles and serve as support of the cell structure. Actin plays a role in short distance transport of vesicles and organelles. It also plays a major role in the cell locomotion and the endocytosis process [10] A drug that inhibits actin filaments by inhibiting the a ddition of actin subunits to the barbed end of the filament is Cytochalasin D (CCD) [ 11, 12 ] Its effects are drug are reversible [ 12 ] A drug that binds to beta tubulin at the C241 and C356 residues with pseudoirreversible kinetics is colchicine [ 13 ] Th e objective of the current study was to assess the impact of vesicular transport on Leishmania development in macrophages. We hypothesized that blocking the actin or microtubule dependent vesicular transport within the cell will deprive Leishmania of nutri ents and other factors vital for its development in macrophages. To carry out the study, either colchicine or Cytochalasin D drugs were used inhibit respectively the microtubule and actin compartments in mouse macrophages infected with L. amazonensis The effects of the drugs on Leishmania development were examined by assessing the parasite load in infected macrophages over time at varying concentrations of the above cytoskeleton inhibiting drugs. The results of the study showed that the parasite loa d (number of parasites per PV) is reduced to half after 48 hour post drug treatment of infected macrophages with L. amazonensis. cytoskeleton plays an important role in Leishmania replication or survival within th e parasitophorous vacuole. MATERIALS AND METHOD S Cell Culture Macrophage s Raw 264.7 murine macrophages were obtained from American Type Culture Collection and cultured in RPMI 1640 medium supplemented with 10% FBS and 1% PennStep. Cells were split every three days under a sterile bio safety hood, when the culture flask is approximately 80% confluent. The cells were scraped off the bottom of a tissue flask using a cell scraper and mixed by pipeting up and down with a serological pipet. One mL of cells was then added to a sterile tissue culture flask using a serological pipet. Nine mL of pre medium was added to the new flask. This fresh culture flask was incubated in a Forma Scientific CO 2 Water Jacked Incubator at 37.0 C and 5.5% CO 2 atmosphere. The rest of the cells were aspirated out into a waste bottle containing the bleach and the old flask discarded into a biological waste bin. Parasite L. amazonensis promastigotes were grown at 23 C in complete medium (Schneider's Drosophila medium) supplemented with 20% heat inactivated fetal bovine serum (FBS) and 10 parasites were split every 5 days in a 1:100 (0.5 mL parasites and 4.5 mL of fresh complete medium) and a 1: 1000 dilution (0.05 mL of parasites and 4.95 mL of fresh complete medium). The parasites are non adherent and swim in the medium. I nfections Plating M acrophages The adhered macrophage cells were scraped from the tissue flask under the hood using a sterile cell scraper. One 50 uL sample of macrophages was then transferred to a 9 6 well plat e. From the 50 uL, a 10 uL sample of macrophages were transferred to a separate well 10 uL of trypan blue dye was added to this 10uL macrophage sample and then mixed by pipetting the mixture up and down. The mixture of macrophages and dye was allowed to sit for 5 minutes at room temp erature to all ow the dye to diffuse into the cell. 10 uL of the mixture was then transferred to a hemocytometer for counting o n light microscope. Living cells had excreted the dye and were observed as being without dye. Dead cells retained the blue dye and were not counted. From the counts an approximation was calculated to estimate the concentration of live macrophages in the or iginal tissue flask. Four sterilized Fisherbrand Microscope Cover glasses (also called coverslips) were added to each of the wells in a tissue culture six well plate under a biosafety hood. The coverslips were transferred from their container to the plate using a sterilized Pasteur pipet that was attached to a vacuum pump which maintained the coverslips sterilized integrity 2 mL of macrophages from the tissue flask of known concentration were then added to the wells. The cells were then incubated overnigh t at 37 C to allow the macrophages to adhere to the coverslips. Co incubation of P arasites and M acrophages The parasite stock was diluted to a 1:20 ratio in 2% paraformaldehyde (PFA) solution; the aliquot was incubated at room temperature (RT) for 15 minutes to immobilize the parasites. A hemocytometer was used to quantify the parasite sample. The volume of par asite stock corresponding to 10 times the amount of macrophages was aseptically added to a centrifuge tube. The parasites were then centrifuged at 2000 rpm for 5 minutes. Under the biosafety hood, the supernatant was then discarded and the parasite pellet was resuspended in RPMI complete medium. The appropriate volume of the parasite solution was then added to each well of the six well plate. The plate was gently swirled by hand to ensure even distribution of parasites, which was confirmed under a light mic roscope. The plate containing the mixture of parasites and

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EFFECTS OF C YTOSKELETON INHIBITING DRUGS ON L AMAZONENSIS DEVELOPMENT University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 3 macrophages was placed at 34 C in an incubator supplemented with 5.5% CO 2 atmosphere. Drug T reatment After a 24 hour infection of Raw 264.7 cells with L. amazonensis colchicine was added at conce ntrations of 1 described [ 14, 15 ] (DMSO) was added to infected Raw 264.7 cells in 2 mL of RPMI 1640 complete medium. Treated cells were incubated at 34 C. Time points were taken by removing a coverslip and fixing in 2% PFA at 0 hr drug treatment (24 hr infection), 12 hr drug treatment (3 6hr infection), 24 hr drug treatment (48 hr infection), and 48 hr drug treatment (72 hr infection). Immuno F luorescence A ssays (IFA) The infections were processed by IFA analysis. The cells on coverslips were fixed in 2% paraformaldehyde (PFA). The PFA wa s aspirated, and then the cells were washed twice in 1x PBS (phosphate buffered saline) and quenched in 50mM of ammonium chloride/ 1xPBS solution for 5 minutes. The cells were then washed twice in 1xPBS, and permeabilized with a blocking buffer of 2% milk and 0.5% saponin in 1xPBS for 30 minutes. The coverslips were then placed on 37uL of a primary antibody mixture for 30 minutes to 1hr in darkness. The primary antibody mixture was composed of 2% ID4B (rat) and blocking buffer for the coverslips of DMSO, 1 ug/mL of CCD and 5 ug/mL of CCD. The primary antibody mixture for DMSO, 1uM colchicine, and 10 uM colchicine was comprised of 2% ID4B IgG (from rat), 2.5% beta tubulin (from mouse), and blocking buffer. After the primary antibody was added, the coverslips were then washed three times with a binding buffer (2% milk, 0.05% saponin, 1xPBS). The coverslips were then mounted onto 37uL of a secondary antibody mixture for 1hr in darkness. A secondary mixture for the coverslips of DMSO, 1 ug/mL of CCD, and 5 ug/mL of CCD was of AlexaFluor 488 chicken anti rat IgG (1:200), 4',6 diamidino 2 phenylindole ( DAPI) (1.2:1000), 2.5% phalloidin, and blocking buffer. A secondary antibody mixture for the coverslips of DMSO, 1uM colchicine, and 10 uM colchicine was composed of AlexaFluor 488 chicken anti rat IgG (1 :200), DAPI (1.2:1000), AlexaFl u o r 568 goat anti mouse IgG (1:100), and blocking buffer. After the secondary antibody treatment, the coverslips were washed with binding buffer three times, followed by a wash with 1xPB three times. The coverslips were mounted onto slides with Fluoro Gel with Tris Buffer (Electron Microscopy Sciences). The slides were then stored at 4 C for future analysis under fluorescence microscope. Fluorescence M icroscopy P reparations of microscope slides were examined using a Zeiss Axiovert 200M fluorescence microscope outfitted with DAPI, fluorescein isothiocyanate (FITC), and tetramethyl rhodamine isothiocyanate (TRITC) filters. The images were observed at a 100x oil immersion objectiv e. Pictures were taken with a Zeiss AxioCam MRm camera attached to the microscope and processed using the AxioVision Rel 4.7 software. A Z stack of images with a slice distance of 0.250 0.700 um between focal planes was collected and then merged to create the final image. Data Collection Samples were collected at 24 hrs of infection and 12 hrs, 24 hrs, and 48 hrs after drug treatment. Non treated samples were used as control. The samples, both treated and non treated, were evaluated by counting at least 60 infected macrophages. The number of parasites within PVs was also counted. Parasite load was obtained by dividing the number of parasites within PVs by the number of infected cells. The infection ratio was calculated as num ber of infected macrophages divid ed by the total number of macrophages counted multiplied by 100. RESULTS I: Infection R ate of Raw264.7 M acrophages by L. amazonensis P arasites Raw 264.7 macrophages were co incubated with L. amazonensis promastigotes and after 24 hours, an infection rate of 51.6% was obtained. This indicated that about half of our total macrophages internalized the Leishmania parasite before the drug treatment. A representative sample of an infected Raw 264.7 macrophage is shown in F igure 1.

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KELLY J OHNSON DR BLAISE NDJAMEN & DR PETER E K IMA University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 4 Figure 1: Images of Raw264.7 infected at (A) 24 h and (B) 72 h. Lysosomal associated membrane protein 1(Lamp 1), in green, shows lysosomes and PVs. The nuclei of both parasite and macrophage are stained in blue (DAPI). Solid arrows point to parasites. Dashed arrows point to the PVs containing the parasite(s). II: Effect of Cytoskeleton I nhibiting D rugs on L. amazonensis Development in Macrophages To determine the impact of vesicular transport on L. amazonensis development, cytoskeleton inhibiting drugs (cytochalasinD and colchicine) were used in our experiments. II A : Effect of C ytochalasin D on L. amazonensis D evelopment in M acrophages After 24 hours of infection and no drugs, the parasite load for DMSO, 1 ug/mL CCD, and 5 ug/mL CCD were varied slightly with values of 1.25, 1.20, and 1.22, respectively. After 24 hours of drug treatment, the parasite load began to vary between the samples. DMSO samples had increased to 1.64, 1 ug/mL CCD had increased to 1.5, and the parasite load f or 5 ug/mL CCD was 1.26. After 48 hours of drug treatment, the parasite load of DMSO had reached 2.0, indicating that the parasites had replicated within the PV. The parasite load of 1 ug/mL CCD dropped to 1.24, and for 5 ug/mL CCD the parasite load dropped to 1.12. Figure 2 and Figure 3 show representative images of the macrophages at the four time points in the experiment. Figure 2 shows the effect of 1 ug/mL of CCD and Figure 3 shows the effect of 5 ug/mL of CCD. In the images, the parasites are not divi ding within the PV after 48 hours like the untreated infected macrophages in Figure 1. Figure 4, a graph of the parasite load, shows that over time the parasites do not divide within the PV when treated with 1 ug/mL of CCD or 5 ug/mL of CCD. Figure 4 shows that when the cell is not treated the parasites are able to divide, as there are two parasites per PV.

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EFFECTS OF C YTOSKELETON INHIBITING DRUGS ON L AMAZONENSIS DEVELOPMENT University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 5 Figure 2: Images of infected cells treated with 1 ug/mL CCD Staining of F actin (red) and Lamp 1 (green) with phalloidin and ID4B, respectively, in L. amazonensis infected Raw 264.7 murine macrophages treated with 1 ug/mL cytochalasin D. Solid arrows point to parasite. DAPI (blue) stains dsDNA of macrophage and pa rasite. (A) 0 hr drug treatment (B) 12 hr drug treatment (C) 24 hr drug treatment (D) 4 hr drug treatment. Figure 3: Images of infected cells treated with 5 ug/mL CCD Staining of F actin and Lamp 1 with phalloidin and ID4B, respectively, in L. amazonensis infected Raw 264.7 murine macrophages treated with 5 ug/mL cytochalasin D. Arrow points to parasite. (A) 0 hr drug treatment (B) 12 hr drug treatment (C) 24 hr drug tre atment (D) 48 hr drug treatment.

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KELLY J OHNSON DR BLAISE NDJAMEN & DR PETER E K IMA University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 6 Figure 4: Effect of cytochalasin D on L. amazonensis development in macrophages. Macrophages incubated with L. amazonensis and DMSO ( ), 1 ug/mL CCD ( is the mean of two experiments. II B. Effect of C olchicine on L. amazonensis D evelopment in M acrophages Over the course of the experiment, 1 uM colchicine and 10 uM colchicine never had parasite loads above 1.4. Prior to the addition of drugs, the parasite load was 1.25, 1.26, and 1.38 for the infections that would receive DMSO, 1 uM colchicine, and 10 uM colchicine, respectively. Throughout the 48 hours of drug trea tment, the parasite load of 1 uM colchicine and 10 uM colchicine remained fairly constant to end with parasite load values of 1.33 and 1.28, respectively, as seen in Figure 5. However, the treatment of DMSO had doubled to reach a parasite load value of 2.0, indicating the replication of parasites within the PV. In Figure 6, the microtubules are inhibited by 1 uM colchicine. The images show that the microtubules are present at 0 hr and 12 hr of drug treatment and have depolymerized by 24 hours of drug tre atment, indicating the drug is working to inhibit microtubules. Figure 6 also shows the representative images of the infection over time. The treated infected cells do not experience any parasite replication within the PV.

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EFFECTS OF C YTOSKELETON INHIBITING DRUGS ON L AMAZONENSIS DEVELOPMENT University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 7 Figure 5: Effect of colchicine on L. amazonensis development in macrophages. Macrophages incubated with L. amazonensis and DMSO (solid black), 1 uM colchicine (few dots), and 10 um colchicine (many dots). Data is the mean of two experiments. Figure 6: Images of infected cells treated with 1 uM Colchicine. Staining of beta tubulin and Lamp 1 in L. amazonensis infected Raw 264.7 murine macrophages treated with 1 uM colchicine. Arrow points to parasite. (A) 0 hr drug treatment (B) 12 hr drug treatment (C) 24 hr drug treatment (D) 48 hr drug treatment

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KELLY J OHNSON DR BLAISE NDJAMEN & DR PETER E K IMA University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 8 Figure 7 show s that 10uM of colchicine works to block microtubules between 0 and 12 hr In Figure 7c, although there are many parasites, the parasites are in separate PVs so no reproduction has occurred. After 48 hours, there is still no parasite development within the PV, as shown in Figure 7d with only one parasite per PV. Figure 7: Images of infected cells treated with 10 uM Colchicine. Staining of beta tubulin and Lamp 1 in L. amazonensis infected Raw 264.7 murine macrophages treated with 10 uM colchicine. Arrow points to parasite. (A) 0 hr drug treatment (B) 12 hr drug treatment (C) 24 hr drug treatment (D) 48 hr drug treatment. DISCUSSION The objective of the study was to determine the effects of cytoskeleton inhibiting drugs on Leishmania development within the PV. The hypothesis was that inhibiting microtubules and actin with drugs would slow the development of Leishmania within the PV. The cell cytoskeleton is made of microtubules and actin filaments. Microtubules and actin filaments are both polar structures with associated motor proteins that can transport material along their tubules or filaments. Microtubules are composed of alpha and beta tubulin subunits that form a tubulin heterodimer or a microtubule subunit. Microtubules grow faster at the end with t he beta tubulin subunit, which is called the plus end, compared to the alpha tubulin subunit, which is called the minus end. Kinesin and dynein are the motor proteins associated with microtubules. The role of microtubules in the mammalian cell is to provid e long distance transport of vesicles and organelles. Microtubules also play a role in cell structure and support [10] Actin is composed of the monomeric molecule G actin that polymerizes into a filamentous polymer, F actin [ 16 ] The roles of actin in the cell include cell migration by way of myosin II and short distance vesicular or organelle transport using myosin I, V, and VI [10] Myosin V has been shown to tr ansport ER vesicles in neurons [ 17 ] Myosin I and myosin VI have been shown to be associated with Golgi derived vesicles and cytoplasmic vesicles [ 17 ] To evaluate the effects the drugs had on Leishmania development, the parasite load and infection rate were analyzed. The main finding of th e study was that the parasite development was significantly hindered when microtubule and actin inhibiting drugs, colchicine and

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EFFECTS OF C YTOSKELETON INHIBITING DRUGS ON L AMAZONENSIS DEVELOPMENT University of Florida | Journal of Undergraduate Research | Volume 12 Issue 3 | Summer 2011 9 CCD, were used, separately, during a Leishmania infection of macrophages. When the cells were not treated with microtubule or actin inhibiting drugs, the parasites multiplied within the PVs. The parasite load (number of parasites per PV) for untreated cells reached 2.0 after 48 hours. The parasite load for all treated cells after 48 hours was less than 1.3, demonstrating that th e pharmacological inhibition of actin and microtubules impacted the development of L. amazonensis within the PV. L. amazonensis replicates within a single PV to form a larger PV with many parasites. Because the large PV with many parasites was not seen aft er 48 hours of drug treatment and was seen after 48 hours without drug treatment, it was cytoskeleton are necessary components for the development of this parasite. Some limitations of the study were tha t the infected cells played a role in the parasite load. It may have made it difficult for the parasites to reproduce. The literature recomme nds that infections using L. amazonensis occur in 8 1 9 20 ] Another limitation of the study was the effect the drugs had on other agment and it became difficult to distinguish between the parasites and the cell after 24 hours of drug treatment. With DMSO, it became difficult to count the cells because the field of view was overpopulated with macrophages, making it difficult to distin guish one In this study we demonstrated that L. amazonensis parasites develop very well in their mammalian host macrophages. However, successful development of Leishmania parasites requires a well functioning cell cyto skeleton. REFERENCES [ 1 ] World Health Organization. 2006. Control of Leishmaniasis, Report by the Secretariat Geneva. WHO Technical Report Series No. 793 [ 2 ] Meinecke C K Schottelius J Os kam L Fleischer B Congenital transmission of visceral leishmaniasis (Kala Azar) from an a s ymptomatic mother to her c hild. 1999. Pediatr 104( 5 ):e65 [3] Desjeux P 1996. Leishmania sis: Pu blic h ealth a spect s and control. Clin Dermatol 14:417 23 [ 4 ] Love DC Kane MM Mosser DM. 1998. Leishmania amazonensis: The p hagocytosis of amastigotes by macrophages. Exp Parasitol 80:161 171 [5] Ade rem A, Underhill, DM. 1999. Mechanisms of phagocytosis in macrophages. Annu Rev Immunol. 17:593 623 [ 6 ] Courret N Frhel C Gouhier N Pouchelet M Prina E Roux P, Antoine JC. 2002. Biogenesis of Leishmania harbouring parasitophorous vacuoles following phagocytosis of the metacyclic promastigote or amastigote stages of the paras ites. J Cell Sci 115:2303 2316 [7] Ndjamen B Kang BH, Hatsuzawa K, Kima P E. 2010. Leishmania parasitophorous vacuoles interact continuously with the host cell's endoplasmic reticulum; parasitophorous vacuoles are hybrid compartments Cell Microbiol 12 :1480 1494 [8] Klumperman J. 2000. Transport between ER and Golgi [Review]. Curr Opin Cell Biol 12.4: 445 449 [9] Toomre D, Keller P, White J Olivo JC Simons K. 1999. Dual color visualization of trans Golgi network to plasma membrane traffic alo ng microtubules in living cells. J Cell Sci 112 : 21 33 [10] Karp G 2007. The cytoskeleton and cell motility. In: Cell and Molecular Biology Concepts and Experiments 5 th ed. New York: Wiley p. 344 412 [11] Schliwa M. 1982. Action of c ytochala sin D on cytoskeletal networks. J Cell Biol. 92:79 91 [12] Stevenson B Begg D. 1994. Concentration dependent effects of cytochalasin D on tight junctions and actin fila ments in MDCK epithelial cells. J Cell Sci 107:367 75 [13] Uppuluri S, Knipling L Sackett DL Wolff J. 1993. Localization of the colchicine binding site of tubulin, Proc Natl Acad Sci USA 90: 11598 1 1602 [14] Harrison RE Bucci C Vieira OV Schroer TA Grinstein S. 2003. Phagosomes fuse with late endosomes and/or lysosomes by extension of membrane protrusions along m icrotubu les: Role of Rab7 and RILP. Mol Cell Biol 23( 1 8): 6494 6 506 [15] Isowa N, Xavier AM Dziak E Opas M McRitchie DI Slutsky AS Keshavjee SH Liu M. 1999. LPS induced depolymerization of cytoskeleton and its role in TNF alpha production by rat pneumocytes, Am J Physiol Lung Cell Mol Physiol 277( 3 ):606 615 [ 16 ] Kabsch W, Vandekerckhove J. 1992. S tructure and function of actin. Annu Rev Biophys Biomol Struct 21: 49 76 [ 17 ] De Pina AS, Langford GM. 1999. Vesicle transport: The role of actin filaments and myosin m otors Microsc Res Techniq 47:93 106 [18] Kima PE, Dunn W. 2005. Exploiting calnexin expression on phagosomes to isolate Leishmania parasitophorous vacuoles Microb Pathogenesis 38( 4 ):139 145 [19] Prina E Lang T Glaichenhaus N Antoine JC. 1996. Presentation of the protective parasite a ntigen LACK by Leishmania infected macrophages. J Immun 156: 4318 4327 [20] Zilberstein D, Shapira M. 1994. The role of pH and temperature in the d evelopment of Leishmania p arasites, Annu Rev Microbiol 48: 449 470


Summer Focus on Medical Research : Effects of Cytoskeleton-inhibiting Drugs on Leishmania amazonensis Development in Mac...
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Title: Summer Focus on Medical Research : Effects of Cytoskeleton-inhibiting Drugs on Leishmania amazonensis Development in Macrophages
Series Title: Journal of Undergraduate Research
Physical Description: Serial
Language: English
Creator: Johnson, Kelly
Ndjamen, Blaise
Kima, Peter E.
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011
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Abstract: Leishmaniasis is a disease that affects approximately 2 million people yearly around the world. This disease is caused by a parasite called Leishmania, which has two hosts: mammals and sandflies. In mammals, Leishmania enters macrophages by phagocytosis and develop in a specific compartment called the parasitophorous vacuole (PV). The main objective of this study was to examine the impact of vesicular transport in the host cell on Leishmania development within PVs. We hypothesized that blocking the host vesicular transport will subsequently impair the interaction between PVs and host cell organelles that may provide important sources of nutrients and factors for Leishmania development within the PV. To achieve our objective, Raw264.7 macrophages were first exposed to L. amazonensis promastigotes and after 24 hours, some cultures were treated with colchicine, a microtubule-inhibiting drug, or cytochalasin D, an actin-inhibiting drug, for a 48-hour time course. Samples were processed by immuno-fluorecence assays and analyzed by fluorescence microscopy. We found that blocking the host cell cytoskeleton function significantly inhibits the development of L. amazonensis within the PVs in Raw 264.7 mouse macrophages.
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Effects of Cytoskeleton-inhibiting Drugs on Leishmania

amazonensis Development in Macrophages

Kelly Johnson, Dr. Blaise Ndjamen, and Dr. Peter E. Kima


College of Agricultural and Life Sciences, University of Florida

Leishmaniasis is a disease that affects approximately 2 million people yearly around the world. This disease is caused by a parasite
called Leishmania, which has two hosts: mammals and sandflies. In mammals, Leishmania enters macrophages by phagocytosis and
develop in a specific compartment called the parasitophorous vacuole (PV). The main objective of this study was to examine the
impact of vesicular transport in the host cell on Leishmania development within PVs. We hypothesized that blocking the host
vesicular transport will subsequently impair the interaction between PVs and host cell organelles that may provide important sources
of nutrients and factors for Leishmania development within the PV. To achieve our objective, Raw264.7 macrophages were first
exposed to L. amazonensis promastigotes and after 24 hours, some cultures were treated with colchicine, a microtubule-inhibiting
drug, or cytochalasin D, an actin-inhibiting drug, for a 48-hour time course. Samples were processed by immuno-fluorecence assays
and analyzed by fluorescence microscopy. We found that blocking the host cell cytoskeleton function significantly inhibits the
development of L. amazonensis within the PVs in Raw 264.7 mouse macrophages.


INTRODUCTION

Leishmania is a protozoan parasite that is transmitted to
mammals during a bite from infected sandflies. Leishmania
causes a disease called Leishmaniasis, which occurs in at
least 88 countries worldwide [1]. There are three clinical
presentations of Leishmaniasis: cutaneous, mucocutaneous,
and visceral. Cutaneous Leishmaniasis causes skin lesions
on the body. According to the Center for Disease Control,
there are an estimated 1.5 million new cases annually of
cutaneous Leishmaniasis. Mucocutaneous Leishmaniasis
affects the mucous membranes, causing mucosal
ulcerations. Visceral Leishmaniasis and kala azar can be
lethal if left untreated. In the visceral form, the parasites
infect the macrophages of internal organs, like the liver and
spleen, and subsequently cause the organ to enlarge [2].
Controlling Leishmaniasis remains a serious public health
issue for many countries. There is no vaccine for
Leishmaniasis [3]. Additionally, co-infection with
HIV/AIDS has led to the spread of Leishmaniasis. The
immune deficiency from the HIV causes the increased
susceptibility of Leishmania infection, and the parasite
accelerates the onset of AIDS in patients [1].
Leishmania has two hosts in its life cycle. The vector of
the parasite is a blood sucking sandfly, which harbors the
promastigote form of the parasite. When the infected
sandfly takes a blood meal from a mammal, Leishmania
transfers to its mammalian hosts, such as dogs and humans.
Within the mammalian host, Leishmania transforms from
the promastigote to the amastigote form within the
macrophages [4]. Macrophages are designed to eliminate
foreign material from the body through the process of


phagocytosis. Phagocytosis begins by the engulfment of
the foreign particle to form a phagosome. Host cell
endocytic components, such as early endosomes, late
endosomes and lysosomes, which contain digestive
enzymes and other microbiocidal factors, fuse with the
phagosome and lead to the destruction of the foreign
material [5].
However, Leishmania parasites are generally not
destroyed by the macrophage. These parasites survive and
replicate in macrophages within a parasitophorous vacuole
(PV) [4]. There are two different types of PVs. The first is
the large communal PV, associated with L. amazonensis
and L. Mexicana, where many parasites occupy a single
vacuole. There is also the tight individual PV, associated
with L. major and L. donovani, which has only one parasite
per vacuole [6]. Mechanisms by which Leishmania
parasites evade the host's defense and/or obtain nutrients
for their development within PVs in macrophages remain
to be established. Recent research has suggested that
endoplasmic reticulum components may be recruited to
Leishmania PVs through the process of vesicular transport
[7].
Intracellular transport of vesicles occurs by using
microtubules from ER to cis-Golgi and post-Golgi
trafficking [8, 9]. The cell cytoskeleton is made of
microtubules and actin filaments. Microtubules and actin
filaments are both polar structures with associated motor
proteins that can transport material along their tubules or
filaments. Microtubules are composed of alpha- and beta-
tubulin subunits that form a tubulin heterodimer or a
microtubule subunit [10]. There are two types of actin
subunits. G-actin is the globular subunits of actin that


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KELLY JOHNSON, DR. BLAISE NDJAMEN, & DR. PETER E. KIMA


assemble into filaments, and F-actin is the filament form of
actin. Like microtubules, actin has a plus and minus end of
the filament, where the plus end grows faster than the
minus end. Myosins are the motor protein associated with
actin. Microtubules in the mammalian cell enable long
distance transport of vesicles and organelles and serve as
support of the cell structure. Actin plays a role in short
distance transport of vesicles and organelles. It also plays a
major role in the cell locomotion and the endocytosis
process [10]. A drug that inhibits actin filaments by
inhibiting the addition of actin subunits to the barbed end
of the filament is Cytochalasin D (CCD) [11, 12]. Its
effects are drug are reversible [12]. A drug that binds to
beta-tubulin at the C241 and C356 residues with
pseudoirreversible kinetics is colchicine [13].
The objective of the current study was to assess the
impact of vesicular transport on Leishmania development
in macrophages. We hypothesized that blocking the actin
or microtubule-dependent vesicular transport within the
cell will deprive Leishmania of nutrients and other factors
vital for its development in macrophages. To carry out the
study, either colchicine or Cytochalasin D drugs were used
inhibit respectively the microtubule and actin
compartments in mouse macrophages infected with L.
amazonensis. The effects of the drugs on Leishmania
development were examined by assessing the parasite load
in infected macrophages over time at varying
concentrations of the above cytoskeleton-inhibiting drugs.
The results of the study showed that the parasite load
(number of parasites per PV) is reduced to half after 48-
hour post drug treatment of infected macrophages with L.
amazonensis. This result suggested that the host cell's
cytoskeleton plays an important role in Leishmania
replication or survival within the parasitophorous vacuole.

MATERIALS AND METHODS

Cell Culture

Macrophages. Raw 264.7 murine macrophages were
obtained from American Type Culture Collection and
cultured in RPMI-1640 medium supplemented with 10%
FBS and 1% PennStep.
Cells were split every three days under a sterile bio
safety hood, when the culture flask is approximately 80%
confluent. The cells were scraped off the bottom of a tissue
flask using a cell scraper and mixed by pipeting up and
down with a serological pipet. One mL of cells was then
added to a sterile tissue culture flask using a serological
pipet. Nine mL of pre-warmed (to 37'C) complete RPMI
medium was added to the new flask. This fresh culture
flask was incubated in a Forma Scientific CO2 Water
Jacked Incubator at 37.0 C and 5.5% CO2 atmosphere.
The rest of the cells were aspirated out into a waste bottle
containing the bleach and the old flask discarded into a
biological waste bin.


Parasite. L. amazonensis promastigotes were grown at
23 C in complete medium (Schneider's Drosophila
medium) supplemented with 20% heat inactivated fetal
bovine serum (FBS) and 10-gg/ml gentamicin. The
parasites were split every 5 days in a 1:100 (0.5 mL
parasites and 4.5 mL of fresh complete medium) and a
1:1000 dilution (0.05 mL of parasites and 4.95 mL of fresh
complete medium). The parasites are non-adherent and
swim in the medium.

Infections

Plating Macrophages. The adhered macrophage cells
were scraped from the tissue flask under the hood using a
sterile cell scraper. One 50 uL sample of macrophages was
then transferred to a 96 well plate. From the 50 uL, a 10 uL
sample of macrophages were transferred to a separate well.
10 uL of trypan blue dye was added to this 10uL
macrophage sample and then mixed by pipetting the
mixture up and down. The mixture of macrophages and
dye was allowed to sit for 5 minutes at room temperature to
allow the dye to diffuse into the cell. 10 uL of the mixture
was then transferred to a hemocytometer for counting on
light microscope. Living cells had excreted the dye and
were observed as being without dye. Dead cells retained
the blue dye and were not counted. From the counts an
approximation was calculated to estimate the concentration
of live macrophages in the original tissue flask. Four
sterilized Fisherbrand Microscope Cover glasses (also
called coverslips) were added to each of the wells in a
tissue culture six well plate under a biosafety hood. The
coverslips were transferred from their container to the plate
using a sterilized Pasteur pipet that was attached to a
vacuum pump, which maintained the coverslips sterilized
integrity. 2 mL of macrophages from the tissue flask of
known concentration were then added to the wells. The
cells were then incubated overnight at 37 C to allow the
macrophages to adhere to the coverslips.

Co-incubation of Parasites and Macrophages. The
parasite stock was diluted to a 1:20 ratio in 2%
paraformaldehyde (PFA) solution; the aliquot was
incubated at room temperature (RT) for 15 minutes to
immobilize the parasites. A hemocytometer was used to
quantify the parasite sample. The volume of parasite stock
corresponding to 10 times the amount of macrophages was
aseptically added to a centrifuge tube. The parasites were
then centrifuged at 2000 rpm for 5 minutes. Under the
biosafety hood, the supernatant was then discarded and the
parasite pellet was resuspended in RPMI complete
medium. The appropriate volume of the parasite solution
was then added to each well of the six-well plate. The plate
was gently swirled by hand to ensure even distribution of
parasites, which was confirmed under a light microscope.
The plate containing the mixture of parasites and


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EFFECTS OF CYTOSKELETON-INHIBITING DRUGS ON L. AMAZONENSIS DEVELOPMENT


macrophages was placed at 34 C in an incubator
supplemented with 5.5% CO2 atmosphere.

Drug Treatment

After a 24-hour infection of Raw 264.7 cells with L.
amazonensis, colchicine was added at concentrations of 1
gM and 10 gM, as previously described [14, 15].
Cytochalasin D was added at concentrations of 1 gg/mL
and 5 gg/mL, as previously described by Parsa et al.
(2006). Two gL of dimethyl sulfoxide (DMSO) was added
to infected Raw 264.7 cells in 2 mL of RPMI 1640
complete medium. Treated cells were incubated at 34 C.
Time points were taken by removing a coverslip and fixing
in 2% PFA at 0 hr drug treatment (24 hr infection), 12 hr
drug treatment (36hr infection), 24 hr drug treatment (48 hr
infection), and 48 hr drug treatment (72 hr infection).

Immuno-Fluorescence Assays (IFA)

The infections were processed by IFA analysis. The cells
on coverslips were fixed in 2% paraformaldehyde (PFA).
The PFA was aspirated, and then the cells were washed
twice in lx PBS (phosphate buffered saline) and quenched
in 50mM of ammonium chloride/ IxPBS solution for 5
minutes. The cells were then washed twice in IxPBS, and
permeabilized with a blocking buffer of 2% milk and 0.5%
saponin in IxPBS for 30 minutes. The coverslips were then
placed on 37uL of a primary antibody mixture for 30
minutes to lhr in darkness. The primary antibody mixture
was composed of 2% ID4B (rat) and blocking buffer for
the coverslips of DMSO, 1 ug/mL of CCD and 5 ug/mL of
CCD. The primary antibody mixture for DMSO, luM
colchicine, and 10 uM colchicine was comprised of 2%
ID4B IgG (from rat), 2.5% beta-tubulin (from mouse), and
blocking buffer. After the primary antibody was added, the
coverslips were then washed three times with a binding
buffer (2% milk, 0.05% saponin, IxPBS). The coverslips
were then mounted onto 37uL of a secondary antibody
mixture for lhr in darkness. A secondary mixture for the
coverslips of DMSO, 1 ug/mL of CCD, and 5 ug/mL of
CCD was of AlexaFluor 488 chicken anti-rat IgG (1:200),
4',6-diamidino-2-phenylindole (DAPI) (1.2:1000), 2.5%
phalloidin, and blocking buffer. A secondary antibody
mixture for the coverslips of DMSO, luM colchicine, and
10 uM colchicine was composed of AlexaFluor 488
chicken anti-rat IgG (1:200), DAPI (1.2:1000), AlexaFluor


568 goat anti-mouse IgG (1:100), and blocking buffer.
After the secondary antibody treatment, the coverslips were
washed with binding buffer three times, followed by a
wash with IxPB three times. The coverslips were mounted
onto slides with Fluoro-Gel with Tris Buffer (Electron
Microscopy Sciences). The slides were then stored at 4 C
for future analysis under fluorescence microscope.

Fluorescence Microscopy

Preparations of microscope slides were examined using
a Zeiss Axiovert 200M fluorescence microscope outfitted
with DAPI, fluorescein isothiocyanate (FITC), and
tetramethyl rhodamine isothiocyanate (TRITC) filters. The
images were observed at a 100x oil immersion objective.
Pictures were taken with a Zeiss AxioCam MRm camera
attached to the microscope and processed using the
AxioVision Rel 4.7 software. A Z-stack of images with a
slice distance of 0.250-0.700 um between focal planes was
collected and then merged to create the final image.

Data Collection

Samples were collected at 24 hrs of infection and 12 hrs,
24 hrs, and 48 hrs after drug treatment. Non-treated
samples were used as control. The samples, both treated
and non-treated, were evaluated by counting at least 60
infected macrophages. The number of parasites within PVs
was also counted. Parasite load was obtained by dividing
the number of parasites within PVs by the number of
infected cells. The infection ratio was calculated as number
of infected macrophages divided by the total number of
macrophages counted multiplied by 100.

RESULTS

I: Infection Rate of Raw264.7 Macrophages by L.
amazonensis Parasites

Raw 264.7 macrophages were co-incubated with L.
amazonensis promastigotes, and after 24 hours, an
infection rate of 51.6% was obtained. This indicated that
about half of our total macrophages internalized the
Leishmania parasite before the drug treatment. A
representative sample of an infected Raw 264.7
macrophage is shown in Figure 1.


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
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KELLY JOHNSON, DR. BLAISE NDJAMEN, & DR. PETER E. KIMA


Figure 1: Images of Raw264.7 infected at (A) 24 h and (B) 72 h. Lysosomal associated membrane protein-
1(Lamp-1), in green, shows lysosomes and PVs. The nuclei of both parasite and macrophage are stained in blue
(DAPI). Solid arrows point to parasites. Dashed arrows point to the PVs containing the parasite(s).


II: Effect of Cytoskeleton-lnhibiting Drugs on L.
amazonensis Development in Macrophages

To determine the impact of vesicular transport on L.
amazonensis development, cytoskeleton-inhibiting drugs
(cytochalasinD and colchicine) were used in our
experiments.

11-A: Effect of Cytochalasin D on L. amazonensis
Development in Macrophages

After 24 hours of infection and no drugs, the parasite
load for DMSO, 1 ug/mL CCD, and 5 ug/mL CCD were
varied slightly with values of 1.25, 1.20, and 1.22,
respectively. After 24 hours of drug treatment, the parasite
load began to vary between the samples. DMSO samples
had increased to 1.64, 1 ug/mL CCD had increased to 1.5,


and the parasite load for 5 ug/mL CCD was 1.26. After
48hours of drug treatment, the parasite load of DMSO had
reached 2.0, indicating that the parasites had replicated
within the PV. The parasite load of 1 ug/mL CCD dropped
to 1.24, and for 5 ug/mL CCD the parasite load dropped to
1.12.
Figure 2 and Figure 3 show representative images of the
macrophages at the four time points in the experiment.
Figure 2 shows the effect of 1 ug/mL of CCD and Figure 3
shows the effect of 5 ug/mL of CCD. In the images, the
parasites are not dividing within the PV after 48 hours like
the untreated infected macrophages in Figure 1. Figure 4, a
graph of the parasite load, shows that over time the
parasites do not divide within the PV when treated with 1
ug/mL of CCD or 5 ug/mL of CCD. Figure 4 shows that
when the cell is not treated the parasites are able to divide,
as there are two parasites per PV.


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EFFECTS OF CYTOSKELETON-INHIBITING DRUGS ON L. AMAZONENSIS DEVELOPMENT


Figure 2: Images of infected cells treated with 1 ug/mL CCD. Staining of F-actin (red) and Lamp-1 (green) with phalloidin
and ID4B, respectively, in L. amazonensis infected Raw 264.7 murine macrophages treated with 1 ug/mL cytochalasin D.
Solid arrows point to parasite. DAPI (blue) stains dsDNA of macrophage and parasite. (A) 0-hr drug treatment (B) 12-hr drug
treatment (C) 24-hr drug treatment (D) 4- hr drug treatment.


Figure 3: Images of infected cells treated with 5 ug/mL CCD. Staining of F-actin and Lamp-1 with phalloidin and ID4B,
respectively, in L. amazonensis infected Raw 264.7 murine macrophages treated with 5 ug/mL cytochalasin D. Arrow points
to parasite. (A) 0-hr drug treatment (B) 12-hr drug treatment (C) 24-hr drug treatment (D) 48 hr-drug treatment.


University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
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KELLY JOHNSON, DR. BLAISE NDJAMEN, & DR. PETER E. KIMA


Figure 4: Effect of cytochalasin D on L. amazonensis development in macrophages. Macrophages incubated
with L. amazonensis and DMSO (), 1 ugmL CD (- -), and 5 ugmL CD (...). Data is the mean of two
experiments.


ll-B. Effect of Colchicine on L. amazonensis
Development in Macrophages

Over the course of the experiment, 1 uM colchicine and
10 uM colchicine never had parasite loads above 1.4. Prior
to the addition of drugs, the parasite load was 1.25, 1.26,
and 1.38 for the infections that would receive DMSO, 1
uM colchicine, and 10 uM colchicine, respectively.
Throughout the 48 hours of drug treatment, the parasite
load of 1 uM colchicine and 10 uM colchicine remained
fairly constant to end with parasite load values of 1.33 and
1.28, respectively, as seen in Figure 5. However, the


treatment of DMSO had doubled to reach a parasite load
value of 2.0, indicating the replication of parasites within
the PV.
In Figure 6, the microtubules are inhibited by 1 uM
colchicine. The images show that the microtubules are
present at 0 hr and 12 hr of drug treatment and have
depolymerized by 24 hours of drug treatment, indicating
the drug is working to inhibit microtubules. Figure 6 also
shows the representative images of the infection over time.
The treated infected cells do not experience any parasite
replication within the PV.


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EFFECTS OF CYTOSKELETON-INHIBITING DRUGS ON L. AMAZONENSIS DEVELOPMENT


25
MDMSO
01uM colchicine
B10uM colchicine






g15





> 1





05






24/0 24/12 24/24 24/48
Time points (initial infection timeldrug treatment time)


Figure 5: Effect of colchicine on L. amazonensis development in macrophages. Macrophages incubated with
L. amazonensis and DMSO (solid black), 1 uM colchicine (few dots), and 10 um colchicine (many dots). Data
is the mean of two experiments.


Figure 6: Images of infected cells treated with 1 uM Colchicine. Staining of beta-tubulin and Lamp-1 in L.
amazonensis infected Raw 264.7 murine macrophages treated with 1 uM colchicine. Arrow points to parasite.
(A) 0-hr drug treatment (B) 12-hr drug treatment (C) 24-hr drug treatment (D) 48-hr drug treatment.

University of Florida I Journal of Undergraduate Research I Volume 12, Issue 3 I Summer 2011
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KELLY JOHNSON, DR. BLAISE NDJAMEN, & DR. PETER E. KIMA


Figure 7 shows that 10uM of colchicine works to block
microtubules between 0 and 12 hr. In Figure 7c, although
there are many parasites, the parasites are in separate PVs


so no reproduction has occurred. After 48 hours, there is
still no parasite development within the PV, as shown in
Figure 7d with only one parasite per PV.


Figure 7: Images of infected cells treated with 10 uM Colchicine. Staining of beta-tubulin and Lamp-1 in L.
amazonensis infected Raw 264.7 murine macrophages treated with 10 uM colchicine. Arrow points to parasite.
(A) 0-hr drug treatment (B) 12-hr drug treatment (C) 24-hr drug treatment (D) 48-hr drug treatment.


DISCUSSION

The objective of the study was to determine the effects
of cytoskeleton-inhibiting drugs on Leishmania
development within the PV. The hypothesis was that
inhibiting microtubules and actin with drugs would slow
the development of Leishmania within the PV. The cell
cytoskeleton is made of microtubules and actin filaments.
Microtubules and actin filaments are both polar structures
with associated motor proteins that can transport material
along their tubules or filaments.
Microtubules are composed of alpha- and beta-tubulin
subunits that form a tubulin heterodimer or a microtubule
subunit. Microtubules grow faster at the end with the beta-
tubulin subunit, which is called the plus end, compared to
the alpha-tubulin subunit, which is called the minus end.
Kinesin and dynein are the motor proteins associated with


microtubules. The role of microtubules in the mammalian
cell is to provide long distance transport of vesicles and
organelles. Microtubules also play a role in cell structure
and support [10].
Actin is composed of the monomeric molecule G-actin
that polymerizes into a filamentous polymer, F-actin [16].
The roles of actin in the cell include cell migration by way
of myosin II and short distance vesicular or organelle
transport using myosin I, V, and VI [10]. Myosin V has
been shown to transport ER vesicles in neurons [17].
Myosin I and myosin VI have been shown to be associated
with Golgi-derived vesicles and cytoplasmic vesicles [17].
To evaluate the effects the drugs had on Leishmania
development, the parasite load and infection rate were
analyzed. The main finding of the study was that the
parasite development was significantly hindered when
microtubule- and actin-inhibiting drugs, colchicine and


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EFFECTS OF CYTOSKELETON-INHIBITING DRUGS ON L. AMAZONENSIS DEVELOPMENT


CCD, were used, separately, during a Leishmania infection
of macrophages. When the cells were not treated with
microtubule- or actin-inhibiting drugs, the parasites
multiplied within the PVs. The parasite load (number of
parasites per PV) for untreated cells reached 2.0 after 48
hours. The parasite load for all treated cells after 48 hours
was less than 1.3, demonstrating that the pharmacological
inhibition of actin and microtubules impacted the
development of L. amazonensis within the PV. L.
amazonensis replicates within a single PV to form a larger
PV with many parasites. Because the large PV with many
parasites was not seen after 48 hours of drug treatment and
was seen after 48 hours without drug treatment, it was
concluded that actin and microtubules of the host's
cytoskeleton are necessary components for the
development of this parasite.
Some limitations of the study were that the infected cells
should have been incubated at 34 C during the infection,


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instead of 37 C. The difference in temperature may have
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parts of the cell. For instance, at 10 agM colchicine, the
cells' nucleus began to fragment and it became difficult to
distinguish between the parasites and the cell after 24 hours
of drug treatment. With DMSO, it became difficult to
count the cells because the field of view was overpopulated
with macrophages, making it difficult to distinguish one
cell's vacuoles from that of another.
In this study we demonstrated that L. amazonensis
parasites develop very well in their mammalian host
macrophages. However, successful development of
Leishmania parasites requires a well-functioning cell
cytoskeleton.


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[13] Uppuluri S, Knipling L, Sackett DL, Wolff J. 1993. Localization of the
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[15] Isowa N, Xavier AM, Dziak E, Opas M, McRitchie DI, Slutsky AS,
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[18] Kima PE, Dunn W. 2005. Exploiting calnexin expression on phagosomes to
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[19] Prina E, Lang T, Glaichenhaus N, Antoine JC. 1996. Presentation of the
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[20] Zilberstein D, Shapira M. 1994. The role of pH and temperature in the
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