Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells

Trypanosoma cruzi, is the etiological agent of a neglected tropical malady known as Chagas' disease, which affects about 8 million people in Latin America. 30–40% of affected individuals develop a symptomatic chronic infection, with cardiomyopathy being the most prevalent condition. T. cruzi utilizes an interesting strategy for entering cells: T. cruzi enhances intracellular calcium levels, which in turn trigger the exocytosis of lysosomal contents. Lysosomes then donate their membrane for the formation of the parasitophorous vacuole. Membrane rafts, cholesterol-enriched microdomains in the host cell plasma membrane, have also been implicated in T. cruzi invasion process. Since both plasma membrane and lysosomes collaborate in parasite invasion, we decided to study the importance of these membrane domains for lysosomal recruitment and fusion during T. cruzi invasion into host cells. Our results show that drug dependent depletion of plasma membrane cholesterol changes raft organization and induces excessive lysosome exocytosis in the earlier stages of treatment, leading to a depletion of lysosomes near the cell cortex, which in turn compromises T. cruzi invasion. Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events of pre-docked lysosomes, reducing lysosome availability at the cell cortex and consequently compromising T. cruzi infection

Correction: LAMP-2 absence interferes with plasma membrane repair and decreases T. cruzi host cell invasion.

[This corrects the article DOI: 10.1371/journal.pntd.0005657.]

LAMP-2 absence interferes with plasma membrane repair and decreases <i>T</i>. <i>cruzi</i> host cell invasion

<div><p><i>Trypanosoma cruzi</i> enters host cells by subverting the mechanism of cell membrane repair. In this process, the parasite induces small injuries in the host cell membrane leading to calcium entry and lysosomal exocytosis, which are followed by compensatory endocytosis events that drive parasites into host cells. We have previously shown that absence of both LAMP-1 and 2, major components of lysosomal membranes, decreases invasion of <i>T</i>. <i>cruzi</i> into host cells, but the mechanism by which they interfere with parasite invasion has not been described. Here we investigated the role of these proteins in parasitophorous vacuole morphology, host cell lysosomal exocytosis, and membrane repair ability. First, we showed that cells lacking only LAMP-2 present the same invasion phenotype as LAMP1/2^-/- cells, indicating that LAMP-2 is an important player during <i>T</i>. <i>cruzi</i> invasion process. Second, neither vacuole morphology nor lysosomal exocytosis was altered in LAMP-2 lacking cells (LAMP2^-/- and LAMP1/2^-/- cells). We then investigated the ability of LAMP-2 deficient cells to perform compensatory endocytosis upon lysosomal secretion, the mechanism by which cells repair their membrane and <i>T</i>. <i>cruzi</i> ultimately enters cells. We observed that these cells perform less endocytosis upon injury when compared to WT cells. This was a consequence of impaired cholesterol traffic in cells lacking LAMP-2 and its influence in the distribution of caveolin-1 at the cell plasma membrane, which is crucial for plasma membrane repair. The results presented here show the major role of LAMP-2 in caveolin traffic and membrane repair and consequently in <i>T</i>. <i>cruzi</i> invasion.</p></div

Absence of LAMP-2 does interfere with membrane repair.

<p>(A-B) WT, LAMP1/2^-/- or LAMP2^-/- were submitted to membrane injury by cell scraping. (A) Cells were scraped in the presence of Propidium Iodide (PI). Histograms show the number of cells presenting PI labeling (PI +), which represent the number of cells that suffered injury during scraping, while cells excluding PI represent those that didn’t suffer membrane injury. Bars above the curve indicate the percentage of injured (PI +) and non-injured cells (PI -). (B) Cells were scraped in the absence of PI, allowed to reseal, and then exposed to PI. Histograms show the number of PI + and PI–cells, which represent the ones that did not or did recover from injury, respectively. Bars above the curve indicate the percentage of non-viable (PI +) and viable cells (PI -). (C) Cells were exposed or not to <i>T</i>. <i>cruzi</i> trypomastigotes for 30 minutes in the presence of Propidium Iodide (PI). Blue curves represent control cultures, without parasite exposure, while red curves represent cell cultures exposed to <i>T</i>. <i>cruzi</i>. Histograms show the number of cells presenting PI labeling (PI +), which represent the number of cells that suffered injury, while cells excluding PI represent those that didn’t suffer membrane injury. Bars above the curve indicate the percentage of injured (PI +) and non-injured cells (PI -) in the presence or absence of <i>T</i>. <i>cruzi</i>. (D) Difference in the percentage of PI+ cells between control and <i>T</i>. <i>cruzi</i> exposed cell cultures. Asteriks indicate statistically significant differences p<0.05. Data shown are representative of three independent experiments.</p

LAMP-2 deficiency is enough to compromise <i>T</i>. <i>cruzi</i> invasion in host cells.

<p>WT, LAMP1/2^-/- or LAMP2^-/- fibroblasts monolayers were exposed to Tissue Culture derived Trypomastigotes (TCT) from Y strain at a MOI of 50 for 20 minutes, washed, fixed and then processed for immunofluorescence detection of total intracellular parasites. Quantitative analysis of parasite infection rates in the three fibroblast cell lines was determined by the number of internalized parasites per 100 counted cells (A), as well as the percentage of infected cells (B). Data are shown as mean of triplicates ±SD. Asterisks indicate statistically significant differences (p<0.05, Student’s t test) between WT and LAMP deficient cells. (C) Representative panels of <i>T</i>. <i>cruzi</i> invasion in the three different fibroblast cell lines revealed by immunofluorescence labeling. Cell and parasite nuclei, as well as parasite kinetoplast DNA, were labeled with DAPI (blue); extracellular parasites in the field were labeled with anti-<i>T</i>. <i>cruzi</i> antibody followed by secondary IgG labeled with Alexa Fluor 546 (red). White arrows indicate intracellular parasites, while red arrows indicate extracellular parasites. Data shown are representative of three independent experiments.</p

Absence of LAMP-2 leads to actin cytoskeleton rearrangement and decrease in cholesterol levels at the cell plasma membrane.

<p>(A) WT, LAMP1/2^-/- or LAMP2^-/- cells were submitted to phalloidin staining and analyzed in a fluorescence microscope. Arrows indicate the presence of the long actin stress fibers. (B) WT, LAMP1/2^-/- or LAMP2^-/- cells were submitted to lipid extraction from plasma membrane and the amount of cholesterol content evaluated. Plasma membrane cholesterol of LAMP2^-/- and LAMP1/2^-/- was measured as a percentage the plasma membrane content of WT cells, which was set as 100. Asterisk above bars indicate statistically significant differences.</p

Absence of LAMP does not affect lysosomal exocytosis, but do interfere with compensatory endocytosis events.

<p>(A) Lysosome exocytosis assay. WT, LAMP1/2^-/- or LAMP2^-/- fibroblasts monolayers were exposed to 5 or 10μM Ionomycin for 10 minutes at 37°C, in the presence of calcium. Both extracellular media and lysates were collected and assayed for beta-hexosaminidase activity. Results are shown as the ratio between extracellular media β-hexosaminidase activity and total β-hexosaminidase activity (extracellular media plus cell lysate hexosaminidase activity). Non treated cells were used as lysosomal exocytosis negative control. Data are shown as mean of triplicates ±SD. Asterisks indicate statistically significant differences (p < 0.05, Student’s t test) between control and treated cells. (B) Measurement of compensatory endocytosis events induced by membrane injury. Cells were labeled with WGA-Alexa Fluor 488, submitted to membrane injury by cell scraping in the presence (red) or absence (blue) of calcium, and then incubated with trypan-blue to eliminate plasma membrane labeling. Only fluorescence from internalized membranes was preserved. The endocytosis was then quantified by FACS analysis. Histograms show the number of cells displaying WGA-Alexa Fluor labeling for the different fibroblast cell lines, WT, LAMP1/2^-/- and LAMP2^-/-. Bars above the curves indicate the median of the fluorescence for each condition (with or without calcium). Data shown are representative of three independent experiments. (C) Detection of ASM in the supernatant of control non-scraped cells, and cells that had been subjected to scrape wounding and membrane repair. Supernatants of WT, LAMP1/2^-/- and LAMP2^-/-, in the different conditions, were run on a gel, blotted onto nitrocellulose membranes and revealed using an anti-ASM antibody. The panel shows the presence of ASM in the supernatant of all three cells, only upon wounding and membrane repair.</p

Absence of LAMP does not interfere with parasitophorous vacuole morphology.

<p>Transmission Electron Microscopy images of parasitophorous vacuoles from WT (A), LAMP1/2^-/- (B) e LAMP2^-/- (C) cells. Red arrows indicate parasite membrane and black arrows indicate parasitophorous vacuole membrane. (D) Graph shows the rate between parasitophorous vacuole and parasite areas. Data are shown as mean from 15 observed vacuoles ±SD from each cell line, WT, LAMP1/2^-/- and LAMP2^-/-. No statistically significant differences were observed (P < 0,05, Student’s T test).</p

MβCD treatment is effective in sequestering cholesterol from the plasma membrane.

<p>(A) Control, (B) 15 mM MβCD treated and (C) 15 mM MβCD followed by 0.05 mM WSC treated cardiomyocytes show significant changes in Filipin labeling. Cholesterol-depleted cells (B) reveal very little Filipin labeling, whereas cholesterol-replenished cells show strong labeling for cholesterol, similar to control cells. (D) Cardiomyocytes treated with 5, 10 or 15 mM of MβCD reveal a substantial decrease in Filipin labeling in a dose-dependent manner whereas cholesterol replenishment with 0.05 mM of WSC returns Filipin fluorescence to control values. Normalized data are shown as mean of triplicates ±SD. Asteriks indicate statistically significant differences (* p < 0.05 and ** p < 0.01, One way ANOVA followed by Newman-Keuls) between control and treated cells. Scale bar: 10 µm.</p

<i>T. cruzi</i> invasion of cells and association with LAMP-1 in cardiomyocytes decreases after cholesterol depletion.

<p>Cardiomyocytes pre-treated or not with different cyclodextrins were washed and challenged with <i>T. cruzi</i> trypomastigotes at a M.O.I of 50, for 40 minutes at 37°C, then fixed and processed for immunofluorescence detection of total intracellular parasites, as well as intracellular parasites associated with LAMP-1 (a lysosomal marker). Both <i>T. cruzi</i> internalization (A) and association with host LAMP-1 (B) diminishes after incubation with 10 and 15 mM of MβCD but not after treatment with 10 and 15 mM of HγCD. Cholesterol replenishment after treatment with 15 mM MβCD reverts the effect of the drug on parasite cell invasion (A), and at least partially on LAMP-1 association (B). The average number of cardiomyocytes ±SD per 10 counted fields in each coverslip is shown above the bars (A). Data are shown as mean of triplicates ±SD. Asteriks indicate statistically significant differences (p < 0.05, Student's t test) between control and treated cells. (C) Representative panels of <i>T. cruzi</i> invasion and association with host cell lysosomes, revealed by immunocytochemistry. Total cell and parasite nuclei, as well as parasite kinteoplast DNA were labeled with DAPI; lysosomes were labeled with anti-LAMP 1 antibody followed by secondary IgG labeled with Alexa Fluor 488; extracellular parasites in the field were labeled with anti-<i>T.cruzi</i> antibody followed by secondary IgG labeled with Alexa Fluor 546. From top to bottom: control cells, 15 mM MβCD treated cells, 15 mM HγCD treated cells and 15 mM MβCD treated cells followed by incubation with 0.05 mM of WSC. Blue arrows show total <i>T. cruzi</i> trypomastigotes in the field, yellow ellipsoids show lysosomal associated trypomastigotes, red triangles points out extracellular trypomastigotes and the last column shows the merge of the three previous. Scale bar: 10 µm.</p