Mean fluorescence intensity obtained from labeling of epimastigote forms of the two <i>T</i>. <i>cruzi</i> clonal populations.

<p>Col1.7G2 (blue) and JG (red) with polyclonal antibodies against different <i>T</i>. <i>cruzi</i> anti—oxidant enzymes: Ascorbate peroxidase, APX; Mitochondrial Peroxiredoxin, MPX; Superoxide Dismutase A, SOD A; Superoxide Dismutase B, SOD B; Trypanothione Redutase, TR and Trypanothione Synthase, TS before (A) and after (B) treatment with 30 μM H₂O₂. Dotted lines represent control non-labeled parasites.</p

(A-B) Quantification of invasion and intracellular multiplication rates of Col1.7G2 and JG in human cardiomyocyte cultures.

<p>Cultures of human cardiomyocytes derived from iPSCs were exposed to Col1.7G2 or JG and the number of infected cells per 100 cells (A), as well as the number of intracellular parasites per infected cell (B), were evaluated for the determination of invasion and intracellular multiplication rates, respectively. The data represent the mean of triplicates ± the standard error of the mean (SEM). (C) Relative fluorescence of oxidized CM-H₂DCFDA present in human cardiomyocyte cultures, 48 hours after infection with Col1.7G2 or JG. Cultures of uninfected human cardiomyocytes maintained for the same time were used as controls. The data represent the mean of duplicates ± the standard error of the mean (SEM). (D) Quantification of intracellular multiplication rates of Col1.7G2 and JG in cultures of human cardiomyocytes treated with catalase. Human cardiomyocyte cultures from iPSCs were treated with catalase and then exposed to Col1.7G2 or JG and the number of intracellular parasites per infected cell was evaluated. The data represent the mean of triplicates ± the standard error of the mean (SEM). (E) Combination of data from graphs shown in B and D, showing JG or Col1.7G2 intracellular growth from independent experiments performed in catalase treated (dashed lines) and non-treated (continuous lines) human cardiomyocyte cultures. Asterisks indicate statistically significant differences (*** p≤0.001, ** p≤0.01 and * p≤0.05—Student's t-test).</p

Quantification of epimastigotes intracellular levels of calcium (A) and superoxide anion (B) upon JG and Col1.7G2 epimastigotes treatment with H₂O₂.

<p>(A) JG and Col1.7G2 cells previously incubated with fura 2-AM, were treated or not (control) with 30 μM H₂O₂ for 30 minutes and the calcium levels in 10⁷ parasites/mL was determined. (B) JG and Col1.7G2 parasites were incubated with MitoSOX and subsequently exposed to 30 μM H₂O₂ for 30 minutes, the oxMitoSOX level was measured in 10⁷ parasites/mL. The data represent the mean of triplicates ± the standard error of the mean (SEM). Asterisks indicate statistically significant differences (**<0.01 *** p≤0.001—Student's t-test).</p

Parasitophorous vacuole escape rate for <i>T</i>. <i>cruzi</i> clonal populations, Col1.7G2 and JG, upon infection in primary mouse cardiomyocytes.

<p>(A) The graph shows the proportion of intracellular parasites associated with LAMP-1, a lysosomal marker, over the time of infection. The data represent the mean of triplicates ± the standard error of the mean (SEM). The data shown are representative of three individual experiments. (Student's t-test). (B) Representative images of intracellular parasites in primary cultures of cardiomyocytes from BALB/c neonatal mice, inside or outside the parasitophorous vacuole, 4 and 12 hours (respectively) after exposure to JG or Col1.7G2 (100X). The nuclear marker, DAPI, was used to identify the genetic material of both the parasite and the host cell and anti-LAMP-1, a lysosomal marker, was used to identify parasites inside vacuole. The scale bar corresponds to 10 μm.</p

Quantification of intracellular multiplication rates of JG and Col1.7G2 in mouse cardiomyocyte cultures treated or not with catalase.

<p>(A and B) Primary BALB/c neonatal cardiomyocyte cultures were treated (dashed line) or not (continuous line) with catalase, exposed to JG (A) or Col1.7G2 (B) and the number of intracellular parasites per infected cell along 72 hours of infection was evaluated. The data represent the mean of triplicates ± the standard error of the mean (SEM). Asterisks indicate statistically significant differences (* p≤0.01—Student t test). (C) Representative images of cell infection in primary BALB/c neonatal cardiomyocytes cultures, treated with catalase, 72 hours after exposure to JG or Col1.7G2. The scale bar corresponds to 10 μm.</p

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

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 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

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