Silencing of the laminin gamma-1 gene blocks Trypanosoma cruzi infection

Abstract

It is thought that Trypanosoma cruzi, the protozoan that causes Chagas' disease, modulates the extracellular matrix network to facilitate infection of human cells. However, direct evidence to document this phenomenon is lacking. Here we show that the T. cruzi gp83 ligand, a cell surface trans-sialidase-like molecule that the parasite uses to attach to host cells, increases the level of laminin gamma-1 transcript and its expression in mammalian cells, leading to an increase in cellular infection. Stable RNA interference (RNAi) with host cell laminin gamma-1 knocks down the levels of laminin gamma-1 transcript and protein expression in mammalian cells, causing a dramatic reduction in cellular infection by T. cruzi. Thus, host laminin gamma-1, which is regulated by the parasite, plays a crucial role in the early process of infection. This is the first report showing that knocking down the expression of a human gene by RNAi inhibits the infection of an intracellular parasite.

FIG. 1.

T. cruzi trypomastigote gp83 ligand up-regulates trypanosome entry into human cells. (A) gp83 ligand up-regulates T. cruzi entry into HCASM cells. HCASM cells were preincubated with increasing concentrations of gp83, gp83 preincubated with antibodies, or boiled gp83 for 30 min, washed, and exposed to trypomastigotes at a ratio of 10 parasites per cell for 2 h, and the number of internalized trypanosomes was determined. (B) gp83 ligand up-regulates T. cruzi entry into HeLa cells. HeLa cells were preincubated with gp83 (0.4 μg/ml) under the same conditions as in panel A, and entry was evaluated similarly. Bars represent the mean results ± 1 standard deviation obtained with triplicate samples in one representative experiment selected from three experiments with similar results. *, significant difference compared to control values (P < 0.05).

FIG. 2.

Exposure of T. cruzi gp83 ligand to HCASM cells up-regulates the expression of laminin γ-1. (A) Exposure of gp83 to HCASM cells up-regulates the expression of laminin γ-1 as determined by cDNA microarray analysis. RNA from HCASM cells exposed or not exposed to gp83 was reverse transcribed to labeled cDNA and hybridized onto cDNA microarray slides. Duplicate hybridizations were normalized against their respective controls and analyzed with GeneSpring Software. Bars represent the mean ± 1 standard deviation of ECM gene expression of duplicate experiments with similar results. *, significant difference between laminin γ-1 gene expression and the expression of the other ECM genes (P < 0.01). (B) Kinetics of laminin γ-1 transcript upon exposure of gp83 to HCASM cells. RNA from cells exposed or not exposed to gp83 was reversed transcribed to cDNA, and the laminin γ-1 transcript levels were evaluated by real-time-PCR. Data are the mean ± 1 standard deviation of normalized levels of laminin γ-1 transcript against human 18S RNA in one representative experiment of three independent experiments performed in triplicate with similar results. Each point is the mean of results for triplicate samples in one representative experiment (±1 standard deviation). The P value was <0.05 for points at 120 and 180 min with respect to points at 0 and 60 min.

FIG. 3.

Laminin γ-1 RNAi constructs used for generating shRNA in HeLa cells. (A) Schematic representation of the predicted shRNA structure. The orientation of the oligonucleotide sequences cloned behind a strong U6 promoter is shown. (B) Design of oligonucleotide constructs from laminin γ-1 cDNA to generate shRNA. The 5′ and 3′ termini underlined show the modification for cloning into the IMG-800 vector. Numbers on oligonucleotides show the locations on the laminin γ-1 cDNA.

FIG. 4.

RNAi with the laminin γ-1 gene reduces laminin γ-1 transcript levels in HeLa cells. RNA from HeLa cells stably transfected with either the IMG-800 vector alone or containing construct L-2 or L-3 was reversed transcribed to cDNA, and the laminin γ-1 transcript level was evaluated by real-time PCR. The results were normalized against human 18S RNA. Bars represent the means of results from triplicate samples in one representative experiment (±1 standard deviation) selected from three independent experiments with similar results. *, significant difference compared to control value, cells transfected with the vector alone (P < 0.05).

FIG. 5.

RNAi with laminin γ-1 knocks down laminin γ-1 expression and blocks T. cruzi infection of HeLa cells. (A) shRNA constructs targeted to the laminin γ-1 gene reduce the level of laminin γ-1 protein expression. Different protein concentrations of HeLa cells stably transfected with either the IMG-800 vector alone or containing construct L-2 or L-3 were probed with either anti-laminin γ-1 antibodies or β-actin antibodies in immunoblot assays. The results shown are from a representative experiment of three performed with similar results. (B) Densitometric scanning of immunoblots normalized to their corresponding β-actin absorbance as shown in panel A. The mean of three independent experiments ± 1 standard deviation is plotted. Open bars and solid bars represent normalized absorbance of laminin γ-1 bands from cells transfected with the vector alone or the chimeric vector containing constructs, respectively. *, significant difference compared to control values, cells transfected with the vector alone (P < 0.05). (C) RNAi with laminin γ-1 significantly reduces trypanosome infection. HeLa cells stably transfected with the IMG-800 vector alone or with the vector containing construct L-2 or L-3 were exposed to trypomastigotes at a ratio of 15 parasites per cell for 2 h, and trypanosome binding was evaluated (top). T. cruzi multiplication in cells transfected with IMG-800, L-2, or L-3 was evaluated at 72 h (bottom). Bars in panel C represent the means ± 1 standard deviation of results from triplicate samples in one representative experiment (±1 standard deviation) selected from three experiments with similar results. *, significant difference compared to control values, cells transfected with the vector alone (P < 0.05).