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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 ␥-1 transcript and its expression in mammalian cells, leading to an increase in cellular infection. Stable RNA interference (RNAi) with host cell laminin ␥-1 knocks down the levels of laminin ␥-1 transcript and protein expression in mammalian cells, causing a dramatic reduction in cellular infection by T. cruzi. Thus, host laminin ␥-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.

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The molecular mechanisms of Trypanosoma cruzi induced cardiac fibrosis remains to be elucidated. Primary human cardiomyoctes (PHCM) exposed to invasive T. cruzi trypomastigotes were used for transcriptome profiling and downstream bioinformatic analysis to determine fibrotic-associated genes regulated early during infection process (0 to 120 minutes). The identification of early molecular host responses to T. cruzi infection can be exploited to delineate important molecular signatures that can be used for the classification of Chagasic patients at risk of developing heart disease. Our results show distinct gene network architecture with multiple gene networks modulated by the parasite with an incline towards progression to a fibrogenic phenotype. Early during infection, T. cruzi significantly upregulated transcription factors including activator protein 1 (AP1) transcription factor network components (including FOSB, FOS and JUNB), early growth response proteins 1 and 3 (EGR1, EGR3), and cytokines/chemokines (IL5, IL6, IL13, CCL11), which have all been implicated in the onset of fibrosis. The changes in our selected genes of interest did not all start at the same time point. The transcriptome microarray data, validated by quantitative Real-Time PCR, was also confirmed by immunoblotting and customized Enzyme Linked Immunosorbent Assays (ELISA) array showing significant increases in the protein expression levels of fibrogenic EGR1, SNAI1 and IL 6. Furthermore, phosphorylated SMAD2/3 which induces a fibrogenic phenotype is also upregulated accompanied by an increased nuclear translocation of JunB. Pathway analysis of the validated genes and phospho-proteins regulated by the parasite provides the very early fibrotic interactome operating when T. cruzi comes in contact with PHCM. The interactome architecture shows that the parasite induces both TGF-β dependent and independent fibrotic pathways, providing an early molecular foundation for Chagasic cardiomyopathy. Examining the very early molecular events of T. cruzi cellular infection may provide disease biomarkers which will aid clinicians in patient assessment and identification of patient subpopulation at risk of developing Chagasic cardiomyopathy.

Human defensins play a fundamental role in the initiation of innate immune responses to some microbial pathogens. Here we show that human defensin ␣-1 displays a trypanocidal role against Trypanosoma cruzi, the causative agent of Chagas' disease. The toxicity of human defensin ␣-1 against T. cruzi is mediated by membrane pore formation and the induction of nuclear and mitochondrial DNA fragmentation, leading to trypanosome destruction. Exposure of trypomastigote and amastigote forms of T. cruzi to defensin ␣-1 significantly reduced parasite viability in a peptide concentration-dependent and saturable manner. The toxicity of defensin ␣-1 against T. cruzi is blocked by anti-defensin ␣-1 immunoglobulin G. Electron microscopic analysis of trypomastigotes exposed to defensin ␣-1 revealed pore formation in the cellular and flagellar membranes, membrane disorganization, and blebbing as well as cytoplasmic vacuolization. Furthermore, human defensin ␣-1 enters the trypanosome when membrane pores are present and is associated with later intracellular damage. Trypanosome membrane depolarization abolished the toxicity of defensin ␣-1 against the parasite. Preincubation of trypomastigotes with defensin ␣-1 followed by exposure to human epithelial cells significantly reduced T. cruzi infection in these cells. Thus, human defensin ␣-1 is an innate immune molecule that causes severe toxicity to T. cruzi and plays an important role in reducing cellular infection. This is the first report showing that human defensin ␣-1 causes membrane pore formation in a human parasite, leading to trypanosome destruction.