Early Trypanosoma cruzi infection reprograms human epithelial cells

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, has the peculiarity, when compared with other intracellular parasites, that it is able to invade almost any type of cell. This property makes Chagas a complex parasitic disease in terms of prophylaxis and therapeutics. The identification of key host cellular factors that play a role in the T. cruzi invasion is important for the understanding of disease pathogenesis. In Chagas disease, most of the focus is on the response of macrophages and cardiomyocytes, since they are responsible for host defenses and cardiac lesions, respectively. In the present work, we studied the early response to infection of T. cruzi in human epithelial cells, which constitute the first barrier for establishment of infection. These studies identified up to 1700 significantly altered genes regulated by the immediate infection. The global analysis indicates that cells are literally reprogrammed by T. cruzi, which affects cellular stress responses (neutrophil chemotaxis, DNA damage response), a great number of transcription factors (including the majority of NF κ B family members), and host metabolism (cholesterol, fatty acids, and phospholipids). These results raise the possibility that early host cell reprogramming is exploited by the parasite to establish the initial infection and posterior systemic dissemination.

Figure 1

Differential gene expression in T. cruzi infected HeLa cells at t ₀, t ₃, and t ₆ compared to noninfected control cells (≥2-fold, P ≤ 0.05). Red bars indicate upregulated genes and green bars indicate downregulated genes. Inset table shows number of differentially expressed genes for each condition.

Figure 2

Venn diagrams comparing (a) upregulated and (b) downregulated genes (fold change ≥ 2, P ≤ 0.05) with respect to control cells; (c) number of genes up- or downregulated, comparing one condition to the previous one (t ₀ versus C, t ₃ versus t ₀, and t ₆ versus t ₃) using fold change ≥ 2 and P ≤ 0.05.

Figure 3

Cellular processes predicted to be modulated during T. cruzi infection. (a) Pathways analysis with upregulated genes from t ₀, t ₃, and t ₆; (b) pathways analysis with upregulated genes comparing t ₀ versus C, t ₃ versus t ₀, and t ₆ versus t ₃, showing characteristic pathways altered at each time point.

Figure 4

Heat maps of some representative biological processes altered during T. cruzi infection. Genes regulated by T. cruzi involved in immune response/inflammation (top) and genes involved in stress response and transcription factor activity (down) are represented.

Figure 5

Quantitation of selected genes by real-time PCR. Mean relative fold changes of upregulated and down-egulated genes analyzed by qPCR in 3 biological replicates of infected HeLa cells at t ₀ relative to that in control cells. Error bar represents SD of replicates. The cross-bars in the y-axis indicate changes in the scale used.

Figure 6

Temporal regulation of host genes related to cell cycle control and DNA repair on infected cells.

Figure 7

(a) Temporal regulation of host genes related to lipid metabolism after infection with T. cruzi. (b) Western blot analysis showing overexpression of PTGS2 and OLR1 proteins and normalization with GADPH. (c) Red Nile staining of control and infected cells at t ₆.