Trypanosoma cruzi utilizes the host low density lipoprotein receptor in invasion

Abstract

Background: Trypanosoma cruzi, an intracellular protozoan parasite that infects humans and other mammalian hosts, is the etiologic agent in Chagas disease. This parasite can invade a wide variety of mammalian cells. The mechanism(s) by which T. cruzi invades its host cell is not completely understood. The activation of many signaling receptors during invasion has been reported; however, the exact mechanism by which parasites cross the host cell membrane barrier and trigger fusion of the parasitophorous vacuole with lysosomes is not understood.

Methodology/principal findings: In order to explore the role of the Low Density Lipoprotein receptor (LDLr) in T. cruzi invasion, we evaluated LDLr parasite interactions using immunoblot and immunofluorescence (IFA) techniques. These experiments demonstrated that T. cruzi infection increases LDLr levels in infected host cells, inhibition or disruption of LDLr reduces parasite load in infected cells, T. cruzi directly binds recombinant LDLr, and LDLr-dependent T. cruzi invasion requires PIP2/3. qPCR analysis demonstrated a massive increase in LDLr mRNA (8000 fold) in the heart of T. cruzi infected mice, which is observed as early as 15 days after infection. IFA shows a co-localization of both LDL and LDLr with parasites in infected heart.

Conclusions/significance: These data highlight, for the first time, that LDLr is involved in host cell invasion by this parasite and the subsequent fusion of the parasitophorous vacuole with the host cell lysosomal compartment. The model suggested by this study unifies previous models of host cell invasion for this pathogenic protozoon. Overall, these data indicate that T. cruzi targets LDLr and its family members during invasion. Binding to LDL likely facilitates parasite entry into host cells. The observations in this report suggest that therapeutic strategies based on the interaction of T. cruzi and the LDLr pathway should be pursued as possible targets to modify the pathogenesis of disease following infection.

Figure 1. LDLr expression in T. cruzi infected host cells.

A. Immunoblot analysis of LDLr in infected cells. Increased LDLr levels were observed in both infected HFF cells (upper panel) and H9c2 cell lines (lower panel) after 1h incubation with parasites. B. Quantitative analysis of immunoblots. Arbitrary units of the expressed LDLr proteins normalized to respective GAPDH levels represented in the bar histogram as quantitated using Alpha Ease FC software. C- control, I- infected, C(HFF)- control HFF cells, I(HFF)- infected HFF cells, C(H9c2)- control H9c2 cells and I(H9c2)- infected H9c2 cells (n = 4 and p<0.05 represented as star). C. Distribution of LDLr in uninfected and infected fibroblast cells. IFA demonstrated an even distribution of LDLr at cell membrane in uninfected cells, but clustering of LDLr in infected cells after 15 minutes of incubation with parasites (arrows). (Host nucleus≈HN, bar represents 50µm).

Figure 2. Inhibition/disruption of LDLr reduces parasite load in infected cells.

A. PCSK9 pretreatment reduced parasite invasion. Incubation of parasites with PCSK9 pretreated HFF cells reduced parasite load by degrading LDLr. The bar histogram demonstrates the parasite load in PCSK9 pretreated and untreated HFF cells (n = 4 and p<0.05 represented as an “*”). B. Immunoblot analysis of LDLr in wild type and LDLr KO cells. Immunoblot analysis using monoclonal LDLr antibodies confirmed the lack of full length LDLr at 120 KD (represented by arrow) in KO cells. However, LDLr KO cells express a truncated LDLr which lacks the LDL binding domain (27). C. Quantitative analysis of parasite load in infected wild type and LDLr KO cells. Reduced parasite load in LDLr KO cells after 68h of infection compared to infected wild type cells demonstrated by real time PCR analysis. Bar histogram represents the parasite load (%) in infected cells. D. Kinetic studies of parasite binding and invasion in LDLr KO cells. Lack of full length LDLr protein in LDLr KO cells reduced the number of parasites attached to cell membrane (Upper line) and also retarded the parasite invasion (lower line), demonstrated by IFA as described under experimental procedures. E. IFA of LDLr in infected LDLr KO cells. IFA using monoclonal LDLr antibodies (red) demonstrated the clustering of disrupted LDLr around the structures similar to parasites (arrow) in KO cells (bar represents 50µm). F. Co-localization of parasites with disrupted LDLr in infected KO and wild type cells. Double staining IFA using infected mouse serum (green) and monoclonal LDLr antibodies (red) demonstrated the co-localization of parasites with disrupted LDLr (top panel) and full length LDLr (lower panel). (bar represents 50µm).

Figure 3. T. cruzi targets LDLr during invasion as demonstrated by IFA.

A. Association of LDLr with T. cruzi during invasion. IFA of LDLr in Infected HFF cells showed the co-localization of LDLr (red) with parasite. The cells were stained with DAPI (blue) to detect nucleus (bar represents 50µm). B. Binding of parasite to recombinant human LDLr. IFA demonstrated the direct binding of parasite with exogenously added recombinant huLDLr using monoclonal antibodies specific for LDLr (bar represents 50µm). C. Direct binding of parasite to Fluorescence labeled recombinant huLDLr. Fluorescent labeled huLDLr bound to the parasites on their cell surface demonstrated by fluorescent microscopy (left) and stained with DAPI (center). Alexa fluor 488 labeled GAPDH dye conjugate is used as a control (right). (bar represents 10µm).

Figure 4. Role of LDLr and its associates in trafficking T. cruzi into host lysosomes.

A. PIP2/3 is associated with LDLr during invasion. Co-localization of PIP2/PIP3 (green) and LDLr (red) with parasite (DAPI) is demonstrated by triple staining IFA. B. Parasite utilizes LDLr/clathrin complex to enter host cells. Triple staining IFA of infected 3T3-L1 cells demonstrated the co-localization of clathrin (green) with LDLr (red) and parasites (DAPI). C. Presence of LDLr during lysosomal fusion with parasites during infection. Parasite trafficking to lysosomes (LAMP1- green) by LDLr (red) is demonstrated by the co-localization lysosome, LDLr and parasite as shown by IFA. (green arrow represents the presence of LAMP1 around parasitophorous vacuoles. D. Co-localization of total LAMP-2 with parasites during infection. Triple staining IFA demonstrated the co-localization of LAMP-2 (green) with LDLr (red) and parasites (DAPI) (bar represents 50µm).

Figure 5. Role of LDL/LDLr in acute stage of T. cruzi infected CD1 mice.

A. Increased mRNA levels of LDLr in heart. qPCR analysis demonstrated a massive increase in LDLr mRNA levels in 15dpi CD1 mouse heart. B. Serum lipid profile of infected mice. A significant reduction in LDL and total triglycerides in the serum of infected mice is detected using colorimetric assay. C. Association of LDLr with parasite nests in infected heart. IFA of hearts demonstrated the co-localization of LDLr (green) with parasites (pseudocysts) in infected (15dpi) CD1 mice (bar represents 10µm). D. Accumulation of LDL with parasites in infected mouse heart. IFA showed the presence of LDL (green) co-localized with parasites (bar represents 10µm).