Our laboratory studies early host-pathogen interactions that occur during the establishment of intracellular infection by the protozoan parasite, Trypanosoma cruzi. As the causative agent of Chagas' disease in humans, T. cruzi represents an important public health problem in Latin America, where several million individuals are chronically infected. While recent advances in the field have provided a new paradigm for host cell invasion by T. cruzi trypomastigotes, little is known regarding the molecular and cellular events required to shape a permissive host cell environment for intracellular growth and survival of this pathogen. It is the understanding of these basic processes that will guide our efforts toward effective prevention or control of Chagas' disease.
Host cell invasion and transient residence within the lysosomal compartment are requirements for successful establishment of infection by T. cruzi. In vitro models of T. cruziinfection (invasion and intracellular replication) have been critical to advancing our knowledge of the molecular and cellular basis of these interactions. More than a decade ago, it was demonstrated that T. cruzi trypomastigotes exploit a unique actin-independent mechanism to enter non-professional phagocytes that involves targeted fusion of host cell lysosomes with the plasma membrane at the site of parasite attachment. While lysosome-dependent entry has long been considered to be the sole mechanism by which this intracellular pathogen gains access to non-professional phagocytic cells, our laboratory recently demonstrated that the majority of invading T. cruzi trypomastigotes do not rely on lysosome recruitment for host cell invasion. Instead, these motile trypomastigotes can penetrate cells by first inducing invagination of the host cell plasma membrane in process that is facilitated by disruption of host cell actin microfilaments. Parasite-containing vacuoles gradually acquire lysosomal properties and beginning at ~8 hours post-infection, vacuole rupture allows T. cruzi to emerge into the host cell cytoplasm, where differentiation to the intracellular amastigote forms is completed and replication occurs.
Employing a combination of biochemical, molecular and cellular approaches we are focusing on the role of host cell protein phosphatidylinositol (PI)-3 kinases in the regulation of T. cruzi invasion and vacuole maturation. DNA microarray analysis is used to define host transcriptional responses to T. cruzi in vitro and in vivo. Interestingly, preliminary findings with T. cruzi-infected human fibroblasts revealed that the early transcriptional response to this pathogen was minimal and significantly delayed as compared to other intracellular pathogens. We have also determined that T. cruzi egress from the lysosomal vacuole at time points >8 hours is a strong trigger for host cell gene expression. These findings suggest that similar to the cellular response to other pathogens that take up residence in the host cell cytoplasm, eg. Listeria monocytogenes and viral pathogens, cytosolic surveillance pathways can alert the host cell to the presence of an intracellular pathogen, by triggering expression of genes involved in the innate immune response. We are currently pursuing the characterization of host cell signaling pathways that trigger this set of responses in parasite-infected cells.
Finally, our laboratory is pursuing the characterization of the unique ability of T. cruzi to downregulate, connective tissue growth factor (CTGF), a cytokine that plays a fundamental role in the development of tissue fibrosis by mediating many of the pro-fibrotic effects of TGFbeta. T. cruzi trypomastigotes release a soluble activity that effectively interferes with the host fibrogenic response on two levels: infection of dermal fibroblasts with T. cruzi inhibits basal expression of CTGF and blocks TGFbeta-dependent induction of this cytokine in both normal and scleroderma fibroblasts. Inhibition of CTGF expression is followed by down-regulation of the extracellular matrix proteins, fibronectin and collagen I a1. Our goal is to elucidate the mechanisms underlying this T. cruzi-mediated response in dermal fibroblasts and to isolate the parasite-generated anti-fibrogenic activity. In addition, studies are underway to determine the role of T. cruzi modulation of ECM protein expression in the parasite infective process using in vitro and in vivo models of infection.