Caveolae mediated entry of pathogens into cells has been reported with a number of viruses, bacteria, parasites and toxins. Examples of these are Ebola, Echovirus
, influenza, FimH-expressing Escherichia coli
, cholera toxin and malaria, which have been shown to co-localize or proposed to utilize caveolae to enter cells [32
]. Caveolar entry is an additional mechanism utilized by pathogens to avoid their destruction by preventing their degradation via the endosomal or lysosomal compartments [34
]. However, cellular invasion by T. cruzi
requires transient residence within an acidic lysosomal vacuole [37
]. This allows activation of TxTox, which plays a role in vacuole disruption [38
]. In addition, lysosomal vacuoles also provide other necessary signals that permit the parasite to complete it’s intracellular life cycle [40
]. Entry of T. cruzi
into host cells is an active rather than passive process. Communication occurs between the host cell’s signaling pathways and the pathogen [41
]. Our in vitro
invasion studies suggest that trypomastigotes of T. cruzi
do not enter through caveolae and that Cav-1 does not play a role in host cell signaling required for its internalization.
Based on the findings presented in this study, it does not appear that caveolar endocytosis plays a major role in the entry of the parasite into cells. Endocytosis via caveolae occurs in a similar manner to that involving clathrin coated pits (CCPs), utilizing much of the same molecular machinery. For instance, the SNARE proteins, which are involved in the docking and fusion of vesicles, are employed by both caveolae and CCPs. In addition, the small GTPase dynamin, which mediated CCP internalization, has also been shown to localize to the necks of caveolae and be involved in their internalization as well [ 14
]. While the exact processes mediating internalization via caveolae remain unknown, several studies have shown that ligand-mediated activation of a tyrosine kinase signaling pathway is an important step [16
]. The list of ligands internalized by caveolae include several pathogenic agents which have been shown to utilize caveolae for entry into host cells [18
Acute infection with the virulent T. cruzi Tulahuen strain resulted in mortality on both the Cav-1 and wild type mice, although death was slightly delayed in the wild-type mice. Interestingly, the mortality observed in infected Cav-1 mice was associated with a significant decrease in blood parasitemia. However, examination of heart tissue by real-time PCR and histopathology revealed no alterations in parasite load. Furthermore, in the absence of Cav-1 the ability of T. cruzi to enter cells was not compromised. These results suggest that the loss of Cav-1 does not alter infectivity and/or intracellular survival of the parasite.
There is limited information regarding the role of Cav-1 in the immune system. This is in part due to the previously contentious issue of its expression in cells of the immune system. We and others have observed that Cav-1 is a component of many immune cells and is expressed in a regulated manner. Expression of Cav-1 is low in unstimulated macrophages and increases with LPS stimulation [30
]. The induction of inflammatory mediators by macrophages during T. cruzi
infection is mediated by glycoinositol phospholipids (GIPLs) [46
]. Signaling is mediated through TLR-2 and possibly TLR-4 based on recent studies [47
]. It is of great importance that macrophages derived from Cav-1 null mice produced significantly lower amounts of chemokines and cytokines during T. cruzi
infection. This suggests that Cav-1 may be involved in regulating their production under certain conditions.
Interestingly, our recent studies examined the role of Cav-1 in the pathogenesis of Salmonella typhimurium
in the mouse model [49
]. S. typhimurium
infected Cav-1 null mice had a significant mortality and an increased bacterial tissue burden compared with wild type mice [49
]. In contradistinction with the present data with T. cruzi
infection, in Salmonella
infected Cav-1 null mice there was an increased production of inflammatory mediators such as cytokines, chemokines, and nitric oxide. Nevertheless, infected Cav-1 null mice were unable to control this infection and the mice died [49
]. In addition, macrophages obtained from Cav-1 null mice had an increased inflammatory responses and nitric oxide production in vitro in response to Salmonella LPS. These results demonstrate that Cav-1 plays a key role in regulating anti-inflammatory responses in macrophages in Salmonella
infection. In addition, these observations suggest the increased production of toxic mediators from Cav-1 null mice may be responsible for the marked susceptibility of Cav-1 null mice to S. typhimurium
]. The differences in the responses of Cav-1 null mice to infection with T. cruzi
vs. S. typhimurium
infection are very intriguing. They are both intracellular organisms that can reside in macrophages for extended periods of time. However, one is an intracellular parasite while the other an intracellular bacterium. In contradistinction to Salmonella
infection, T. cruzi
may interdict important signaling pathways in infected cells. These experiments are ongoing in our laboratory. It also underscores the fact that the response of Cav-1 null mice to infection is not the same for all microorganisms.
The production of chemokines and cytokines has been shown to play a role in controlling T. cruzi. A thorough examination of their levels in infected mice revealed that they were downregulated during infection in Cav-1 null mice compared to wild-type mice. The reason for the reduced peripheral parasitemia in infected Cav-1 null mice is not known since the reduction in inflammatory mediators in these mice might suggest that the parasitemia should be increased, There was no correlation between extent of parasite load in tissue and peripheral parasitemia. However, this is not always the case. Importantly, all mice wild type and null mice still died.
Our results showed that the decrease in chemokine and cytokine expression was not due to the limited number of parasites in Cav-1 null mice, but rather an intrinsic defect in Cav-1 null macrophages. However, we cannot exclude the contribution of other immune cells in the overall reduction in serum chemokine and cytokine production in infected Cav-1 null mice. In addition, although immune cells play a major role in the production of inflammatory mediators during T. cruzi
infection, other cells may also be involved. Infection of endothelial cells, which express high levels of Cav-1, with T. cruzi
causes direct induction of IL-1β and IL-6 [50
Despite the impaired immune response in Cav-1 null mice during an acute infection with T. cruzi, they do not display an increased mortality rate. This suggests that the lack of chemokine and cytokine production in this mouse model is not a primary cause of death. It is possible that Cav-1 may play a role in cytokine/cytokine receptor systems. Surprisingly, despite the deficiency in serum chemokine production we did not observe any significant difference in recruitment of immune populations to the heart of Cav-1 null mice compared to wild-type mice.
To our knowledge this is the first report examining the role of Cav-1 in the host mediated response to infection with the protozoan parasite, T. cruzi. The results in this study show that Cav-1 null mice did not display an increased susceptibility to T. cruzi despite their reduced inflammatory profile. The role of cytokines and chemokines in the host defense against this parasite requires additional study.