New data from our laboratory have recently revealed a connection between T. cruzi
and the host cell autophagic pathway. Using epithelial chinese hamster ovary cells (CHO cells) over-expressing green fluorescent protein tagged LC3 (GFP-LC3), the best characterized autophagosome marker, colocalization between the TcPV and LC3 was observed by confocal microscopy (4
). Quantification studies showed that the maximal recruitment of LC3 occurs between 1 and 6 h after infection, related to the intrinsic capacity of the particular Try strain to escape the vacuole. At earlier times (i.e., less than 1 h) we observed by real time video microscopy, numerous GFP-LC3 decorated autophagosomes concentrated near the plasma membrane in the vicinity of the Try contact site. This observation, associated with the fact that GFP-LC3 was specifically recruited to the cytosolic face of the plasma membrane at T. cruzi
contact sites, indicates that parasite interaction with LC3 positive compartments occurs at very early times in the TcPV formation and persists during vacuole transport until parasite escape. At later times, 48 or 72 h after infection, when amastigotes were distributed in the cytoplasm, interaction with GFP-LC3 or endogenous LC3 proteins was no longer observed (4
The recruitment of LC3 to the TcPV is modified in relation to the level of autophagic activity of the host cell. Under conditions that induce autophagy, such as starvation or rapamycin treatment, the extent of colocalization significantly increases. In contrast, the LC3 acquisition was impaired under conditions that inhibit autophagy. Furthermore, recent results from ourlaboratory have confirmed that this protein is present at the membrane that envelops T. cruzi Try during invasion and TcPV formation by different T. cruzi strains in distinct classes of host cells. shows parasites surrounded by this autophagic protein at the time that they are entering the cells. Different examples of epithelial and cardiac muscle derived host cells (CHO cells, rat heart myoblast H9C2 cells and mouse cardiomyocyte HL1 cells) overexpressing GFP-LC3 were used and also different T. cruzi strains including Brazil heart, K98 (not shown) and CL Brener clone. These observations suggest that T. cruzi–host autophagy interaction represents a widespread phenomenon that proceeds in cells relevant for the pathophysiology of the infection and Chagas disease. Moreover, we extended our studies to phagocytic cells and observed in RAW264.7 macrophages the same interaction events () with a percentage of colocalization that increased after starvation induction (data not shown).
Figure 1 Interaction of T. cruzi trypomastigotes and host cell autophagic pathway proceeds in different classes of host cells and T. cruzi strains. Stably transfected cells overexpressing GFP-LC3 were infected for 1 h with TCT of T. cruzi (Multiplicity of infection, (more ...)
The crucial role of the T. cruzi
–host autophagy interaction was demonstrated when quantification studies showed that autophagy induction significantly increased the percentage of infected cells at 1–3 h after infection. Interestingly, this percentage was markedly reduced in the presence of the PI3K inhibitors wortmannin or 3-methyladenine or in the absence of the specific autophagy genes Beclin-1 or Atg5, necessary for the first steps of the autophagic pathway (4
). In addition, in the new host cell systems analyzed, increased host cell colonization by T. cruzi
was always observed under autophagy induction (data not shown). Nevertheless, different parasitic forms could engage different host cell responses during invasion as shown for MT, which activate signaling cascades involving mammalian target of rapamycin and/or PI3K/PKC (9
), which are opposite to previously demonstrated for T. cruzi
We have also shown that when autophagy is induced, the localization of lysosomal markers like Lamp-1 and Cathepsin D increase on TcPV, whereas in Atg 5 KO cells the localization of these markers are significantly reduced at 15 min or 1 h after infection. Taking into account that T. cruzi
needs the lysosomal environment to effectively infect host cells (20
), this result provides an explanation for why these autophagy impaired cells (or cells pretreated with autophagy inhibitors) have a reduced capacity to become infected. Although Lamp-1 and Cathepsin D are typically used as lysosomal markers, a strong body of evidences shows that these proteins are located in other cellular compartments like the trans-Golgi network, late endosomes, and even in the plasma membrane in the case of Lamp-1 or Lamp-2. For this reason, to further analyze the specific localization of lysosomes and autophagosomes during T. cruzi
invasion, we preloaded CHO GFP-LC3 cells with a self-quenched bovine albumin (DQ-BSA, Molecular Probes, Eugene, Oregon) and performed time-lapse live images of the infection process. The advantage of this probe is that it only emits fluorescence when albumin is degraded to small peptides; this reaction occurs in the presence of acidic proteases restricted to lysosomes. Thus, under starvation, compartments labeled with GFP-LC3 and DQ-BSA, representing autophagolysosomes (or autolysosomes), are transported and fuse to plasma membrane domains that contain invading Try (, Copyright Landes Bioscience). Originally published in Ref. 4
, PMID: 19115481; http://dx.doi.org/10.4161/auto.5.1.7160
. Furthermore, it is possible to see the parasite body inside the cell wrapped by a membrane containing GFP-LC3 and autolysosomes in close proximity to this large structure that is forming. GFP-LC3 could have been left behind in this locale after autolysosomes have fused with the plasma membrane. Nevertheless, we cannot exclude the possibility that the GFP-LC3 protein may be recruited directly from the cytosol before the arrival of autolysosomes. Both situations support the crucial role of this pathway in the T. cruzi
Figure 2 Autolysosomes associate to parasites during T. cruzi infection. Stably transfected CHO cells overexpressing GFP-LC3 were labeled with DQ-BSA (10 μg/mL) for 1 h and incubated for 2 h in starvation medium before infection with TCT of the CL Brener (more ...)
Considering the aforementioned results, we propose a general model in which under autophagic induction host cells produce a new set of autolysosomal vesicles that are available to migrate to the cell surface and to shelter the parasites after fusion with the plasma membrane (). To invade host cells, T. cruzi
exploits the lysosomal exocytosis machinery to attract these compartments to the cell surface, but under normal conditions when cells are maintained in full nutrient media, the number of lysosomes available for T. cruzi
is more limited, compared to the numerous acidic vesicles present under starvation conditions (47
). T. cruzi
has also the capacity to recruit the autophagolysosomes specifically to the invasion sites. The final result is an enhanced infection rate in starved compared to control cells. According to our working model, all conditions that activate the autophagic flux (and the autophagolysosome generation) would benefit T. cruzi
colonization of cells. Interestingly, cells cotransfected to overexpress both LC3 and VAMP-7 proteins present an elevated degree of infection in both full nutrient and starvation conditions (data not shown). As VAMP-7 is a SNARE protein that favors fusion between autophagosomes and late endosomes/lysosomes, activating the autophagic flux (48
), these results support the proposed model.
Figure 3 A proposed model of T. cruzi infection involving the host cell autophagic pathway. Panel A: the parasite subverts the lysosomal exocytic process to invade host cells in basal conditions. Note that basal autophagy also would provide acidic vesicles (i.e., (more ...)
The autophagolysosomal nature of the recently formed TcPV could have other benefits for the parasites within, compared with the canonical lysosomal compartments. In principle, autophagolysosomes are compartments enriched in a high variety of simple nutrients originated from complex intracellular components previously entrapped by autophagic transport and degraded by lysosomal hydrolases. Furthermore, the membrane of autophagosomes has particular physical properties that permit the interaction (and concentration) of specific compounds, like monodansyl cadaverine (MDC), a fluorescent polyamine used to label late autophagosomes/autolysosomes (49
). In a previous work, we have demonstrated that polyamines are required for T. cruzi
); thus, it is possible that other physiological polyamines (e.g., putrescine, spermidine, and spermine) could be concentrated in these compartments in the same way as MDC generating a more favorable environment to initiate Try differentiation.
Another interesting point to consider is that invading trypomastigotes cause a substantial mechanical lesion of the plasma membrane that should be rapidly repaired to maintain the internal electrolyte equilibrium and cell viability. Considering that autophagy is the main process involved in the clearance of damaged organelles, the rapid recruitment and accumulation of LC3 at the specific T. cruzi
entry sites and the subsequent fusion with autolysosomes could be interpreted as a mechanism to repair the recently produced injury. In this sense, as mentioned above the work from the group of Andrews (41
) revealed that Try wound the host cell membrane and trigger a Ca-dependent plasma membrane repair mechanism mediated by lysosomal exocytosis. Although the majority of the processes described in this report reinforce the lysosomal-dependent T. cruzi
entry model, none of them discard the participation of the autophagic pathway.
Another interesting consideration is that some pathogens enter phagocytic cells using mechanisms to avoid the activation of the respiratory burst. The lipophosphoglycan, the major surface glycoconjugate of Leishmania
promastigotes, has been reported to play an active role in protecting parasites within phagolysosomes via the impairment of killing mechanisms (51
). It could be possible that the generation of a vacuole with autophagosomal characteristics in macrophages () can help T. cruzi
Try to prevent the generation of NO and peroxynitrites that can kill them.
The new model presented here proposes that T. cruzi
exploits a previously induced autophagic pathway to efficiently colonize the host cell. Although T. cruzi
has the capacity to infect the majority of the cells, it was demonstrated that cardiac and smooth muscle myocytes are the more susceptible cells to this parasitic infection (52
). Indeed, heart or digestive lesions characterize the chronic stage of Chagas disease (54
). On the other hand, using mice models it was demonstrated that tissues like cardiac or smooth muscles activate autophagy 24–48 h after starvation and that contrary to organs like liver that quickly returns to the basal level, this response is sustained (55
). In these conditions, it could be possible that contact with T. cruzi
favors the rapid colonization of these target tissues increasing the probability to develop heart disease. We do not exactly know which is the specific role of autophagy in the natural human infection. However, epidemiological data show that only 30% of infected people present the chronic clinical manifestations. Although many factors could be participating in the development of chronic disease, one of the most important is the life conditions including the nutritional status of the infected people. It is tempting to speculate that a nutritional deficit produces in humans a strong induction of autophagy that could benefit parasite infection as well as the persistence in target organs and the generation of chronic disease. Although the connection between a low nutrition and the presence of infections may result obvious, to date the molecular aspect of this relationship has not been addressed.
Considering all the concepts and experimental data mentioned above and taking into account the remarkable versatility displayed by T. cruzi to infect cells, it is highly probable that the real paradigm will be a hybrid situation where some features of the proposed models of T. cruzi entry in nonphagocytic cells occur simultaneously. Moreover, if we consider that host cells have a different subset of molecular components depending of the cell type and the specific environment in which they are immersed, it is likely that T. cruzi may induce a specific mechanism by engaging the components available at that particular moment, and that in a different cell type or situation, the parasite will activate another set of cellular responses. Further experiments using powerful technologies available nowadays will be necessary to elucidate all the intrinsic aspects of T. cruzi–host cell interplay displayed during the establishment of the chagasic infection.