Escape from the lysosome
Once it has invaded the host cell, T. cruzi escapes from the phagolysosome to gain access to the cytoplasm, and this process is dependent on phagolysosome acidifica- tion.64 Lysis of the phagolysosome membrane is believed to be mediated by a parasite-derived secreted pore-forming molecule known as Tc-Tox, which works optimally at low pH.65,66 Tc-Tox cross-reacts with antibodies against complement C9 protein,66 and this property was used to screen an expression library and identify a T. cruzi gene (LYT1) coding for a hemolytic protein acting at low pH, which cross-reacts with anti-C9 antibodies.67 In addition, T. cruzi neuraminidase
Figure 26.2 Intracellular invasion by T. cruzi trypomastigotes. TOP. Trypomastigotes are highly motile and they attach to the surface of nonphagocytic mammalian cells (1a). The parasite causes increased intracellular Ca21 levels by triggering signaling cascades (1b), and by inducing plasma membrane lesions, through which extracellular Ca21 can flow into the cell (1c). Increased intracellular Ca21 concentrations facilitate parasite entry by inducing transient depolymerization of the cortical actin cytoskeleton (1d) and lysosome anterograde (toward the “ 1 ” end) translocation on microtubules to reach the parasite entry site in the cell periphery (1e).
BOTTOM. At the parasite entry site, lysosomes exocytose while sealing membrane wounds, and release acid sphingomyelinase, ASM (2a). ASM generates ceramide domains in the outer leaflet of the plasma membrane and favors its invagination, allowing parasites to enter the cell enveloped in a plasma membrane-derived vacuole, in most cases devoid of lysosomal markers (2b). Phagosomes, formed by the fusion of phagophores with lysosomes, may fuse to the nascent parasitophorous vacuole (2c), which may also fuse with endosomes and additional lysosomes as it matures (2d). The parasitophorous vacuole undergoes retrograde (away from the “plus” end) translocation on microtubules toward the perinuclear region (2e).
(trans-sialidase), which has also been shown to be active at low pH, has also been implicated in the disruption of parasitophorous vacuoles.68 Interestingly, coinmuno- precipitation experiments suggest that Tc-Tox/LYT1 protein is capable of physically interacting with trans-sialidase.69 However, direct proof of the proposed functions of these proteins in egress from the parasitophorous vacuole is still lacking.
Host cellular processes required for T. cruzi invasion
An RNAi-based genome-wide functional screen designed to identify mammalian cell genes and processes which support establishment of intracellular infection and
intracellular growth by T. cruzi was performed by Caradonna et al.70 The screen employed a multistep process, with an initial library of >25,000 siRNA pools which was assayed on an in vitro culture system using HeLa cells, to determine the effect of silencing individual genes over the T. cruzi intracellular infection process. The initial screen was followed by secondary screens with endpoints at 18 h and 72 h postinfection, corresponding to the prereplication phase of infection (i.e., invasion, <24 h after cell infection) and intracellular growth (i.e., amastigote multiplication, >24—90 h after cell infection). Although functional confirmation experiments performed in this study concentrated on host pathways which support the parasites intracellular growth (host metabolic networks and cellular signaling pathways were identified as important for this phase of the parasite life cycle), the screen did identify several dozen candidate host genes involved in the establishment of intracellular residence by T. cruzi. Genes that were found to affect parasite invasion included host cell signaling molecules, cytoskeletal proteins (CLASP1, cofilin-1), extracellular matrix proteins (laminin, collagen 1a), and genes involved in protein trafficking and organelle biosynthesis, among others. These findings are in line with what would be expected based on the knowledge regarding mammalian cell invasion described in previous sections of this chapter. The identification of CLASP1 as a protein required for parasite invasion in the RNAi knockdown screen was immediately followed by functional confirmation in subsequent studies,62 highlighting the validity of the use of unbiased screens to identify functional leads that can be subsequently confirmed and characterized in the pursuit of unveiling the details of the infection process by T. cruzi, and intracellular pathogens in general.