Novel PI 3-kinase-dependent mechanisms of trypanosome invasion and vacuole maturation

Abstract

Mammalian cell invasion by the protozoan parasite, Trypanosoma cruzi, is facilitated by the activation of host cell phosphatidylinositol 3 (PI 3)-kinases. We demonstrate that the well-characterized Ca2+-regulated lysosome-mediated parasite entry pathway is abolished by wortmannin pretreatment. In addition, we have characterized a novel route of T. cruzi invasion unexpectedly revealed in the course of this study. For over a decade, targeted exocytosis of lysosomes at the host cell plasma membrane was considered as the primary mechanism for T. cruzi entry into non-professional phagocytic cells. We now provide evidence that a significant fraction (50% or greater) of invading T. cruzi trypomastigotes exploit an alternate actin-independent entry pathway that involves formation of a tightly associated host cell plasma membrane-derived vacuole enriched in the lipid products of class I PI 3-kinases, PtdInsP3/PtdIns(3,4)P2. Initially devoid of lysosomal markers, the resultant parasite-containing vacuoles gradually acquire lysosome associated membrane protein 1 (lamp-1) and fluid phase endocytic tracer from the lysosomal compartment. In striking contrast to latex bead phagosomes, few T. cruzi vacuoles associate with the early endosomal marker, EEA1 and the 'maturation' process becomes refractory to PI 3-kinase inhibition immediately following parasite internalization. Jointly, these data provide a new paradigm for T. cruzi invasion of non-professional phagocytic cells and reveal a novel vacuole maturation process that appears to bypass the requirement for EEA1.

The autophagic pathway is a key component in the lysosomal dependent entry ofTrypanosoma cruziinto the host cell

“…3,9 A more recent work shows the participation of class I of PI3K in the formation of an early plasma membrane derived vesicle where the parasite resides prior lysosomal fusion. 8 Therefore, we next asked about the by incubating TCT in acidic pH, therefore D-MEM stabilized (buffered) at pH = 5 was used as a positive control medium to induce differentiation. 38 Furthermore, D-MEM pH = 7 was used as control for normal nutritional conditions.…”

“…To date, two main invasion process involving different signaling pathways have been described: the calcium-dependent fusion of lysosomes with the host plasma membrane, 6,7 and the activation of class I of PI3-kinases that generates a plasma membrane derived vacuole initially devoided of lysosomal markers. 8 Although these two ways require the disruption of the host cell actin cytoskeleton, the lysosomal independent T. cruzi entry model appears to be more significant early after internalization (50% of intracellular parasites colocalize with plasma membrane markers against 20% that acquire lysosomal components). 8 However, the lysosomal fusion step is essential for the establishment of a productive infection, 9 and for progression and completion of T. cruzi intracellular cycle, 10,11 since the parasite Tc-Tox, a pore-forming toxin produced by T. cruzi, is activated in the acid environment provided by the lysosomes.…”

“…8 Although these two ways require the disruption of the host cell actin cytoskeleton, the lysosomal independent T. cruzi entry model appears to be more significant early after internalization (50% of intracellular parasites colocalize with plasma membrane markers against 20% that acquire lysosomal components). 8 However, the lysosomal fusion step is essential for the establishment of a productive infection, 9 and for progression and completion of T. cruzi intracellular cycle, 10,11 since the parasite Tc-Tox, a pore-forming toxin produced by T. cruzi, is activated in the acid environment provided by the lysosomes. 12 Furthermore, recent work shows that MT and extracellular amastigotes require cholesterol and the ganglioside GM1 from membrane rafts to invade nonphagocytic cells.…”

Tumour necrosis factor (TNF)-mediated NF-κB activation facilitates cellular invasion of non-professional phagocytic epithelial cell lines by Trypanosoma cruzi

“…The increased cytosolic Ca 2+ levels induce the recruitment of lysosomes to the site of T. cruzi adhesion and its subsequent internalization (reviewed by Andrade and Andrews, 2005). An alternative pathway has been proposed by Woolsey and co-workers in 2003, which leads to the enrichment of cell membrane with lipids produced by PI3K (phosphoinositide 3-kinase) as one step for cell invasion, where fusion with lysosomes would occur after parasite invasion (Woolsey et al, 2003).…”

Critical role for phosphoinositide 3-kinase gamma in parasite invasion and disease progression of cutaneous leishmaniasis

“…Neutrophils from PI3Kγ −/− mice display impaired activation of Rac and reduced Factin accumulation at the leading edge, which correlate with their reduced ability to migrate in response to chemotactic stimuli (10,11). Studies using PI3K inhibitors, such as wortmannin or LY294002, show that type I PI3Ks are involved in phagocytosis (12)(13)(14)(15) and mediate the entry of parasites, such as Trypanosoma cruzi, into host cells (16)(17)(18). The precise role of each isoform, however, remains unclear.…”