Surface-associated antigen induces permeabilization of primary mouse B-cells and lysosome exocytosis facilitating antigen uptake and presentation to T-cells.

B-cell receptor (BCR)-mediated antigen internalization and presentation are essential for humoral memory immune responses. Antigen encountered by B-cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. Internalization of such antigens requires myosin-mediated traction forces and extracellular release of lysosomal enzymes, but the mechanism triggering lysosomal exocytosis is unknown. Here, we show that BCR-mediated recognition of antigen tethered to beads, to planar lipid-bilayers or expressed on cell surfaces causes localized plasma membrane (PM) permeabilization, a process that requires BCR signaling and non-muscle myosin II activity. B-cell permeabilization triggers PM repair responses involving lysosomal exocytosis, and B-cells permeabilized by surface-associated antigen internalize more antigen than cells that remain intact. Higher affinity antigens cause more B-cell permeabilization and lysosomal exocytosis and are more efficiently presented to T-cells. Thus, PM permeabilization by surface-associated antigen triggers a lysosome-mediated B-cell resealing response, providing the extracellular hydrolases that facilitate antigen internalization and presentation.

Endophilin-A2-dependent tubular endocytosis promotes plasma membrane repair and parasite invasion.

Endocytosis of caveolae has previously been implicated in the repair of plasma membrane wounds. Here, we show that caveolin-1-deficient fibroblasts lacking caveolae upregulate a tubular endocytic pathway and have a reduced capacity to reseal after permeabilization with pore-forming toxins compared with wild-type cells. Silencing endophilin-A2 expression inhibited fission of endocytic tubules and further reduced plasma membrane repair in cells lacking caveolin-1, supporting a role for tubular endocytosis as an alternative pathway for the removal of membrane lesions. Endophilin-A2 was visualized in association with cholera toxin B-containing endosomes and was recruited to recently formed intracellular vacuoles containing Trypanosoma cruzi, a parasite that utilizes the plasma membrane wounding repair pathway to invade host cells. Endophilin-A2 deficiency inhibited T. cruzi invasion, and fibroblasts deficient in both caveolin-1 and endophilin-A2 did not survive prolonged exposure to the parasites. These findings reveal a novel crosstalk between caveolin-1 and endophilin-A2 in the regulation of clathrin-independent endocytosis and plasma membrane repair, a process that is subverted by T. cruzi parasites for cell invasion.

Ascorbate-Dependent Peroxidase (APX) from Leishmania amazonensis Is a Reactive Oxygen Species-Induced Essential Enzyme That Regulates Virulence.

The molecular mechanisms underlying biological differences between two Leishmania species that cause cutaneous disease, L. major and L. amazonensis, are poorly understood. In L. amazonensis, reactive oxygen species (ROS) signaling drives differentiation of nonvirulent promastigotes into forms capable of infecting host macrophages. Tight spatial and temporal regulation of H2O2 is key to this signaling mechanism, suggesting a role for ascorbate-dependent peroxidase (APX), which degrades mitochondrial H2O2 Earlier studies showed that APX-null L. major parasites are viable, accumulate higher levels of H2O2, generate a greater yield of infective metacyclic promastigotes, and have increased virulence. In contrast, we found that in L. amazonensis, the ROS-inducible APX is essential for survival of all life cycle stages. APX-null promastigotes could not be generated, and parasites carrying a single APX allele were impaired in their ability to infect macrophages and induce cutaneous lesions in mice. Similar to what was reported for L. major, APX depletion in L. amazonensis enhanced differentiation of metacyclic promastigotes and amastigotes, but the parasites failed to replicate after infecting macrophages. APX expression restored APX single-knockout infectivity, while expression of catalytically inactive APX drastically reduced virulence. APX overexpression in wild-type promastigotes reduced metacyclogenesis, but enhanced intracellular survival following macrophage infection or inoculation into mice. Collectively, our data support a role for APX-regulated mitochondrial H2O2 in promoting differentiation of virulent forms in both L. major and L. amazonensis Our results also uncover a unique requirement for APX-mediated control of ROS levels for survival and successful intracellular replication of L. amazonensis.

A MFS-like plasma membrane transporter required for Leishmania virulence protects the parasites from iron toxicity.

Iron is essential for many cellular processes, but can generate highly toxic hydroxyl radicals in the presence of oxygen. Therefore, intracellular iron accumulation must be tightly regulated, by balancing uptake with storage or export. Iron uptake in Leishmania is mediated by the coordinated action of two plasma membrane proteins, the ferric iron reductase LFR1 and the ferrous iron transporter LIT1. However, how these parasites regulate their cytosolic iron concentration to prevent toxicity remains unknown. Here we characterize Leishmania Iron Regulator 1 (LIR1), an iron responsive protein with similarity to membrane transporters of the major facilitator superfamily (MFS) and plant nodulin-like proteins. LIR1 localizes on the plasma membrane of L. amazonensis promastigotes and intracellular amastigotes. After heterologous expression in Arabidopsis thaliana, LIR1 decreases the iron content of leaves and worsens the chlorotic phenotype of plants lacking the iron importer IRT1. Consistent with a role in iron efflux, LIR1 deficiency does not affect iron uptake by L. amazonensis but significantly increases the amount of iron retained intracellularly in the parasites. LIR1 null parasites are more sensitive to iron toxicity and have drastically impaired infectivity, phenotypes that are reversed by LIR1 complementation. We conclude that LIR1 functions as a plasma membrane iron exporter with a critical role in maintaining iron homeostasis and promoting infectivity in L. amazonensis.

The gp82 surface molecule of Trypanosoma cruzi metacyclic forms.

Gp82 is a surface glycoprotein expressed in Trypanosoma cruzi metacyclic trypomastigotes, the parasite forms from the insect vector that initiate infection in the mammalian host. Studies with metacyclic forms generated in vitro, as counterparts of insect-borne parasites, have shown that gp82 plays an essential role in host cell invasion and in the establishment of infection by the oral route. Among the gp82 properties relevant for infection are the gastric mucin-binding capacity and the ability to induce the target cell signaling cascades that result in actin cytoskeleton disruption and lysosome exocytosis, events that facilitate parasite internalization. The gp82 sequences from genetically divergent T. cruzi strains are highly conserved, displaying >90 % identity. Both the host cell-binding sites, as well as the gastric mucin-binding sequence of gp82, are localized in the C-terminal domain of the molecule. In the gp82 structure model, the main cell-binding site consists of an α-helix, which connects the N-terminal ß-propeller domain to the C-terminal ß-sandwich domain, where the second cell binding site is nested. The two cell binding sites are fully exposed on gp82 surface. Downstream and close to the α-helix is the gp82 gastric mucin-binding site, which is partially exposed. All available data support the notion that gp82 is structurally suited for metacyclic trypomastigote invasion of host cells and for initiating infection by the oral route.

Cell signaling during Trypanosoma cruzi invasion.

Cell signaling is an essential requirement for mammalian cell invasion by Trypanosoma cruzi. Depending on the parasite strain and the parasite developmental form, distinct signaling pathways may be induced. In this short review, we focus on the data coming from studies with metacyclic trypomastigotes (MT) generated in vitro and tissue culture-derived trypomastigotes (TCT), used as counterparts of insect-borne and bloodstream parasites, respectively. During invasion of host cells by MT or TCT, intracellular Ca(2) (+) mobilization and host cell lysosomal exocytosis are triggered. Invasion mediated by MT surface molecule gp82 requires the activation of mammalian target of rapamycin (mTOR), phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC) in the host cell, associated with Ca(2) (+)-dependent disruption of the actin cytoskeleton. In MT, protein tyrosine kinase, PI3K, phospholipase C, and PKC appear to be activated. TCT invasion, on the other hand, does not rely on mTOR activation, rather on target cell PI3K, and may involve the host cell autophagy for parasite internalization. Enzymes, such as oligopeptidase B and the major T. cruzi cysteine proteinase cruzipain, have been shown to generate molecules that induce target cell Ca(2) (+) signal. In addition, TCT may trigger host cell responses mediated by transforming growth factor ß receptor or integrin family member. Further investigations are needed for a more complete and detailed picture of T. cruzi invasion.

Co-infection with Trypanosoma cruzi protects mice against early death by neurological or pulmonary disorders induced by Plasmodium berghei ANKA.

OBJECTIVE: The objective of this study was to investigate whether the infection of C57BL/6 mice by P. berghei ANKA, which causes severe malaria, was modulated by co-infection with Trypanosoma cruzi. METHODS: Groups of C57BL/6 mice were infected either with P. berghei ANKA, T. cruzi strain G, or with both parasites. The presence of parasites was checked by microscopic examination of blood samples. Symptoms of neurological or respiratory disorders, as well as mortality, were registered. Breakdown of the blood brain barrier was determined by injecting the dye Evans blue. Histological sections of the lung were prepared and stained with hematoxilin-eosin. RESULTS: All mice infected only with P. berghei ANKA died within 7-11 days post-infection, either with symptoms of cerebral malaria or with respiratory abnormalities. The animals co-infected with T. cruzi strain G survived longer, without any of the referred to symptoms. Protection against the early death by severe malaria was effective when mice were given T. cruzi 15 days before P. berghei inoculation. Breakdown of the blood brain barrier and extensive pulmonary oedema, caused by malaria parasites, were much less pronounced in co-infected mice. The degree of protection to severe malaria and early death, conferred by co-infection with T. cruzi, was comparable to that conferred by treatment with anti-CD8 antibodies. CONCLUSION: Co-infection with T. cruzi protects C57BL/6 against the early death by malaria infection, by partially preventing either the breakdown of the blood brain, and cerebral malaria as a consequence, or the pulmonary oedema.