Proteome-wide analysis reveals molecular pathways affected by AgNP in a ROS-dependent manner.

The use of mass spectrometry-based proteomics has been increasingly applied in nanomaterials risk assessments as it provides a proteome-wide overview of the molecular disturbances induced by its exposure. Here, we used this technique to gain detailed molecular insights into the role of ROS as an effector of AgNP toxicity, by incubating Bend3 cells with AgNP in the absence or presence of an antioxidant N-acetyl L-cystein (NAC). ROS generation is a key player in AgNP-induced toxicity, as cellular homeostasis was kept in the presence of NAC. By integrating MS/MS data with bioinformatics tools, in the absence of NAC, we were able to pinpoint precisely which biological pathways were affected by AgNP. Cells respond to AgNP-induced ROS generation by increasing their antioxidant pool, via NRF2 pathway activation. Additionally, cell proliferation-related pathways were strongly inhibited in a ROS-dependent manner. These findings reveal important aspects of the AgNP mechanism of action at the protein level.

Biological and Molecular Effects of Trypanosoma cruzi Residence in a LAMP-Deficient Intracellular Environment.

Trypanosoma cruzi invades non-professional phagocytic cells by subverting their membrane repair process, which is dependent on membrane injury and cell signaling, intracellular calcium increase, and lysosome recruitment. Cells lacking lysosome-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) are less permissive to parasite invasion but more prone to parasite intracellular multiplication. Several passages through a different intracellular environment can significantly change T. cruzi's gene expression profile. Here, we evaluated whether one single passage through LAMP-deficient (KO) or wild-type (WT) fibroblasts, thus different intracellular environments, could influence T. cruzi Y strain trypomastigotes' ability to invade L6 myoblasts and WT fibroblasts host cells. Parasites released from LAMP2 KO cells (TcY-L2-/-) showed higher invasion, calcium signaling, and membrane injury rates, for the assays in L6 myoblasts, when compared to those released from WT (TcY-WT) or LAMP1/2 KO cells (TcY-L1/2-/-). On the other hand, TcY-L1/2-/- showed higher invasion, calcium signaling, and cell membrane injury rates, for the assays in WT fibroblasts, compared to TcY-WT and TcY-L1/2-/-. Albeit TcY-WT presented an intermediary invasion and calcium signaling rates, compared to the others, in WT fibroblasts, they induced lower levels of injury, which reinforces that signals mediated by surface membrane protein interactions also have a significant contribution to trigger host cell calcium signals. These results clearly show that parasites released from WT or LAMP KO cells are distinct from each other. Additionally, these parasites' ability to invade the cell may be distinct depending on which cell type they interact with. Since these alterations most likely would reflect differences among parasite surface molecules, we also evaluated their proteome. We identified few protein complexes, membrane, and secreted proteins regulated in our dataset. Among those are some members of MASP, mucins, trans-sialidases, and gp63 proteins family, which are known to play an important role during parasite infection and could correlate to TcY-WT, TcY-L1/2-/-, and TcY-L2-/- biological behavior.

Neuronal Parasitism, Early Myenteric Neurons Depopulation and Continuous Axonal Networking Damage as Underlying Mechanisms of the Experimental Intestinal Chagas' Disease.

There is a growing consensus that the balance between the persistence of infection and the host immune response is crucial for chronification of Chagas heart disease. Extrapolation for chagasic megacolon is hampered because research in humans and animal models that reproduce intestinal pathology is lacking. The parasite-host relationship and its consequence to the disease are not well-known. Our model describes the temporal changes in the mice intestine wall throughout the infection, parasitism, and the development of megacolon. It also presents the consequence of the infection of primary myenteric neurons in culture with Trypanosoma cruzi (T. cruzi). Oxidative neuronal damage, involving reactive nitrogen species induced by parasite infection and cytokine production, results in the denervation of the myenteric ganglia in the acute phase. The long-term inflammation induced by the parasite's DNA causes intramuscular axonal damage, smooth muscle hypertrophy, and inconsistent innervation, affecting contractility. Acute phase neuronal loss may be irreversible. However, the dynamics of the damages revealed herein indicate that neuroprotection interventions in acute and chronic phases may help to eradicate the parasite and control the inflammatory-induced increase of the intestinal wall thickness and axonal loss. Our model is a powerful approach to integrate the acute and chronic events triggered by T. cruzi, leading to megacolon.

The Influence of Recombinational Processes to Induce Dormancy in Trypanosoma cruzi.

The protozoan Trypanosoma cruzi is the causative agent of Chagas disease, a neglected tropical disease that affects around 8 million people worldwide. Chagas disease can be divided into two stages: an acute stage with high parasitemia followed by a low parasitemia chronic stage. Recently, the importance of dormancy concerning drug resistance in T. cruzi amastigotes has been shown. Here, we quantify the percentage of dormant parasites from different T. cruzi DTUs during their replicative epimastigote and amastigote stages. For this study, cells of T. cruzi CL Brener (DTU TcVI); Bug (DTU TcV); Y (DTU TcII); and Dm28c (DTU TcI) were used. In order to determine the proliferation rate and percentage of dormancy in epimastigotes, fluorescent-labeled cells were collected every 24 h for flow cytometer analysis, and cells showing maximum fluorescence after 144 h of growth were considered dormant. For the quantification of dormant amastigotes, fluorescent-labeled trypomastigotes were used for infection of LLC-MK2 cells. The number of amastigotes per infected LLC-MK2 cell was determined, and those parasites that presented fluorescent staining after 96 h of infection were considered dormant. A higher number of dormant cells was observed in hybrid strains when compared to non-hybrid strains for both epimastigote and amastigote forms. In order to investigate, the involvement of homologous recombination in the determination of dormancy in T. cruzi, we treated CL Brener cells with gamma radiation, which generates DNA lesions repaired by this process. Interestingly, the dormancy percentage was increased in gamma-irradiated cells. Since, we have previously shown that naturally-occurring hybrid T. cruzi strains present higher transcription of RAD51-a key gene in recombination process -we also measured the percentage of dormant cells from T. cruzi clone CL Brener harboring single knockout for RAD51. Our results showed a significative reduction of dormant cells in this T. cruzi CL Brener RAD51 mutant, evidencing a role of homologous recombination in the process of dormancy in this parasite. Altogether, our data suggest the existence of an adaptive difference between T. cruzi strains to generate dormant cells, and that homologous recombination may be important for dormancy in this parasite.

The MASP family of Trypanosoma cruzi: changes in gene expression and antigenic profile during the acute phase of experimental infection.

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host-parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms. METHODS: By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice. FINDINGS AND CONCLUSIONS: We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.