Impairment of Infectivity and Immunoprotective Effect of a LYT1 Null Mutant of Trypanosoma cruzi

Abstract

Trypanosoma cruzi is the causative agent of Chagas' disease and infects an estimated 18 million people in the American continents (27). This protozoan parasite is transmitted by three main routes: via the feces of infected triatomine insects, via blood transfusions, and from mother to fetus during pregnancy. Parasites persist through the entire life of the patient, and the disease normally progresses from an acute stage characterized only by systemic parasitemia to a virtually asymptomatic stage that can last for several decades. Eventually, onethird of chronically infected patients manifest symptoms of Chagas' disease, developing severe cardiac and/or gastrointestinal disease, which leads to death in most cases. Nowadays there is neither an effective cure nor a vaccine against T. cruzi infection, and the only methods for combating the disease are the control of the vector in areas of endemicity and the prevention of transmission via blood supply.The invasion of host cells by T. cruzi involves several steps: attachment of the parasite to the cell surface, internalization mediated by the recruitment and fusion of host cell lysosomes, and escape of the parasite from the parasitophorous vacuole to multiply freely in the cytosol as amastigotes (2,3,26). Though many parasite proteins are very important for T. cruzi infection, surprisingly, only a few have been identified experimentally. One parasite factor likely involved in the infection is the protein product of the LYT1 gene. Manning-Cela et al. (22) isolated a LYT1 cDNA clone from a Y strain T. cruzi amastigote library by virtue of its cross-reactivity to antibodies against the human complement component C9. On this basis, the researchers proposed that the 552-amino-acid protein encoded by the LYT1 open reading frame could be involved in a lytic pathway, mediating the escape of T. cruzi from the acidic parasitophorous vacuole into the cytosol, as was previously shown for the T. cruzi protein Tc-TOX with pore-forming activity at a low pH (4). However, a bioinformatics analysis of all available DNA and protein databases failed to identify paralogs. To gain insight into the possible role of the LYT1 gene product, the researchers generated a biallelic, LYT1 deletion in the CL strain and the biological characteristics of a mutant clone (clone L16) were assessed. The deletion of both alleles did not impair the capacity of epimastigote parasites to proliferate in axenic cultures. Nevertheless, the LYT1 null parasites exhibited reduced infectivity in cell culture experiments and also displayed accelerated in vitro-stage transition and diminished hemolytic activity at an acidic pH (23). These distinct phenotypes were attributed to the fact that different forms of the protein are expressed as a result of alternative trans splicing. Through this genetic regulation process of the primary transcript, two LYT1 protein derivates were obtained, differing only in the presence or absence of the first 28 amino acids. The trans-splicing processing was developmentally regulated, sinc...

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“…While the molecular identity of TcTOX remains unknown, TcLYT1, a protein with similar characteristics and suspected to be identical to TcTOX, appears to contribute to the hemolytic activity. A TcLYT1 Ϫ/Ϫ strain showed diminished infectivity in cell cultures (93) and attenuated virulence in the mouse model (159). The vacuolar escape of T. cruzi may depend on the differentiation of metacyclic trypomastigotes into amastigotes (93,99), as hemolytic activity has been detected for only some developmental stages.…”