A Monoallelic Deletion of the TcCRT Gene Increases the Attenuation of a Cultured Trypanosoma cruzi Strain, Protecting against an In Vivo Virulent Challenge

Abstract

Trypanosoma cruzi calreticulin (TcCRT) is a virulence factor that binds complement C1, thus inhibiting the activation of the classical complement pathway and generating pro-phagocytic signals that increase parasite infectivity. In a previous work, we characterized a clonal cell line lacking one TcCRT allele (TcCRT+/−) and another overexpressing it (TcCRT+), both derived from the attenuated TCC T. cruzi strain. The TcCRT+/− mutant was highly susceptible to killing by the complement machinery and presented a remarkable reduced propagation and differentiation rate both in vitro and in vivo. In this report, we have extended these studies to assess, in a mouse model of disease, the virulence, immunogenicity and safety of the mutant as an experimental vaccine. Balb/c mice were inoculated with TcCRT+/− parasites and followed-up during a 6-month period. Mutant parasites were not detected by sensitive techniques, even after mice immune suppression. Total anti-T. cruzi IgG levels were undetectable in TcCRT+/− inoculated mice and the genetic alteration was stable after long-term infection and it did not revert back to wild type form. Most importantly, immunization with TcCRT+/− parasites induces a highly protective response after challenge with a virulent T. cruzi strain, as evidenced by lower parasite density, mortality, spleen index and tissue inflammatory response. TcCRT+/− clones are restricted in two important properties conferred by TcCRT and indirectly by C1q: their ability to evade the host immune response and their virulence. Therefore, deletion of one copy of the TcCRT gene in the attenuated TCC strain generated a safe and irreversibly gene-deleted live attenuated parasite with high immunoprotective properties. Our results also contribute to endorse the important role of TcCRT as a T. cruzi virulence factor.

Gene-deleted live-attenuatedTrypanosoma cruziparasites as vaccines to protect against Chagas disease

“…This study showed that the mutants not only retained the deletion of one allele but also remained in the original locus. Thus, the mutation was stable, without demonstrable reversion to the wild-type genotype, even after longterm infection cycle in the mammalian host [145]. Similar results using this approach were obtained by Zago et al for lyt1 mutants [131] and by Basombrío et al [129] for a gp72 mutant.…”

“…Necropsies at day 60 post infection revealed that mice immunized with mutant parasites showed reduced inflammatory response in heart and muscle tissues, compared to controls. Also, the spleen index was significantly reduced in mutant and wild-type TCC immunized mice [145]. Taken together, these results showed that TcCRT+/À parasites were limited in two major properties conferred by TcCRT and indirectly by C1q: the capacity to evade the host immune response and their virulence status.…”

“…Moreover, the concentration of parasites in feces of each group was different [124]. The TcCRT+/À impaired differentiation in insect vectors, together with the undetectable parasitemia in the mouse blood system could contribute to the reduction of the strain transmission [145]. Interestingly, the differentiation capacity was also evaluated for gp72 mutant parasites, and the same evidence was obtained for this gene.…”

Trypanosoma cruzi Evades the Complement System as an Efficient Strategy to Survive in the Mammalian Host: The Specific Roles of Host/Parasite Molecules and Trypanosoma cruzi Calreticulin

“…Furthermore, parasites carrying a monoallelic deletion of the TcCRT gene, are significantly susceptible to complement-mediated lysis. As expected, parasites overexpressing TcCRT are significantly more resistant to the complement-mediated lysis by CP and LP ( Sanchez-Valdez et al, 2013 , 2014 ).…”

Parasite Infection, Carcinogenesis and Human Malignancy

“…In addition, TcCRT plays a central role during the host-parasite interplay by interacting with complement proteins such as complement factor ( C 1), mannose-binding lectin (MBL), and ficolins to inhibit activation of the complement system that leads to increased infectivity of the parasite (Lopez et al, 2010, Ramirez et al, 2011b, Sosoniuk et al, 2014). This indicates that TcCRT does not only act as a virulence factor (Sanchez-Valdez et al, 2014) to persist, sustain and promote infectivity but also inhibits growth and metastasis of tumors. A further study has demonstrated that some T. cruzi strains are able to respond to oxidative stress caused by DNA damaging agents (Campos et al, 2011, Grazielle-Silva et al, 2015).…”