Evaluation of high efficiency gene knockout strategies for Trypanosoma cruzi

<p>Abstract</p> <p>Background</p> <p><it>Trypanosoma cruzi</it>, a kinetoplastid protozoan parasite that causes Chagas disease, infects approximately 15 million people in Central and South America. In contrast to the substantial <it>in silico </it>studies of the <it>T. cruzi </it>genome, transcriptome, and proteome, only a few genes have been experimentally characterized and validated, mainly due to the lack of facile methods for gene manipulation needed for reverse genetic studies. Current strategies for gene disruption in <it>T. cruzi </it>are tedious and time consuming. In this study we have compared the conventional multi-step cloning technique with two knockout strategies that have been proven to work in other organisms, one-step-PCR- and Multisite Gateway-based systems.</p> <p>Results</p> <p>While the one-step-PCR strategy was found to be the fastest method for production of knockout constructs, it does not efficiently target genes of interest using gene-specific sequences of less than 80 nucleotides. Alternatively, the Multisite Gateway based approach is less time-consuming than conventional methods and is able to efficiently and reproducibly delete target genes.</p> <p>Conclusion</p> <p>Using the Multisite Gateway strategy, we have rapidly produced constructs that successfully produce specific gene deletions in epimastigotes of <it>T. cruzi</it>. This methodology should greatly facilitate reverse genetic studies in <it>T. cruzi</it>.</p

Effects of IFN-γ coding plasmid supplementation in the immune response and protection elicited by Trypanosoma cruzi attenuated parasites

Abstract Background Previous studies showed that a naturally attenuated strain from Trypanosoma cruzi triggers an immune response mainly related to a Th2-type profile. Albeit this, a strong protection against virulent challenge was obtained after priming mice with this attenuated strain. However, this protection is not enough to completely clear parasites from the host. In T. cruzi infection, early Interferon-gamma (IFN-γ) is critical to lead type 1 responses able to control intracellular parasites. Therefore we evaluated whether the co-administration of a plasmid encoding murine IFN-γ could modify the immune response induced by infection with attenuated parasites and improve protection against further infections. Methods C57BL/6J mice were infected intraperitoneally with three doses of live attenuated parasites in combination with plasmid pVXVR-mIFN-γ. Before each infection dose, sera samples were collected for parasite specific antibodies determination and cytokine quantification. To evaluate the recall response to T. cruzi, mice were challenged with virulent parasites 30 days after the last dose and parasite load in peripheral blood and heart was evaluated. Results As determined by ELISA, significantly increase in T. cruzi specific antibodies response was detected in the group in which pVXVR-mIFN-γ was incorporated, with a higher predominance of IgG2a subtype in comparison to the group of mice only inoculated with attenuated parasites. At our limit of detection, serum levels of IFN-γ were not detected, however a slight decrease in IL-10 concentrations was observed in groups in which pVXVR-mIFN-γ was supplemented. To analyze if the administration of pVXVR-mIFN-γ has any beneficial effect in protection against subsequent infections, all experimental groups were submitted to a lethal challenge with virulent bloodstream trypomastigotes. Similar levels of challenge parasites were detected in peripheral blood and heart of mice primed with attenuated parasites alone or combined with plasmid DNA. Expansion of IgG antibodies was not significant in TCC+ pVXVR-mIFN-γ; however, the overall tendency to sustain a Th2 profile was maintained. Conclusions Overall, these results suggest that administration of plasmid pVXVR-mIFN-γ could have beneficial effects on host specific antibody production in response to T. cruzi attenuated infection; however, this outcome is not reflected in an improved protection against further virulent infections

Additional file 1: Figure S1. of Effects of IFN-γ coding plasmid supplementation in the immune response and protection elicited by Trypanosoma cruzi attenuated parasites

pVXVR-mIFN-γ administration alters the parasite-specific immune response elicited by infection with attenuated parasites by the intramuscular and oral route. Mice (n = 4) were infected (4 weeks apart) with 3 doses of 105 metacyclic trypomatigotes of the attenuated TCC strain, 50 μg of plasmid pVXVR-mIFN-γ or a combination of both by the intramuscular (A-C) or oral (B-D) route. After each infection dose, serum samples were collected for (A-B) T. cruzi specific total IgGs levels and (C-D) serum levels of parasite specific IgG subtypes. (PDF 48 kb

Gene-deleted live-attenuated Trypanosoma cruzi parasites as vaccines to protect against Chagas disease

© Informa UK, Ltd. Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. This illness is now becoming global, mainly due to congenital transmission, and so far, there are no prophylactic or therapeutic vaccines available to either prevent or treat Chagas disease. Therefore, different approaches aimed at identifying new protective immunogens are urgently needed. Live vaccines are likely to be more efficient in inducing protection, but safety issues linked with their use have been raised. The development of improved protozoan genetic manipulation tools and genomic and biological information has helped to increase the safety of live vaccines. These advances have generated a renewed interest in the use of genetically attenuated parasites as vaccines against Chagas disease. This review discusses the protective capacity of genetically attenuated parasite vaccines and the challenges and perspectives for the development of an effective whole-parasi

Evaluation of pathogen P21 protein as a potential modulator of the protective immunity induced by Trypanosoma cruzi attenuated parasites

BACKGROUND TcP21 is a ubiquitous secreted protein of Trypanosoma cruzi and its recombinant form (rP21) promotes parasite cell invasion and acts as a phagocytosis inducer by activating actin polymerisation in the host cell. OBJECTIVE Our goal was to evaluate if the additional supplementation of rP21 during a prime/boost/challenge scheme with T. cruzi TCC attenuated parasites could modify the well-known protective behavior conferred by these parasites. METHODS The humoral immune response was evaluated through the assessment of total anti-T. cruzi antibodies as well as IgG subtypes. IFN-γ, TNF-α and IL-10 were measured in supernatants of splenic cells stimulated with total parasite homogenate or rP21. FINDINGS Our results demonstrated that, when comparing TCC+rP21 vs. TCC vaccinated animals, the levels of IFN-γ were significantly higher in the former group, while the levels of IL-10 and TNF-α were significantly lower. Further, the measurement of parasite load after lethal challenge showed an exacerbated infection and parasite load in heart and skeletal muscle after pre-treatment with rP21, suggesting the important role of this protein during parasite natural invasion process. MAIN CONCLUSION Our results demonstrated that rP21 may have adjuvant capacity able to modify the cytokine immune profile elicited by attenuated parasites

A monoallelic deletion of the TcCRT gene increases the attenuation of a cultured Trypanosoma cruzi strain, protecting against an in vivo virulent challenge.

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

Biological behavior of different Trypanosoma cruzi isolates circulating in an endemic area for Chagas disease in the Gran Chaco region of Argentina

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Infectivity and persistence of TcCRT mutants in Balb/c and nude mice.

<p>Infectivity in mice infected with 5×10⁵ metacyclic TcCRT+/–, wild type and TcCRT+ trypomastigote clones in Balb/c and nude mice, as detected by hemoculture and PCR. ND =  not done. Numbers in the third line refer to days p.i. (*) samples obtained after cyclophosphamide immunosuppression.</p

TcCRT+/– mutant parasites are stable after prolonged infection in mice.

<p>(A) Schematic representation of the TcCRT genomic locus in TcCRT+/– parasites (B) PCR analysis carried out from genomic DNA of TcCRT+/– and wild type parasites recovered from hemocultures of chronically infected mice. Primers: Pair CRT1-CRT2 and H1-H2 amplify the TcCRT (1.2 KB) and HYG CDS (0.96 kb), respectively. Pair CRT7-H4 amplifies the 5' UTR of TcCRT together with a fragment of the HYG CDS (1.45 kb). Pair CRT93-H6 amplifies the 3' UTR of TcCRT together with a fragment of the HYG CDS (1.4 kb). The sizes of the fragments correspond to those predicted for the replacement of TcCRT by the HYG gene.</p

TcCRT+/– immunization decreases tissue inflammatory response and spleen indexes in challenged mice.

<p>Autopsies were performed on mice 4 months post-priming and 2 months after virulent challenge. Dispersion diagrams of histopathological alterations in hearth muscle (A) and skeletal tissue (B). The inflammatory responses were graded as absent (–), slight (+), moderate (++), and severe (+++). Each dot represents a mouse. Representative images of H&E staining (blue: nuclear, pink: muscle/cytoplasm/keratin) of heart tissue (C and D) and skeletal muscle sections (E and F) from non-immunized and TcCRT+/– mice respectively (magnification, 25X). Li, Lymphocytic infiltrates. (G) Spleen indexes on day 60 post-challenge. Mice inoculated with TcCRT+/– parasites present lower spleen indexes compared with non-immunized controls (p = 0.02) and TcCRT+ parasites (p = 0.004).</p