Chagas’ disease has become a progressive emerging disease in the United States. In South and Central America, the disease may affect over 18 million people. Typically, the causative agent, Trypanosomacruzi, may not immediately kill the host, but by its definition, the parasite keeps the host in check, in order to maximize transmission and therefore, prolong its life cycle. The mammalian host can gradually develop illness from an acute or mild form of the disease to a latent stage, to a chronic stage. There is significant mortality due to the Acute and Chronic stages of this disease1. In particular, Chronic Chagas Cardiomyopathy (CCC) is thought to be caused by an autoimmune attack upon nerve and/or heart tissue.
Recognition of this disease as a potential threat to the US blood supply was in 1992 when several biopharmaceutical companies developed diagnostic tests to identify this disease2. In 1995 the FDA accepted a 510(k) diagnostic test for Chagas’ disease3,4. Progressive improvements to identify several highly antigenic T. cruzi molecules reactive with human chagasic antiserum were made in subsequent years (2006-2011); the American Association of Blood Banks (AABB), the FDA, CDC, and WHO have now recognized this disease as one of the 13 donor screening assays for Infectious Agents.
Presently there are no vaccines against Chagas’ disease and therapeutic agents (benznidazole) have only recently (2017) been FDA approved for use in children ages 2 to 12 years old with Chagas’ disease. Side effects are common, frequent, and severe with increasing age. In this regard, alternative methods for treatment are necessary. Our primary focus has been to search and identify highly antigenic T. cruzi molecules reactive with human chagasic antiserum2,3,5,6. To this end, one particular cloned gene from T. cruzi amastigotes was sequenced and found to be identical to the repetitive antigen Clone 36 ("Antigen 36")7.
Search of GenBank using our DNA sequence translated by the TFASTA program revealed a high degree of homology of Human Ro52 with the translated sequence in the second reading frame of Ag 36. Direct comparison of the Ag 36 DNA sequence with Ro52 DNA sequence revealed a significant homology of the Ag 36 DNA sequence to TRIM21, the gene for human Ro528. The TRIM21 (tripartite motif containing-21) gene protein product Ro52 was subsequently shown to be an E3 Ubiquitin ligase that modifies transcription factors for alpha and beta interferons and other cytokines9. Knock-out of Ro2 in mice strains showed that they were susceptible to tissue inflammation and systemic autoimmunity after injury induced by skin tagging10. However, the TRIM21 protein product Ro52 also can stimulate innate immunity, for example, in macrophages. There it acts in a non-degradation pathway of ubiquitination, as an E3 ligase for IRF-8 that upregulates cytokines such as IL-12/p40, and therefore contributes to innate immunity in macrophages11. TRIM21-deficient bone marrow-derived macrophages showed a reduced response to Toll-like receptor agonist Bacillus Calmette-Guerin12.
TRIM21 is also an antibody receptor and cytosolic ubiquitin ligase that provides a line of defense against invading viruses by acting as a sensor that intercepts antibody-coated viruses that have evaded extracellular neutralization and breached the cell membrane13-15. After interaction with the Fc of antibodies bound to the virus, the TRIM21 receptor triggers a coordinated effector and signaling response that prevents viral replication while at the same time inducing an anti-viral cellular state16. This dual effector function is tightly regulated by auto-ubiquitination and phosphorylation. In addition, TRIM21 has been studied extensively with antibody receptors bound to viruses but minimally with parasites16. We proposed that the partial gene homology between TRIM21 and Ag 36 may cause RNA interference, mRNA silencing, or other down-regulation of Ro52, leading to the autoimmunity of Chronic Chagas Cardiomyopathy (CCC)17. We also suggested that the interference could be beneficial for the parasite, during its invasion of macrophages by blocking TRIM21 stimulation of innate immunity in these phagocytic cells. In this work, we extend the comparison of DNA sequences of Ag 36 to eighteen additional mammalian TRIM21 sequences, using online bioinformatics tools to determine significant partial homologies and to test whether the homology is associated with CCC.