Local association of Trypanosoma cruzi chronic infection foci and enteric neuropathic lesions at the tissue micro-domain scale.

Digestive Chagas disease (DCD) is an enteric neuropathy caused by Trypanosoma cruzi infection. The mechanism of pathogenesis is poorly understood and the lack of a robust, predictive animal model has held back research. We screened a series of mouse models using gastrointestinal tracer assays and in vivo infection imaging systems to discover a subset exhibiting chronic digestive transit dysfunction and significant retention of faeces in both sated and fasted conditions. The colon was a specific site of both tissue parasite persistence, delayed transit and dramatic loss of myenteric neurons as revealed by whole-mount immunofluorescence analysis. DCD mice therefore recapitulated key clinical manifestations of human disease. We also exploited dual reporter transgenic parasites to home in on locations of rare chronic infection foci in the colon by ex vivo bioluminescence imaging and then used fluorescence imaging in tissue microdomains to reveal co-localisation of infection and enteric nervous system lesions. This indicates that long-term T. cruzi-host interactions in the colon drive DCD pathogenesis, suggesting that the efficacy of anti-parasitic chemotherapy against chronic disease progression warrants further pre-clinical investigation.

Bioluminescent:Fluorescent Trypanosoma cruzi Reporter Strains as Tools for Exploring Chagas Disease Pathogenesis and Drug Activity.

Chagas disease results from infection with the trypanosomatid parasite Trypanosoma cruzi. Progress in developing new drugs has been hampered by the long term and complex nature of the condition and by our limited understanding of parasite biology. Technical difficulties in assessing the parasite burden during the chronic stage of infection have also proven to be a particular challenge. In this context, the development of noninvasive, highly sensitive bioluminescence imaging procedures based on parasites that express a red-shifted luciferase has greatly enhanced our ability to monitor infections in experimental models. Applications of this methodology have led to new insights into tissue tropism and infection dynamics and have been a major driver in drug development. The system has been further modified by the generation of parasite reporter lines that express bioluminescent:fluorescent fusion proteins, an advancement that has allowed chronic infections in mice to be examined at a cellular level. By exploiting bioluminescence, to identify the rare sites of tissue infection, and fluorescence to detect T. cruzi at the level of individual host cells in histological sections, it has been possible to investigate the replication and differentiation status of parasites in vivo and to examine the cellular environment of infection foci. In combination, these data provide a framework for the detailed dissection of disease pathogenesis and drug activity.

Optimising genetic transformation of Trypanosoma cruzi using hydroxyurea-induced cell-cycle synchronisation.

The limited flexibility and time-consuming nature of the genetic manipulation procedures applicable to Trypanosoma cruzi continue to restrict the functional dissection of this parasite. We hypothesised that transformation efficiency could be enhanced if electroporation was timed to coincide with DNA replication. To test this, we generated epimastigote cultures enriched at the G1/S boundary using hydroxyurea-induced cell-cycle synchronisation, and then electroporated parasites at various time points after release from the cell-cycle block. We found a significant increase in transformation efficiency, with both episomal and integrative constructs, when cultures were electroporated 1 h after hydroxyurea removal. It was possible to generate genetically modified populations in less than 2 weeks, compared to the normal 4-6 weeks, with a 5 to 8-fold increase in the number of stably transformed clones. This straightforward optimisation step can be widely applied and should help streamline functional studies in T. cruzi.

Human Regulatory Protein Ki-1/57 Is a Target of SUMOylation and Affects PML Nuclear Body Formation.

Ki-1/57 is a nuclear and cytoplasmic regulatory protein first identified in malignant cells from Hodgkin's lymphoma. It is involved in gene expression regulation on both transcriptional and mRNA metabolism levels. Ki-1/57 belongs to the family of intrinsically unstructured proteins and undergoes phosphorylation by PKC and methylation by PRMT1. Previous characterization of its protein interaction profile by yeast two-hybrid screening showed that Ki-1/57 interacts with proteins of the SUMOylation machinery, the SUMO E2 conjugating enzyme UBC9 and the SUMO E3 ligase PIAS3, which suggested that Ki-1/57 could be involved with this process. Here we identified seven potential SUMO target sites (lysine residues) on Ki-1/57 sequence and observed that Ki-1/57 is modified by SUMO proteins in vitro and in vivo. We showed that SUMOylation of Ki-1/57 occurred on lysines 213, 276, and 336. In transfected cells expressing FLAG-Ki-1/57 wild-type, its paralog FLAG-CGI-55 wild-type, or their non-SUMOylated triple mutants, the number of PML-nuclear bodies (PML-NBs) is reduced compared with the control cells not expressing the constructs. More interestingly, after treating cells with arsenic trioxide (As2O3), the number of PML-NBs is no longer reduced when the non-SUMOylated triple mutant Ki-1/57 is expressed, suggesting that the SUMOylation of Ki-1/57 has a role in the control of As2O3-induced PML-NB formation. A proteome-wide analysis of Ki-1/57 partners in the presence of either SUMO-1 or SUMO-2 suggests that the involvement of Ki-1/57 with the regulation of gene expression is independent of the presence of either SUMO-1 or SUMO-2; however, the presence of SUMO-1 strongly influences the interaction of Ki-1/57 with proteins associated with cellular metabolism, maintenance, and cell cycle.

Ki-1/57 and CGI-55 ectopic expression impact cellular pathways involved in proliferation and stress response regulation.

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.