Gene deletion suggests a role for Trypanosoma Cruzi surface glycoprotein gp72 in the insect and mammalian stages of the life cycle

Abstract

We have explored the biological function of a surface glycoprotein (GP72) of Trypanosoma cruzi by studying a null mutant parasite, generated by targeted gene deletion. GP72 deletion affected parasite morphology in several stages of the life cycle. Insect midgut (epimastigote) forms had a detached flagellum (apomastigote) in the null mutant. The abnormal flagellar phenotype persisted during development of the infective (metacyclic) forms but there was no impairment in the acquisition of complement resistance, sialidase expression or cell infectivity. The GP72 null mutant could efficiently infect and proliferate in mouse macrophages and non-phagocytic L6E9 cells. The mammalian stages of the life cycle also showed major morphological abnormalities. During early subcultures in L6E9 cells, few extracellular fully flagellated forms, expressing markers characteristic of trypomastigotes, were seen. The extracellular population consisted almost exclusively of rounded forms with short flagella (micromastigote), which expressed an amastigote-specific surface marker and no sialidase. The propagation of the parasite was not affected, despite the apparent lack of the trypomastigote forms, which are thought to be primarily responsible for cell invasion. After some subcultures, the extracellular population changed to about equal numbers of micromastigotes and a range of flagellated forms that still did not include true trypomastigotes. Instead, the kinetoplast remained close to the nucleus and the flagellum emerged from the middle of the cell (mesomastigote). Half of the flagellum adhered to the cell body and the remainder was free at the anterior end. In Triatoma infestans, the survival of the mutant was dramatically reduced, suggesting that either GP72 itself, or the altered properties of the flagellum, were critical for establishment in the insect vector.

Comprehensive Proteomic Analysis of Trypanosoma cruzi Epimastigote Cell Surface Proteins by Two Complementary Methods

“…Little is reported about proteins mediating the interaction between T. cruzi epimastigotes and the insect hosts, but it has been observed that, on the anterior midgut surface, parasites were bound through the cell body or flagellum and, on the posterior midgut, only flagellar attachment to perimicrovillar membranes occurred . This attachment is presumably due to the involvement of glycoinositol phospholipid molecules. − When a gene encoding a surface glycoprotein was deleted in those epimastigotes, a significant decrease of the parasite population attached to the triatomine vector Rhodnius prolixus was observed. , This may suggest the involvement of glycoproteins that are concentrated in the flagellar attachment zone in the process. In our study we could identify several putative uncharacterized proteins that are annotated and described by Blast2GO software as flagellar associated proteins (SI Table 1), which leads us to believe that, by the cell surface trypsinization and biotinylation methodologies employed here to analyze the cell surface proteome, one can detect the proteins that are involved in the parasite–vector interaction.…”

“…41−43 When a gene encoding a surface glycoprotein was deleted in those epimastigotes, a significant decrease of the parasite population attached to the triatomine vector Rhodnius prolixus was observed. 44,45 This may suggest the involvement of glycoproteins that are concentrated in the flagellar attachment zone in the process. In our study we could identify several putative uncharacterized proteins that are annotated and described by Blast2GO software as flagellar associated proteins (SI Table 1), which leads us to believe that, by the cell surface trypsinization and biotinylation methodologies employed here to analyze the cell surface proteome, one can detect the proteins that are involved in the parasite−vector interaction.…”

Trypanosoma cruzi: Attenuation of virulence and protective immunogenicity after monoallelic disruption of the cub gene

“…cruzi have been obtained in previous studies after targeted deletion of apparently unrelated, single-copy genes (Caler et al, 1998;Manning-Cela et al, 2001;Ribeiro de Jesus et al, 1993;Basombrı ´o et al, 2002;Garzon et al, 2003).…”

Trypanin Disruption Affects the Motility and Infectivity of the Protozoan Trypanosoma cruzi

“…Whether or not motility is essential for T. cruzi cytokinesis might be further explored by deleting genes such as PF16 or another CMF with a severe motility defect (Baron et al, 2007). A previous work with gp72-KO is also informative (de Jesus et al, 1993). In this report, the authors show that gp72 (FLA-1 in T. brucei) mutants have detached flagella, and the epimastigote mutants are able to differentiate into infective metacyclic forms (reduced compared with WT).…”

“…The lack of gp72 caused detached flagella and reduced metacyclogenesis. Though they were able to infect host cells in culture, they were not able to generate bloodstream trypomastigotes; instead, only amastigote forms were released (de Jesus et al, 1993;Cooper et al, 1993). The multicopy genes PFR-1 and PFR-2 were edited using CRISPR/Cas9 technology, and the null mutant also showed a detached flagellum, and they were unable to build the paraflagellar rod structure as shown by transmission microscopy (Lander et al, 2015).…”