Homology, paralogy and function of DGF-1, a highly dispersed Trypanosoma cruzi specific gene family and its implications for information entropy of its encoded proteins.

Surface adhesion proteins are essential for Trypanosoma cruzi invasion of mammalian cells. Here we show that Dispersed Gene Family-1 (DGF-1) members, previously identified as nuclear repeated sequences present in several chromosomes and comprising the third largest T. cruzi specific gene family, have conserved adhesin motifs including four segments with significant similarity to human beta 7 integrin. Flow cytometry and biotinylation assays with anti-DGF-1 antibodies indicated that, as expected, DGF-1 members are expressed on the trypomastigote surface. The DGF-1 genealogy, inferred using T. cruzi Genome Project data and network phylogeny algorithms, suggests that this gene family is separated in at least three groups with differential distribution of functional domains. To identify which members of this gene family are expressed we used a combined approach of RT-PCR and codon usage profiles, showing that expressed members have a very biased codon usage favoring GC, whereas non-expressed members have a homogeneous distribution. Shannon information entropy was used to measure sequence variability and revealed four major high entropy segments in the extracellular domain of DGF-1 overlapping with important putative functional modules of the predicted proteins. Testing for natural selection, however, indicated that these high entropy segments were not under positive selection, which contradicts the notion that positive selection is the cause of high variability in specific domains of a protein relative to other less variable regions in the same molecule. We conjectured that members of the DGF-1 family might be associated with the ability of T. cruzi to bind extracellular matrix proteins, such as fibronectin and laminin, and speculated on mechanisms that would be generating the localized diversity in these molecules in the absence of selection.

Differential infectivity by the oral route of Trypanosoma cruzi lineages derived from Y strain.

BACKGROUND: Diversity of T. cruzi strains is a central problem in Chagas disease research because of its correlation with the wide range of clinical manifestations and the biogeographical parasite distribution. The role played by parasite microdiversity in Chagas disease epidemiology is still debatable. Also awaits clarification whether such diversity is associated with the outcome of oral T. cruzi infection, responsible for frequent outbreaks of acute Chagas disease. METHODS AND FINDINGS: We addressed the impact of microdiversity in oral T. cruzi infection, by comparative analysis of two strains, Y30 and Y82, both derived from Y strain, a widely used experimental model. Network genealogies of four nuclear genes (SSU rDNA, actin, DHFR-TS, EF1α) revealed that Y30 is closely related to Discrete Typing Unit TcII while Y82 is more closely related to TcVI, a group containing hybrid strains. Nevertheless, excepting one A-G transition at position 1463, Y30 and Y82 SSU rDNAs were identical. Y82 strain, expressing the surface molecule gp82, infected mice orally more efficiently than Y30, which expresses a related gp30 molecule. Both molecules are involved in lysosome exocytosis-dependent host cell invasion, but exhibit differential gastric mucin-binding capacity, a property critical for parasite migration toward the gastric mucosal epithelium. Upon oral infection of mice, the number of Y30 and Y82 parasites in gastric epithelial cells differed widely. CONCLUSIONS: We conclude that metacyclic forms of gp82-expressing Y82 strain, closely related to TcVI, are better adapted than Y30 strain (TcII) to traverse the stomach mucous layer and establish oral route infection. The efficiency to infect target cell is the same because gp82 and gp30 strains have similar invasion-promoting properties. Unknown is whether differences in Y30 and Y82 are natural parasite adaptations or a product of lab-induced evolution by differential selection along the 60 years elapsed since the Y strain isolation.

Characterization of a 21kDa protein from Trypanosoma cruzi associated with mammalian cell invasion.

Trypanosoma cruzi genomic database was screened for hypothetical proteins that showed high probability of being secreted or membrane anchored and thus, likely involved in host-cell invasion. A sequence that codes for a 21kDa protein that showed high probability of being secreted was selected. After cloning this protein sequence, the results showed that it was a ubiquitous protein and secreted by extracellular amastigotes. The recombinant form (P21-His(6)) adhered to HeLa cells in a dose-dependent manner. Pretreatment of host cells with P21-His(6) inhibited cell invasion by extracellular amastigotes from G and CL strains. On the other hand, when the protein was added to host cells at the same time as amastigotes, an increase in cell invasion was observed. Host-cell pretreatment with P21-His(6) augmented invasion by metacyclic trypomastigotes. Moreover, polyclonal antibody anti-P21 inhibited invasion only by extracellular amastigotes and metacyclic trypomastigotes from G strain. These results suggested that P21 might be involved in T. cruzi cell invasion. We hypothesize that P21 could be secreted in the juxtaposition parasite-host cell and triggers signaling events yet unknown that lead to parasite internalization.

Experimental phylogeny of neutrally evolving DNA sequences generated by a bifurcate series of nested polymerase chain reactions.

A known phylogeny was generated using a four-step serial bifurcate PCR method. The ancestor sequence (SSU rDNA) evolved in vitro for 280 nested PCR cycles, and the resulting 15 ancestor and 16 terminal sequences (2,238 bp each) were determined. Parsimony, distance, and maximum likelihood analysis of the terminal sequences reconstructed the topology of the real phylogeny and branch lengths accurately. Divergence dates and ancestor sequences were estimated with very small error, particularly at the base of the phylogeny, mostly due to insertion and deletion changes. The substitution patterns along the known phylogeny are not described by reversible models, and accordingly, the probability substitution matrix, based on the observed substitutions from ancestor to terminal nodes along the known phylogeny, was calculated. This approach is an extension of previous studies using bacteriophage serial propagation, because here mutations were allowed to occur neutrally rather than by addition of a mutagenic agent, which produced biased mutational changes. These results provide for the first time biochemical experimental support for phylogenies, divergence date estimates, and an irreversible substitution model based on neutrally evolving DNA sequences. The substitution preferences observed here (A to G and T to C) are consistent with the high G+C content of the Thermus aquaticus genome. This suggests, at least in part, that the method here described, which explores the high Taq DNA polymerase error rate, simulates the evolution of a DNA segment in a thermophilic organism. These organisms include the bacterial rod T. aquaticus and several Archaea, and thus, the method and data set described here may well contribute new insights about the genome evolution of these organisms.