Functional complementation of a yeast knockout strain by Schistosoma mansoni Rho1 GTPase in the presence of caffeine, an agent that affects mutants defective in the protein kinase C signal transduction pathway

Abstract

In a previous study, the Schistosoma The digenetic trematode, Schistosoma mansoni, is the causative agent of schistosomiasis, a disease that is still one of the most prevalent parasitic infections around the world (Engels et al. 2002). The understanding of parasite biology, mechanisms of drug resistance and antigenic variation that determine escape from the host immune system are the main reasons for studying parasites genomes. Therefore, S. mansoni transcriptome projects have contributed to the discovery of thousands of new genes in this parasite (Verjovski-Almeida et al. 2004 The retention of S. mansoni eggs in the liver triggers inflammatory reactions that eventually may lead to liver fibrosis, the most serious pathological lesion of the disease (Dunne et al. 1995). A possible way to prevent these symptoms could be to inhibit egg laying. Several lines of evidence suggest the involvement of low-molecular weight GTP-binding proteins (LMWGPs) of the Ras su- perfamily in the maturation process and egg production of S. mansoni female worms (Schussler et al. 1997, VandeWaa et al. 1989). Thus, this superfamily of LMWGPs might represent an interesting target for the development of new anti-schistosomiasis drugs.The Rho GTPase gene (SmRho1) from S. mansoni, encodes a 193 amino acids protein that is highly homologous to the Rho-type l LMWGPs of several species, has been previously characterized by Santos et al. (2002). The SmRho1 gene was able to complement a S. cerevisiae Rho1 null mutant strain even under temperature (37ºC) and osmotic (300 mM CaCl 2 ) stress conditions, contrasting with the human RhoA GTPase that was not able to provide complementation in such conditions (Qadota et al. 1994). The α3-helix loop7 had been previously defined by Qadota et al. (1994) as the region of the human RHOA protein responsible for its inability to complement yeast rho1 null mutants at the conditions described above. After comparison of this region of the amino acid sequences from S. cerevisiae Rho1 (ScRho1), Candida albicans Rho1, human RhoA, and SmRho1, Santos et al. (2002) identified the proline 96 and threonine 100 residues of the human RhoA as the most probable determinants of the complementation differences between the human and worm genes. Three SmRho1 point mutants (smrho1 E97P , smrho1 L101T , and smrho1 E97P, L101T ) were generated by site directed mutagenesis and the conditional lethality phenotype at high temperature was reproduced in all mutants. This confirmed the importance of proline 96 and threonine 100 for the temperature-sensitive phenotype seen in 324 324 324 324 324The SmRho1 protein from S. mansoni • Pedro HN de Aguiar et al.yeast Rho1 null mutant complemented by the human RhoA GTPase, and provided strong evidence that the related amino acid positions (Gln101 and Ile105) in the ScRho1 GTPase are indeed important for regulation of the cell wall synthesis performed by this protein in yeast. The ScRho1 protein participates in two classic pathways to perform its cellular functions in response to osmotic stress...

Functional Expression of Parasite Drug Targets and Their Human Orthologs in Yeast

“…By performing genome-wide drug sensitivity screens (chemogenomic profiling) [12], [13], [14], [15], [16], [17] of yeast mutants with the antimalarials quinine [18], St. John's Wort [19] and artemisinin [20], researchers were able to identify their primary targets as well as identify potential side effects. Furthermore, several groups have been able to complement yeast loss-of function mutations by expressing coding sequences from parasites such as Plasmodium [21], [22], Schistosoma [23], [24], [25], Leishmania [26] or Trypanosoma [6], [27], [28], [29], [30], [31].…”

Cloning and in vitro characterization of a Schistosoma japonicum aquaglyceroporin that functions in osmoregulation

“…4 ). This differs from Sm AQP, which shows peak expression in adults 32 S. japonicum infection occurs when cercariae are released by snails into fresh water, and then cercariae penetrate human skin to achieve invasion 43 . Our finding of peak Sj AQP expression in cercariae suggests that Sj AQP probably protects parasites from osmolality changes when moving from fresh water (zero osmolality) to hosts and vectors (physiological osmolalities) and vice versa.…”