Dataset Information

Novel Activity of a Synthetic Decapeptide Against Toxoplasma gondii Tachyzoites.

ABSTRACT: The killer peptide KP is a synthetic decapeptide derived from the sequence of the variable region of a recombinant yeast killer toxin-like microbicidal single-chain antibody. KP proved to exert significant activities against diverse microbial and viral pathogens through different mechanisms of action, but little is known of its effect on apicomplexan protozoa. The aim of the present study was to evaluate the in vitro activity of KP against Toxoplasma gondii, a globally widespread protozoan parasite of great medical interest. The effect of KP treatment and its potential mechanism of action on T. gondii were evaluated by various methods, including light microscopy, quantitative PCR, flow cytometry, confocal microscopy, and transmission electron microscopy. In the presence of KP, the number of T. gondii tachyzoites able to invade Vero cells and the parasite intracellular proliferation were significantly reduced. Morphological observation and analysis of apoptotic markers suggested that KP is able to trigger an apoptosis-like cell death in T. gondii. Overall, our results indicate that KP could be a promising candidate for the development of new anti-Toxoplasma drugs with a novel mechanism of action.

SUBMITTER: Giovati L

PROVIDER: S-EPMC5920037 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Novel Activity of a Synthetic Decapeptide Against Toxoplasma gondii Tachyzoites.

Giovati Laura L Santinoli Claudia C Mangia Carlo C Vismarra Alice A Belletti Silvana S D'Adda Tiziana T Fumarola Claudia C Ciociola Tecla T Bacci Cristina C Magliani Walter W Polonelli Luciano L Conti Stefania S Kramer Laura H LH

Frontiers in microbiology 20180420

The killer peptide KP is a synthetic decapeptide derived from the sequence of the variable region of a recombinant yeast killer toxin-like microbicidal single-chain antibody. KP proved to exert significant activities against diverse microbial and viral pathogens through different mechanisms of action, but little is known of its effect on apicomplexan protozoa. The aim of the present study was to evaluate the in vitro activity of KP against Toxoplasma gondii, a globally widespread p ...[more]

PMID: 29731744

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Project description:The triazine WR99210 [4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichlorophenoxypropyloxy)-1,3,5 triazine] inhibits Toxoplasma gondii in vitro at nanomolar levels (P < 0.05). The 50% inhibitory concentration (IC(50)) was approximately 50 nM. It is a potent inhibitor in vitro and is also effective in vivo. Administration of WR99210 parenterally (i.e., intraperitoneally) reduced the mean number of RH strain tachyzoites present in peritoneal fluid substantially 4 days after intraperitoneal infection of mice. There was a mean of approximately 35 million parasites in control mice as contrasted with approximately 2 million parasites in mice treated with 1.25 mg WR99210/kg of body weight in a representative experiment (P < 0.05). In addition the prodrug PS-15 N'-[3-(2,4, 5-trichlorophenoxy)propyloxy]-N9-(1-methylethyl) imidocarbonimidicdiamide is converted to 4,6-diamino-1,2-dihydro-2,2-dimethyl-1-(2,4,5-trichlorophenoxypropyloxy)-1,3,5 triazine in vivo when the prodrug is administered orally. PS-15 administered by gavage also reduced intraperitoneal RH strain T. gondii tachyzoite numbers. WR99210 has high efficacy and relatively low toxicity because of its substantial effect on T. gondii dihydrofolate reductase (DHFR) but not the mammalian host DHFR. Amino acid sequences of T. gondii, Plasmodium falciparum, and Homo sapiens DHFRs were compared. It is of interest that of the DHFR amino acids considered to be interacting with WR99210 in P. falciparum within interatomic distances within 3 to 5 A, four of eight were shared with T. gondii DHFR. H. sapiens also shared four amino acids thought to be interacting with WR99210. Efficacy of intraperitoneal administration of WR99210 and peroral administration of PS-15 demonstrate the potential usefulness of this class of compounds in treatment of toxoplasmosis administered either parenterally or perorally. The recent development program for this class of antimicrobials as antimalarials makes our proof of principle of improved efficacy of triazines (compared with the gold standard treatment, pyrimethamine) against T. gondii especially promising.

Project description:Toxoplasma motility is both activated and suppressed by 3',5'-cyclic nucleotide signaling. Cyclic GMP (cGMP) signaling through Toxoplasma gondii protein kinase G (TgPKG) activates motility, whereas cyclic AMP (cAMP) signaling through TgPKAc1 inhibits motility. Despite their importance, it remains unclear how cGMP and cAMP levels are maintained in Toxoplasma. Phosphodiesterases (PDEs) are known to inactivate cyclic nucleotides and are highly expanded in the Toxoplasma genome. Here, we analyzed the expression and function of the 18-member TgPDE family in tachyzoites, the virulent life stage of Toxoplasma. We detected the expression of 11 of 18 TgPDEs, confirming prior expression studies. A knockdown screen of the TgPDE family revealed four TgPDEs that contribute to lytic Toxoplasma growth (TgPDE1, TgPDE2, TgPDE5, and TgPDE9). Depletion of TgPDE1 or TgPDE2 caused severe growth defects, prompting further investigation. While TgPDE1 was important for extracellular motility, TgPDE2 was important for host cell invasion, parasite replication, host cell egress, and extracellular motility. TgPDE1 displayed a plasma membrane/cytomembranous distribution, whereas TgPDE2 displayed an endoplasmic reticulum/cytomembranous distribution. Biochemical analysis of TgPDE1 and TgPDE2 purified from Toxoplasma lysates revealed that TgPDE1 hydrolyzes both cGMP and cAMP, whereas TgPDE2 was cAMP specific. Interactome studies of TgPDE1 and TgPDE2 indicated that they do not physically interact with each other or other TgPDEs but may be regulated by kinases and proteases. Our studies have identified TgPDE1 and TgPDE2 as central regulators of tachyzoite cyclic nucleotide levels and enable future studies aimed at determining how these enzymes are regulated and cooperate to control Toxoplasma motility and growth. IMPORTANCE Apicomplexan parasites require motility to actively infect host cells and cause disease. Cyclic nucleotide signaling governs apicomplexan motility, but it is unclear how cyclic nucleotide levels are maintained in these parasites. In search of novel regulators of cyclic nucleotides in the model apicomplexan Toxoplasma, we identified and characterized two catalytically active phosphodiesterases, TgPDE1 and TgPDE2, that are important for Toxoplasma's virulent tachyzoite life cycle. Enzymes that generate, sense, or degrade cyclic nucleotides make attractive targets for therapies aimed at paralyzing and killing apicomplexan parasites.

Dataset Information

Novel Activity of a Synthetic Decapeptide Against Toxoplasma gondii Tachyzoites.

Publications

Novel Activity of a Synthetic Decapeptide Against <i>Toxoplasma gondii</i> Tachyzoites.

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