Chemical genetic screen identifies Toxoplasma DJ-1 as a regulator of parasite secretion, attachment, and invasion.

Toxoplasma gondii is a member of the phylum Apicomplexa that includes several important human pathogens, such as Cryptosporidium and Plasmodium falciparum, the causative agent of human malaria. It is an obligate intracellular parasite that can cause severe disease in congenitally infected neonates and immunocompromised individuals. Despite the importance of attachment and invasion to the success of the parasite, little is known about the underlying mechanisms that drive these processes. Here we describe a screen to identify small molecules that block the process of host cell invasion by the T. gondii parasite. We identified a small molecule that specifically and irreversibly blocks parasite attachment and subsequent invasion of host cells. Using tandem orthogonal proteolysis-activity-based protein profiling, we determined that this compound covalently modifies a single cysteine residue in a poorly characterized protein homologous to the human protein DJ-1. Mutation of this key cysteine residue in the native gene sequence resulted in parasites that were resistant to inhibition of host cell attachment and invasion by the compound. Further analysis of the invasion phenotype confirmed that modification of Cys127 on TgDJ-1 resulted in a block of microneme secretion and motility, even in the presence of direct stimulators of calcium release. Together, our results suggest that TgDJ-1 plays an important role that is likely downstream of the calcium flux required for microneme secretion, parasite motility, and subsequent invasion of host cells.

IDENTIFICATION OF CONOIDIN A AS A COVALENT INHIBITOR OF PEROXIREDOXIN II.

Conoidin A (1) is an inhibitor of host cell invasion by the protozoan parasite Toxoplasma gondii. In the course of studies aimed at identifying potential targets of this compound, we determined that it binds to the T. gondii enzyme peroxiredoxin II (TgPrxII). Peroxiredoxins are a widely conserved family of enzymes that function in antioxidant defense and signal transduction, and changes in PrxII expression are associated with a variety of human diseases, including cancer. Disruption of the TgPrxII gene by homologous recombination had no effect on the sensitivity of the parasites to 1, suggesting that TgPrxII is not the invasion-relevant target of 1. However, we showed that 1 binds covalently to the peroxidatic cysteine of TgPrxII, inhibiting its enzymatic activity in vitro. Studies with human epithelial cells showed that 1 also inhibits hyperoxidation of human PrxII. These data identify Conoidin A as a novel inhibitor of this important class of antioxidant and redox signaling enzymes.

Synthesis and chemical characterisation of target identification reagents based on an inhibitor of human cell invasion by the parasite Toxoplasma gondii.

The use of phenotype-based screens as an approach for identifying novel small molecule tools is reliant on successful protein target identification strategies. Here we report on the synthesis and chemical characterisation of a novel reagent for protein target identification based on a small molecule inhibitor of human cell invasion by the parasite Toxoplasma gondii. A detailed (1)H NMR study and biological testing confirmed that incorporation of an amino-containing functional group into the aryl ring of this inhibitor was possible without loss of biological activity. Interesting chemical reactivity differences were identified resulting from incorporation of the new substituent. The amine functionality was then used to prepare a biotinylated reagent that is central to our current protein target identification studies with this inhibitor.

Efficient sporulation in Clostridium difficile requires disruption of the sigmaK gene.

A 14.6 kb prophage-like insertion, termed skinCd, was found to interrupt the sigK gene, which encodes an RNA polymerase sigma factor essential for sporulation, in six strains of Clostridium difficile. Until now, Bacillus subtilis was the only spore-former shown to carry such an insertion, and the presence of the insertion is not required for efficient sporulation in this organism. The B. subtilis and C. difficile skin elements proved to be divergent in sequence, inserted at different sites within the sigK gene and in opposite orientations. The skinCd element was excised from the chromosome specifically during sporulation, forming a circular molecule. Two natural isolates of C. difficile lacked the skinCd element and were defective in sporulation. When a merodiploid strain was created that carries both interrupted and uninterrupted versions of the sigK gene, the cells became Spo-, showing that the uninterrupted gene is dominant and inhibits sporulation. C. difficile sigK genes, whether skinCd+ or skinCd-, lack the N-terminal pro-sequence found in all other sigK genes studied to date. Thus, regulated excision of skinCd appears to be a critical mechanism for achieving proper temporal activation of sigmaK.