The inhibition of Hsp90 in cancerous cells has been correlated with the reduction in double-strand break (DSB repair) activity. However, the precise effect of Hsp90 on the DSB repair pathway in normal cells has remained enigmatic. Our results show that the Hsp82 chaperone, the ortholog of mammalian Hsp90, is indispensable for homologous-recombination (

Repairing double strand breaks (DSBs) is absolutely essential for the survival of obligate intracellular parasite Toxoplasma gondii. Thus, DSB repair mechanisms could be excellent targets for chemotherapeutic interventions. Recent genetic and bioinformatics analyses confirm the presence of both homologous recombination (HR) as well as non homologous end joining (NHEJ) proteins in this lower eukaryote. In order to get mechanistic insights into the HR mediated DSB repair pathway in this parasite, we have characterized the key protein involved in homologous recombination, namely TgRad51, at the biochemical and genetic levels. We have purified recombinant TgRad51 protein to 99% homogeneity and have characterized it biochemically. The ATP hydrolysis activity of TgRad51 shows a higher KM and much lower kcat compared to bacterial RecA or Rad51 from other related protozoan parasites. Taking yeast as a surrogate model system we have shown that TgRad51 is less efficient in gene conversion mechanism. Further, we have found that TgRad51 mediated gene integration is more prone towards random genetic loci rather than targeted locus. We hypothesize that compromised ATPase activity of TgRad51 is responsible for inefficient gene targeting and poor gene conversion efficiency in this protozoan parasite. With increase in homologous flanking regions almost three fold increments in targeted gene integration is observed, which is similar to the trend found with ScRad51. Our findings not only help us in understanding the reason behind inefficient gene targeting in T. gondii but also could be exploited to facilitate high throughput knockout as well as epitope tagging of Toxoplasma genes.

Sirtuins (PfSIR2A and PfSIR2B) are implicated to play pivotal roles in the silencing of sub‐telomeric genes and the maintenance of telomere length in P. falciparum 3D7 strain. Here, we identify the key factors that regulate the cellular abundance and activity of these two histone deacetylases. Our results demonstrate that PfSIR2A and PfSIR2B are transcriptionally downregulated at the mid‐ring stage in response to febrile temperature. We found that the molecular chaperone PfHsp90 acts as a repressor of PfSIR2A & B transcription. By virtue of its presence in the PfSIR2A & B promoter proximal regions PfHsp90 helps recruiting H3K9me3, conferring heterochromatic state, and thereby leading to the downregulation of PfSIR2A & B transcription. Such transcriptional downregulation can be reversed by the addition of 17‐(allylamino)‐17‐demethoxygeldanamycin or Radicicol, two potent inhibitors of PfHsp90. The reduced occupancy of PfSir2 at sub‐telomeric var promoters leads to the de‐repression of var genes. Thus, here we uncover how exposure to febrile temperature, a hallmark of malaria, enables the parasites to manipulate the expression of the two prominent epigenetic modifiers PfSir2A and PfSir2B.

Tropical theileriosis is a lymphoproliferative disease caused by the intracellular schizonts of Theileria annulata, an apicomplexan parasite. It causes severe infection in cattle and the untreated cattle would possibly die within 3–4 weeks of infection. The chemotherapy for this disease is largely dependent on the use of hydroxynaphthoquinone, namely buparvaquone. There have been reports recently of the development of resistance against this drug in T. annulata . Hence, identification of new drug molecule(s) or repurposing of existing drug molecule(s) against T. annulata is quite important. Here, we present the screening of 400 compounds included in the open-access Pathogen box from Medicine for Malaria Venture (MMV) to discover the novel compounds with potential inhibitory activity against T. annulata infected bovine leucocytes. We identified two compounds, MMV000062 and MMV560185, with IC 50 values of 2.97 μM and 3.07 μM, respectively. MMV000062 and MMV560185 were found non-toxic to BoMac cells with CC 50 values 34 μM and > 100 μM, respectively. The therapeutic indices of these compounds, MMV000062 and MMV560185, were calculated as more than 33 and 11, respectively. Further, it was observed that the parasite-infected cells under long-term culture were unable to recover with these compounds. We further deciphered that MMV560185 kills the infected cell by activation of TNFR-1 mediated extrinsic pathway of the apoptosis. The phenotypic characteristics of apoptosis were confirmed by Transmission Electron Microscopy. Our results suggest that it may be possible to develop MMV560185 further for chemotherapeutics of tropical theilerosis.

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