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The β-clamp is the processivity-promoting factor for most of the enzymes in prokaryotic DNA replication; hence, it is a crucial drug target. In the present study, we investigated the β-clamp from Helicobacter pylori, aiming to seek potential drug molecules against this gastric-cancer-causing bacterium. An in silico screening of Food and Drug Administration (FDA) approved drugs against the H. pylori β-clamp, followed by its in vitro inhibition using a surface competition approach, yielded the drug diflunisal as a positive initial hit. Diflunisal inhibits the growth of H. pylori in the micromolar range. We determined the structure of diflunisal in complex with the β-clamp to show that the drug binds at subsite I, which is a protein-protein interaction site. Successful identification of FDA-approved molecules against H. pylori may lead to better and faster drug development.

The role of TatD DNases as DNA repair enzymes or cell death (apoptotic) nucleases is well established in prokaryotes as well as eukaryotes. The current study aims to characterize the TatD nuclease from Bacillus anthracis (Ba TatD) and to explore its key histidine catalytic residues. Ba TatD was found to be a metal-dependent, nonspecific endonuclease which could efficiently cleave double-stranded DNA substrates. Moreover, Ba TatD nuclease was observed to be thermostable up to 55°C and act in a wide pH range indicating its industrial applicability. Diethyl pyrocarbonate-based histidine-selective alkylation of the Ba TatD resulted in a loss of its nuclease activity suggesting a crucial role of the histidine residues in its activity. The key residues of Ba TatD were predicted using sequence analysis and structure-based approaches, and then the predicted residues were further tested by mutational analysis. Upon mutational analysis, H128 and H153 have been found to be crucial for Ba TatD activity, though H153 seems to bear an important but a dispensable role for the Ba TatD nuclease. Ba TatD had a uniform expression in the cytosol of B. anthracis, which indicates a significant role of the protein in the pathogen's life cycle. This is the first study to identify and characterize the TatD DNase from B. anthracis and will be helpful in gaining more insights on the role of TatD proteins in Gram-positive bacteria where it remains unexplored.

Motility and phagocytosis are key processes that are involved in invasive amoebiasis disease caused by intestinal parasite Entamoeba histolytica. Previous studies have reported unconventional myosins to play significant role in membrane based motility as well as endocytic processes. EhMyosin IB is the only unconventional myosin present in E. histolytica, is thought to be involved in both of these processes. Here, we report an interaction between the SH3 domain of EhMyosin IB and c-terminal domain of EhFP10, a Rho guanine nucleotide exchange factor. EhFP10 was found to be confined to Entamoeba species only, and to contain a c-terminal domain that binds and bundles actin filaments. EhFP10 was observed to localize in the membrane ruffles, phagocytic and macropinocytic cups of E. histolytica trophozoites. It was also found in early pinosomes but not early phagosomes. A crystal structure of the c-terminal SH3 domain of EhMyosin IB (EhMySH3) in complex with an EhFP10 peptide and co-localization studies established the interaction of EhMySH3 with EhFP10. This interaction was shown to lead to inhibition of actin bundling activity and to thereby regulate actin dynamics during endocytosis. We hypothesize that unique domain architecture of EhFP10 might be compensating the absence of Wasp and related proteins in Entamoeba, which are known partners of myosin SH3 domains in other eukaryotes. Our findings also highlights the role of actin bundling during endocytosis., Author summary Entamoeba histolyica is a highly motile human pathogen which eats the blood cells and immune cells by phagocytosis during progression of Amoebiasis disease. E. histolytica infections are a major concern in the developing countries. Myosins are motor proteins that move over actin cytoskeleton to drive the cellular processes. Unconventional myosins are a type of myosin which are different from myosin present in muscles, and are involved in regulation of membrane dependent processes crucial for cellular movement and endocytosis. In contrast to other eukaryotes, Entamoeba has only one unconventional myosin, Myosin IB which shows more similarity with metazoan myosins rather than amoeboid myosins. Myosin IB has been shown to be involved in phagocytosis. The exact role played by Myosin IB in the phagocytic process is still not fully understood. SH3 domain is present at the c-terminal tail of Myosin IB which has been found to interact with proteins that regulate the actin cytoskeleton in other organisms. In this work, we have identified EhFP10 as one of the interacting proteins of EhMyosin IB SH3 domain through a co-crystal structure and biophysical experiments. Our localisation studies demonstrated the involvement of EhFP10 in phagocytosis and pinocytosis. This is the first report of the involvement of a FYVE domain containing GEF in pinocytosis. We have also analysed that EhFP10 has a unique c-terminal domain not present in any other FYVE family GEFs in Entamoeba as well as in other organisms. Actin binding studies indicated that the c-terminal domain of EhFP10 binds to actin filaments and leads to formation of thicker actin bundles. Myosin IB interaction with EhFP10 inhibits the formation of actin bundles. Through our results, we could hypothesize that the presence of a unique GEF like EhFP10 could compensate for the absence of WASP proteins in E. histolytica which have been found to interact with the myosin I SH3 domain in other organisms and regulate actin dynamics during endocytosis. Our study reveals a rare interaction of a myosin with a GEF, which interact to regulate actin bundling. EhMyosin IB is different from other amoeboid myosins and lies between the metazoan and amoeboid myosins like human myosin IE. Hence, the findings have broader implication to completely understand the closure stage of a phagocytic and pinocytic cup.