The Heme Biosynthetic Pathway of the Obligate Wolbachia Endosymbiont of Brugia malayi as a Potential Anti-filarial Drug Target

Human filarial nematodes are causative agents of elephantiasis and African river blindness, which are among the most debilitating tropical diseases. Currently used drugs mainly affect microfilariae (mf) and have less effect on adult filarial nematodes, which can live in the human host for more than a decade. Filariasis drug control strategy relies on recurrent mass drug administration for many years. Development of novel drugs is also urgently needed due to the threat of drug resistance occurrence. Most filarial worms harbor an obligate endosymbiotic bacterium, Wolbachia, whose presence has been identified as a potential drug target. Comparative genomics had suggested Wolbachia heme biosynthesis as a potential drug target, and we present an analysis of selected enzymes alongside their human homologues from several different aspects—gene phylogenetic analyses, in vitro enzyme kinetic and inhibition assays and heme-deficient E. coli complementation assays. We also conducted ex vivo Brugia malayi viability assays using heme pathway inhibitors. These experiments demonstrate that heme biosynthesis could be critical for filarial worm survival and thus is a potential anti-filarial drug target set

Outline of the chloramphenicol (Cat) gene mutagenesis and assembly experiment

<p><b>Copyright information:</b></p><p>Taken from "USER™ friendly DNA engineering and cloning method by uracil excision"</p><p></p><p>Nucleic Acids Research 2007;35(6):1992-2002.</p><p>Published online 6 Mar 2007</p><p>PMCID:PMC1874603.</p><p>© 2007 The Author(s)</p> () PCR primer design strategy. Lines with the arrowheads symbolize priming sequences in the PCR primers. Left and Right cloning primers contain 5′ extensions compatible with the single-stranded extensions on the linearized pNEB206A vector. The overlapping primers P1/P2 and P3/P4 are selected in the vicinity of the targeted mutations. Asterisks designate the point mismatches in the primers compared to the template sequence. Overlapping primers are complementary to each other within the boxed sequences. The 3′ dT of the boxed sequence in the primer sequences is replaced by dU. () Schematic representation of six PCR products amplified using the indicated primers. Cycling conditions are described in Materials and Methods. The label, Cat926 etc. shows the PCR fragment size in bp. The ends of the PCR products are flanked by the compatible overlapping sequences with a single dU residue at the junction. () Four assembly reactions were carried out using the indicated PCR fragments to assemble a full-length Cat gene carrying the indicated phenotype into pNEB206A

Nondestructive enzymatic deamination enables single-molecule long-read amplicon sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution

The predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA, leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite-treated DNA, we applied a new enzymatic deamination approach, termed enzymatic methyl-seq (EM-seq), to long-range sequencing technologies. Our methodology, named long-read enzymatic modification sequencing (LR-EM-seq), preserves the integrity of DNA, allowing long-range methylation profiling of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) over multikilobase length of genomic DNA. When applied to known differentially methylated regions (DMRs), LR-EM-seq achieves phasing of >5 kb, resulting in broader and better defined DMRs compared with that previously reported. This result showed the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long-range epigenetic analysis at single-molecule and single-nucleotide resolution