Nicardipine HCl To better understand Wolbachia s dependence
To better understand Wolbachia’s dependence on these enzymes, RNAi experiments targeting these transcripts will be required to examine the consequences of their down-regulation on Wolbachia in the different tissues of the parasite. Initial studies have shown that decreases in Bm-cpl-3 and Bm-cpl-6 transcript levels after 96hr of siRNA treatment resulted in a significant reduction of Wolbachia in the Bm-cpl-3 and Bm-cpl-6 siRNA treated worms. It was also accompanied by a significant reduction in the number of microfilariae produced within the uteri of the treated female worms and in the number of microfilaria released into the culture medium. This supports our hypothesis that there is a definite link between the Bm-CPL-3 and Bm-CPL-6 enzymes and Wolbachia. This link might be directly or, more likely, indirectly involved in the maintenance of the symbiotic relationship and, consequently, proper embryonic development leading to the release of stretch microfilaria. The mechanism mediating this effect is still unknown. One possible indirect biochemical link may be secondary: for example, these enzymes may be used by Wolbachia for amino Nicardipine HCl provisioning as genome analysis of Wolbachia has shown that they lack the capability for amino acid biosynthesis (Foster et al., 2005a).
Further characterization of the Bm-CPL-3 and -6 function(s) may lead to an improved understanding of filarial nematode biology and identify these proteases as anti-filarial drug targets. Cysteine proteases are known to have essential functions in a variety of parasitic systems, including other nematodes, and are being developed as potential targets for drugs and vaccines (McKerrow, 1999, Newton and Meeusen, 2003, Sajid et al., 2011, Marco and Coteron, 2012, Vermeire et al., 2012).
Acknowledgments We thank the NIAID/NIH Filariasis Research Reagent Repository Center (www.filariasiscenter.org) for the adult female B. malayi worm supply. We thank Drs. Mark Blaxter and Georgio Koutsovoulos from The University of Edinburgh, UK, for providing us with the putative sequences of the A. viteae CPL family members before they were available on the 959 nematodes website (http://nematodes.org/genomes/acanthocheilonema_viteae/). We thank Dr. Kenneth M. Pfarr, University Clinic Bonn, Germany, for providing us with the cDNA samples used for the analyses of the A. viteaecpl transcripts from female adult worms recovered from infected M. unguiculatus that were treated with tetracycline. We also thank Saheed Bachu for technical assistance. This study was funded by NIAID/NIH Grant No. AI072465 to T.R.U., E.G. and S.L. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript. The authors report no conflict of interest.
Introduction Autophagy is an evolutionarily conserved, multistep process, whereby cellular components and damaged organelles are sequestered within autophagosomes for lysosomal degradation. Among them, autophagosome biogenesis requires two ubiquitin-like conjugation systems: the Atg12–Atg5 and the Atg8–phosphatidylethanolamine (PE) systems. Atg4 is a cysteine protease of the C54 family and plays an important role in the Atg8/LC3 lipid conjugation system (Marino et al., 2003). Atg4 was first found to physically interact with Atg8 in the yeast Saccharomyces cerevisiae in 1998 (Lang et al., 1998). There is only one single member of Atg4 proteins in yeast, and deletion of Atg4 impairs the autophagy process (Kirisako et al., 2000). In mammals, there are four Atg4 homologues, Atg4A, Atg4B, Atg4C, and Atg4D (Marino et al., 2003). There are eight human Atg8 homologues belonging to two subfamilies: the LC3 (microtubule-associated protein 1 light chain 3) subfamily, which is comprised of LC3A (isoform a and b), LC3B, and LC3C; and the GABARAP (GABAA receptor-associated protein) subfamily, including GABARAP, GABARAPL1/Atg8L/GEC1, GABARAPL2/GATE-16/GEF2, and GABARAPL3 (Le Grand et al., 2011). All substrates have a conserved cleavage site for Atg4 (Fig. 1). The substrate specificity of different Atg4 homologues is not identical. Studies have shown Atg4B is able to cleave most of the human Atg8 homologues tested so far (Hemelaar et al., 2003, Kabeya et al., 2004, Li et al., 2011, Tanida et al., 2004). Atg4A is a potent protease for the GABARAP subfamily, but not the LC3 subfamily, whereas Atg4C and Atg4D seem to possess marginal activities until they are activated by the cleavage of their N-termini via a caspase (Li et al., 2011, Scherz-Shouval et al., 2003).