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Brugia malayi , a parasitic nematode that causes lymphatic filariasis, harbors endosymbiotic intracellular bacteria, Wolbachia , that are required for the development and reproduction of the worm. The essential nature of this endosymbiosis led to the development of anti- Wolbachia chemotherapeutic approaches for the treatment of human filarial infections. Our study is aimed at identifying specific proteins that play a critical role in this endosymbiotic relationship leading to the identification of potential targets in the adult worms. Filarial cysteine proteases are known to be involved in molting and embryogenesis, processes shown to also be Wolbachia dependent. Based on the observation that cysteine protease transcripts are differentially regulated in response to tetracycline treatment, we focused on defining their role in symbiosis. We observe a bimodal regulation pattern of transcripts encoding cysteine proteases when in vitro tetracycline treated worms were examined. Using tetracycline-treated infertile female worms and purified embryos we established that the first peak of the bimodal pattern corresponds to embryonic transcripts while the second takes place within the hypodermis of the adult worms. Localization studies of the native proteins corresponding to Bm-cpl-3 and Bm-cpl-6 indicate that they are present in the area surrounding Wolbachia , and, in some cases, the proteins appear localized within the bacteria. Both proteins were also found in the inner bodies of microfilariae. The possible role of these cysteine proteases during development and endosymbiosis was further characterized using RNAi. Reduction in Bm-cpl-3 and Bm-cpl-6 transcript levels was accompanied by hindered microfilarial development and release, and reduced Wolbachia DNA levels, making these enzymes strong drug target candidates. [ABSTRACT FROM AUTHOR]

In the present study, we characterized a sterile cpi-2a(ok1256) deletion mutant in Caenorhabditis elegans and showed that CPI-2a has an essential regulatory role during oogenesis and fertilization. We have also shown that the CPI2a inhibitor and both Ce-CPL-1 and Ce-CPZ-1 enzymes are present in the myo-epithelial sheath surrounding germ cells, oocytes, and embryos as well as in the yolk granules within normal oocytes. Staining of mutant worms with anti-yolk protein antibodies has indicted that the proteins are not present in the mature oocytes. Moreover, green fluorescent protein expression was absence or reduced in cpi-2a/yp170:gfp mutant oocytes, although it was expressed in one of the successfully developed embryos. Based on these results, we hypothesize that the sterility in cpi-2a(ok1256) mutant worms is potentially caused by two possible mechanisms: 1) defects in the uptake and/or processing of yolk proteins by the growing oocytes and 2) indirect induction of defects in cell-cell signaling that is critical for promoting germ line development, oocyte maturation, ovulation, and fertilization. A defect in any of these processes would have detrimental effects on the development of normal embryos and consequently normal production of progenies as we observed in cpi-2a mutant worms. This is the first study that demonstrates the expression of cysteine proteases and their endogenous inhibitor in the gonadal sheath cells surrounding germ cells and oocytes, which indirectly have established their potential involvement in proteolytic processing of molecules within the gonadal sheath cells, such as components of the extracellular matrix or the cytoskeletal proteins, which are essential for proper cell-cell signaling activities of the gonadal sheath cells during normal maturation and ovulation processes. [ABSTRACT FROM AUTHOR]

A novel filarial serine protease inhibitor (SPI) from the human parasitic nematode Onchocerca volvulus, Ov-SPI-1, was identified through the analysis of a molting third-stage larvae expressed sequence tag dataset. Subsequent analysis of the expressed sequence tag datasets of O. volvulus and other filariae identified four other members of this family. These proteins are related to the low molecular weight SPIs originally isolated from Ascaris suum where they are believed to protect the parasite from host intestinal proteases. The two Ov-spi transcripts are up-regulated in the molting larvae and adult stages of the development of the parasite. Recombinant Ov-SPI-1 is an active inhibitor of serine proteases, specifically elastase, chymotrypsin, and cathepsin G. Immunolocalization of the Ov-SPI proteins demonstrates that the endogenous proteins are localized to the basal layer of the cuticle of third-stage, molting third-stage, and fourth-stage larvae, the body channels and multivesicular bodies of third-stage larvae and the processed material found between the two cuticles during molting. In O. volvulus adult worms the Ov-SPI proteins are localized to the sperm and to eggshells surrounding the developing embryos. RNA interference targeting the Ov-spi genes resulted in the specific knockdown of the transcript levels of both Ov-spi-1 and Ov-spi-2, a loss of native proteins, and a significant reduction in both molting and viability of third-stage larvae. We suggest the Ov-SPI proteins play a vital role in nematode molting by controlling the activity of an endogenous serine protease(s). The localization data in adults also indicate that these inhibitors may be involved in other processes such as embryogenesis and spermatogenesis. [ABSTRACT FROM AUTHOR]

We have analyzed the expression and function of Cecpz-1, a Caenorhabditis elegans cathepsin Z-like cysteine protease gene, during development of the worm. The cpz-1 gene is expressed in various hypodermal cells of all developmental stages and is specifically expressed in the gonads and the pharynx of adult worms. Disruption of cpz-1 function by RNA interference or cpz1(ok497) deletion mutant suggests that cpz-1 has a role in the molting pathways. The presence of the native CPZ-1 protein in the hypodermis/cuticle of larval and adult stages and along the length of the pharynx of adult worms, as well as the cyclic expression of the transcript during larval development, supports such function. We hypothesize that the CPZ-1 enzyme functions directly as a proteolytic enzyme degrading cuticular proteins before ecdysis and/or indirectly by processing other proteins such as proenzymes and/or other proteins that have an essential role during molting. Notably, an impressive level of the CPZ-1 native protein is present in both the new and the old cuticles during larval molting, in particular in the regions that are degraded prior to shedding and ecdysis. The similar localization of the related Onchocerca volvulus cathepsin Z protein suggests that the function of CPZ-1 during molting might be conserved in other nematodes. Based on the cpz-1 RNA interference and cpz-1 (ok497) deletion mutant phenotypes, it appears that cpz-1 have additional roles during morphogenesis. Deletion of cpz-1 coding sequence or inhibition of cpz-1 function by RNA interference also caused morphological defects in the head or tail region of larvae, improperly developed gonad in adult worms and embryonic lethality. The CPZ-1 native protein in these affected regions may have a role in the cuticular and the basement membrane extracellular matrix assembly process. The present findings have defined a critical role for cathepsin Z in nematode biology. [ABSTRACT FROM AUTHOR]

The geographic spread and rapid increase in the cases of Middle East respiratory syndrome (MERS) caused by a novel coronavirus (MERS-CoV) during the past two months have raised concern about its pandemic potential. Here we call for the rapid development of an effective and safe MERS vaccine to control the spread of MERS-CoV. [ABSTRACT FROM AUTHOR]