suramin br Materials and methods br Results

Materials and methods
Results
Discussion In this study we characterized in more detail the interrelationship between the expression levels of the B. malayi Bm-cpl-3 and Bm-cpl-6 cysteine proteases and the fitness of Wolbachia, and consequently that of the parasite. Our present in vitro data support our previous in vivo studies showing that killing Wolbachia using tetracycline treatment of B. malayi worms resulted in differential expression of these two enzymes (Ghedin et al., 2009). In suramin to B. malayi, tetracycline treatment of A. viteae, which lacks a Wolbachia endosymbiont, did not affect the expression of these enzymes. That being said, the comparison of cpl expression in B. malayi worms cultured for 6days in vitro in the presence of tetracycline with A. viteae, which were recovered from jirds after six weeks of treatment with 0.5% (w/v) tetracycline in drinking water (Strubing et al., 2010) was not optimal. However, six weeks of similar oral tetracycline treatment of Litomosoidessigmodontis-infected jirds has been shown to lead to a persistent depletion of Wolbachia, demonstrating sufficient tetracycline delivery with this treatment (Arumugam et al., 2008). In contrast, similar tetracycline treatment of A. viteae, a Wolbachia-free filarial nematode did not show any phenotypic effects on the worm (Hoerauf et al., 1999). In our previous studies we have shown that treatment of B. malayi in vitro with 40μg/ml tetracycline caused 100% degradation of Wolbachia by day 5 without affecting the worms’ motility (Ghedin et al., 2009). The in vitro studies support our previous observations of a bimodal expression pattern of B. malayi transcripts after tetracycline treatment, including the two enzymes we studied here. We hypothesize that this bimodal expression may be due to stage-specific responses of the host in response to Wolbachia death; the first effect is in the pre-embryonic and embryonic stages of the worms and the later effect is due to reduced fitness of the worms. The differential expression patterns in female worms at various stages of fertility were not surprising because adult female worms (>70days after infection) can produce eggs and contain developing embryonic stages, which would account for the differential expression profiles in comparison to those in unfertile females. Clinical studies in humans have also shown that in filaria-infected patients treated with antibiotics the first phenotype is reduction in the levels of microfilariae followed by a long-term sterility and eventual death of adult worms (Taylor et al., 2010). However, it is possible that some of the responses observed upon tetracycline treatment may also be unrelated to its anti-bacterial effects. Tetracycline is known to have pleiotropic effects that are unrelated to its antibiotic activity. For example, bacteriologically inactive analogs of tetracycline have been shown to inhibit molting in B. malayi larvae, suggesting that the effect on molting observed in tetracycline treated larvae is not related to its anti-Wolbachia activity (Rajan, 2004). Similarly, tetracycline has been shown to exhibit anti-collagenase activity, an enzymatic activity that is likely important in molting, as collagens are an important component of the parasite cuticle (Griffin et al., 2010). Dissection of the exact mechanism of the effect of tetracycline on adult worms will require additional studies utilizing bacteriologically inactive analogs in addition to tetracycline, perhaps coupled with RNAi studies that specifically target the enzymes in question in parallel of both B. malayi and A. viteae worms. To better understand the possible functions of the Ic cysteine proteases in B. malayi, we analyzed the localization of these proteins. Contrary to what was observed for cathepsin L-like cysteine proteases in the Ia group (Guiliano et al., 2004), the proteins in the Ic group were not localized to the hypodermal lamellae or the eggshell surrounding all stages of the developing microfilariae. They were instead found in the inner bodies of microfilariae and co-localized with Wolbachia. In some cases, they were found within the Wolbachia cells. As for our qRT-PCR analysis, RNAseq transcriptome analysis of B. malayi stages (Choi et al., 2011) indicate that Bm-cpl-3 is specifically up-regulated in immature microfilariae while Bm-cpl-6 is up-regulated in both immature and mature microfilariae. However, Bm- cpl-1, Bm-cpl-4 and Bm-cpl-5 are up-regulated in the L3 stage, further indicating their potential differing roles during development of the parasite. Our work supports the hypothesis that there is tissue-specific differential regulation of CPL gene expression in B. malayi.