Cytotoxic necrotizing factor 1 (CNF1), a virulence factor expressed by pathogenic Escherichia coli, acts on Rho-GTPases and specifically deamidates a single glutamine residue (Gln-63 in RhoA) required for GTP hydrolysis. This modification constitutively activates the effector binding function of Rho-GTPases and eventually leads to their proteasome-mediated degradation. Previous structural investigation revealed that the CNF1 active site is located in a deep and narrow pocket and that the entrance to this pocket is formed by nine loop segments. We have examined the functional importance of five of these loops (2, 6, 7, 8, and 9) by deleting them individually. We find that deletion of proximally located loops 8 and 9 in the 32 kDa catalytic domain of CNF1 (CNF1-C) nearly or completely abolishes deamidation of RhoA in vitro, identifying a potential Rho-GTPase recognition site. Deletion of loop 7 causes protein folding errors, and deletion of loop 6 has a small effect on deamidation. In contrast, deletion of loop 2 is found to increase deamidation 5-7-fold, implying that this loop rearranges in binding RhoA. None of the loop deletions or wild-type CNF1-C is able to deamidate RhoA containing Asn-63 instead of Gln-63, suggesting that the fit between the toxin and its target is highly precise. In addition, we show that the specificity constant (k(cat)/K(m)) of CNF1-C for RhoA is 825 +/- 3 M(-)(1) s(-)(1). This modest value is consistent with the confining size of the active site pocket acting to exclude nonspecific targets but also limiting reactivity toward intended targets.
This review summarizes the progress towards control of lymphatic filariasis (LF) and onchocerciasis, focussing on the impact of mass drug administration (MDA) programmes, in particular those that have developed following the donation of ivermectin and albendazole. The contrasting strategies and objectives of the different programmes are compared, and the impact on transmission, clinical disease and public health assessed. The constraints on programme success are: (i) the absence of a macrofilaricide, which can be used in a public health context; (ii) the sustainability of high coverage of ivermectin over many years in onchocerciasis control; and (iii) the problem of treatment in areas where Loa loa (tropical eye worm) is co-endemic with onchocerciasis because of the rare severe adverse events. LF programmes are expanding rapidly in over 30 countries, where circa 60 million people received treatments in 2002. No serious adverse events have been associated with MDAs for LF elimination. Research on new approaches to treatment using antibiotics are showing promising results in pilot settings because doxycyline has been shown to have long-term embryostatic effects and sustained reductions of microfilaria loads in onchocerciasis and bancroftian filariasis.