CRY
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.