As shown in sand fly transmission studies, LmxMPK3 is essential for the Leishmania parasite to pass through the insect stage of its life cycle, since LmxMPK3 null mutants fail to establish sand fly infection (see 5.1.2). LmxMPK3 might thus be a good target for blocking parasite transmission to the insect vector.
Especially in malaria research the development of drugs and vaccines with a strong transmission-blocking potential has become increasingly important as programmes for the elimination of the disease are strongly supported by several influential organisations (Greenwood, 2008; Targett and Greenwood, 2008). While malaria control describes the reduction in cases of clinical malaria and mortality (with the parasite still persisting in the community as asymptomatic infections), malaria elimination aims at stopping transmission completely within a defined region. The latter can only be achieved by killing all of the parasites within the target population. The concept of transmission-blocking vaccines (TBVs) is the induction of antibodies against sexual-stage/zygote-specific surface molecules of the malaria parasite which are passively transferred to blood-feeding mosquitoes where they block further parasite development. There is also an enhanced research priority for transmission-blocking drugs that could be used safely for mass drug administration (MDA) which describes the administration of drugs to the whole of a population at risk whether or not individuals are infected. This chemopreventive method had successfully contributed to the elimination of malaria from the island Aneityum in Vanuatu (Kaneko et al., 2000).
Likewise, modern trends in Leishmania vaccine development aim at transmission-blocking activities (Tabbara, 2006). Indeed, LPG has been found to be a promising TBV candidate against CL (Tonui et al., 2001a; Tonui et al., 2001b). Moreover, the FML (fucose mannose ligand) vaccine Leishmune® is a TBV against zoonotic VL which has been shown to inhibit the binding of procyclic promastigotes to the insect midgut (Saraiva et al., 2006). It has been suggested that the decrease in dog infectivity to the insect vector by prophylactic vaccination reduces human infections (Dye, 1996).
As LmxMPK3 is an intracellular molecule it is qualified rather as a transmission-blocking drug target than as a TBV candidate. As it has already been discussed in 5.4, a cell-permeable synthetic inhibitor selectively inhibits an inhibitor-sensitised version of LmxMPK3 in the
LmxMPK3 deletion background and thus induces flagellar shortening which eventually leads to parasites revealing the LmxMPK3 null mutant phenotype. Referring to this observation, a selective inhibitor for wild type LmxMPK3 which must not interfere with mammalian MAP kinases could be used as a transmission-blocking drug. Preventive administration of such an inhibitor to a target population might contribute to the elimination of CL - and possibly also other forms of leishmaniasis - in this population. The inhibitor would either enter the parasite in the mammalian host or would be passively transferred to the blood-feeding sand fly and reach the parasite there. However, this approach requires the development of a cell-permeable inhibitor which is highly specific for LmxMPK3 and extremely safe as the majority of individuals to whom it would be administered will not be infected.
The inhibitor might also be applied as a spray alternatively to the chemical insecticides used for spraying of houses as a vector control intervention for reducing and interrupting parasite transmission (WHO, 2006). It is uncertain if such an inhibitor spray would induce a rapid and effective response before the sand fly would feed on the next host. Moreover, promastigotes would have to be attacked in early developmental insect stages to interrupt their life cycle.
Nevertheless, a main advantage of a highly specific inhibitor over chemical insecticides is a less harmful effect on the environment and on human health.
5.10 LmxMPK3 mutants as model systems to study human ciliopathies
It has been shown that lack of LmxMPK3 in L. mexicana leads to short-flagellated parasites (Erdmann, diploma thesis, 2004; see 5.1.1) which lost ability to establish sand fly infection and thus to complete its life cycle (see 5.1.2). As pointed out in 5.1.1, the MAP kinase is likely to be critically involved in intraflagellar transport (IFT), a process essential for the construction and maintenance of flagella and cilia.
A loss of the physiological function of cells/tissues following cilia defects is also reflected by numerous different human ciliopathies. Cilia are actually present in a number of organs (kidneys, liver, pancreas, lungs, thyroid gland) and cells (endothelial cells, photoreceptors in the retina, sperm cells, myocardium, odontoblasts, cortical and hypothalamic neurons) of the human body (Badano et al., 2006). Those cilia confer the motility of sperm cells, the transport of fluids over epithelial cells and also sensory perception (Pazour and Rosenbaum, 2002b).
Depending on the mutant protein the resulting disorders range from organ-specific to pleiotropic phenotypes. It can be distinguished between motile and sensory cilia dysfunctions. The former affect motile cilia with a “9+2” axonemal organisation of microtubule pairs (see 1.2.1), while the latter refer to primary, non-motile cilia with a “9+0” axonemal construction. This classification is, however, strongly simplified as examples of motile primary cilia (e.g. in the renal epithelium; Ong and Wagner, 2005) as well as motile cilia with sensory
roles exist. The latter include cilia in the female reproductive tract containing TRP channels (cation channels) which have been suggested to relay physiochemical changes in the oviduct (Teilmann et al., 2005). Several human ciliopathies have been mainly ascribed to mutations in IFT components including the IFT motor proteins.
The laterality abnormality situs inversus is caused by the paralysis of the otherwise motile primary cilia on the embryonic node which normally generate a leftward flow of extraembryonic fluid by which the left-right axis of symmetry is established. It has been shown that targeted knock-outs of KIF3A and KIF3B, subunits of the heterotrimeric kinesin complex, and Tg737, the mouse orthologue of IFT complex B protein IFT88, indeed prevent assembly of nodal cilia and produce situs inversus in 50% of mouse embryos (Nonaka et al., 1998; Marszalek et al., 1999; Murcia et al., 2000).
In addition, Tg737 mutations are responsible for the development of polycystic kidney disease (PKD) - characterised by cystic, enlarged kidneys - and hepatic fibrosis (Moyer et al., 1994). The primary cilia of kidney epithelial cells project into the lumen of the ducts/tubules of the nephrons and might have a role in detecting fluid flow through the nephrons. Polycystins, membrane proteins which have been suggested to form cation channels, might be involved in Ca2+ signalling which detects the state of the epithelium and regulates its proliferation and differentiation (Somlo and Ehrlich, 2001). PKD can thus result from a defect in the polycystins themselves, in their IFT or in the cilium where those membrane proteins are localised.
Furthermore, retinal degeneration - leading to retinitis pigmentosa (RP) and blindness - can develop as a result of defects in the IFT machinery. Vertebrate photoreceptor rods and cones are polarised sensory neurons in which the outer segments are formed from primary cilia.
The synthesis of proteins and lipids required for the assembly and maintenance of the outer segment occurs in the inner segment. Consequently, IFT is required for moving the cargo through the connecting cilium, the only direct link between the inner and the outer segment.
Indeed, studies of a KIF3A knock-out in photoreceptor cells (Marszalek et al., 2000) and Tg737 mutant mice (Pazour et al., 2002) revealed accumulations of opsin in the inner segments, degenerated outer segments and also apoptotic photoreceptor cells, an indicator of RP.
It has been realised that several human pleiotropic disorders are also caused by ciliary dysfunctions. Those include not only kidney and retinal defects, but also affect other tissues such as the limb and nervous system, as is the case in Bardet-Biedl syndrome (BBS) (Badano et al., 2006). Indeed, the orthologue of the BBS protein BBS3 in C. elegans has been shown to be involved in IFT (Fan et al., 2004) and moreover, loss of BBS7 and BBS8 resulted in the formation of short cilia (Blacque et al., 2004).
To fully understand the molecular background of those different ciliophathies, the exact mechanisms by which IFT controls ciliary assembly and maintenance will have to be elucidated. Moreover, the role of individual IFT components within this mechanism will have to be analysed. Badano et al. (2006) therefore claimed that in vivo models that allow the specific blocking and unblocking of ciliogenesis are invaluable. LmxMPK3 mutants of L. mexicana, in particular the inhibitor-sensitised LmxMPK3 mutant, could provide such an in vivo model as the reversible inhibition of the MAP kinase results in a reversible disassembly of the flagellum (see 4.4.4 and 5.4).