An understanding of the parasite's biology will help control and eradicate this deadly disease. Visceral leishmaniasis is referred to as Kala azar (due to hyperpigmentation) in the Indian subcontinent.
Amastigotes in the mammalian host 7
During the fusion of the phagosome with the lysosome, acidification in the phagosome is prevented by the parasite for a short period of time. In immature DCs, RAB7 recruitment is inhibited by the parasite which ensures long-term survival in the phagosome.
Evasion of host immune response 10
Miltefosine 15
Miltefosine is readily available in the Indian subcontinent and is a good alternative to antimony-resistant VL and CL. Furthermore, gradual increase in the concentration of Miltefosine and exposure for a long time leads to the development of resistance in the Leishmania parasite (Moreira et al., 2011).
Paromomycin 16
Miltefosine can induce drug resistance in the parasite, as it has a long half-life (150 hours), so fears of the emergence of resistant strains of the parasite cannot be ruled out (Perez-Victoria et al., 2006). Some studies have pointed out a transporter-independent entry of sitamaquin into the parasitic cells (López-Martín et al., 2008).
Vaccines for Leishmaniasis 18-20
- Third generation vaccines 19
- Vaccines based on Sandfly saliva 20
- Live attenuated vaccines 20
- Synthetic polyvalent peptide vaccines 20
Another biopterin transporter null mutant (BT1-/-) generated Th1 response with increased levels of IFN-γ and IL12 ( Papadopoulou et al., 2002 ). LCR1 and HASPB1 are membrane proteins that elicited a good response against visceral leishmaniasis (Wilson et al., 1995).
Isolation of Leishmania 22
Organization of control 22
Serious events related to the use of drugs for leishmaniasis need to be highlighted. There is a need to improve detection techniques based on the molecular biology of the parasite.
Leishmaniasis elimination programme 24
Biosynthesis of pyrimidines 24
Resting cells are mostly dependent on the salvage pathway, while the de novo pathway is inhibited (Fairbanks et al., 1995). With the exception of dihydroorotate dehydrogenase, all the enzymes of the de novo pyrimidine pathway are localized in the cytoplasm.
Regulation of de novo pyrimidine pathway in mammalian cells 26
Ser-1859 site is located in the linker region connecting the aspartate transcarbamoylase and dihydroorotase domains in the CAD complex in mammalian cells. Ser-1859 phosphorylation mark on CAD aided the progression through S phase of cell cycle through enhancement in pyrimidine pool synthesis (Robitaille et al., 2013).
Regulation of de novo pyrimidine pathway in E. coli 27
A clearer picture of the regulation of the CAD complex emerged in a landmark publication in 2013, in which it was shown that the CAD complex is activated by Ser-Thr kinase-based mTOR-mediated phosphorylation of Ser-1859 via S6 kinase. A detailed scheme representing the regulation of the de novo pyrimidine biosynthesis pathway can be found in (Figure 1.4).
De novo pyrimidine pathway mediates viral growth 29
2016 reported that the de novo biosynthesis of pyrimidines depends on pyrimidine salvage enzymes in Trypanosoma brucei (Leija et al., 2016). Whole-cell metabolic analysis of a human airway epithelial cell line infected with Staphylococcus aureus showed a drastic reduction in de novo pyrimidine biosynthesis, which was also confirmed by labeling experiments, which supported reduced precursor uptake (Gierok et al., 2016).
Pyrimidine degradation 30
Dihydroorotate dehydrogenase is also emerging as a potential drug candidate for the treatment of malaria, suggesting a broader role played by this enzyme beyond pyrimidine synthesis (Hortua Triana et al., 2016). Starvation of pyrimidine in cells could also lead to apoptosis, as shown in a metabolic screen on myeloma cells treated with AICAr (5-aminoimidazole-4-carboxamide-1-β-riboside) (Bardeleben et al., 2013).
Pyrimidine pathway in Leishmania donovani 31
Another degradation pathway has been discovered in the last decade, leading to the formation of 3-hydroxypropionic acid along with NH3 and CO2. First, the first three enzymes of the mammalian pyrimidine biosynthetic pathway are clubbed together in the form of a trifunctional enzyme called CAD, while the parasite has three independent enzymes for the initial three steps of the pathway (Figure 1.5 represents the de novo pyrimidine pathway in L. donovani). Second, the localization of Ld DHOD is cytoplasmic, while the mammalian counterpart localizes in the inner mitochondrial membrane with dependence on ubiquinone.
Similarly, N-(phosphonoacetyl)-L-aspartic acid (PALA), a specific inhibitor of ATCase, inhibited the growth of both life forms of L.
Dihydroorotase 35
Structure of E. coli dihydroorotase 35
Both subunits showed the same structure except for the tetrapeptide (residues 109-112) orientation lying in the loop region. While the tetrapeptide in subunit A formed part of the protein structure and did not form any hydrogen bonds with DHO in the active site, indicating a loop out conformation. Both zincs in the B subunit are bridged by a carboxylated lysine (Lys 102) as well as the carboxyl group of CA-asp.
In the absence of substrate, the two zinc atoms of subunit B are linked by carboxylated lysine from the inner side and the hydroxyl group in the solvent from the outer side.
Reaction mechanism of dihydroorotase 37
Aquifex aeolicus dihydroorotase 37
Structure and reaction mechanism of mammalian dihydroorotase 38
The directivity of dihydroorotase is dependent on pH, as the cyclization of CA-asp is favored in the slightly acidic pH, while the hydrolysis of DHO is favored in slightly alkaline pH.
Inhibitors of de novo pyrimidine biosynthesis pathway 39
Among them, pyrazofurin (antimalarial) and nucleoside 5' monophosphate derivative of barbiturate (BMP) are potent inhibitors of ODCase (Scott et al., 1986). The A3 compound works by inhibiting pyrimidine biosynthesis and thus starves the virus (Hoffman et al., 2011). Among the flavonols, kaempferol, myricetin and quercitin are aromatic compounds with a heterocyclic pyran-4-one ring and are known to be potent inhibitors of amidohydrolases.
Protection against various types of cancers has also been observed by the administration of kaempferol (Dang et al., 2015).
L-Asparaginase 40
Asparaginase in the form of drug formulations is administered to the patient suffering from ALL. Asparaginases exert their toxic effects because they have an intrinsic glutaminase activity that causes a reduced intake of L-glutamine by the cells of the liver and pancreas. Another study pointed out that the acidic environment in the host cell was weakened by the M.
The coding sequence of chromosome 15 relates to the type I asparaginase and is annotated as cytoplasmic asparaginase-like protein, while the coding sequence of chromosome 36 also relates to type I asparaginase annotated as asparaginase-like protein in the GeneDB database.
Structure and reaction mechanism of asparaginase 41
Human type III asparaginase is an Ntn (N-terminal nucleophilic) hydrolase which requires autoclaving to gain activity; however the Km for asparagine is very high making this enzyme inefficient in catalyzing asparagine. In the first step of the reaction the nucleophile is activated by the adjacent base which then attacks the carbon atom of the amide substrate. In the second step of the reaction, water acting as a nucleophile attacks the carbon atom of the ester following a similar series of steps including the transition state (Michalska and Jaskolski., 2006).
The first threonine is located in the loop region, which mediates the first nucleophilic attack on the substrate, while the second attacking threonine is part of a more rigid structure.
Role of asparaginase in pathogenesis 43
Expression of Type II asparaginase is monitored by cAMP and the oxygen-sensing mechanism regulator of fumarate and nitrate reduction (FNR) protein. The study has provided sufficient evidence that asparagine serves as a key nutrient in the survival of M. In a recently reported study, Type II L-asparaginase has been shown to be a key player in the infectivity of Salmonella typhimurium (McLaughlin et al., 2016).
Although ammonium has been suggested as the preferred nitrogen source for bacteria, but increasing evidence suggests that asparagine may be the preferred nitrogen source (Williams et al., 2015).
Significance of the current work 45
If the other enzyme in the pathway is not inhibited, the feasibility of the reverse reaction remains dim. Flow cytometry-based studies furthered our previous conclusions that both de novo and salvage pathways play a vital role in parasite growth and survival. Whether these asparagine variants confer any advantage in parasite growth or infectivity remains to be addressed.
Further studies will reveal the functional aspects of the two asparaginase variants in L. Leishmania donovani cytoplasmic asparaginase-like protein (LDBPK_150440) is clearly localized in the cytosol of the parasite. Leishmania donovani cytoplasmic asparaginase-like protein (LDBPK_364650) is clearly localized in the parasite cytosol.
Leishmania donovani asparaginase variants tagged with GFP were found to be localized in the cytosol of the parasite. The localization patterns of variants of asparaginase, a key enzyme in aspartate metabolism that provides precursors to the de novo pyrimidine pathway, were found to be cytosolic in L.
Cloning, expression and biochemical characterization of
Cloning of L. donovani dihydroorotase 49
Expression and purification of L. donovani dihydroorotase 50
When the optical density of the culture reached 0.4-0.6, the culture was cooled to 18°C and induced by adding 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) to it and kept for 14 hours in an incubator shaker kept at 18°. C and 180 rpm. The Ni NTA column was prepared by loading it with 1M NiSO4 and pre-equilibrating it with 10 mM imidazole prepared in binding buffer (20 mM Tris-HCl, 250 mM NaCl, pH 8.0). The protein mixture bound to Ni NTA was loaded onto the column and the protein was eluted by applying an imidazole gradient of 20 mM to 300 mM.
The recombinant protein was then subjected to dialysis overnight in dialysis buffer (20 mM Tris-HCl, 250 mM NaCl, pH 8.0).
Determination of oligomeric state 51
Enzyme kinetic characterization 52
Results 53-57
- Kinetic characterization of recombinant L. donovani dihydroorotase 55
All rescue pathway genes were found to be overexpressed in the kaempferol-treated parasites (Figure 4.3A). Kaempferol and zebularine-mediated inhibition of the de novo and salvage pathway led to a change in the gene expression of the pyrimidine pathway, and significant DNA damage was also induced. Asparaginase, a key enzyme in the aspartate metabolism pathway, is present in the form of two variants in the Leishmania donovani parasite, which is the cause of visceral leishmaniasis (VL).
The growing concern in the scientific community regarding drug-resistant strains of Leishmania provides a new impetus to investigate the metabolic pathways of the parasite. Whatever the case may be, the bulk of the asparaginase variants in the parasites will be ruled out by genetic and biochemical studies. The existence of the parasite in the dual host system (sandfly and mammalian host) in varying pH (alkaline in sandfly and acidic in the phagolysosome of macrophage) led us to determine the pH optima for dihydroorotase in both directions.
Asparaginases in many intracellular parasites such as M. typhimurium have been implicated to exert their effects on parasite infectivity. These findings have increased interest in L. donovani asparaginases as to what role they may play in parasite growth and infectivity.
