| dc.description.abstract | The disease malaria afflicts more than a billion people and kills almost one
to three million of them every year. Of the four species of Plasmodium affecting man viz., P. falciparum, P. vivax, P. ovale and P. malariae, Plasmodium falciparum is the deadliest as it causes cerebral malaria. The situation has become worse with the continuous emergence of drug resistance in the parasite. Therefore, improving existing drugs and deciphering new pathways for drug development are the need of the hour. The discovery of the type II fatty acid biosynthesis pathway in Plasmodium falciparum (Surolia and Surolia, 2001) has opened up new avenues for the development of new antimalarials as this pathway is entirely
different from the human host in which type I pathway exists. Although many biochemical pathways such as the purine, pyrimidine and carbohydrate metabolic pathways, and the phospholipid, folate and heme biosynthetic pathways operate in the malaria parasite and are being investigated for their amenability as antimalarial therapeutic targets, no antimalarial of commercial use based on the
direct use of these biochemical pathways as targets has emerged so far. This is due
to the fact that the structure and function of the targets of these drugs overlaps
with that of the human host. A description of such pathways forms the Chapter 1
of the thesis. This is followed by a description of the discovery and the importance of fatty acid biosynthesis pathway and the available literature on the various enzymes that are targets of potential antimalarials. Three isoforms are known for condensing enzymes - FabH which functions in initiation, and FabB and FabF
which function in elongation. These isoforms differ in their biochemical properties and have unique roles to play in deciding the membrane composition of any organism. This aspect is also discussed in this chapter.
Cloning and expression of -ketoacyl-ACP synthase, FabB/F from Plasmodium falciparum is described in Chapter 2. PfFabB/F is coded by the nuclear genome and is targeted to the apicoplast. The gene is coded by the locus
MAL6P1.165 and the putative amino acid sequence of the protein exists in PlasmoDB. All apicoplast targeted proteins have a characteristic bipartite leader sequence consisting of a signal and a transit peptide sequence (Waller et al., 1998). Since the mature protein start site was not known and none of the software packages could predict the site, I aligned the PfFabB/F sequence with the sequences of other -ketoacyl-ACP synthases. On the basis of similarity with E.
coli synthases and the mature protein start site of plant synthases, I cloned the first
construct of PfFabB/F. The sequence was amplified by PCR and ligated in pET as
well as pGEX vector. Expression in various hosts under different temperature and
induction conditions could not solubilize the protein in significant quantities and
most of the protein was found in inclusion bodies. Next I expressed the sequence
with five more amino acids towards the N-terminal and expressed it as an N-
terminal NusA fusion. The protein was purified by single step Ni-NTA affinity
chromatography. Along with the full length protein (108 kDa), a truncated version
of the protein was also obtained. The identity of the protein was confirmed by
western blotting using anti-His antibody and anti-FabB/F antibody.
In Chapter 3, the substrate specificity of PfFabB/F has been elucidated. PfFabB/F condenses malonyl-ACP with a range of acyl-ACPs. In vivo, acyl carrier protein (ACP) shuttles the acyl substrates between various enzymes of the fatty acid biosynthesis pathway. Enzymes of the pathway other than synthases can accept substrate analogs like acyl-CoA and acyl-NAC’s also in vitro. Acyl-ACPs are not very stable species and thus are not commercially available. Therefore,
they have to be synthesized. Since malonyl-ACP could not be synthesized by chemical means, enzymatic synthesis of acyl-ACPs was done. Acyl-ACP synthetase (Aas) or holo-ACP synthase (ACPS) can be used for enzymatic
synthesis. Aas is specific only for longer chain substrates; therefore, I decided to
use holo-ACP synthase, an enzyme responsible for converting apo-ACP to holo-ACP in the presence of CoA in vivo (Lambalot and Walsch, 1995). When acyl-CoAs are supplied in place of CoA, acyl-ACP is produced. Malonyl-ACP and acyl-ACPs (C4-C16:1) were thus synthesized using holo-ACP synthase from E. coli. The reaction went to almost 95% completion, indicating broad substrate
specificity of this enzyme. Bacterial or plant acyl-ACPs of different chain lengths
can be resolved by Conformation Sensitive PAGE (Heath and Rock, 1995, Post-
Beittenmiller et al., 1991). However, Pfacyl-ACPs synthesized using ACPS did not show any significant shift on CS-PAGE. Therefore I resorted to MALDI-TOF (Matrix Assisted Laser Desorption and Ionization- Time Of Flight) for monitoring the PfFabB/F condensation reactions. PfFabB/F condensed C4-C12-ACPs with malonyl-ACP to their corresponding -ketoacyl-ACP products, with C6, C8 and
C10-ACPs being most readily elongated. C14-ACP was very sluggishly elongated, and C16 and C16:1-ACPs were not elongated at all. The condensation reaction was also followed by autoradiography using14C labeled malonyl-ACP, exploiting the clear mobility shift between malonyl-ACP and the other acyl-ACPs.
The inhibitory effect of cerulenin, a known inhibitor of condensing enzymes was also checked. PfFabB/F also exhibited malonyl decarboxylase activity resulting in
the production of acetyl-ACP in the absence of any significant condensation activity.
All the enzymes of fatty acid synthesis pathway required to complete a cycle were assembled together for the in vitro reconstitution of Plasmodium fatty acid synthesis cycle which is described in Chapter 4. Earlier studies of Surolia &
Surolia have shown that C12 and C14 fatty acids are the major constituents of
Plasmodium lipids. One of my objectives was to determine the maximum length of the acyl ACP product that is synthesized when all the functionally active enzymes of fatty acid synthesis are put together (Kapoor et. al, 2001, Sharma et al., 2003, Karmodiya and Surolia, 2006). Condensing enzymes have a
deterministic role in the fatty acid composition as they catalyze the only
irreversible step in fatty acid biosynthesis. By analyzing products of the
elongation cycle by mass spectrometry it was apparent that C14-ACP is the longest species formed. As already mentioned, PfFabB/F readily elongates C12-ACP but C14-ACP is weakly elongated. Thus the end product of the Plasmodium FAB pathway is influenced by the substrate specificity of PfFabB/F. This
confirms the role of PfFabB/F as a decisive enzyme in determining the length of
fatty acids synthesized. The inhibition of the cycle by cerulenin and triclosan is
also described in this chapter.
Chapter 5 describes the ability of the PffabB/F gene to complement for the mutation of condensing enzymes in CY244 cells (fabBtsfabF-, Yasuno et al., 2004). CY244 cells were transformed with pBAD alone or PfFabB/F cloned in pBAD vector (pBADPffabB/F) and the growth was monitored at non-permissive temperature. The product of PfFabB/F could rescue the growth of mutant cells at high temperature but only in the presence of oleic acid. FabB and FabF are the isoforms of condensing enzymes involved in elongation of the fatty acid synthesis cycle but they have a unique role to play (Garwin et al., 1980). FabB is
responsible for unsaturated fatty acid synthesis, and fabB-mutants require oleic
acid supplementation for growth. FabF is utilized in temperature regulation of
membrane fluidity and E. coli FabF elevates the level of C18:1 or cis-vaccenic
acid at lower growth temperature but FabF-mutants have no growth phenotype
(Ulrich et al., 1983). Rescue of CY244 cells in the presence of oleic acid
supplementation indicated that the PffabB/F gene behaves like FabF and not FabB. Analysis of the fatty acid composition of membrane lipids of CY244 cells transformed with pBAD vector or pBADPffabB/F by GC-MS demonstrated no elevated levels of cis-vaccenic acid in transformed cells. This observation is in agreement with the in vitro determined substrate specificity data which shows that PfFabB/F does not elongate C16:1ACP.
The thesis ends with a summary of the findings in Chapter 6 in the context of FabB and FabF enzymes known from other sources.
2, 4, 4’-Trichloro-2’hydroxydiphenylether, commonly known as triclosan, has been used as a topical antibacterial agent for decades. I determined its efficacy
in treating acute systemic bacterial infection in mouse model. Triclosan, as
compared to other well known antibiotics, could extend the survival time of mice
by 48 hours. This work is described in Appendix I. (Sharma et al., 2003) | en_US |
