2.10 Canales de distribución
3.1.1. Factores determinantes del proyecto
3.1.1.5 Disponibilidad de materia prima
The results obtained from LA analogue experiments indicate that in L. major promastigotes, LA biosynthesis and salvage pathways might be redundant. In order to test this possibility further, a gene replacement strategy was taken, with the hypothesis that it would be possible to replace genes encoding either LIPA or LPLA. The results were very surprising, since every attempt to replace either gene in L. major promastigotes resulted in the duplication of the target gene by genome amplification. Leishmania are renowned for their genomic plasticity (Cruz et al., 1993), and it is known that although Leishmania are mostly diploid, some chromosomes are aneuploid, such as chromosome 1 in L. major (Martinez-Calvillo et al., 2005; Sunkin et al., 2000). Also, it has been established in attempted knockout studies of L. major dihydrofolate reductase-thymidylate synthase (DHFR- TS) (Cruz et al., 1993), a hypothetical gene (LmjF01.0750) (Martinez-Calvillo et al., 2005), L. mexicana cdc2-related kinase 1 (CRK1) (Mottram et al., 1996) and L. tarentolae J-binding protein 1 (JBP1) (Genest et al., 2005) that targeting of essential genes in Leishmania frequently results in aneuploidy for the chromosome in question, tetraploidy, or the creation of ectopic DNA fragments called amplicons (Feng et al., 2009b; Genest et al., 2005).
The efficiency of correct targeting of the LIPA and LPLA genes was high, which rules out the possibility of either gene occupying a non-recombinogenic region of chromosomal DNA. Indeed, after integration of a stable re-expressing copy of LPLA into the 18S SSU RNA locus it was possible to obtain a LPLA null mutant, which suggests that the endogenous LPLA gene is essential in promastigotes. However, despite the expression of an ectopic copy of LPLA, the growth of the LPLA null mutant is significantly slower than that of wild-type at all time points on the growth curve. Unfortunately, the immunogenic response raised against recombinant LPLA protein in rabbit was poor, and as such the α-LmjLPLA polyclonal antibody generated was not useful in western blot analyses, despite multiple attempts to purify and concentrate the antibody (data not shown). It is therefore not possible to make any conclusions with regards to the expression level of LPLA under normal conditions or in the null mutant containing an ectopic
copy of LPLA. In terms of LIPA, it was not possible to generate a null mutant, even in the presence of an integrated ectopic copy of the gene. It is likely that LIPA is essential, however it is not possible to make as firm a conclusion as it is for LPLA.
The obvious question is, why would both LIPA and LPLA proteins be essential for survival of L. major promastigotes? In E. coli, LA biosynthesis and salvage pathways are redundant (Morris et al., 1995), and based on the results gained from the effect of LA analogue experiments in this thesis, I expected the same to be true in L. major. The first possible explanation is that in L. major promastigotes, LIPA and LPLA are important but not essential, and that the inability to successfully replace both alleles could be due to limitation(s) in the methods used to achieve this. For example, promastigotes may be able to survive without salvage of LA, yet after replacement of the second LPLA allele, the parasites may take some time to up-regulate the biosynthesis pathway. The procedure I followed involved incubating transfected parasites for 24 h before cloning. Upon cloning it is clear that competition with other parasites is not an issue, however it could be that during the 24 h period preceding the initiation of cloning, the LPLA null mutants are out-competed/over-grown by parasites with duplicated genomes that do not need to switch metabolic pathways to survive. I would argue against this because promastigotes do not grow exponentially during the 24 h post transfection (data not shown). In terms of the attempted creation of a LIPA null mutant, 10 µM LA was included in each step of selection of the second-round allele replacement, yet it was still not possible to replace the remaining allele without the parasites duplicating their genome. This again provides support for the notion that both LIPA and LPLA are essential, since as explained above, if LPLA is important when LA is not limiting, replacement of the LIPA gene should be feasible.
A second possibility to explain the essentiality of both LIPA and LPLA is that only one of the two pathways used to acquire LA is active at any one time, and that the expression of the genes involved in these pathways is regulated by LA availability in the medium. It seems peculiar that if an organism has the genetic capacity to encode a compensatory enzyme/set of enzymes to complement a growth defect, it would not do so. For example, an ACP knock-down mutant in human HEK293 T cells resulted in decreased cell growth and eventually death by 72 h in culture, and the principal cause was due to an almost complete loss of protein lipoylation (Feng et al., 2009a). This study highlighted the importance of LA biosynthesis in lipoylation of α-KADHs and the GCC (Feng et al., 2009a). Also, it has previously
been shown in mouse that deletion of lipA has an embryonic-lethal phenotype (Yi & Maeda, 2005; Yi et al., 2009), which further emphases the importance of LipA in mammals. Nevertheless, in the HEK293 T cell ACP knock-down line, addition of 2 µM exogenous LA in the growth medium did not rescue growth or result in increased lipoylation of α-KADHs and H-protein (Feng et al., 2009a). This result argues against the possibility that LA availability in the medium was a limiting factor affecting the capacity of LA salvage to compensate for lack of LA biosynthesis. Also, in this thesis 10 µM LA was included in the growth medium at all steps during the selection of a LIPA null mutant, and yet LA salvage could still not compensate for a loss of LIPA protein. This fact thus opposes the theory that the LA salvage pathway is 'switched off' in LA-poor conditions.
A third possibility to explain the essentiality of both LIPA and LPLA is that the pathways for LA biosynthesis and salvage are indeed redundant in terms of lipoylating α-KADHs and the GCC, yet the enzymes involved could have essential roles other than catalysing lipoylation reactions. For example, in E. coli, LipB has a surprising additional role as a negative regulator of dam gene expression. The dam gene encodes a DNA methyltransferase which transfers methyl groups from SAM to adenine residues in the sequence 5'-GATC-3' in double stranded DNA, and has a large impact on the chromosome replication, gene expression and mismatch repair (Barras & Marinus, 1989). Also, in P. falciparum, an unexpected result was obtained whereby LipB null mutants exhibited a faster growth phenotype that was mainly due to accelerated progression through the intraerythrocytic cell cycle (Gunther et al., 2007). Again, it could be that LipB has a role other than octanoylation of the PDH in P. falciparum (although it cannot be ruled out that in the P. falciparum LipB null line, increased growth rate is due to an increase in myristate production through type II FASI) (Gunther et al., 2007).
A body of data is also accumulating that implicates LA biosynthesis in mitochondrial RNA processing (Hiltunen et al., 2009). In 1993, a screen for mutants with altered mitochondrial tRNA precursor/product ratios resulted in the identification of the lip5 mutant (Sulo & Martin, 1993). The lip5 mutant had a non- functional lipA gene and as a result completely lacked lipoylated α-KADHs and GCC. Other phenotypes included accumulation of mitochondrial tRNA precursors, destabilisation of the mitochondrial genome and poor cell growth, which was not rescued by the addition of 2 µM LA (Sulo & Martin, 1993). In S. cerevisiae,
mutants in type II FAS genes lack lipoylated α-KADHs and GCC (Schonauer et al., 2008). It had been hypothesised that the main role of type II FAS is the provision of octanoyl-ACP as a substrate for LA biosynthesis (Brody et al., 1997; Hiltunen et al., 2009; Wada et al., 1997), however this particular study went a step further to show that FASII mutants had similar RNA processing phenotypes to that of the lip5 mutant (Schonauer et al., 2008). The authors illustrated that mutants of the pdh,
α
-kgdh and gcc genes did not cause RNA processing phenotypes, and it was proposed that biosynthesis of LA is not just involved in lipoylating α-KADHs and the GCC, but is also somehow implicated in RNA processing (Schonauer et al., 2008). Interestingly, a similar phenotype of LA depletion and changes in mitochondrial ultrastructure was observed in a T. brucei PCF ACP knock-down line (Stephens et al., 2007). A follow-up study subsequently showed that RNAi of ACP resulted in malformation of the mitochondrial membrane, and the authors concluded that the principle cause of the RNAi phenotype was due to a change in phospholipid composition of the mitochondrial membrane (Guler et al., 2008). It would be interesting to determine whether RNA processing is also affected in this line, and whether LA is also the cause of this phenotype. Unfortunately, it was outside the scope of this thesis to investigate the exciting possibility that enzymes involved in LA metabolism play key roles in cellular functions other than lipoylation of α-KADHs and the GCC.A fourth and final possibility to explain the essentiality of both LIPA and LPLA in L. major is that the LA biosynthesis and salvage pathways lipoylate their apoproteins in a substrate-specific manner. Given that all four apoproteins (H-protein, E2k, E2p and E2b) are lipoylated (and assumedly active) during logarithmic growth phase in L. major (see Figure 3.1), there is a possibility that three of them are essential to parasite survival (excluding the H-protein, which has been shown to be dispensable in L. major promastigotes (Scott et al., 2008)). If LA biosynthesis and salvage have differential substrate specificities for the α-KADHs, one would then expect that these genes be essential to promastigote survival. An interesting observation in E. coli is that the lipB null mutant KER184 can grow on any medium that bypasses the need for the α-KGDH (for example, on succinate-containing medium), however acetate-containing minimal medium is not sufficient to sustain growth. This result infers that in the absence of LipB, LplA is not able to sufficiently lipoylate the α-KGDH to permit cell growth, yet can lipoylate the PDH enough to allow colony formation (Reed & Cronan, 1993). Another interesting study showed
that in L. monocytogenes, which encodes two LplA enzymes (LplA1 and LplA2) but lacks LA biosynthesis enzymes, LplA1 is essential for intracellular survival of the bacterium, and that LplA1 and LplA2 are not redundant (O'Riordan et al., 2003). This interesting phenomenon was shown to be due to the fact that LplA1 can use lipoyl-peptides and ATP as substrates for lipoylation, whereas LplA2 requires free LA and ATP (Keeney et al., 2007).