Heme A biosynthesis is essential for Trypanosoma cruzi

MARCELO L MERLI; BRENDA A. CIRULLI; LUCAS PAGURA; JULIA A CRICCO

Newport, RI

Conferencia; Chemistry & Biology of Tetrapyrroles Gordon Research Conference; 2014

Gordon research Conference

Heme A biosynthesis is essential for Trypanosoma cruzi Heme is an essential cofactor for aerobics organisms, most of them synthesize it by a conserved route through all domains of life. However, several aerobic organisms do not produce heme but contain heme-proteins involved in essential metabolic pathways. Within this group we can find several pathogenic microorganisms, relevant for human health like trypanosomatids (Trypanosoma cruzi, Trypanosoma brucei and Leishmania spp., responsible of Chagas disease, sleeping sickness and visceral or cutaneous or mucocutaneous leishmaniasis respectively). Trypanosoma cruzi lacks of any heme biosynthesis pathway but presents several essential heme-proteins (Tripodi et al., 2011). It contains a mitochondrial respiratory chain with a CcO as a terminal oxidase. Based on several biochemical assays it was proposed that T. cruzi includes a branched respiratory chain with a aa3-type cytochrome c oxidase as main terminal oxidase. In this model, the other putitave oxidases might be a Trypanosomatid Alternative Oxidase (TAO) as it was described in T. brucei or another terminal oxidase named as cytochrome o oxidase (cyt o). These other oxidases might be the responsible for the remanent O2 consumption when CcO was inhibited by cyanide. At the moment none of them have being identified and characterized (Stoppani et al.,1980; Silva et al., 2011). In our group we are interested to elucidate how T. cruzi transport heme to the mitochondrion (Trypanosomatids present only one mitochondrion per cell) to be used on the mitochondrial heme proteins, how heme A is produced and how it is inserted into the CcO complex. We identified two genes in T. cruzi genomic sequence that encode for proteins with homology sequence to Cox10 and Cox15 enzymes. These genes, named TcCOX10 and TcCOX15, were expressed in yeast Δcox10 and Δcox15 cells, respectively, suppressing their respiratory deficiency. These and other results obtained in our lab confirmed the HOS activity for TcCox10 protein and the HAS activity for TcCox15 protein (Buchensky et al., 2010). Both proteins were expressed as fusion proteins with a C-terminal His tag and they were detected by western blot assays on mitochondrial fractions obtained from the corresponding yeast cell cultures (Buchensky et al., 2010). In our previous results, the expression of TcCOX10 gene (TcCOX10.6xHIS) was under control of a constitutive promoter (ADH1 promoter in a high copy number vector). However, when we expressed TcCOX10.6xHIS gene under the control of an inducible promoter (MET25 promoter, in a high copy number vector) we did not observed suppression of the respiratory deficiency of Δcox10 yeast cells. However, when the Δcox10 containing TcCOC10.6xHIS cells were grown at lower methionine concentrations (MET25 promoter is repressed at high methionine concentrations), a weak suppression of the respiratory deficiency was observed. The presence of TcCox10.His protein was verified by western blot analysis. Based on these results we suggest different hypothesis for the lower HOS activity of TcCox10.His protein in Δcox10 yeast cells. One of them could imply a less efficient mitochondrial importation and/or folding processes for TcCox10. Another, an incorrect folding of this protein in the mitochondrial inner membrane, being less stable compared to the native Cox10. A third possibility could imply a weaker interaction between TcCox10 and the rest of yeast mitochondrial proteins involved in heme A biosynthesis and/or hemilation of CcO as it was postulated for Cox10 protein (Khalimonchuk et al., 2012 and Bestwick et al., 2010). These scenarios were not evident for TcCox15 because the expression of TcCOX15 gene (TcCOX15.6xHIS) always suppressed the respiratory deficiency of Δcox15 yeast cells (TcCOX15.6xHIS, cloned in the same vector under MET25 promoter). One approach followed to study TcCox10 and TcCox15 in the parasite itself implied the cloning of both genes in a T. cruzi inducible expression vector (pTcIndex vector inducible by tetracycline, Taylor and Kelly, 2006). Both genes were cloned to be expressed as fusion proteins with a C- terminal his tag or with his tag plus GFP (TcCox10.His, TcCox15.His, TcCox10.His.GFP and TcCox15.His.GFP). Prior to use these new constructions to transfect T. cruzi epimastigotes (TcCOX10.6xHIS.GFP and TcCOX15.6xHIS.GFP), we tested them in Δcox10 and Δcox15 yeast cells, respectively. Surprisingly, we observed that Δcox10 containing TcCOX10.6xHIS.GFP were able to restore respiratory deficiency more efficiently than Δcox10 containing TcCOX10.6xHIS. The inclusion of C-terminal GFP tag enhance the performance of TcCox10 (in yeast cells) stabilizing TcCox10 or helping to fold and maintain TcCox10 in the mitochondrial inner membrane. By the other hand, Δcox15 cells containing TcCOX15.6xHIS.GFP gene were much less efficient to restore respiratory deficiency than Δcox15 cells containing TcCOX15.6xHIS. This fact suggested that the C-terminal GFP affect the activity of TcCox15 (in yeast), destabilizing the protein or the interactions with other mitochondrial proteins involved in hemilation of CcO as was recently proposed by Bareth et al. (Bareth et. al, 2013). This new scenario opened new questions we will try to answer to understand similarities and differences in heme A biosynthesis between a non heme producer organism like T. cruzi and other that can produce heme like yeast. Considering the impossibility of partial or total silencing T. cruzi´s genes to evaluate the effect of loss of function, we designed several TcCox10 and TcCox15 mutants to analyze how affect parasite proliferation in a WT context. We changed some residues reported relevant for catalytic activity in both proteins (Buchensky et al., 2010) obtaining the following mutants: TcCox10 N128K, TcCox10 H248A and TcCox10 R144-148A, TcCox15 H129A, TcCox15 H206S and TcCox15 H307A. The activity of these mutants were tested in yeast Δcox10 and Δcox15 cells, respectively. They were inactive, because they did not restore the respiratory deficiency of mutants yeast cells, confirming their presence by western blot assays using anti-TcCox10 or anti-TcCox15 antibodies (previously obtained) and commercial anti-His antibodies. Later, the TcCOX10 , TcCOX15 WT and mutant genes were cloned in the inducible vector for T. cruzi, pTcIndex, and these constructions were used to transfect epimastigotes. After the selection processes, we analyzed the effect caused on parasite proliferation by the induction of TcCOX15 WT and mutants genes. It was observed that the presence of no active TcCox15 mutant proteins (H129A and H307A) caused a negative effect on epimastigote growth, verifying by western blot and indirect immunofluorescence assays. The production of heme A was analyzed spectrophotometrically and our preliminary results confirmed a lower heme A concentration in cells containing the mutant TcCox15 proteins. At the moment we are analyzing the expression of TcCOX10 WT and mutants genes in epimastigotes. Also we are studying the effect of the expression of TcCOX15 WT and mutants genes on metaclyclogenesis (from epimastigotes to metacyclic trypomastigotes) and infection capabilities. Together all the results presented allowed us to postulate that TcCox15 is HAS synthase in T. cruzi. Heme A is an essential cofactor for T. cruzi epimastigotes and the CcO activity is relevant, at least in this life stage. Considering the presence or absence of other terminal oxidases (branched respiratory chain) was not confirmed, our results support the hypothesis that present CcO as the main and essential mitochondrial oxidase. Although T. cruzi showed a conserved function for HOS and HAS in heme A biosynthesis played by TcCox10 and TcCox15 proteins, our results let us postulate a new scenario where the regulation of this pathway and the interactions between Cox10 and Cox15 (HOS and HAS) and other proteins involved in heme A insertion into CcO in T. cruzi (a no-heme producer organism) could be different compared to other organisms like yeast. We cannot exclude the possibility that these proteins might be playing another role in this parasite along with the HOS and HAS function. References: Bareth, B., Dennerlein, S., Mick, D. U., Nikolov, M., Urlaub, H., and Rehling, P. 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