Trypanosoma cruzi, could restrict the intracellular heme content through its transport

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

Newport RI, United States.

Conferencia; 2016 Chemistry & Biology of Tetrapyrroles Gordon Research Conference. Salve Regina University,; 2016

Gordon Research Conference

Heme is an essential cofactor for aerobic 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 a heme biosynthesis pathway but presents several essential heme-proteins (Tripodi et al., 2011). It contains a mitochondrial respiratory chain with an aa3-type cytochrome c oxidase as a main terminal oxidase. T. cruzi also contains heme-proteins involved in ergosterol and unsaturated fatty acid synthesis (CYP450, cyt b5, etc) (Tripodi et al., 2011)It was previously proven that T. cruzi epimastigotes? proliferation depends on heme in a dose dependent manner and that the heme uptake might occur via a specific porphyrin transporter, possibly a member of the ABC-transporter family (Cupello, et. al, 2011). Our group is interested in elucidating how T. cruzi takes heme from different hosts, how heme transport is regulated, how heme is distributed inside the cell and how heme is transported to the mitochondrion (Trypanosomatids present only one mitochondrion per cell) to be used by the mitochondrial heme proteins, how heme A is produced and how it is inserted into the CcO complex. We used several fluorescent heme analogs as a tool to elucidate the heme transport along the different life-cycle stages. First, we demonstrated that T. cruzi transports heme in the replicative life-cycle stages, the epimastigotes in the midgut of the insect vector and the intracellular amastigote in the mammalian host, but not in the infective form, the trypomastigote stage. Our results revealed the existence of a protein transporter that might discriminate between structurally related compounds (Merli, et al., 2016). Also, we characterized a T. cruzi protein, named TcHTE for T. cruzi Heme Transport Enhancer, which is conserved in trypanosomatids, presenting sequences homology to LHR1 of L. amazonensis and Ce-HGR4 of C. elegans. Our results demonstrate that TcHTE plays a critical role in T. cruzi´s heme transport. Surprisingly, TcHTE was not homogenously distributed along the plasma membrane or in internal vesicles as it was observed for LHR1 on L. amazonensis promastigotes (Huynh, et. al., 2012). These observations might indicate differences in heme transport between these trypanosomatids, which could be mediated by different transporters and/or involving different mechanisms. Although many transporters have been biochemically characterized in T. cruzi, their locations remain unknown (Silber, et. al, 2002, Canepa, et. al, 2009); however it is established that the trypanosomatids? transport proceeds through the flagellar pocket (Krishnamurthy G, et. al, 2005, Landfear SM, et. al, 2001) precisely where TcHTE was found. Recently, we obtained polyclonal antibodies that specifically recognize TcHTE, which were used to evaluate the presence of TcHTE along the different T. cruzi life stages. Our recent and unpublished results show that TcHTE was present primarily in the epimastigote and amastigotes stages, and it was barely being detected in the trypomastigotes stage. The protein was present in the flagellar pocket region during the life-stages were heme is imported from the environment. Also, the TcHTE protein level changes accordingly to the variation in the intracellular heme (and/or selectively heme analogs) content. These finding support the hypothesis that TcHTE is critical for heme transport. Taking these results into account we postulate that TcHTE plays a relevant role in T. cruzi heme transport/trafficking enhancement regulating this activity, without excluding it as part of or associated with the transporter complex. Considering the critical role of heme as an essential cofactor in T. cruzi, and the fact that its uptake inhibition results deleterious for the parasite, new approaches will be required for the complete identification of the molecular partners involved in its transport and distribution. Their identification and characterization might result in new molecular targets to inhibit parasite proliferation.