Leishmania LABCG1 and LABCG2 transporters are involved in virulence and oxidative stress: functional linkage with autophagy

Background: The G subfamily of ABC (ATP-binding cassette) transporters of Leishmania include 6 genes (ABCG1-G6), some with relevant biological functions associated with drug resistance and phospholipid transport. Several studies have shown that Leishmania LABCG2 transporter plays a role in the exposure of phosphatidylserine (PS), in virulence and in resistance to antimonials. However, the involvement of this transporter in other key biological processes has not been studied. Methods: To better understand the biological function of LABCG2 and its nearly identical tandem-repeated transporter LABCG1, we have generated Leishmania major null mutant parasites for both genes (Delta LABCG1-2). NBD-PS uptake, infectivity, metacyclogenesis, autophagy and thiols were measured. Results: Leishmania major Delta LABCG1-2 parasites present a reduction in NBD-PS uptake, infectivity and virulence. In addition, we have shown that Delta LABCG1-2 parasites in stationary phase growth underwent less metacyclogenesis and presented differences in the plasma membrane's lipophosphoglycan composition. Considering that autophagy is an important process in terms of parasite virulence and cell differentiation, we have shown an autophagy defect in Delta LABCG1-2 parasites, detected by monitoring expression of the autophagosome marker RFP-ATG8. This defect correlates with increased levels of reactive oxygen species and higher non-protein thiol content in Delta LABCG1-2 parasites. HPLC analysis revealed that trypanothione and glutathione were the main molecules accumulated in these Delta LABCG1-2 parasites. The decrease in non-protein thiol levels due to preincubation with buthionine sulphoximide (a gamma-glutamylcysteine synthetase inhibitor) restored the autophagy process in Delta LABCG1-2 parasites, indicating a relationship between autophagy and thiol content. Conclusions: LABCG1-2 transporters from Leishmania could be considered as phosphatidylserine and nonprotein thiol transporters. They probably accomplish transportation in conjunction with other molecules that are involved in oxidative stress, autophagy, metacyclogenesis and infectivity processes. The overall conclusion is that LABCG1-2 transporters could play a key role in Leishmania cell survival and infectivity.