Proline transport inhibitors trigger differential responses in Trypanosoma cruzi growth inhibition

Background: Proline is a fundamental amino acid for Trypanosoma cruzi, the etiological agent of Chagas disease. Proline is mainly incorporated from the extracellular medium by amino acid transport systems. Different proline analogues proved to interact with the proline permease TcAAAP069 and inhibit the proline uptake by T. cruzi. Methods: Decyl- (1), oleyl- (2) and farnesyl- (3) substituted proline analogues were evaluated on six T. cruzi DTUs. Cell death type was determined by flow cytometry, and the effect on the parasite metabolism was analysed by directed NMR exometabolomics. Structural modifications of 1 (compounds 4 – 6) were implemented to have more information on the mode of action (MoA). Results: The compounds showed broad-spectrum activity against all DTUs. Compound 3 at high concentration (116 µM) induced necrosis. The removal of the triazole from 1 proved to be important for the activity. Compounds 1 and 2 induced deep changes in the exometabolome, diminishing the amounts of succinate, lactate, acetate, and ethanol excreted. The fluorescent labelling and subsequent microscopy showed that compound 1 can be taken up by epimastigotes. Conclusions: Two different MoA related to proline transport for decyl and farnesyl-substituted proline analogues are proposed. The former presented an in-cell action while the latter was not taken up by the parasites but interacted with the extracellular side of the proline permease. General Significance: Subtle structural variations in the compounds determine differences in the MoA. This finding opens new perspectives that should be examined on the development of new drugs targeting metabolite permeases.

Low-proline environments impair growth, proline transport and in vivo survival of Staphylococcus aureus strain-specific putP mutants

Staphylococcus aureus is a common cause of disease in humans, particularly in hospitalized patients. This species needs to import several amino acids to survive, including proline. Previously, it was shown that an insertion mutation in the high-affinity proline uptake gene putP in strain RN6390 affected proline uptake by the bacteria as well as reducing their ability to survive in vivo. To further delineate the effect of the putP mutation on growth of S. aureus strain RN6390, a proline uptake assay that spanned less than 1 min was done to measure transport. An eightfold difference in proline levels was observed between the wild-type strain and the high-affinity proline transport mutant strain after 15 s, indicating that the defect was only in proline transport and not a combination of proline transport, metabolism and accumulation that would have been assessed with longer assays. A putP mutant of S. aureus strain RN4220 was then grown in minimal medium with different concentrations of proline. When compared to the wild-type strain, the putP mutant strain was significantly growth impaired when the level of proline was decreased to 1·74 μM. An assessment of proline concentrations in mouse livers and spleens showed proline concentrations of 7·5 μmol per spleen and 88·4 μmol per liver. To verify that the effects on proline transport and bacterial survival were indeed caused solely by a mutation in putP, the putP mutation was complemented by cloning a full-length putP gene on a plasmid that replicates in S. aureus. Complementation of the putP mutant strains restored proline transport, in vitro growth in low-proline medium, and in vivo survival within mice. These results show that the mutation in putP led to attenuated growth in low-proline media and by corollary low-proline murine organ tissues due to less efficient transport of proline into the bacteria.