LEISHMANIASIS: A RE-EMERGING NEGLECTED DISEASE. BIOMOLECULAR AND METABOLOMIC STUDIES AIMED AT IMPLANTING ITS CONTROL IN EMILIA-ROMAGNA

Background. Leishmaniases (VL) represents a major health problem. The first part of the thesis evaluates by molecular techniques and cytokine analysis the prevalence of asymptomatic Leishmania infections in autoimmune rheumatic patients treated with biological drugs and living in Leishmania endemic foci in Italy. In the second part, the effect of the niacin analogue, 6-AN on Leishmania parasite growth and metabolism using the metabolomics technology was investigated. The pentose phosphate pathway (PPP), has been reported as a target of 6-AN, thus PPP might represent a good target. Methods. VL qualitative and real-time PCR were performed on DNA extracted from PBMCs from 50 autoimmune rheumatic patients treated with immunosuppressive biologic drugs for at least 5 years. Plasma cytokine concentrations were also measured in plasma from Leishmania DNA-positive and -negative rheumatic patients as well as from the healthy control group. In the 6-AN study, L. mexicana M379 and L. infantum PCM5 promastigotes were treated with 7.8 mM 6-AN and 2.17% DMSO for 24 hours. After vitality, infectivity of 6-AN-treated promastigotes to mouse macrophages, and 6-AN interactions with oxidizing compounds were also studied. Small metabolites were extracted and analysed by pHILIC-LC-MS in polarity switching mode and data were analysed with IDEOMv19 and MetaboAnalyst 3.0. Results.Eighteen out the 50 (36%) autoimmune rheumatic patients were positive for Leishmania DNA by conventional and/or quantitative PCR with a detection of high parasite burdens (1 to 136 parasite/ml in 4 patients, 1.000 to 40.000 in 11 patients and over 1.000.000 in 3 patients). Patients that were taking a steroid in association with the biological drug showed a higher positivity for circulating L. infantum kDNA than those given the biological drug only (p<0.05). Pro-inflammatory IL-1, IL-6, IL-12(p70), IL-7, IL-15, IFN-γ and TNF-α; anti-inflammatory IL-4, IL-13; and regulatory IL-10 cytokines were markedly elevated in all autoimmune rheumatic patients with additional increases in inflammatory mediators in autoimmune rheumatic patients positive for Leishmania DNA. In both L. mexicana and L. infantum, 6-AN caused significant depletion of phosphoribosylpyrophosphate (PRPP) and nicotinate (Na) and as a result purine and pyrimidine nucleotides were reduced and their nucleobases accumulated. Glutathione, ribose-5-phosphate, 6-phosphogluconate levels and downstream PPP intermediates were similar to controls. For L. infantum, it was possible to analyse NAD+ and NADPH, which were found decreased together with the PPP intermediate D-sedoheptulose 7-phosphate. Moreover, 6-AN treatment caused a marked elongation in parasite body. 6-AN in combination with the oxidizing compounds has additive effects against Leishmania and did not affect the infectivity of the treated promastigotes to mouse macrophages. Conclusions.VL molecular screening and cytokine analysis should be taken into account before treating autoimmune rheumatic patients with biologic drugs, especially in rural areas. In mammals 6-AN is converted to abnormal 6-ANAD/P by NAD+ glycohydrolase, however, in Leishmania its toxicity is only seen in millimolar range, in which 6-AN is responsible for the depletion of cellular phosphoribosyl pyrophosphate (PRPP) content probably in the Preiss-Handler NAD+ salvage pathway, resulting in depletion of nucleotides required for nucleic acid biosynthesis. The marked elongation in the 6-AN-treated parasite bodies confirms nucleotide starvation. Leishmania NAD+ glycohydrolase might decompose NAD+ but might not catalyze exchange reactions, as found in other microrganisms, however, combined 13C-glucose labeling and flux analysis might be useful to ascertain the fate and action mechanism of 6-AN in Leishmania. In addition, PRPP synthetase should also be a good target for new potential drugs against leishmaniasis pointing to the growth-inhibitory effect of PRPP depletion.