Silver and Nitrate Oppositely Modulate Antimony Susceptibility through Aquaglyceroporin 1 in Leishmania (Viannia) Species

Abstract

d Antimony (Sb) resistance in leishmaniasis chemotherapy has become one of the major challenges to the control of this spreading worldwide public health problem. Since the plasma membrane pore-forming protein aquaglyceroporin 1 (AQP1) is the major route of Sb uptake in Leishmania, functional studies are relevant to characterize drug transport pathways in the parasite. We generated AQP1-overexpressing Leishmania guyanensis and L. braziliensis mutants and investigated their susceptibility to the trivalent form of Sb (Sb III ) in the presence of silver and nitrate salts. Both AQP1-overexpressing lines presented 3-to 4-fold increased AQP1 expression levels compared with those of their untransfected counterparts, leading to an increased Sb III susceptibility of about 2-fold. Competition assays using silver nitrate, silver sulfadiazine, or silver acetate prior to Sb III exposure increased parasite growth, especially in AQP1-overexpressing mutants. Surprisingly, Sb III -sodium nitrate or Sb III -potassium nitrate combinations showed significantly enhanced antileishmanial activities compared to those of Sb III alone, especially against AQP1-overexpressing mutants, suggesting a putative nitrate-dependent modulation of AQP1 activity. The intracellular level of antimony quantified by graphite furnace atomic absorption spectrometry showed that the concomitant exposure to Sb III and nitrate favors antimony accumulation in the parasite, increasing the toxicity of the drug and culminating with parasite death. This is the first report showing evidence of AQP1-mediated Sb III susceptibility modulation by silver in Leishmania and suggests the potential antileishmanial activity of the combination of nitrate salts and Sb III .

Comparative transcriptomic analysis of antimony resistant and susceptible Leishmania infantum lines

“…Treatment failure with Sb V has been reported in Bihar (India), where more than 60% of patients with VL are unresponsive to Sb V [ 4 , 13 ]. Different mechanisms of drug resistance have been reported [ 11 ], including decreased Sb III entry into the cell due to reduced expression of aquaglyceroporin (AQP1) [ 14 – 16 ] or sequestration of the metal–thiol conjugate into vesicular membranes of Leishmania by the ATP-binding cassette transporter [ 17 , 18 ] and greater Sb III efflux due to amplification of ABC transporters [ 19 ] .…”

Application of CRISPR/Cas9-Based Reverse Genetics in Leishmania braziliensis: Conserved Roles for HSP100 and HSP23

“…Studies reporting on the use of homologous recombination in L. braziliensis demonstrate the generation of stable transgenic parasite lines from integration of DNA constructs into the SSU rDNA genomic locus. These include L. braziliensis lines expressing reporter genes, e.g., luciferase or eGFP, which hold potential for parasite tracking and monitoring effects of antileishmanial compounds in vitro and in vivo [ 67 , 68 , 69 ], and over expressing parasite lines for the analysis of gene products, e.g., to assess antimony susceptibility and resistance mechanisms [ 70 , 71 , 72 ]. Moreover, circular extrachromosomal cosmids can be stably introduced into L. braziliensis to over-express stretches of genomic DNA and connect the over expression phenotypes to biological processes such as virulence [ 73 ] and antimony resistance [ 59 ].…”

Comparative transcriptomic analysis of antimony resistant and susceptible Leishmania infantum lines

“…The chemotherapy against leishmaniasis remains the main strategy to manage the disease control, but several implications regarding the treatment should be considered [11][12][13][14][15][16][17]. Pentavalent antimonials are considered one of the main options of treatment, however these drugs have several toxic side effects and high resistance rates [9,[11][12][13]16,17]. Thus, the comprehension of resistance molecular mechanisms in Leishmania spp.…”

“…Treatment failure with Sb V has been reported in Bihar (India), where more than 60% of patients with VL are unresponsive to Sb V [4,13]. Different mechanisms of drug resistance have been reported [11], including decreased Sb III entry into the cell due to reduced expression of aquaglyceroporin (AQP1) [14][15][16], or sequestration of the metal-thiol conjugate into vesicular membranes of Leishmania by the ATP Binding Cassette transporter [17,18], and greater Sb III e ux due to ampli cation of ABC transporters [19].…”