Infectivity and Drug Susceptibility Profiling of Different Leishmania-Host Cell Combinations

Abstract

Protozoan parasites of the genus Leishmania are the causative agents of leishmaniasis, a spectrum of a disease that threatens public health worldwide. Although next-generation therapeutics are urgently needed, the early stage of the drug discovery process is hampered by very low hit rates from intracellular Leishmania phenotypic high-throughput screenings. Designing and applying a physiologically relevant in vitro assay is therefore in high demand. In this study, we characterized the infectivity, morphology, and drug susceptibility of different Leishmania and host cell infection combinations. Primary bone marrow-derived macrophage (BMDM) and differentiated human acute monocytic leukemia (THP-1) cells were infected with amastigote or promastigote forms of Leishmania amazonensis and Leishmania donovani. Regardless of host cell types, amastigotes were generally well phagocytosed and showed high infectivity, whereas promastigotes, especially those of L. donovani, had predominantly remained in the extracellular space. In the drug susceptibility test, miltefosine and sodium stibogluconate (SSG) showed varying ranges of activity with 14 and >10-fold differences in susceptibility, depending on the host-parasite pairs, indicating the importance of assay conditions for evaluating antileishmanial activity. Overall, our results suggest that combinations of Leishmania species, infection forms, and host cells must be carefully optimized to evaluate the activity of potential therapeutic compounds against Leishmania.

In Vivo Efficacy of SQ109 against Leishmania donovani, Trypanosoma spp. and Toxoplasma gondii and In Vitro Activity of SQ109 Metabolites

“…One possible reason for this difference in activity may be due to the use of different host cells. The previous report utilized J774 macrophages that originated from lymphoma cells from BALB/c mice, whereas phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1 cells were used in the current study, and host celldependent in vitro activity of anti-leishmanial drugs in L. donovani, as well as in other species, is well documented [20,28]. In the case of T. gondii, the activity was evaluated for the first time and the IC 50 for SQ109 in the intracellular tachyzoite assay was 1.82 µM, generally comparable to that found with the intracellular stages of L. donovani and T. cruzi.…”

“…For the imaging of infected cells and parasites, Draq-5 was observed under a 20× air objective. An image analysis algorithm (Columbus, Perkin Elmer Technology) was used to detect the Draq-5 signal in the nuclei of cells and parasites as described in a previous article and shown in Supplementary Figure S1 [20]. The number of parasites was defined by the value of the number of parasites in infected cells of the acquired image.…”

“…The THP-1 human monocyte cell line was grown in RPMI-1640 medium (Welgene, Gyeongsangbuk-do, Republic of Korea) supplemented with 10% FBS and 1% P/S. For differentiation, THP-1 cells were treated for 3 days with 50 ng/mL of PMA [20]. After being plated in 384-well plates, THP-1 cells were infected with amastigotes or stationary phase promastigotes at MOI of 1:10 and treated with drugs after 1 day of infection.…”

Antileishmanial Activity of Flavones-Rich Fraction From Arrabidaea chica Verlot (Bignoniaceae)

“…Our findings against the promastigote form recommend selecting the ACFF and luteolin, the main component of the fraction—to verify the activity against the forms of L. amazonesis intracelular amastigote. In the search for new drugs against Leishmania spp., intracellular amastigote is the stage of the parasite considered as the most relevant target for the primary screening of new compounds ( de Muylder et al, 2011 ), and the most consistent indicator of in vivo activity ( Croft, 1986 ), additionally considered "the gold standard" of in in vitro studies ( Baek et al, 2020 ). It is therefore of interest to test the effectiveness of A. chica compounds in intracellular amastigotes.…”

Antileishmanial Activity of the Genus Piper: A Systematic Review

“…Most studies included in this systematic review, direct counting was commonly used as the methodology of choice to evaluate antipromastigote and anti-amastigote activities, together with the use of axenic amastigotes. Differences in drug sensitivities exist between axenic amastigotes and intracellular amastigotes [89]. As Leishmania is an intracellular pathogen, the use of an intracellular assay to confirm the effects of potential drugs is recommended.…”