Gene deletion as a possible strategy adopted by New World Leishmania infantum to maximize geographic dispersion

Abstract

Background: The present study investigates implications of a sub-chromosomal deletion in Leishmania infantum strains, the causative agent of American Visceral Leishmaniasis (AVL). Primarily found in New World strains, the deletion leads to the absence of the ecto-3'-nucleotidase/nuclease enzyme, impacting parasite virulence, pathogenicity, and drug susceptibility. The factors favoring prevalence and the widespread geographic distribution of these deleted mutant parasites (DEL) in the NW (NW) are discussed under the generated data.

Methods: We conducted phenotypic assessments of the sub-chromosomal deletion through in vitro assays with axenic parasites and experimental infections in both in vitro and in vivo models of vertebrate and invertebrate hosts using geographically diverse mutant field isolates.

Results: Despite reduced pathogenicity, the DEL strains efficiently infect vertebrate hosts and exhibit relevant differences, including enhanced metacyclogenesis and colonization rates in sand flies, potentially facilitating transmission. This combination may represent a more effective way to maintain and disperse the transmission cycle of DEL strains.

Conclusions: Phenotypic assessments reveal altered parasite fitness, with potential enhanced transmissibility at the population level. Reduced susceptibility of DEL strains to miltefosine, a key drug in VL treatment, further complicates control efforts. The study underscores the importance of typing parasite genomes for surveillance and control, advocating for the sub-chromosomal deletion as a molecular marker in AVL management.

Fig 1. In vitro phenotypic characterization of DEL, NonDEL and HTZ strains.

A) Normalized 3’NT activity for NonDEL, HTZ and DEL strains. The 3’NT activity was measured for DEL (n = 08), HTZ (n = 03) and NonDEL (n = 11) parasites harvested at stationary phase. The original values, expressed as nmol Pi x h^-1 x 10 ^-7 cells, were normalized by the values obtained with the L. amazonensis assays. Compared to the L. amazonensis control, four NonDEL samples presented higher activity (a); three NonDEL and two HTZ strains presented lower activity (b); all DEL and one HTZ samples presented the lowest enzyme activity (c). Unpaired t-test. B) 3’NT activity average expressed as nmol Pi x h^-1 x 10 ^-7 for each genotype group composed by the strains individually presented in graph A. Kruskal-Wallis test followed by Mann-Whitney U test. C) 3’NT activity expressed as nmol Pi x h^-1 x 10 ^-7 cells in cultures from: stationary phase promastigotes = Pro; metacyclic enriched fraction of stationary phase culture = PNA−; SC = stressed culture parasites “axenic amastigotes”. D) Percentage of metacyclic at stationary phase. Dots represent the average value of two independent experiments for each strain (DEL = 07 and NonDEl = 09). Unpaired t-Test. E-F) Percentage of metacyclic at stationary phase of less-than-20 passages (<20P) culture and culture-adapted parasites - more than 30 passages (>30P). Percentage of metacyclic was determined 72 hours (late stationary phase) after an initial inoculum of 10⁶ parasites/ml. Metacyclic enrichment was obtained by Peanut agglutinin (PNA); percentage of cells from the PNA− fraction was determined in relation to the total cell count. Red = DEL; Blue = NonDEL; Grey = HTZ. Unpaired t test. E-F) Paired t-test for DEL and NonDEL groups of parasites in <20P and >30P conditions. ns = not-significant. Moderate to strong negative correlation (Pearson r = 0.64)was detected for 3’NT activity (A) and metacyclic enrichment (D) (n =14 strains; S1 Data).

Fig 2. Fold change of transcripts from culture adapted promastigotes harvested from the late stationary phase (72 hours).

The targets 3’NU/NT = 3’ecto-nucleotidase; NT1 = nucleoside transporter 1, Amastin and Paraflagellar Rod Protein were chosen based on previous data pointing to differences in CNV between DEL and NonDEL [4]. SHERP, META1 and META2 were included as potential markers for metacyclic [26] and for resistance to oxidative stress [27]. Total RNA was reversed transcribed in cDNA and targets quantified by Real Time qPCR. Delta-Delta Ct method was applied using alpha-tubulin as endogenous control and the OW sample NonDEL 3124 as calibrator. Mann Whitney U test (unpaired) for DEL (n = 5) and NonDEL (n = 9) samples. ns = not significant.

Fig 3. Fold change of transcripts from metacyclic-enriched cultures obtained by PNA from the late stationary phase (PNA−).

The targets 3’NU/NT = 3’ecto-nucleotidase; NT1 = nucleoside transporter 1, Amastin and Paraflagellar were chosen based on previous data pointing to differences in CNV between DEL and NonDEL [4]. SHERP, META1 and META2 were included as potential markers for metacyclic and for resistance to oxidative stress [27]. Total RNA from both PNA+ and PNA− cultures were reversed transcribed in cDNA and targets quantified by Real Time qPCR. Delta-Delta Ct method was applied using alpha-tubulin as reference gene and the sample in PNA+ used as a calibrator for the correspondent sample in PNA− culture. Mann Whitney U Test (unpaired) for DEL (n = 6) and NonDEL (n = 9) samples. ns = not significant.

Fig 4. Infection outcome with DEL, Non-DEL and HTZ strains in L. longipalpis.

(A) Number of Parasite per midgut. In red is represented the merged result of independent infection assays with three DEL strains (n = 3): Mato Grosso (MT_3223), Piauí (DEL_PI_2976) and Rio de Janeiro (DE_RJ_3598). In blue is represented the merged result of independent infection assays with three NonDEL strains (n = 3): MS_2666, PI_2972 and MT_3210. In grey is depicted the result obtained with one HTZ strain HTZ_MT_3134. (B) Percentage of metacyclic parasite forms detected in insect gut after infection with two DEL (n = 2; DEL_3223_MT, DEL_2976_PI) and two NonDEL (n = 2; NonDEL_3210_MT, NonDEL_2972_PI.) strains. All sand fly infection parameters were assessed at 192h (day 8) post-infection. Mann-Whitney test was used for pair-wise comparisons of parasite numbers expressed as the average of the biological replicates, and parasite forms.

Fig 5. DEL parasites have a reduced ability to infect macrophages and escape killing from neutrophil NETs.

(A-C) L. infantum metacyclics [5 × 10⁵] were incubated in the presence or absence of supernatants enriched in NETs for 4h at 35^oC. Alamar blue (10% v/v) was added, and cells were incubated for more 4h at 35^oC. Alamar blue fluorescence was read at 540/590 nm excitation/emission on a SpectraMax fluorimeter. Results are expressed as relative to control (parasites without NETs). Results of at least 3 independent experiments are shown as mean ± SEM. One-way ANOVA followed by Fisher’s LSD post-hoc comparison tests was performed. *p < 0.05 and ****p < 0.01. (D-F) Raw macrophages (2x10⁵) were seeded onto coverslips and then infected with Leishmania parasites at a cell ratio of 1 macrophage to 5 parasites. After 24h at 35^oC, free parasites were washed out and coverslips were either fixed or incubated with medium (RPMI + 2.5% FBS) for more 24h (48h time point). Cells were stained with Panoptic dye kit and the number of infected macrophages (D-F) and the number of amastigotes/macrophage (G-I) were counted. Results are shown as mean ± SEM. Wilcoxon t-test was performed. *p < 0.05. Blue bars = NonDEL; red bars = DEL strains.

Fig 6. Neutrophil and monocyte recruitment to the site of infection by DEL and NonDEL parasites and parasite load from the draining lymph nodes.

A - B) percentage of CD11b+ cells obtained from inoculated ear 12–15 hours PI (neutrophil and monocyte recruitment, respectively). Blue = NonDEL strains; Red = DEL strains. L. infantum metacyclic (10⁵) was inoculated in the inner ear of anesthetized BALB/c mice. 12–15 hours post-infection animals were euthanized, and ears were collected in RPMI. Ordinary one-way ANOVA. C) Parasite load expressed by equivalent parasite per mg of the draining lymph nodes from the ear. Material was collected and preserved in DNAShied for further DNA isolation and qPCR. Strains used in the assays were geographically unrelated: RJ = Rio de Janeiro; MS = Mato Grosso do Sul; MT = Mato Grosso; PI = Piauí.