Importance of polyphosphate in the Leishmania life cycle

Abstract

Protozoan parasites contain negatively charged polymers of a few up to several hundreds of phosphate residues. In other organisms, these poly-phosphate (polyP) chains serve as an energy source and phosphate reservoir, and have been implicated in adaptation to stress and virulence of pathogenic organisms. In this study, we confirmed first that the polyP polymerase vacuolar transporter chaperone 4 (VTC4) is responsible for polyP synthesis in Leishmania parasites. During Leishmaniain vitro culture, polyP is accumulated in logarithmic growth phase and subsequently consumed once stationary phase is reached. However, polyP is not essential since VTC4-deficient (vtc4^- ) Leishmania proliferated normally in culture and differentiated into infective metacyclic parasites and into intracellular and axenic amastigotes. In in vivo mouse infections, L. majorVTC4 knockout showed a delay in lesion formation but ultimately gave rise to strong pathology, although we were unable to restore virulence by complementation to confirm this phenotype. Knockdown of VTC4 did not alter the course of L. guyanensis infections in mice, suggesting that polyP was not required for infection, or that very low levels of it suffice for lesion development. At higher temperatures, Leishmania promastigotes highly consumed polyP, and both knockdown or deletion of VTC4 diminished parasite survival. Thus, although polyP was not essential in the life cycle of the parasite, our data suggests a role for polyP in increasing parasite survival at higher temperatures, a situation faced by the parasite when transmitted to humans.

Keywords: Leishmania; VTC4; infectivity; life cycle; polyphosphate; temperature stress.

Figure 1. FIGURE 1: High variability of polyP abundance in different Leishmania species.

Parasites were grown in complete Schneider’s medium. PolyP was extracted from 2 x 10⁷ logarithmic phase (day 3 post dilution) promastigotes, digested with the polyphosphatase Ppx1, and released P_i was quantified colorimetrically. Results of a pool of 2 independent experiments were expressed as mean ± SD of polyP level relative to L. major. Statistical significance was assessed by Student’s t-test; * p < 0.05, ** p < 0.01, ns: non-significant. Lmj, L. major (IR75); Lae, L. aethiopica (LDS372); Lme, L. mexicana (M379); Ldo, L. donovani (AG83); Lgy, L. guyanensis (M5313); Lbr, L. braziliensis (LTB325); Lpa, L. panamensis (1166).

Figure 2. FIGURE 2: RNA interference on VTC4 in L. guyanensis.

(A) Schematic map of SSU rRNA locus in Leishmania and targeting of VTC4. Regions are the SSU rRNA (grey box), VTC4 region (black arrow), puromycin-resistance gene ORF (PAC, white arrow) and the stem loop stuffer fragment (striped box). (B) Relative VTC4 mRNA levels were assessed by qRT-PCR with VTC4 and KMP11 gene primers. Primer sequences are presented in Table S2. (C) Relative polyP was quantified by digesting the extracted polyP and measuring the released Pi colorimetrically. A and B; 21, 22, 23 and 24; 83 and 85 represented recombinant Leishmania clones selected by antibiotic resistances, for more detail see Fig. S2. Results of a pool of 3 independent experiments were expressed as mean ± SD. Statistical significance was assessed by Student’s t-test (B and C); * p < 0.05, *** p < 0.001, ns: non-significant.

Figure 3. FIGURE 3: PolyP and VTC4 fluctuation during promastigote growth of L. major and L. mexicana.

L. major (A) and L. mexicana (C) promastigote cell concentrations were counted in independent cultures. (B, D) PolyP was extracted from cell lines, digested with polyphosphatase and P_i were quantified colorimetrically using malachite green. P_i concentrations represented polyP content of 3 x 10⁷ cells for L. major (B) and 8 x 10⁶ cells for L. mexicana (D). Results of a pool of minimum 3 independent experiments were expressed as mean ± SD. (E) Western blot analysis of 20 µg of L. major promastigotes pellet protein using the anti-cd-LmjVTC4 antibody and anti-α-tubulin as loading control.

Figure 4. FIGURE 4: In vitro proliferation of VTC4 knockout L. major parasites.

L. major WT or vtc4^- promastigotes were diluted at a concentration of 5 x 10⁵ parasites per ml and cultured in complete medium. Cell concentrations were counted in independent cultures. Results of a pool of 4 independent experiments were expressed as mean ± SD. Statistical significance was assessed by Student’s t-test; * p < 0.05, *** p < 0.01, ns: non-significant.

Figure 5. FIGURE 5: Normal metacyclogenesis in absence of polyP in L. major.

Metacyclic promastigotes of WT and vtc4^- L. major clones were isolated by PNA assay (A) or by Ficoll centrifugation gradient (B). Results of a pool of 2 independent experiments were expressed as mean ± SD. Statistical significance was assessed by Student’s t-test (A and B); ns: non-significant.

Figure 6. FIGURE 6: VTC4 and polyP levels at different life cycle stages of L. major and L. mexicana.

(A) VTC4 abundance in 20 µg of pellet protein at different L. major life cycle stages, detected by cd-LmjVTC4 antibody. (B and C) Relative polyP quantification in L. major and L. mexicana promastigotes and amastigotes, by staining P_i residues after polyP digestion. Results of a pool of 2 independent experiments were expressed as mean ± SD. Statistical significance was assessed by Student’s t-test (B and C); ** p < 0.01, *** p < 0.001. (D) PolyP gel displaying polyP abundance in 6 x 10⁷ logarithmic and stationary promastigotes as well as macrophage isolated L. major amastigotes. Chains were separated by electrophoresis on a 35% polyacrylamide gel and visualized by negative DAPI staining. log, logarithmic; stat, stationary; ama, amastigotes; pro, promastigotes; RD, RNase/DNase; P, Polyphosphatase. PolyP standards represent an average of the respective sizes ranging from 13 up to 60 residues (P13 and P60).

Figure 7. FIGURE 7: In vivo infection with VTC4 knockdown and knockout Leishmania.

(A) C57BL/6 mice hind footpads were infected with 3 x 10⁶ late stationary phase L. guyanensis parasites and disease progression was monitored by measuring footpad swelling over time. (B) Parasite load was quantified by in vivo imaging (Xenogen) measuring the luminescence signal (photon flux/10min/footpad). (C) C57BL/6 or (D) BALB/c mice hind footpads were infected with 3 x 10⁶ late stationary phase L. major parasites and disease progression was monitored by measuring footpad swelling over time. Results of a pool of 2 independent experiments (A and B) or one representative of 3 independent experiments (C and D) were expressed as mean ± SD (n ≥ 5). Statistical significance was assessed by Repeated measure ANOVA (A and C) or Student’s t-test (B and D); ***p < 0.001, ns: non-significant.

Figure 8. FIGURE 8: Lack of VTC4 and polyP results in increased cell death upon heat exposure.

Logarithmic phase (day 3 post dilution) or stationary phase (day 6 post dilution) promastigotes of L. guyanensis (A and B) and L. major (C and D) were incubated at different temperatures (26°C and 37°C). The percentage of dead cells was assessed at the indicated time points by uptake of propidium iodide (PI). Results of a pool of 3 independent experiments were expressed as mean ± SD. Statistical significance was assessed by Two-way ANOVA (A - D); * p < 0.05, ** p < 0.01, *** p < 0.001, ns: non-significant.

Figure 9. FIGURE 9: Higher temperature increases Leishmania consumption of polyP.

(A) L. major logarithmic phase (day 3 post dilution) promastigotes were incubated for 6 hours at 26°C or 37°C. PolyP was quantified in attomole/cell (amol/cell) and represented as absolute values. Results of a pool of 3 independent experiments were expressed as mean ± SD of polyP levels. Statistical significance was assessed by Two-way ANOVA; ** p < 0.01, ns: non-significant. (B) PolyP was extracted from logarithmic phase (day 3 post dilution) L. major promastigotes exposed at different temperatures for the indicated time. PolyP chains were separated by gel electrophoresis and visualized by negative DAPI staining on a 35% polyacrylamide gel. PolyP standards represent an average of the respective sizes ranging from 13 up to 300 residues (P13, P60 and P300). (C) Quantification of polyP chains consumption by ImageJ of lanes of Fig. 9B representing polyP chains in Leishmania parasites cultured at 26°C (blue line) or exposed to a 37°C heat shock for 17h (red line). The black line represents the P300 standard and was included for comparison. The x axis represents the distance from the top of the gel and the y axis the relative intensity of the signal.