Fig 1.
Stage-specific changes in Leishmania growth rates.
A. Schematic overview of 2H2O labeling protocol. Parasite stages were cultivated axenically in the presence 5% 2H2O, or isolated from infected macrophages or BALB/c lesion incubated or infused with 2H2O (final concentration 5%). Parasite stages were harvested at multiple time points and extracts containing total DNA/RNA, or total proteins and lipids generated from purified parasite fraction. Levels of deuterium enrichment in constituent dRib/Rib, amino acids and fatty acids were subsequently quantitated by GC-MS. B. Kinetics of 2H-labeling of DNA dRib in cultured promastigotes (Prolog, Prostat) and amastigotes (Amaaxenic), and in amastigotes isolated from macrophages (AmaMø) and murine lesions (Amalesion). The fraction of new cells (Y-axis) was calculated from the level of 2H-enrichment in dRib relative to maximum labeling observed in each parasite stage after long term (equilibrium) labeling. Calculated doubling times for each stage are shown in inset boxes. C. Comparative growth rates of different Leishmania stages, calculated from 2H-enrichment in dRib. D. Section of stained cutaneous lesion (with detail in insert) and calculated range of parasite numbers/phagolysosome. Abbreviations: dRib; deoxyribose, Rib; ribose.
Fig 2.
Rates of RNA turnover in cultured and intracellular Leishmania stages.
Kinetics of 2H-labeling of RNA ribose in (A) cultured parasite stages (Prolog, Prostat, Amaaxenic) (B) amastigotes isolated from infected J774 macrophages (AmaMø) and (C) amastigotes isolated from BALB/c lesions (Amalesion). The fraction of new molecules (Y-axis) was calculated from the level of 2H-enrichment in Rib relative to maximum labeling observed in each parasite stage after long term labeling. Inset boxes shows estimated RNA turnover (t1/2 in days) in each stage. D. Comparative rates of RNA turnover in different Leishmania developmental stages.
Fig 3.
Rates of protein turnover in cultured and intracellular Leishmania stages.
Parasite stages were 2H2O-labeled in culture or in situ in infected BALB/c mice and harvested at the indicated time points. Kinetics of 2H-labeling of proteinogenic alanine in (A) cultured parasite stages (Prolog, Prostat, Amaaxenic) and (B) amastigotes isolated from BALB/c lesion (Amalesion). The fraction of new molecules (Y-axis) was calculated from the level of 2H-enrichment in alanine relative to maximum labeling observed in each parasite stage after long term labeling. Inset boxes in A and B show turnover (t1/2) in days. C. Comparative rates of protein turnover in different Leishmania developmental stages. Note that similar estimates of protein turnover were obtained by measuring deuterium incorporation into other proteinogenic amino acids (S6 Fig.).
Fig 4.
Stage-specific changes in fatty acid synthesis.
Parasite stages were 2H2O-labeled in culture or in situ in infected BALB/c mice and harvested at the indicated time points. Kinetics of 2H-labeling of the major fatty acid, C18:0 (stearic acid) in (A) cultured parasite stages (Prolog, Prostat, Amaaxenic) and (B) amastigotes isolated from BALB/c lesion (Amalesion). The fraction of new molecules (Y-axis) was calculated from the level of 2H-enrichment in stearate relative to maximum labeling observed in each parasite stage after long term labeling. Inset boxes show turnover (t1/2) in days. C. Comparative rates of stearic acid turnover in different Leishmania developmental stages.
Fig 5.
Lesion amastigotes utilize both salvage and de novo biosynthetic pathways to supply their fatty acid needs.
A. The maximum level of 2H-enrichment (EM1, %) in major cellular fatty acids of Prolog and Amalesion were determined after labeling for 7 days and >6 weeks, respectively. 2H-enrichment in the total plasma lipid of infected mice was also measured to determine the potential contribution of labeled host fatty acids to the parasite labeling. Note that while saturated and unsaturated C18 fatty acids are predominant fatty acids in both stages, the fatty acid composition of Amalesion differs from cultured promastigotes in containing elevated levels of C20:4 n-6, and polyunsaturated very long chain fatty acids (C22:4 n-6, C22:6 n-3) (S8 Fig.). The C:D nomenclature refers to overall chain length and number of double bonds in each fatty acid, respectively. n-3 and n-6 refers to the two major biosynthetic pathways involved in unsaturated fatty acid biosynthesis (where-3 and-6 refer to the position of double bond relative to the methyl carbon). B. Stage-specific differences in the levels of 2H-enrichment in fatty acid pools can be used to infer the contributions of de novo biosynthesis and salvage pathways. In particular, levels of 2H-enrichment in the major Amalesion C18 fatty acids, C18:1 (oleic acid) and C18:2 (linoleic acid), were appreciably higher than in host plasma, indicating that these stages are dependent on de novo biosynthesis. Conversely, the elevated levels of 2H-enrichment in C20:4 n-6 and C22:6n-3 compared to C18:1 precursor (comparable to plasma pools) indicate that these very long chain polyunsaturated fatty acids are primarily scavenged from the host cell.