Plasmodium falciparum ensures its amino acid supply with multiple acquisition pathways and redundant proteolytic enzyme systems

Abstract

Degradation of host hemoglobin by the human malaria parasite Plasmodium falciparum is a massive metabolic process. What role this degradation plays and whether it is essential for parasite survival have not been established, nor have the roles of the various degradative enzymes been clearly defined. We report that P. falciparum can grow in medium containing a single amino acid (isoleucine, the only amino acid missing from human hemoglobin). In this medium, growth of hemoglobin-degrading enzyme gene knockout lines (missing falcipain-2 and plasmepsins alone or in combination) is impaired. Blockade of plasmepsins with the potent inhibitor pepstatin A has a minimal effect on WT parasite growth but kills falcipain-2 knockout parasites at low concentrations and is even more potent on falcipain-2, plasmepsin I and IV triple knockout parasites. We conclude that: (i) hemoglobin degradation is necessary for parasite survival; (ii) hemoglobin degradation is sufficient to supply most of the parasite's amino acid requirements; (iii) external amino acid acquisition and hemoglobin digestion are partially redundant nutrient pathways; (iv) hemoglobin degradation uses dual protease families with overlapping function; and (v) hemoglobin-degrading plasmepsins are not promising drug targets.

Fig. 1.

Amino acid requirements for cultured WT parasites. (A) Flow cytometry of the starting culture and cultures incubated in different media for 25 h. FITC-H channel histograms are shown. The truncated, low fluorescence peaks correspond to uninfected RBCs. RBCs infected with mature parasites (schizonts) have high fluorescence intensity and correspond to the far-right peaks. RBCs infected with younger (ring and trophozoite) parasites are closer to the uninfected RBC peak. A single amino acid was omitted from the 5AA as indicated. No AA was used as a control (Lower Right). Parasitemia gates (defined in Materials and Methods) are shown as horizontal bars, and parasitemias are listed underneath the bar. Except for the starting culture in Upper Left, media used are listed above the bar. (B) Isoleucine dose dependence. Parasites were incubated for 51 h in no AA supplemented with varying concentrations of isoleucine. The y axis is percent growth relative to that in full RPMI medium 1640. Error bars indicate the SD of triplicate measurements. (C) Effect of 4-thiaisoleucine. Parasites were cultured in no AA supplemented as follows: lane a, no addition; lane b, 15 μM isoleucine; and lane c, 15 μM isoleucine plus 100 μM 4-thiaisoleucine. Gray bars indicate 51-h incubation; black bars indicate 124-h incubation. Representative experiments that were repeated more than three times are shown.

Fig. 2.

Creation of a chromosomal falcipain-2 disruption. (A) Schematic of the falcipain locus on chromosome 11. PacI (P) digestion results in fragments of 4.0 kb (falcipain-2, FP2) and 16.8 kb (falcipain-2′, FP2′). FP3, falcipain-3. (B) Schematic of the falcipain-2 knockout plasmid. FP2–5′ is a 590-bp fragment homologous to the 5′ coding region of the falcipain-2 gene. FP2–3′ is a 605-bp fragment homologous to the 3′ coding region of the falcipain-2 gene. The 7.9-kb plasmid has no PacI site. BSD, blasticidin S-deaminase. (C) Schematic of the falcipain-2 knockout resulting from double crossover. PacI digestion of the integrant with two concatenated plasmids yields a 13.2-kb fragment. (D) Schematic of the falcipain-2 knockout resulting from single crossover. This integration results in a promoter-less copy of falcipain-2, as indicated by FP2*. PacI digestion of the integrant with three concatenated plasmids results in a 26-kb fragment. (E) Southern blot of PacI digested total DNA from untransfected parasites (WT), falcipain-2 single knockout clones (FP2a, FP2b, FP2c, and FP2d), and plasmepsins I and IV, and falcipain-2 triple knockout clones (Triple 1 and Triple 2). Wt-FP2 indicates the fragment corresponding to the WT falcipain-2 locus, d-K/O indicates the falcipain-2 knockout resulting from a double crossover, s-K/O indicates the falcipain-2 knockout resulting from a single crossover. FP2–5′ was used for the Southern probe. Falcipain-2′, an almost identical copy of falcipain-2 that is expressed later in the life cycle (11), cross-hybridizes with this probe. DNA size ladders (in kb) are shown on the right of the blots.

Fig. 3.

Growth of knockout clones in RPMI medium 1640 (Lower) and I medium (Upper). Parasitemias of asynchronous cultures were measured by flow cytometry. Fold increases of parasites were calculated and graphed against time. Black diamonds indicate WT; blue diamonds indicate plasmepsin IV/I double knockout; red triangles indicate falcipain-2 single knockout clone b; green inverse triangles indicate falcipain-2 single knockout clone c; purple triangles indicate Triple 1; yellow inverse triangles indicate Triple 2. Error bars indicate the SD of triplicate measurements. Curves are simulated by an exponential growth model. The same colors for symbols are used for curves. Doubling times were calculated and are listed in Table 1.

Fig. 4.

Sensitivities to protease inhibitors. (A) E64 sensitivities. Parasites were incubated with E64 for 62 h. The percent growth of cultures compared with the untreated culture is graphed against E64 concentration in linear scale. Solid diamonds and solid lines indicate I medium; open diamonds and dotted lines indicate RPMI medium 1640. Error bars indicate the SD of triplicate measurements. PM4/1, plasmepsin I/IV double knockout; FP2b, falcipain-2 single knockout clone b; FP2c, falcipain-2 single knockout clone c. (B) Pepstatin A sensitivities. Parasites were incubated with pepstatin A for 63 h. The percent growth of cultures compared with the untreated culture is graphed against pepstatin A concentration in log₁₀ scale. Symbols and lines are the same as in A. E64 and pepstatin A IC₅₀ values are listed in Table 2.