Suppressor screen connects HAD protein function to metabolic control in Plasmodium falciparum

In the malaria parasite Plasmodium falciparum, isoprenoid synthesis from glycolytic intermediates is essential for survival. The antibiotic and antimalarial fosmidomycin (FSM) inhibits isoprenoid synthesis. In FSM-resistant P. falciparum, we identify a loss-of-function mutation in HAD2 as causative for resistance. Enzymatic characterization shows that HAD2, a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, functions as a nucleotidase. Harnessing a growth defect in HAD2-mutant parasites, we select for suppression of HAD2-mediated FSM resistance and uncover hypomorphic suppressor mutations in the locus encoding the glycolytic enzyme phosphofructokinase. Metabolic profiling demonstrates that FSM resistance is achieved via increased steady-state levels of MEP pathway and glycolytic intermediates and confirms reduced PFK9 function in the suppressed strains. We identify HAD2 as a novel regulator of malaria glycolytic metabolism and drug sensitivity. Our study informs the biological functions of an evolutionarily conserved family of metabolic regulators and reveal a previously undescribed strategy for cellular glycolytic regulation.


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As an obligate intracellular parasite of human erythrocytes, the malaria parasite Plasmodium 48 falciparum has unique metabolic features that may be exploited to discover new drug targets and 49 develop new therapies. In the red blood cell niche, Plasmodium parasites depend on glucose 50 metabolism. Infection with Plasmodium spp. results in a nearly 100-fold increase in glucose 51 import in red blood cells (Mehta, Sonawat, & Sharma, 2006; E. Roth, 1990; E. F. Roth, 1987). 52 Despite these energy requirements, the parasite demonstrates little aerobic respiration via the 53 TCA cycle. Instead, it relies on anaerobic glycolysis to produce ATP ( Scheibel & Miller, 1969). 56 57 Besides ATP production, glucose also has a number of anabolic fates in P. falciparum. One such 58 fate is the synthesis of isoprenoids. Isoprenoids are a large class of hydrocarbons with extensive 59 structural and functional diversity (Gershenzon & Dudareva, 2007). In the malaria parasite,

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We hypothesized that these FSM S E2 clones, driven by a fitness advantage, had acquired new 173 suppressor mutation(s) at an additional locus, resulting in loss of FSM resistance and an increase 174 in growth rate. We performed whole genome sequencing on the five E2 clones to identify any 175 genetic changes that segregated with the FSM R and FSM S (suppressed) phenotypes. Indeed, we 176 find that a mutation (pfk9 T1206I ) in the locus encoding phosphofructokinase-9 (PFK9, 177 We evaluated the effects of the pfk9 T1206I allele on native PFK activity in P. falciparum (Beutler, 191 1984

Loss of HAD2 leads to dysregulation of glycolysis at PFK9
196 Since decreased PFK9 activity restored FSM sensitivity to had2 mutant strains, we postulated 197 that HAD2 normally regulates glycolysis, perhaps at the step catalyzed by PFK9. To evaluate 198 this possibility, we performed targeted metabolic profiling on the parental parasite strain as well 199 as E2 clones R1-R3 and S1-S2 ( Figure 5D) To understand the role of PFK9 in suppressing FSM resistance, we also determined the steady-207 state levels of glycolytic intermediates ( Figure 5D). We find levels of DOXP and MEcPP are 208 tightly correlated with cellular levels of the PFK9 product, FBP (Pearson r≥0.9, p≤0.01). 209 Clustering indicates that resistant clones are characterized by a metabolic signature of increased 210 levels of FBP, DOXP, and MEcPP ( Figure 5D). Our results suggest that HAD2 controls 211 metabolic homeostasis at the PFK step. Of note, the pfk9 T1206I suppressor allele restores nearly 212 parental levels of FBP and downstream MEP pathway intermediates ( Figure 5D). Thus, the 213 hypomorphic allele of PFK9 suppresses the metabolic dysregulation caused by loss of HAD2. indicate that HAD2 modulates metabolism through nucleotide dephosphorylation and ultimately 219 restricts glycolysis at the level of PFK9. Loss of HAD2 misroutes metabolism, leading to 220 increased substrate availability to the MEP pathway. This results in a substantial fitness cost in 221 asexual growth, which is rescued by reduced PFK9 activity. 222

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In further support of this model, we independently repeated our genetic selection with three 224 FSM R E2 clones (had2 R157X , PFK9 WT ), cultured without FSM for >1 month ( Figure 4). As before, 225 these strains also lost their FSM resistance phenotype ( Figure 4) and restored normal growth 226 ( Figure 3). We sequenced HAD2 and PFK9 in these three suppressed strains (E2-S3, -S4, and -227 S5). We find that all strains again maintain the had2 R157X mutation and acquire new, independent 228 PFK9 mutations, correlating with increased growth rate and FSM sensitivity (Figures 3 and 4). 229 Of the four PFK9 variants identified in this study, three variants map to the alpha domain, while 230 one variant (S335L) maps to the beta domain ( Figure 5A). As with the original T1206I variant 231 found in strains S1 and S2, these additional variants show a significant reduction in PFK activity 232 from lysate ( Figure 5B). 233

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Our model also predicted that restoration of functional HAD2 in FSM R strain E2 should restore 235 FSM sensitivity. Using a piggyback transposon system to express GFP-tagged HAD2 (Balu,  Lindquist, & Goldberg, 2012), we were repeatedly unable to obtain successful transfectants from 238 the FSM R clones (had2 R157X , PFK9), perhaps due to reduced overall fitness. However, we were 239 able to rescue loss of HAD2 in a suppressed FSM S E2 clone (had2 R157X , pfk9 T1206I ) ( The existence of the pfk9 T1206I allele in the presence of functional HAD2 seems to reduce parasite 243 fitness, as evidenced by reduced growth (Figure 3 parasites display a unique dependence on glycolysis for energy and biosynthesis.

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Using resistance to a metabolic inhibitor, we identify a nucleotidase, HAD2, as a novel regulator 265 of metabolism. Cells lacking HAD2 exhibit marked dysregulation of central carbon metabolism, 266 including altered steady-state levels of glycolytic intermediates and isoprenoid precursors. 267 HAD2 is necessary for optimum parasite fitness, and strains lacking HAD2 acquire additional 268 genetic changes that suppress HAD2-mediated drug resistance and growth defect. We find that 269 mutations in phosphofructokinase (PFK9) restore wild-type growth rates and FSM sensitivity. 270 Our study thus directly genetically connect the function of HAD2, a HAD superfamily member, 271 to control of essential central carbon metabolism, as outlined in our model ( Figure 6). 272

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In our study, we find that inhibitor resistance is defined by a distinct metabolic signature, which notably also employ the MEP pathway, suggesting a common function of HADs. As P. 296 falciparum HADs influence easily quantified phenotypes (drug tolerance, growth, metabolite 297 levels), the malaria parasite remains an attractive system to study general HAD biology and 298 Melt curve analysis was used to verify that all primer sets produce single products. 364 365 Relative expression levels were calculated using the ΔΔC t method. ΔC t represents the C t value of 366 the reference genes (average of beta tubulin and 18s rRNA) subtracted from the target.