Biology subjects

Odom John, A. R.

Publications and source records attributed to Odom John, A. R..

11 recordsLinked to original sources

bioRxiv2024

Prodrug activation in malaria parasites mediated by an imported erythrocyte esterase, acylpeptide hydrolase (APEH)

The continued emergence of antimalarial drug resistance highlights the need to develop new antimalarial therapies. Unfortunately, new drug development is often hampered by poor drug-like properties of lead compounds. Prodrugging temporarily masks undesirable compound features, improving bioavailability and target penetration. We have found that lipophilic diester prodrugs of phosphonic acid antibiotics, such as fosmidomycin, exhibit significantly higher antimalarial potency than their parent compounds (1). However, the activating enzymes for these prodrugs were unknown. Here, we show that an erythrocyte enzyme, acylpeptide hydrolase (APEH) is the major activating enzyme of multiple lipophilic ester prodrugs. Surprisingly, this enzyme is taken up by the malaria parasite, Plasmodium falciparum, where it localizes to the parasite cytoplasm and retains enzymatic activity. Using a novel fluorogenic ester library, we characterize the structure activity relationship of APEH, and compare it to that of P. falciparum esterases. We show that parasite-internalized APEH plays an important role in the activation of substrates with branching at the alpha carbon, in keeping with its exopeptidase activity. Our findings highlight a novel mechanism for antimicrobial prodrug activation, relying on a host-derived enzyme to yield activation at a microbial target. Mutations in prodrug activating enzymes are a common mechanism for antimicrobial drug resistance (2-4). Leveraging an internalized host enzyme would circumvent this, enabling the design of prodrugs with higher barriers to drug resistance. SignificanceRising antimalarial drug resistance threatens current gains in malaria control. New antimalarial drugs are urgently needed. Unfortunately, many drug candidates have poor drug-like properties, such as poor absorbability in the gastrointestinal tract, or poor accumulation at the site of action. This can be overcome by prodrugging, the addition of prodrug groups which mask poor drug features until they are removed by an activating enzyme. Here, we show that a red blood cell enzyme, acylpeptide hydrolase, is taken up by malaria parasites and serves as the activating enzyme for multiple lipophilic ester prodrugs. Our findings highlight a novel mechanism for prodrug activation, which could be leveraged to design novel prodrugs with high barriers to drug resistance.

microbiology↗

bioRxiv2021

Cooperativity mediates drug resistance and metabolism in Plasmodium falciparum malaria parasites

Efforts to control the global malaria health crisis are undermined by antimalarial resistance. Iden-tifying mechanisms of resistance will uncover the underlying biology of the Plasmodium falciparum malaria parasites that allow evasion of our most promising therapeutics and may reveal new drug targets. We utilized fosmidomycin (FSM) as a chemical inhibitor of plastidial isoprenoid biosynthesis through the methylerythritol phosphate (MEP) pathway. We have thus identified an unusual metabolic regulation scheme in the malaria parasite through the essential glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Two parallel genetic screens converged on independent but functionally analogous resistance alleles in GAPDH. Metabolic profiling of FSM-resistant gapdh mutant parasites indicates that neither of these mutations disrupt overall glycolytic output. While FSM-resistant GAPDH variant proteins are catalytically active, they have reduced assembly into the homotetrameric state favored by wild-type GAPDH. Disrupted oligomerization of FSM-resistant GAPDH variant proteins is accompanied by altered enzymatic cooperativity and reduced susceptibility to inhibition by free heme. Together, our data identifies a new genetic biomarker of FSM-resistance and reveals the central role of GAPDH cooperativity in MEP pathway control and antimalarial sensitivity.

microbiology↗

bioRxiv2021

Enzymatic and structural characterization of HAD5, an essential phosphomannomutase of malaria parasites

The malaria parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of GPI anchor biosynthesis, the putative phosphomannomutase (EC 5.4.2.8) of the parasites, HAD5 (PF3D7_1017400). We confirm the phosphomannomutase and phosphoglucomutase activity of purified recombinant HAD5. By regulating expression of HAD5 in transgenic parasites, we demonstrate that HAD5 is required for malaria parasite egress and erythrocyte reinvasion. Finally, we determine the three-dimensional crystal structure of HAD5 and identify a substrate analog that specifically inhibits HAD5, compared to orthologous human phosphomannomutases. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition, and that HAD5 has potential as a multi-stage antimalarial target.

microbiology↗

bioRxiv2021

Red Blood Cells Function as DNA Sensors

Erythrocytes have long been mistaken as exclusively inert oxygen carriers lacking immune function. Here we show that red blood cells (RBCs) serve as immune sensors through surface expression of the nucleic acid-sensing toll-like receptor 9 (TLR9), a classically endosomal receptor that initiates immune responses following the detection of unmethylated CpG motifs present in pathogen and mitochondrial DNA. Mammalian RBCs express TLR9 on their surface and bind CpG-containing bacterial, malarial, and mitochondrial DNA. Erythrocyte-bound CpG DNA increases during infection, and CpG-carrying RBCs trigger accelerated erythrophagocytosis and innate immune activation characterized by RBC-TLR9 dependent local and systemic cytokine production. Thus, RBC nucleic acid detection and capture regulates red cell clearance and immune responses and provides evidence for RBCs as innate immune sentinels during pathologic states. One Sentence SummaryThe ability of RBCs to detect and bind cell-free nucleic acids contributes to immunity during acute inflammatory states.

immunology↗

bioRxiv2020

Structure-guided microbial targeting of antistaphylococcal prodrugs

Carboxy ester prodrugs have been widely employed as a means to increase oral absorption and potency of phosphonate antibiotics. Prodrugging can successfully mask problematic chemical features that prevent cellular uptake and can be used to target delivery of compounds to specific tissues. However, many carboxy ester promoieties are rapidly hydrolyzed by serum esterases, curbing their potential therapeutic applications. While carboxy ester-based prodrug targeting is feasible, it has seen limited use in microbes due to a paucity of information about the selectivity of microbial esterases. Here we identify the bacterial esterases, GloB and FrmB, that are required for carboxy ester prodrug activation in Staphylococcus aureus. Additionally, we determine the substrate specificities for FrmB and GloB and demonstrate the structural basis of these preferences. Finally, we establish the carboxy ester substrate specificities of human and mouse sera, which revealed several promoieties likely to be serum esterase-resistant while still being microbially labile. These studies lay the groundwork for structure-guided design of anti-staphyloccal promoieties and expand the range of molecules to target staphyloccal pathogens.

biochemistry↗

bioRxiv2020

Accelerated erythrocyte senescence causes dose-limiting anemia of antimalarial enolase inhibitors

Glycolysis controls cellular energy, redox balance, and biosynthesis. Antiglycolytic therapies are under investigation for treatment of obesity, cancer, aging, autoimmunity, and microbial diseases. Interrupting glycolysis is highly valued as a therapeutic strategy, because glycolytic disruption is generally tolerated in mammals. Unfortunately, anemia is a known dose-limiting side effect of these inhibitors and presents a major caveat to development of antiglycolytic therapies. We developed specific inhibitors of enolase - a critical enzyme in glycolysis - and validated their metabolic and cellular effects on human erythrocytes. Enolase inhibition increases erythrocyte susceptibility to oxidative damage and induces rapid and premature erythrocyte senescence, rather than direct hemolysis. We apply our model of red cell toxicity to address questions regarding erythrocyte glycolytic disruption in the context of Plasmodium falciparum malaria pathogenesis. Our study provides a framework for understanding red blood cell homeostasis under normal and disease states and clarifies the importance of erythrocyte reductive capacity in malaria parasite growth.

pharmacology and toxicology↗

bioRxiv2020

The malaria metabolite HMBPP does not trigger erythrocyte terpene production

Infection with malarial parasites renders hosts more mosquito attractive than their uninfected, healthy, counterparts. One volatile organic compound, -pinene, is associated with Plasmodium spp. infection in multiple studies and is a known mosquito attractant. However, how malarial infection results in elevated levels of host-associated -pinene remains unclear. One study suggests that erythrocyte exposure to the malarial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), results in increased levels of -pinene. Here, we establish that endogenous levels of -pinene are present in human erythrocytes, that these levels vary widely by erythrocyte donor, and that -pinene levels are not altered by HMBPP treatment.

biochemistry↗

bioRxiv2020

Antimicrobial prodrug activation by the staphylococcal glyoxalase GloB

With the rising prevalence of multidrug-resistance, there is an urgent need to develop novel antibiotics. Many putative antibiotics demonstrate promising in vitro potency but fail in vivo due to poor drug-like qualities (e.g. serum half-life, oral absorption, solubility, toxicity). These drug-like properties can be modified through the addition of chemical protecting groups, creating "prodrugs" that are activated prior to target inhibition. Lipophilic prodrugging techniques, including the attachment of a pivaloyloxymethyl group, have garnered attention for their ability to increase cellular permeability by masking charged residues and the relative ease of the chemical prodrugging process. Unfortunately, pivaloyloxymethyl prodrugs are rapidly activated by human sera, rendering any membrane permeability qualities absent during clinical treatment. Identification of the bacterial prodrug activation pathway(s) will allow for the development of host-stable and microbe-targeted prodrug therapies. Here, we use two zoonotic staphylococcal species, S. schleiferi and S. pseudintermedius, to establish the mechanism of carboxy ester prodrug activation. Using a forward genetic screen, we identify a conserved locus in both species encoding the enzyme hydroxyacylglutathione hydrolase (GloB), whose loss-of-function confers resistance to carboxy ester prodrugs. We enzymatically characterize GloB and demonstrate that it is a functional glyoxalase II enzyme, which has the capacity to activate carboxy ester prodrugs. As GloB homologs are both widespread and diverse in sequence, our findings suggest that GloB may be a useful mechanism for developing species-or genus-level prodrug targeting strategies.

microbiology↗

bioRxiv2019

Protein prenylation and Hsp40 in thermotolerance of Plasmodium falciparum malaria parasites

During its complex life cycle, the malaria parasite survives dramatic changes in environmental temperature. Protein prenylation is required during asexual replication of Plasmodium falciparum, and heat shock protein 40 (HSP40; PF3D7_1437900) is post-translationally modified with a 15-carbon farnesyl isoprenyl group. In other organisms, farnesylation of Hsp40 orthologs controls its localization and function, including temperature stress survival. In this work, we find that plastidial isopentenyl pyrophosphate (IPP) synthesis and protein farnesylation are required for malaria parasite survival after cold and heat shock. Furthermore, loss of HSP40 farnesylation alters its membrane attachment and interaction with proteins involved in crucial biological processes, such as glycolysis and cytoskeletal organization. Together, this work reveals that farnesylation of HSP40 in P. falciparum is a novel essential function of plastidial isoprenoid biosynthesis. We propose a model by which farnesyl-HSP40 promotes parasite thermotolerance and facilitates vesicular trafficking through its interaction with client proteins.

microbiology↗

bioRxiv2019

Potent, specific MEPicides for treatment of zoonotic staphylococci

Coagulase-positive staphylococci, which frequently colonize the mucosal surfaces of animals, also cause a spectrum of opportunistic infections including skin and soft tissue infections, urinary tract infections, pneumonia, and bacteremia. However, recent advances in bacterial identification have revealed that these common veterinary pathogens are in fact, zoonoses that cause serious infections in human patients. The global spread of multidrug-resistant zoonotic staphylococci, in particular the emergence of methicillin-resistant organisms, is now a serious threat to both animal and human welfare. Accordingly, new therapeutic targets that can be exploited to combat staphylococcal infections are urgently needed. Enzymes of the methylerythritol phosphate pathway (MEP) of isoprenoid biosynthesis represent potential targets for treating zoonotic staphylococci. Here we demonstrate that fosmidomycin (FSM) inhibits the first step of the isoprenoid biosynthetic pathway catalyzed by deoxyxylulose phosphate reductoisomerase (DXR) in staphylococci. In addition, we have both enzymatically and structurally determined the mechanism by which FSM elicits its effect. Using a forward genetic screen, the glycerol-3-phosphate transporter GlpT that facilitates FSM uptake was identified in two zoonotic staphylococci, Staphylococcus schleiferi and Staphylococcus pseudintermedius. A series of lipophilic ester prodrugs (termed MEPicides) structurally related to FSM were synthesized, and data indicate that the presence of the prodrug moiety not only substantially increased potency of the inhibitors against staphylococci, but also bypassed the need for GlpT-mediated cellular transport. Collectively, our data indicate that the prodrug MEPicides selectively and robustly inhibit DXR in zoonotic staphylococci, and further, DXR represents a promising, druggable target for future development.\n\nAuthor SummaryThe proliferation of microbial pathogens resistant to the current pool of antibiotics is a major threat to public health. Drug resistance is pervasive in staphylococci, including several species that can cause serious zoonotic infections in humans. Thus, new antimicrobial agents are urgently need to combat these life-threatening, resistant infections. Here we establish the MEP pathway as a promising new target against zoonotic staphylococci. We determine that fosmidomycin (FSM) selectively targets the isoprenoid biosynthesis pathway in zoonotic staphylococci, and use forward genetics to identify the transporter that facilitates phosphonate antibiotic uptake. Employing this knowledge, we synthesized a series of potent antibacterial prodrugs that circumvent the transporter. Together, these novel prodrug inhibitors represent promising leads for further drug development against zoonotic staphylococci.

microbiology↗

bioRxiv2017

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

In the malaria parasite Plasmodium falciparum, synthesis of isoprenoids from glycolytic intermediates is essential for survival. The antimalarial fosmidomycin (FSM) inhibits isoprenoid synthesis. In P. falciparum, we identify a loss-of-function mutation in HAD2 (PF3D7_1226300) as necessary for FSM resistance. Enzymatic characterization reveals that HAD2, a member of the haloacid dehalogenase-like hydrolase (HAD) superfamily, is a phosphatase. Harnessing a growth defect in resistant parasites, we select for suppression of HAD2-mediated FSM resistance and uncover hypomorphic suppressor mutations in the locus encoding the glycolytic enzyme phosphofructokinase (PFK9). 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 parasite metabolism and drug sensitivity and uncover PFK9 as a novel site of genetic metabolic plasticity in the parasite. Our study informs the biological functions of an evolutionarily conserved family of metabolic regulators and reveals a previously undescribed strategy by which malaria parasites adapt to cellular metabolic dysregulation.\n\nIMPORTANCEUnique and essential aspects of parasite metabolism are excellent targets for development of new antimalarials. An improved understanding of parasite metabolism and drug resistance mechanisms are urgently needed. The antibiotic fosmidomycin targets the synthesis of essential isoprenoid compounds from glucose and is a candidate for antimalarial development. Our study identifies a novel mechanism of drug resistance and further describes a family of metabolic regulators in the parasite. Using a novel forward genetic approach, we also uncover mutations that suppress drug resistance in the glycolytic enzyme PFK9. Thus, we identify an unexpected genetic mechanism of adaptation to metabolic insult that influences parasite fitness and tolerance to antimalarials.

microbiology↗