Babesia duncani multi-omics identifies virulence factors and drug targets

Babesiosis is a malaria-like disease in humans and animals that is caused by Babesia species, which are tick-transmitted apicomplexan pathogens. Babesia duncani causes severe to lethal infection in humans, but despite the risk that this parasite poses as an emerging pathogen, little is known about its biology, metabolic requirements or pathogenesis. Unlike other apicomplexan parasites that infect red blood cells, B. duncani can be continuously cultured in vitro in human erythrocytes and can infect mice resulting in fulminant babesiosis and death. We report comprehensive, detailed molecular, genomic, transcriptomic and epigenetic analyses to gain insights into the biology of B. duncani. We completed the assembly, 3D structure and annotation of its nuclear genome, and analysed its transcriptomic and epigenetics profiles during its asexual life cycle stages in human erythrocytes. We used RNA-seq data to produce an atlas of parasite metabolism during its intraerythrocytic life cycle. Characterization of the B. duncani genome, epigenome and transcriptome identified classes of candidate virulence factors, antigens for diagnosis of active infection and several attractive drug targets. Furthermore, metabolic reconstitutions from genome annotation and in vitro efficacy studies identified antifolates, pyrimethamine and WR-99210 as potent inhibitors of B. duncani to establish a pipeline of small molecules that could be developed as effective therapies for the treatment of human babesiosis.We thank R. Gao for her contribution to the initial eforts to sequence the B. duncani genome. C.B.M.’s research was supported by grants from the National Institutes of Health (AI097218, GM110506, AI123321 and R43AI136118), the Steven and Alexandra Cohen Foundation (Lyme 62 2020), and the Global Lyme Alliance. S.L.’s research was supported by grants by the US National Science Foundation (IIS 1814359) and the National Institutes of Health (1R01AI169543-01). K.G.L.R.’s research was supported by the National Institutes of Allergy and Infectious Diseases (R01 AI136511, R01 AI142743-01 and R21 AI142506-01), the University of California, Riverside (NIFA-Hatch-225935) and the Health Institute Carlos III (PI20CIII/00037).S

The Structure of Bilirubin Oxidase from Bacillus pumilus Reveals a Unique Disulfide Bond for Site-Specific Direct Electron Transfer

Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme’s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes

The Structure of Bilirubin Oxidase from Bacillus pumilus Reveals a Unique Disulfide Bond for Site-Specific Direct Electron Transfer

Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme’s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes

Evidence for a Conserved Function of Eukaryotic Pantothenate Kinases in the Regulation of Mitochondrial Homeostasis and Oxidative Stress

Human PANK1, PANK2, and PANK3 genes encode several pantothenate kinase isoforms that catalyze the phosphorylation of vitamin B5 (pantothenic acid) to phosphopantothenate, a critical step in the biosynthesis of the major cellular cofactor, Coenzyme A (CoA). Mutations in the PANK2 gene, which encodes the mitochondrial pantothenate kinase (PanK) isoform, have been linked to pantothenate-kinase associated neurodegeneration (PKAN), a debilitating and often fatal progressive neurodegeneration of children and young adults. While the biochemical properties of these enzymes have been well-characterized in vitro, their expression in a model organism such as yeast in order to probe their function under cellular conditions have never been achieved. Here we used three yeast mutants carrying missense mutations in the yeast PanK gene, CAB1, which are associated with defective growth at high temperature and iron, mitochondrial dysfunction, increased iron content, and oxidative stress, to assess the cellular function of human PANK genes and functional conservation of the CoA-controlled processes between humans and yeast. Overexpression of human PANK1 and PANK3 in these mutants restored normal cellular activity whereas complementation with PANK2 was partial and could only be achieved with an isoform, PanK2mtmΔ, lacking the mitochondrial transit peptide. These data, which demonstrate functional conservation of PanK activity between humans and yeast, set the stage for the use of yeast as a model system to investigate the impact of PKAN-associated mutations on the metabolic pathways altered in this disease

Babesia BdFE1 Esterase is Required for the Anti-parasitic Activity of the ACE Inhibitor Fosinopril

Effective and safe therapies for the treatment of diseases caused by intraerythrocytic parasites are impeded by the rapid emergence of drug resistance and the lack of novel drug targets. One such disease is human babesiosis, which is a rapidly emerging tick-borne illness caused by Babesia parasites. In this study, we identified fosinopril, a phosphonate-containing, FDA-approved Angiotensin Converting Enzyme (ACE) inhibitor commonly used as a prodrug for hypertension and heart failure, as a potent inhibitor of B. duncani parasite development within human erythrocytes. Cell biological and mass spectrometry analyses revealed that the conversion of fosinopril to its active diacid molecule, fosinoprilat, is essential for its antiparasitic activity. We show that this conversion is mediated by a parasite-encoded esterase, BdFE1, which is highly conserved among apicomplexan parasites. Parasites carrying the L238H mutation in the active site of BdFE1 failed to convert the prodrug to its active moiety and became resistant to the drug. Our data set the stage for the development of this class of drugs for therapy of vector-borne parasitic diseases

The COP9 signalosome mediates the Spt23 regulated fatty acid desaturation and ergosterol biosynthesis

The COP9 signalosome (CSN) is a conserved eukaryotic complex, essential for vitality in all multicellular organisms and critical for the turnover of key cellular proteins through catalytic and non-catalytic activities. Saccharomyces cerevisiae is a powerful model organism for studying fundamental aspects of the CSN complex. This CSN includes a conserved enzymatic core but lacks few subunits that participate in non-catalytic activities, probably explaining its non-essentiality for life. A previous transcriptomic analysis of an S. cerevisiae strain deleted in the CSN5/RRI1 gene lacking CSN catalytic subunit, revealed a downregulation of genes involved in lipid metabolism. We now show that the S. cerevisiae CSN holocomplex is essential for cellular lipid homeostasis. Defects in CSN assembly or activity lead to decreased quantities of ergosterol and unsaturated fatty acids; vacuole defects; diminished lipid droplets size; and to accumulation of endoplasmic reticulum stress. The molecular mechanism behind these findings depends on CSN involvement in upregulating mRNA expression of SPT23. Spt23 is a novel activator of lipid desaturation and ergosterol biosynthesis. Our data reveal for the first time a functional link between the CSN holocomplex and Spt23. Moreover, CSN-dependent upregulation of SPT23 transcription is necessary for the fine-tuning of lipid homeostasis and for cellular health

High-throughput-feasible screening tool for determining enzyme stabilities against organic solvents directly from crude extracts

Wedde S, Kleusch C, Bakonyi D, Gröger H. High-throughput-feasible screening tool for determining enzyme stabilities against organic solvents directly from crude extracts. Chembiochem : a European journal of chemical biology. 2017;18(24):2399-2403