Profiling the Essential Nature of Lipid Metabolism in Asexual Blood and Gametocyte Stages of Plasmodium falciparum

SummaryDuring its life cycle, Plasmodium falciparum undergoes rapid proliferation fueled by de novo synthesis and acquisition of host cell lipids. Consistent with this essential role, Plasmodium lipid synthesis enzymes are emerging as potential drug targets. To explore their broader potential for therapeutic interventions, we assayed the global lipid landscape during P. falciparum sexual and asexual blood stage (ABS) development. Using liquid chromatography-mass spectrometry, we analyzed 304 lipids constituting 24 classes in ABS parasites, infected red blood cell (RBC)-derived microvesicles, gametocytes, and uninfected RBCs. Ten lipid classes were previously uncharacterized in P. falciparum, and 70%–75% of the lipid classes exhibited changes in abundance during ABS and gametocyte development. Utilizing compounds that target lipid metabolism, we affirmed the essentiality of major classes, including triacylglycerols. These studies highlight the interplay between host and parasite lipid metabolism and provide a comprehensive analysis of P. falciparum lipids with candidate pathways for drug discovery efforts

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Plasmid-Mediated Ciprofloxacin Resistance in Escherichia coli Isolates from Nigeria

Ciprofloxacin is a broad-spectrum antimicrobial and a member of the quinolone drug family.\ud After Ciprofloxacin’s patent expired in 2003 generic forms of the drug have quickly become some of the most popular antibacterials in Nigeria. With increased use has come increased\ud resistance. The traditional vertical transmission mechanism, progenitor cell to daughter cell, does not adequately explain the rapid increase and wide dispersion of resistance that has been observed. Our lab posits that horizontal gene transfer is likely responsible for this resistance pattern. We have shown that the plasmid-carried resistance genes account for 17.4% of the instances of Ciprofloxacin resistance in our sample set, the most significant of which were oqxA (9.7%), followed by aac(6’)-Ib-cr (3.2%), qnrS (2.2%) and qepA(2.2 %), no isolates were positive for either qnrA or qnrB. We also characterized the plasmid pMB2, which was isolated\ud from a sample from a Nigerian woman and carries one of the Ciprofloxacin resistance genes, aac(6’)-Ib-cr. We determined that the plasmid is transposase rich, contains a full conjugation system, and carries three molecular transport systems. pMB2 also contains seven other\ud resistance genes including the extended-spectrum ß-lactamase, blaCTX-M-15, which is frequently reported on plasmids with aac(6’)-Ib-cr. These findings contribute to the further understanding\ud the issues surrounding the global antimicrobial resistance phenomenon by elucidating both the\ud nature and importance of PMQR genes in Southwestern Nigeria and the genetic context of the\ud aac(6’)-Ib-cr gene within the large plasmid that carries the allele

Yeast-Based High-Throughput Screen Identifies <i>Plasmodium falciparum</i> Equilibrative Nucleoside Transporter 1 Inhibitors That Kill Malaria Parasites

Equilibrative transporters are potential drug targets; however, most functional assays involve radioactive substrate uptake that is unsuitable for high-throughput screens (HTS). We developed a robust yeast-based growth assay that is potentially applicable to many equilibrative transporters. As proof of principle, we applied our approach to Equilibrative Nucleoside Transporter 1 of the malarial parasite <i>Plasmodium falciparum</i> (PfENT1). PfENT1 inhibitors might serve as novel antimalarial drugs since PfENT1-mediated purine import is essential for parasite proliferation. To identify PfENT1 inhibitors, we screened 64 560 compounds and identified 171 by their ability to rescue the growth of PfENT1-expressing <i>fui1</i>Δ yeast in the presence of a cytotoxic PfENT1 substrate, 5-fluorouridine (5-FUrd). In secondary assays, nine of the highest activity compounds inhibited PfENT1-dependent growth of a purine auxotrophic yeast strain with adenosine as the sole purine source (IC₅₀ 0.2–2 μM). These nine compounds completely blocked [³H]­adenosine uptake into PfENT1-expressing yeast and erythrocyte-free trophozoite-stage parasites (IC₅₀ 5–50 nM), and inhibited chloroquine-sensitive and -resistant parasite proliferation (IC₅₀ 5–50 μM). Wild-type (WT) parasite IC₅₀ values were up to 4-fold lower compared to PfENT1-knockout (<i>pfent1</i>Δ) parasites. <i>pfent1</i>Δ parasite killing showed a delayed-death phenotype not observed with WT. We infer that, in parasites, the compounds inhibit both PfENT1 and a secondary target with similar efficacy. The secondary target identity is unknown, but its existence may reduce the likelihood of parasites developing resistance to PfENT1 inhibitors. Our data support the hypothesis that blocking purine transport through PfENT1 may be a novel and compelling approach for antimalarial drug development

Prev Chronic Dis

IntroductionHealth-risk behaviors such as eating poorly, being physically inactive, and smoking contribute to the leading causes of morbidity and mortality in the United States and are often established during adolescence and young adulthood. The objectives of this study were to characterize the health-risk behaviors of young adults (aged 18\u201324 years) using a large population-based survey of Americans and to determine if behaviors of this group differ by weight category, as assessed by body mass index (BMI).MethodsPrevalence estimates for selected health-risk behaviors were calculated for respondents aged 18 to 24 years to the 2003 Behavioral Risk Factor Surveillance System (BRFSS). Respondents were categorized by BMI, and comparisons between sex and race and ethnicity were made within the overweight and obese categories.ResultsMore than three quarters (78.4%) of respondents consumed fewer than five fruits and vegetables per day, 43.2% reported insufficient or no physical activity, 28.9% were current smokers, 30.1% reported binge drinking, and 11.9% reported frequent mental distress. One quarter (26.1%) of respondents were overweight, and 13.6% were obese. Of obese young adults, 67.2% reported that they currently were trying to lose weight; however, only 24.3% reported having received professional advice to lose weight. More obese women (34.2%) than obese men (16.7%) reported having received professional advice to lose weight. Only 19.1% of obese non-Hispanic white respondents had received professional advice to lose weight compared with 28.0% of obese Hispanic respondents and 30.6% of obese non-Hispanic black respondents.ConclusionMany young adults engage in unhealthy behaviors, and differences exist in health-risk behaviors by BMI category and specifically by sex and race and ethnicity within BMI categories. The transition from adolescence to adulthood may be an opportune time for intervening to prevent future chronic disease.2007720

A broad analysis of resistance development in the malaria parasite

Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance.Bill and Melinda Gates FoundationNational Institute of Allergy and Infectious DiseasesUniversity of California San Diego Genetics Training ProgramNational Institute of General Medical SciencesDepto. de Bioquímica y Biología MolecularFac. de FarmaciaTRUEpu

Mapping the malaria parasite drug-able genome using in vitro evolution and chemogenomics

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify novel antimalarial drug targets and drug resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. This study reveals 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with resistance acquisition where gene amplifications contribute to 1/3 of drug resistance acquisition events. Beyond confirming previously identified multidrug resistance mechanisms we find new drug target-inhibitor pairs, including: thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and drug-able genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite

The Plasmodium falciparum ABC transporter ABCI3 confers parasite strain-dependent pleiotropic antimalarial drug resistance.

Widespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains

Mapping the malaria parasite drug-able genome using<i>in vitro</i>evolution and chemogenomics

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target–inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.Bill and Melinda Gates FoundationThe National Institutes of HealthThe National Institute of Allergy and Infectious DiseasesThe National Institute of General Medical SciencesDepto. de Bioquímica y Biología MolecularFac. de FarmaciaTRUEpu