Influential parameters for the analysis of intracellular parasite metabolomics

Carey, Maureen A.; Covelli, Vincent; Brown, Audrey C.; Medlock, Gregory L.; Haaren, Mareike; Cooper, Jessica G.; Papin, Jason A.; Guler, Jennifer L.

doi:10.1101/190421

Cited by 4 publications

(3 citation statements)

References 49 publications

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“…Other 'omics-level methods also suffer from difficulty identifying parasite signal over human host material. An abundance of host proteins and metabolites leads to difficulty, even in vitro, in generating high-quality proteomics [74] and metabolomics [75], respectively. Thus, proteomics and metabolomics on samples directly from hosts have focused on analyzing human serum to infer biology of disease diversity and drug resistance effects through host-parasite interactions [76][77][78][79][80][81].…”

Section: Direct-from-host Methodologies Exhibit Distinct Challenges Amentioning

confidence: 99%

From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro

Brown

Guler

2020

Trends in Parasitology

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Research on Plasmodium parasites has driven breakthroughs in reducing malaria morbidity and mortality. Experimental analysis of in vivo/ex vivo versus in vitro samples serve unique roles in Plasmodium research. However, these distinctly different environments lead to discordant biology between parasites in host circulation and those under laboratory cultivation. Here, we review how in vitro factors, such as nutrient levels and physical forces, differ from those in the human host and the resulting implications for parasite growth, survival, and virulence. Additionally, we discuss the current utility of direct-from-host methodologies, which avoid the potentially confounding effects of in vitro cultivation. Finally, we make the case for methodological improvements that will drive research progress of physiologically relevant phenotypes. Highlights Standard in vitro culture environments differ dramatically from in vivo conditions in nutrient levels, hematocrit, and rheology and have lower variability in gas levels and temperature. Nutritional and physical differences lead to pronounced, and often rapid, changes in phenomenon, important for understanding virulence in Plasmodium. Parasite drug sensitivity may be altered due to culture adaptation selection, supraphysiological metabolite concentrations, and in vitro media formulations. Parasites propagated in vitro, versus in vivo, show altered transcriptomic and genomic patterns related to virulence factors, metabolism, gametocytogenesis, and more. Direct-from-host methodologies avoid the impacts of in vitro culture adaptation but limit the types of assessments that can be performed as many experiments either require equipment not readily available in endemic settings or necessitate long-term manipulation.

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Section: Direct-from-host Methodologies Exhibit Distinct Challenges Amentioning

confidence: 99%

From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro

Brown

Guler

2020

Trends in Parasitology

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“…The extraction methods and mass spectrometry platforms used in previous studies precluded them from detecting a large number of lipid metabolites [4,8]. Several recent studies [6,23,24] quantified more metabolites than in the studies by Babbitt et al [8] or Olszewski et al [4]. However, they either did not examine all parasite stages or did not include matched uninfected erythrocyte cultures during the IDC (see Table 1).…”

Section: Metabolomics Of Blood-stage Malaria Parasitesmentioning

confidence: 99%

Metabolic alterations in the erythrocyte during blood-stage development of the malaria parasite

Tewari

Swift

Reifman

et al. 2020

Malar J

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Background: Human blood cells (erythrocytes) serve as hosts for the malaria parasite Plasmodium falciparum during its 48-h intraerythrocytic developmental cycle (IDC). Established in vitro protocols allow for the study of host-parasite interactions during this phase and, in particular, high-resolution metabolomics can provide a window into host-parasite interactions that support parasite development. Methods: Uninfected and parasite-infected erythrocyte cultures were maintained at 2% haematocrit for the duration of the IDC, while parasitaemia was maintained at 7% in the infected cultures. The parasite-infected cultures were synchronized to obtain stage-dependent information of parasite development during the IDC. Samples were collected in quadruplicate at six time points from the uninfected and parasite-infected cultures and global metabolomics was used to analyse cell fractions of these cultures. Results: In uninfected and parasite-infected cultures during the IDC, 501 intracellular metabolites, including 223 lipid metabolites, were successfully quantified. Of these, 19 distinct metabolites were present only in the parasite-infected culture, 10 of which increased to twofold in abundance during the IDC. This work quantified approximately five times the metabolites measured in previous studies of similar research scope, which allowed for more detailed analyses. Enrichment in lipid metabolism pathways exhibited a time-dependent association with different classes of lipids during the IDC. Specifically, enrichment occurred in sphingolipids at the earlier stages, and subsequently in lysophospholipid and phospholipid metabolites at the intermediate and end stages of the IDC, respectively. In addition, there was an accumulation of 18-, 20-, and 22-carbon polyunsaturated fatty acids, which produce eicosanoids and promote gametocytogenesis in infected erythrocyte cultures. Conclusions: The current study revealed a number of heretofore unidentified metabolic components of the hostparasite system, which the parasite may exploit in a time-dependent manner to grow over the course of its development in the blood stage. Notably, the analyses identified components, such as precursors of immunomodulatory molecules, stage-dependent lipid dynamics, and metabolites, unique to parasite-infected cultures. These conclusions are reinforced by the metabolic alterations that were characterized during the IDC, which were in close agreement with those known from previous studies of blood-stage infection.

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“…29 In a recent study on the use of purified in vitro-grown ringstage intraerythrocytic Plasmodium falciparum parasites in untargeted metabolomics studies, the authors investigated confounding factors that influence data interpretation, including host contamination and normalization approaches (including double-stranded DNA, total protein, and parasite numbers), and revealed that normalization does not remove the contribution from the parasite's extracellular environment (culture media and host erythrocyte). 30 While analyzing the cell-secretome (i.e. metabolites secreted into the media), cell count-or culture volume-based normalization can be challenging.…”

Section: Cell-based Methodsmentioning

confidence: 99%

Data normalization strategies in metabolomics: Current challenges, approaches, and tools

Misra

2020

Eur J Mass Spectrom (Chichester)

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Data normalization is a big challenge in quantitative metabolomics approaches, whether targeted or untargeted. Without proper normalization, the mass-spectrometry and spectroscopy data can provide erroneous, sub-optimal data, which can lead to misleading and confusing biological results and thereby result in failed application to human healthcare, clinical, and other research avenues. To address this issue, a number of statistical approaches and software tools have been proposed in the literature and implemented over the years, thereby providing a multitude of approaches to choose from – either sample-based or data-based normalization strategies. In recent years, new dedicated software tools for metabolomics data normalization have surfaced as well. In this account article, I summarize the existing approaches and the new discoveries and research findings in this area of metabolomics data normalization, and I introduce some recent tools that aid in data normalization.

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Influential parameters for the analysis of intracellular parasite metabolomics

Cited by 4 publications

References 49 publications

From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro

From Circulation to Cultivation: Plasmodium In Vivo versus In Vitro

Metabolic alterations in the erythrocyte during blood-stage development of the malaria parasite

Data normalization strategies in metabolomics: Current challenges, approaches, and tools

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