Influential Parameters for the Analysis of Intracellular Parasite Metabolomics

Moore

Guler

2020

Sci Rep

Self Cite

for intracellular pathogens, the host cell provides needed protection and nutrients. A major challenge of intracellular parasite research is collection of high parasite numbers separated from host contamination. This situation is exemplified by the malaria parasite, which spends a substantial part of its life cycle inside erythrocytes as rings, trophozoites, and schizonts, before egress and reinvasion. erythrocytic Plasmodium parasite forms refractory to enrichment remain understudied due to high host contamination relative to low parasite numbers. Here, we present a method for separating all stages of Plasmodium-infected erythrocytes through lysis and removal of uninfected erythrocytes. The Streptolysin O-Percoll (SLOPE) method is effective on previously inaccessible forms, including circulating rings from malaria-infected patients and artemisinin-induced quiescent parasites. SLope can be used on multiple parasite species, under multiple media formulations, and lacks measurable impacts on parasite viability. We demonstrate erythrocyte membrane cholesterol levels modulate the preferential lysis of uninfected host cells by SLO, and therefore modulate the effectiveness of SLOPE. Targeted metabolomics of SLOPE-enriched ring stage samples confirms parasite-derived metabolites are increased and contaminating host material is reduced compared to non-enriched samples. Due to consumption of cholesterol by other intracellular bacteria and protozoa, SLope holds potential for improving research on organisms beyond Plasmodium. Malaria, caused by protozoan parasites of the Plasmodium genus, is a continuing threat to global health. A total of five Plasmodium species cause malaria in humans, with Plasmodium falciparum being responsible for the large majority of malaria morbidity and mortality 1. While the global malaria burden has decreased over the past decade, the emergence and spread of antimalarial resistant Plasmodium threatens to undo this progress and emphasizes the dire need to understand more about the biology of this parasite. The current World Health Organization recommendation for treatment of malaria is artemisinin combination therapy 2. However, clinical resistance has now been reported to both artemisinin and almost all of its partner drugs 3-5. All symptoms of malaria, including cyclical fevers and hypoglycemia, occur due to the asexual replication cycle of the parasite within human erythrocytes (Fig. 1a). Parasites undergo rounds of replication progressing from the ring stage, to trophozoites and schizonts, before rupturing from host erythrocytes to release merozoites, which go on to invade new erythrocytes and continue the cycle of infection 6. Many studies aiming to understand the biology of asexual Plasmodium are performed only on late stage parasite samples (trophozoites and schizonts). This is due in part to the larger biomass of these stages but also to the existence of effective enrichment methods 7 ; erythrocytes infected with late stage parasites can be separated from uninfected erythrocytes by density...

Section: Discussionmentioning

confidence: 99%

Cholesterol-dependent enrichment of understudied erythrocytic stages of human Plasmodium parasites

Moore

Guler

2020

Sci Rep

Self Cite

“…The inability to enrich for ring stage parasites has exacerbated host contamination of samples and limited our ability to discern interesting parasites phenotypes (18,21). Successful ring stage enrichment will beneficially reduce contribution of non-parasite factors not only for metabolomics studies, but for a variety of ring stage related experiments.…”

Section: Discussionmentioning

confidence: 99%

“…We previously explored parameters important for metabolomics studies of P. falciparum (21). Not surprisingly, the metabolomes of non-enriched ring stage samples were heavily influenced by host erythrocyte contamination (about a third of 375 total metabolites detected were associated with the blood cells used for parasite propagation).…”

Section: Introductionmentioning

confidence: 99%

“…Results from these two studies emphasize the introduction of 'noise' from a large excess of human host erythrocyte material present in ring stage samples; without an effective way to separate infected from uninfected erythrocytes, only a small fraction of the total cells contain ring-stage parasites (typical parasitemia ranges between 1%-10%). The challenge of detecting parasitespecific material is further exacerbated by leftover erythrocyte membrane material following commonly used lysis steps (21). Data analysis steps (i.e.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Cholesterol-dependent enrichment of understudied erythrocytic stages of humanPlasmodiumparasites

Moore

Guler

2018

Preprint

Self Cite

9 Plasmodium protozoan parasites undergo rounds of asexual replication inside human 10 erythrocytes, progressing from ring stage, to trophozoites and schizonts, before egress and 11 reinvasion. Given the discovery of ring-specific artemisinin tolerance and quiescence in 12Plasmodium falciparum, there is great urgency to better understand ring stage biology. However, 13the lack of an effective enrichment method has left rings and related parasite stages 14 understudied compared to their late stage counterparts, which can be easily isolated due to their 15 paramagnetic properties. Here, a method for separating all Plasmodium infected erythrocytes 16 from uninfected erythrocytes is presented. This approach takes advantage of streptolysin-O 17(SLO) to preferentially lyse uninfected erythrocytes as previously shown by Jackson, et al. 18Following lytic treatment, Percoll gradient centrifugation removes lysed cells, leaving an intact 19 cell population enriched in infected erythrocytes. This SLO-Percoll (SLOPE) method is effective 20 on stages from the entire erythrocytic cycle, including previously inaccessible forms such as 21circulating rings from malaria-infected patients and artemisinin-induced quiescent parasites. 22Furthermore, the utility of SLOPE is extended to multiple media formulations used for the 23 propagation of two human Plasmodium species. The alteration of external cholesterol levels 24modulates SLOPE effectiveness, demonstrating the role of erythrocyte membrane cholesterol 25 in lytic discrimination. Importantly, enrichment does not impact parasite viability, which 26 establishes the non-toxic nature of SLOPE. Targeted metabolomics of SLOPE-enriched ring 27 stage samples confirms the impact on treated samples; parasite-derived metabolites are 28 increased and contaminating host material is reduced compared to non-enriched samples. 30Importance 31Malaria is caused by infection with protozoan Plasmodium parasites and is responsible for over 32 400,000 deaths annually. The availability of effective antimalarial drugs is critical to the reduction 33 of malaria-related mortality, yet widespread resistance highlights the need for the continued 34 study of Plasmodium biology. The SLOPE method is an accessible, scalable, rapid (30-40min), 35and non-toxic enrichment method that is broadly effective on many erythrocytic stages. This 36 method is ideal for use upstream of a variety of sensitive analyses, which will increase 37 experimental quality in virtually all areas of asexual Plasmodium parasite research. Further, 38because the consumption of cholesterol is a common characteristic of other intracellular 39 parasites (both bacteria and other protozoa), SLOPE holds potential for extension to other 40 relevant pathogens. 41 45 falciparum being responsible for the large majority of malaria morbidity and mortality (1). While 46 the global malaria burden has decreased over the past decade, the emergence and spread of 47 antimalarial resistant Plasmodium threatens to undo this progress and emphasizes the dire nee...

“…The major contaminating DNA source that we detected in our samples was from humans ( Figure 574 2A). This was not surprising given that, in our experimental system, the culture and host 575 environments are rich in human DNA [79,80,91]. It is also possible that human DNA was 576 introduced during the single cell isolation or WGA steps [55].…”

mentioning

confidence: 93%

Single cell sequencing of the small and AT-skewed genome of malaria parasites

Liu

Huckaby

et al. 2020

Preprint

24Single cell genomics is a rapidly advancing field; however, most techniques are designed for 25 mammalian cells. Here, we present a single cell sequencing pipeline for the intracellular parasite, 26 Plasmodium falciparum, which harbors a relatively small genome with an extremely skewed 27 base content. Through optimization of a quasi-linear genome amplification method, we achieve 28 more even genome coverage and better targeting of the parasite genome over contaminants. 29 These improvements are particularly important for expanding the accessibility of single cell 30 approaches to new organisms and cell types and for improving the study of genetic mechanisms 31 of adaptation. 32 33 Keywords: whole-genome amplification, AT-skewed genome, malaria, single cell 34 sequencing, MALBAC 35 36 Background 37 Malaria is a life-threatening disease caused by protozoan Plasmodium parasites. P. falciparum 38 causes the greatest number of human malaria deaths [1]. The clinical symptoms of malaria occur 39 when parasites invade human erythrocytes and undergo rounds of asexual reproduction by 40 maturing from early forms into late stage parasites and bursting from erythrocytes to begin the 41 cycle again [2]. In this asexual cycle, parasites possess a single haploid genome during the early 42 stages; rapid genome replication in the later stages leads to an average of 16 genome copies [2].43 44Due to a lack of an effective vaccine, antimalarial drugs are required to treat malaria. However, 45 drug efficacy is threatened by the frequent emergence of resistant populations [3]. Copy number variations (CNVs), or the amplification and deletion of a genomic element, is one of the major 47 sources of genomic variation in P. falciparum that contribute to antimalarial resistance [4][5][6][7][8][9][10][11][12][13][14][15]. 48Similar to bacteria and viruses [16][17][18], a high rate of CNVs may initiate genomic changes that 49 contribute to the rapid adaptation of this organism [7,19]. Despite the importance of CNVs, their 50 dynamics in evolving populations are not well understood. 52The majority of CNVs in P. falciparum have been identified by analyzing bulk DNA in which 53 the CNVs are present in a substantial fraction of individual parasites in the population due to 54 positive selection [8,10,11,20,21]. However, many CNVs likely remain undetected because 55 they are presumably either deleterious or offer no advantages for parasite growth or transmission 56 [22] and are therefore present in low frequency [20,22]. Currently, most CNVs are identified 57 using read-depth analysis of short read sequencing data, which derives an average signal across 58 the population. For this reason, genetic variants must be present in a high frequency (i.e. ~50%) 59 in the population to be detected [23][24][25]. Sequencing at very high depth improves the detection 60 of low frequency CNVs, but the sensitivity is limited to large-scale CNVs present in > 5% cells 61 [26][27][28]. Analysis methods that rely on the detection of reads that span CNV junctions h...