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Malian children infected with Plasmodium ovale and Plasmodium falciparum display very similar gene expression profiles [1]

['Kieran Tebben', 'Institute For Genome Sciences', 'University Of Maryland School Of Medicine', 'Baltimore', 'Maryland', 'United States Of America', 'Department Of Microbiology', 'Immunology', 'Baltimore Maryland', 'Salif Yirampo']

Date: 2023-02

Abstract Plasmodium parasites caused 241 million cases of malaria and over 600,000 deaths in 2020. Both P. falciparum and P. ovale are endemic to Mali and cause clinical malaria, with P. falciparum infections typically being more severe. Here, we sequenced RNA from nine pediatric blood samples collected during infections with either P. falciparum or P. ovale, and characterized the host and parasite gene expression profiles. We found that human gene expression varies more between individuals than according to the parasite species causing the infection, while parasite gene expression profiles cluster by species. Additionally, we characterized DNA polymorphisms of the parasites directly from the RNA-seq reads and found comparable levels of genetic diversity in both species, despite dramatic differences in prevalence. Our results provide unique insights into host-pathogen interactions during malaria infections and their variations according to the infecting Plasmodium species, which will be critical to develop better elimination strategies against all human Plasmodium parasites.

Author summary Multiple species of Plasmodium parasites can cause human malaria. Most studies and elimination efforts target P. falciparum, the most common cause of malaria worldwide and the species responsible for the vast majority of the mortality. Other Plasmodium species, such as P. ovale, typically lead to less severe forms of the disease but little is known about the molecular mechanisms at play during malaria infections with different parasites. We analyzed host and parasite gene expression from children successively infected with P. ovale and P. falciparum and found that, while the parasite gene expression differed significantly, the transcriptional profiles of the host immune cells were similar in P. ovale or P. falciparum infections. This suggests that infected individuals respond to uncomplicated malaria infections similarly, regardless of the Plasmodium species causing the infection, and that alternative immune processes may become important during the progression to severe P. falciparum malaria rather than being inherent features of P. falciparum infections. Additionally, we observed similar levels of genetic diversity among P. ovale and P. falciparum parasites, suggesting that the P. ovale population might be larger than currently thought, possibly due to extensive misdiagnosis or the existence of hidden reservoirs of parasites.

Citation: Tebben K, Yirampo S, Coulibaly D, Koné AK, Laurens MB, Stucke EM, et al. (2023) Malian children infected with Plasmodium ovale and Plasmodium falciparum display very similar gene expression profiles. PLoS Negl Trop Dis 17(1): e0010802. https://doi.org/10.1371/journal.pntd.0010802 Editor: Paul O. Mireji, Kenya Agricultural and Livestock Research Organization, KENYA Received: September 8, 2022; Accepted: January 16, 2023; Published: January 25, 2023 Copyright: © 2023 Tebben et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All sequence data generated in this study are deposited in the Sequence Read Archive under the BioProject PRJNA878485. Custom scripts are available at https://github.com/tebbenk/PfPo_RNAseq. Funding: This work was supported by awards from the National Institutes of Health (R21AI146853 to DS and MAT and R01HL146377 to MAT) and an NIAID-funded predoctoral fellowship (T32 AI095190 to KT). Participant enrollment and sample collection were supported by NIH grants U01AI065683, R01HL130750 and D43TW001589 to CVP and R01AI099628 to MATh. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: None

Introduction Plasmodium parasites caused 241 million cases of malaria and over 600,000 deaths in 2020 [1], a partial reversal of decades of progress towards elimination. Malaria symptoms derive from the asexual replication of Plasmodium parasites in human red blood cells (RBCs)[2]. At least five species of Plasmodium parasites commonly cause human malaria: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi [2]. P. falciparum is responsible for the majority of infections worldwide and is the dominant species in Sub-Saharan Africa [2,3]. P. falciparum can cause severe malaria [2] and is responsible for the majority of malaria deaths due to complications such as severe anemia or cerebral malaria. This high pathogenicity is thought to be, at least partially, due to the sequestration of mature asexual P. falciparum parasites in the microvasculature [4]. Post-mortem analyses of brain [5] and kidney [6] from individuals with severe malaria show parasite accumulation in the tissue microvasculature. This accumulation can lead to obstructions and focal hypoxia, and local increases in inflammatory cells and molecules responding to the parasites [5]. Rupture of adhered infected RBCs after schizont maturation can also lead to focal release of parasite antigens and immune-activating factors, contributing to localized tissue damage at the site of adherence [6]. While most studies focus on P. falciparum, other Plasmodium species also cause significant public health burden and the lack of specific knowledge about these parasites is becoming increasingly problematic. P. vivax is common in South Asia and South America but rare in Sub-Saharan Africa [1,7], while P. knowlesi is a recent zoonotic parasite causing infections in Southeast Asia [8]. P. ovale and P. malariae are widely distributed parasites that have typically been considered as relatively rare [9] and causing milder infections than P. falciparum [3,10,11]. Infection with these parasites typically leads to lower fevers [10] and parasitemia [10,11] than those with P. falciparum, possibly due to a slower intraerythrocytic replication with fewer merozoites produced per cycle [11] and a preference for specific RBCs [10,11]. Because these parasites are difficult to detect on peripheral blood smear [10,11] and often occur in coinfections with more virulent species [9], they are likely underdiagnosed [9]. P. ovale is further categorized into two phenotypically indistinguishable sub-species or species [12], P. ovale curtisi and P. ovale wallikeri, that are often co-endemic, notably in West Africa [2,3,13]. Since blood-stage parasites play a central role in disease severity, transmission and immunity/immune-evasion, analyses of host and parasite gene expression from infected blood samples could provide critical information on these processes and their regulation. Such studies have been performed from P. falciparum infections, either separately to examine changes in host [14–16] or parasite [17–22] transcriptional regulation during infections, or jointly [23–26] from the same samples to investigate host-pathogen interactions. By contrast, a single study has characterized the gene expression profiles of P. ovale parasites during an infection [27], and none have examined the host transcriptome during infections with this parasite. Here, we used dual RNA-sequencing (dual RNA-seq) to examine whole blood samples from three Malian children successively infected with P. falciparum and P. ovale, and simultaneously characterized the gene expression profiles of the host and parasites. Our analyses provide novel transcriptomic and genetic insights on P. ovale infections and allow a first examination of the host immune response to these infections and how this response differs from the immune response to P. falciparum infections.

Discussion Our data indicate that host gene expression does not differ dramatically between uncomplicated malaria infections caused by P. falciparum or P. ovale. This finding is somewhat surprising since species-specific immune responses during Plasmodium infections have been described in rodent [52,53] and human [54–56] studies. One possible explanation for this discrepancy is that previous studies compared infections with different severities and/or different infected individuals. The large differences in gene expression observed between infections caused by distinct Plasmodium species in those studies might therefore have been confounded by host gene expression differences associated with different disease presentations (e.g., severity or symptoms) and/or inter-individual variations. By contrast, our study compared the host gene expression profiles during similar uncomplicated malaria infections and in the same individuals, and clearly showed that host factors contribute more, quantitatively, to the host gene expression profiles during malaria infections than the infecting Plasmodium species (based on the number of differentially expressed human and Plasmodium genes). This result is consistent with reports of important interindividual differences [57] in the susceptibility to [58–60], and immune response against [61,62], P. falciparum infections. Alternatively, previous studies of host gene expression may have been confounded by differences in parasitemia since the parasite load typically differ between parasite species [3] and has been shown, within one species, to be associated with variations in host gene expression [26]. (Our analyses may also suffer from differences in parasitemia between infections, but those variations are not entirely confounded with the infecting species in our sample). We observed that the age of the individual significantly contributed to the differences in host gene expression. This observation could reflect the maturation of the immune system in young children [63], although, given our small sample size of this study, it is difficult to rigorously evaluate the individual contribution of different host factors (e.g., age, sex or ethnicity) which are confounded in this study. Future studies including more samples are needed to fully disentangle the role of these host factors, and other clinical variables, on the host gene expression during malaria infections. Despite the overwhelming importance of individual factors on the host gene expression, we detected statistically significant differences associated with the infecting species for a small number of human genes, possibly reflecting differences in the host response to these two Plasmodium species. We found a higher expression of genes related to dendritic cell development during P. falciparum infections, possibly influencing the effective bridging of the innate and adaptive immune system during infections by this species. P. falciparum parasites have been shown to lead to atypical activation of dendritic cells [64], but the comparison of dendritic cell responses to infection by different Plasmodium species may reveal important species-specific interactions. By contrast, we found that genes involved in activation of the innate immune system and T-cell suppression were expressed at higher levels in P. ovale infections, compared to P. falciparum infections. This is consistent with reports that, per parasitized RBC, P. ovale induces a stronger immune response than P. falciparum [65]. However, given our small sample size, statistical results for specific individual genes should be interpreted with caution. In contrast to the human gene expression results, we found that parasite gene expression vastly differs by species. This could be due to inherent differences in disease features (e.g., parasitemia) or due to true species-specific differences in blood-stage parasite regulation. Several studies have described species-specific gene expression between different species of Plasmodium parasites [66,67] but have primarily examined species-specific genes [67] and proteins [66]. While expression of species-specific variant surface antigens [68] and invasion machinery [69] has been documented, particularly for parasites such as P. falciparum [70] and P. vivax [71], our data suggest that there may also be species-specific expression of genes present in both genomes (i.e., orthologous genes), including genes involved in gametocytogenesis or immune modulation. We chose here to use CIBERSORTx [30] to estimate the relative proportion of each parasite developmental stage, including sexual stages, present in each infection. In contrast to methods developed to estimate the developmental age of parasites [18,72], which work well on relatively homogeneous parasite populations, this method [45] allows characterization of complex mixtures of stages present in a sample (including the gametocytes), and allows for correction of statistical tests for these proportions. This correction is critical for analyzing parasite RNA-seq data generated directly from blood samples since even rare parasite stages can dramatically impact the overall gene expression profile due to the stage-specific differences in transcriptional activity. The RNA-seq data generated also enabled a first glance at the genetic diversity of P. ovale in Mali using characterization of the DNA polymorphisms present in expressed transcripts. Despite the small sample size (only four P. ovale infections analyzed), our study revealed the presence of both sub-species of P. ovale. Interestingly, both the parasite and host gene expression profiles of infections caused by these highly divergent parasites were very similar compared to the profiles of P. falciparum infections. Indeed, we observed greater variation among the parasite gene expression profiles of P. falciparum infections than between those of P. ovale curtisi and P. ovale wallikeri infections. In addition, we observed comparable levels of genetic diversity among P. falciparum parasites as among P. ovale curtisi parasites. This is surprising given the stark difference in prevalence between the two species (and therefore in their population size). While it is difficult to precisely determine the prevalence of P. ovale, due to under-detection and species misidentification, P. ovale has been reported at about 2% prevalence in Mali compared to ~50% for P. falciparum [73]. The observation of similar genetic diversity despite drastic differences in (census) population size is puzzling and suggests that i) the prevalence of P. ovale in Mali is widely underestimated, due to misdiagnosis or high proportion of asymptomatic infections, or ii) that there is a large hidden reservoir of P. ovale parasites. This observation will require validation using larger cohorts but is important to consider as we move closer towards malaria elimination, as it may indicate that some parasite populations are able to maintain a high level of genetic diversity despite little circulation in the population.

Conclusions Here, we described the transcriptional profiles of host and parasites during malaria infections caused by P. ovale or P. falciparum. We found that host factors contribute more to the human gene expression profiles than the species causing the infection, suggesting i) that age, sex or other individual host characteristics play a key role in determining the regulation of white blood cells during malaria infections, and ii) that the host responses to P. ovale and P. falciparum infections are not drastically different (for uncomplicated malaria infections). Despite this overall similarity in response, we detected a few human genes differentially regulated in infections with P. ovale vs P. falciparum suggesting that the host adaptive immune response to these parasites may differ. In addition to insights on the transcriptional regulation of the parasites, this study enabled rigorous characterization of DNA polymorphisms, which revealed the presence of both sub-species of P. ovale and a surprisingly high level of genetic diversity in P. ovale (comparable to that of P. falciparum). Overall, this study provides new insights on the regulation and diversity of P. ovale infections that have important implications for the development of pan-malaria vaccines and for developing approaches to eliminate malaria.

Methods Ethics approval and consent Individual informed consent/assent was collected from all children and their parents. The study protocol and consent/assent process were approved by the institutional review boards of the Faculty of Medicine, Pharmacy and Dentistry of the University of Maryland, Baltimore and of the University of Sciences, Techniques and Technologies of Bamako, Mali (IRB numbers HCR-HP-00041382 and HP-00085882). Samples Samples included in this study were collected from uncomplicated malaria infections from treatment-seeking children from Bandiagara, Mali [28]. Briefly, blood samples were collected from children during unscheduled, patient-initiated visits with i) presentation of symptoms consistent with malaria (fever, headaches, joint pain, vomiting, diarrhea, or abdominal pain) and ii) identification of Plasmodium parasites by thick smear. All infections were successfully treated with antimalarial drugs. Whole-blood samples were collected and preserved in PAXgene blood RNA tubes and stored at -80°C until extraction [28]. We selected, for these analyses, nine blood samples collected from three children successively infected with P. falciparum and P. ovale (determined by light microscopy [28]). Generation of RNA-seq data We extracted RNA from whole blood using MagMax blood RNA kits (Themo Fisher) (between 0.24 μg and 3.16 μg total from each sample). Total RNA was subjected to rRNA depletion and polyA selection (NEB) before preparation of stranded libraries using the NEBNext Ultra II Directional RNA Library Prep Kit (NEB). cDNA libraries were sequenced on an Illumina NovaSeq 6000 to generate ~55–130 million paired-end reads of 75 bp per sample (S2 Table). To confirm the Plasmodium species responsible for the malaria episode, we first aligned all reads from each sample using hisat2 v2.1.0 [74] to a fasta file containing the genomes of P. falciparum 3D7, P. vivax PvP01, P. malariae UG01, and P. ovale curtisi GH01 genomes downloaded from PlasmoDB [75] v55. For the remaining analyses described in this study, we relied on the alignment of all reads using hisat2 to a fasta file containing the P. falciparum 3D7, P. ovale GH01 and human hg38 genomes i) using default parameters and ii) using (—max-intronlen 5000). Reads mapping uniquely to the hg38 genome were selected from the BAM files generated with the default parameters. Reads mapping uniquely to either Plasmodium genome were selected from the BAM files generated with a maximum intron length of 5,000 bp. PCR duplicates were removed from all files using custom scripts. We then calculated read counts per gene using gene annotations downloaded from PlasmoDB (Plasmodium genes) and NCBI (human genes) and the subread featureCounts v1.6.4 [76]. Gene expression analysis We excluded one sample, B4, from all analyses due to a high percent of duplicated reads (96.1% of human reads, 94.5% of P. falciparum reads, S2 Table). For all other samples, read counts per gene were normalized into counts per million (CPM), separately for human and Plasmodium genes. To filter out lowly expressed genes, only human genes that were expressed at least at 10 CPM in > 50% of the samples were retained for further analyses (9,884 genes). Plasmodium genes were filtered using the same criteria, and additionally selected to only include 1:1 orthologs between P. falciparum and P. ovale (2,631 genes). Read counts were normalized via TMM for differential expression analyses. Statistical assessment of differential expression was conducted, separately for the human and Plasmodium genes, in edgeR (v 3.32.1) [77] using a quasi-likelihood negative binomial generalized model i) without covariates for human genes and ii) with and without correcting for proportion of each parasite developmental stage for Plasmodium reads. All results were corrected for multiple testing using false discovery rate (FDR) [78]. Gene expression deconvolution CIBERSORTx [30] was used to estimate, in each sample, the proportion of i) human immune cell subtypes and ii) Plasmodium developmental stages. To deconvolute human reads, we used as a reference LM22 [79], a validated leukocyte gene signature matrix using 547 genes to differentiate 22 immune subtypes (collapsed to eight categories in our analysis). A custom signature matrix derived from P. berghei scRNA-seq data was used for P. falciparum and P. ovale stage deconvolution, using orthologous genes for the appropriate species [45]. Complexity of infection and genotyping To assess the complexity of each infection (i.e., monoclonal vs. polyclonal), allele frequency plots [80] were generated for each sample by calculating the proportion of reads with a given reference allele at each nucleotide position covered at > 50X. We also calculated F ws for all P. falciparum and P. ovale infections using moimix, excluding multi-gene families, according to the methodology described in Bradwell et al. [23]. Pairwise nucleotide differences were determined using each position covered at > 20X in a given pair of samples, separately for P. falciparum and P. ovale infections. Phylogenetic analysis We reconstructed the entire Plasmodium Cytochrome B sequence from each sample using the mpileup file generated from the RNA-seq data and using, at each nucleotide position covered by at least 20 reads, the allele present in most reads. We then generated a neighbor-joining tree with MEGA 11 [81] using the cytochrome B sequences from all sequenced isolates and publicly available sequences for P. ovale curtisi, P. ovale wallikeri and P. vivax in and 500 bootstraps.

Acknowledgments We thank the participants and their families for participating in this study, as well as the community of Bandiagara, Mali.

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