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Genetic control of parasite clearance leads to resistance to Plasmodium berghei ANKA infection and confers immunity

Abstract

Unprecedented cure after infection with the lethal Plasmodium berghei ANKA was observed in an F2 progeny generated by intercrossing the wild-derived WLA and the laboratory C57BL/6 mouse strains. Resistant mice were able to clear parasitaemia and establish immunity. The observed resistance was disclosed as a combinatorial effect of genetic factors derived from the two parental strains. Genetic mapping of survival time showed that the WLA allele at a locus on chromosome 1 (colocalizing with Berghei resistance 1 (Berr1), a locus associated with resistance to experimental cerebral malaria) increases the probability to resist early death. Also, the C57Bl/6 allele at a novel locus on chromosome 9 (Berr3) confers overall resistance to this lethal Plasmodium infection. This report underlines the value of using wild-derived mouse strains to identify novel genetic factors in the aetiology of disease phenotypes, and provides a unique model for studying parasite clearance and immunity associated with malaria.

Introduction

Malaria is a major world-scale health problem being responsible for 1–2 million deaths every year, mainly in tropical Africa where it is endemic. Plasmodium falciparum is the leading cause of death and is responsible for the more severe forms of the disease, including the neurological syndrome cerebral malaria (CM). Efforts to find an effective way to control the disease have been thwarted by the emergence of parasite resistance to the commonly used drugs and by mosquito resistance to insecticides. Given the high impact of malaria as a public health problem, new targets for therapeutic intervention are fundamental. The understanding of the natural mechanisms of host defense against the disease could point out novel intervention targets.

The influence of the host genetic background on malaria has been verified in several studies, both in humans and in murine models. Epidemiological studies and genetic linkage analyses in endemic areas have underscored the importance of the host genetic component against the disease.1, 2, 3 The influence of genetic background on disease outcome is equally well established in murine models. This difference in susceptibility to malaria infection between mouse strains has prompted genetic studies in informative backcross and F2 progenies, as well as in recombinant inbred and congenic strains. These studies have confirmed that genetic susceptibility to malaria is indeed complex and have resulted in the mapping of several loci associated with susceptibility to P. chabaudi (Char18),4, 5, 6, 7, 8, 9 P. yoelii (Pmyr)10 and P. berghei.11 Furthermore, we recently identified two novel loci (Berghei resistance 1 and 2 (Berr1 and 2)) associated with resistance to experimental cerebral malaria (ECM) induced by P. berghei ANKA.12

To further analyse the genetic basis for the control of resistance to P. berghei ANKA, we exploited the natural genetic diversity provided by inbred wild-derived mouse strains, and studied the segregation of malaria-related phenotypes in a (WLA × C57BL/6J)F2 cross. A novel phenotype, characterized by spontaneous cure to malaria infection induced by the otherwise lethal P. berghei ANKA, was identified in this F2 progeny representing the first description of mice capable of survival and cure from this Plasmodium infection. The segregation analysis of the F2 cohort revealed three major loci on chromosomes 1, 4 and 9 that control the survival time and parasitaemia kinetics during P. berghei ANKA infection.

Results

All laboratory and wild-derived inbred strains tested thus far for P. berghei ANKA infection die of ECM or hyperparasitaemia (HP), including the C57BL/6 and the WLA strains used in this study.12 After infection, C57BL/6 mice show neurological symptoms typical of ECM and low parasitaemia rate (on average less than 15%) with death occurring 5 to 9 days postinfection. The C57BL/6 strain is commonly used as a prototype of an ECM susceptible strain. On the other hand, as we described previously, WLA and (WLAxC57BL/6)F1 mice do not show any ECM symptoms, but invariably die later with high levels of parasitaemia (70–85% parasitized red blood cells) (Figure 1).12

Figure 1
figure1

Course of infection by P. berghei ANKA. Survival curves and parasitaemia levels were determined using 11 C57Bl/6 and 11 WLA mice. All individuals succumbed to infection.

The analysis of peak parasitaemia and survival time in 219 (WLAxC57BL/6)F2 animals showed that the response to malaria in F2 mice is segregating in three different phenotypic groups (Figure 2). The majority (159) of the F2 mice (72.6%) died between days 11 and 33 due to the high levels of parasitaemia, similar to the WLA and F1 mice. A second group of 38 (17.4%) F2 mice showed a severe form of disease and died early with low parasitaemia. Within the latter group, eight individuals were identified with clear signs of ECM resembling the parental C57BL/6 strain. Unexpectedly, a third group of 22 F2 mice (10%) were found to survive from infection, representing a new phenotype not observed either in the parental strains or any other inbred mouse strains tested so far. These individuals reached limited parasitaemia peaks of 14–45% between days 10 and 14, but were able to clear parasitaemia and cure (Figure 3). These animals kept asymptomatic for at least 1 year and had lifespans comparable to noninfected mice. They were also able to breed and give rise to normal progeny. Some of these mice were reinfected 1-year later and survived a reinfection experiment showing significantly lower peak parasitaemia levels (one sided P-value=0.0195, Wilcoxon's signed-rank test for paired samples) than observed during the first infection (Table 1). To our knowledge this is the first observation of survival following P. berghei ANKA infection, including parasite clearance and induction of immunity in mice.

Figure 2
figure2

Segregation of malaria phenotypes in a (WLA × C57BL/6)F2 progeny. F2 mice were scored according to the survival time and the peak of parasitaemia.

Figure 3
figure3

Parasite clearance and spontaneous cure after P. berghei ANKA infection. The lines represent the course of parasitaemia of each (WLA × C57BL/6)F2 mice that resist to infection (22 mice total).

Table 1 Parasitaemia peaks in (WLA × C57BL/6)F2 malaria resistant mice

In order to disclose the genetic factors underlying these different malaria phenotypes we next performed a genome-wide scan in the F2 progeny. Cosegregation of each of the three observed phenotypes with genetic markers covering all the mouse genome was evaluated by performing the Pearson's χ2 test of association in contingency tables. Two loci, on distal chromosomes 1 (D1Mit221 P-value=4.91 × 10−7) and 9 (D9Mit18 P-value=3.6 × 10−5), revealed association above the proposed empirically determined genome-wide 5% threshold of significance (P-value <5.2 × 10−5).13

To evaluate the relative contribution of each of these loci to the segregation of malaria phenotypes in the F2 progeny, we searched for quantitative trait loci (QTL) controlling the survival time after infection. This phenotype in our F2 progeny did not approach a normal distribution required for traditional QTL tools, rather it showed a spike in the distribution constituted by the animals that cured from infection. We, therefore, analysed these data using the two-part QTL model as proposed by Broman et al.14, 15 This analysis revealed that the time of survival in the F2 progeny was associated to both loci on chromosomes 1 and 9 (Figure 4). No evidence for linkage was found in any other chromosomal regions.

Figure 4
figure4

QTL analysis of the survival time in the (WLA × C57BL/6)F2 progeny. LOD score plots were generated with the R/QTL software. Survival time showed significant association with chromosomes 1 and 9. Horizontal line represents the proposed empirically determined significance threshold (LOD=4.1). The ticks in the X-axis represent the relative position of microsatellite markers along chromosomes 1 and 9, respectively, from left to right D1Mit211, DiMit156, D1Mit81, D1Mit245, D1Mit202, D1Mit221, D1Mit262 and D9Mit91, D9Mit171, D9Mit353, D9Mit35, D9Mit24, D9Mit212, D9Mit18 and D9Mit279.

The locus on the distal region of chromosome 9 reached the highest significance level close to D9Mit18 (logarithms of the odds (LOD)=4.9) and is here referred to as Berr3. The C57BL/6 allele at this locus appears to confer increased probability to survival following infection by P. berghei ANKA. In particular, 26.1% of the individuals C57BL/6 homozygous at the D9Mit18 marker were found to cure from infection (54.4% of all resistant mice), while 96.7% of the mice homozygous for the WLA allele died within 35 days postinfection (Tables 2Table 2 and 3).

Table 2 Relative penetrance of malaria cure and death in the (WLA × C57BL/6) F2 mice
Table 3 Genotypes of the survivors (WLAxC57BL/6)F2 mice

The locus on distal chromosome 1 reached maximum significance over the marker D1Mit221 (LOD=6.4) that is mapping within the previously reported Berr1 locus.10 An over-representation of the WLA allele at this locus was observed among the mice that resisted early death (Table 3). In fact, only 5.3% of the mice that died early (before 13 days postinfection) were homozygous for the WLA allele, while 55% were C57BL/6 homozygous at the marker D1Mit221. This suggests that WLA genetic factors increase the probability of F2 mice to resist early death, resembling the parental WLA strain (Figure 5).

Figure 5
figure5

Effect of the Berr1 locus on the survival time after P. bergheri ANKA infection of the F2 cohort. Mice that died of infection were classified according to their genotype at the marker D1Mit221 and the average survival time is represented for each genotype group. In all, 34 mice were homozygous for C57BL/6 (bb), 59 homozygous for WLA (ww) and 104 were heterozygous (wb). Error bars represent the s.e.m.

By analysing independently the group of mice that died due to HP, we also observed a difference in parasitaemia kinetics. Two distinct groups could be identified, each reaching similar peaks of parasitaemia but with different kinetics (Figure 2). Thus, one group of animals developed high parasitaemia levels around 2-3 weeks after infection, while the other group reached similar levels only at 4–5 weeks postinfection. To search for QTLs controlling the survival time in these groups, we used the nonparametric QTL model (for non-normal distributed data).15 The analysis revealed evidence of association of this trait to a novel locus on chromosome 4. This locus was designated Berr4 and reached the highest association over the marker locus D4Mit27 (LOD=3.42). An over-representation of the WLA alleles at this marker locus among the mice that exhibited extended resistance to HP (68% of all WLA homozygous) (Table 4.) suggested that the WLA allele at this locus conferred resistance. This locus was not identified when analysing survival time after infection in all F2 progeny. In fact, we observed that at this locus, the genotypes of the mice that cure from infection were distributed according to Mendel's law (Table 3), enforcing that this locus only influences time of death due to HP. In contrast, we noted a deviation of the distribution at the chromosomes 1 and 9 loci, with an over-representation of the resistant alleles (WLA for D1Mit221 and C57Bl/6 for D9Mit18). This observation implies that the survival phenotype results to large extent from the joint effect of two resistance loci.

Table 4 Relative penetrance of early and late death due to HP in the (WLA × C57BL/6)F2 mice

Discussion

The use of murine models has been instrumental in the genetic analysis of susceptibility/resistance to malaria. However, this approach is restricted by the use of common laboratory mouse strains that comprise a limited pool of natural genetic diversity. This limitation can be overcome by the use of inbred strains derived from wild progenitors, which provide a source for genetic variation.16, 17, 18 We have previously exploited the genetic diversity represented by these strains in order to identify phenotypes related to malaria susceptibility/resistance and to dissect their genetic control.12, 19 Using this approach, we reported a novel phenotype constituted by survival and cure from infection by P. berghei ANKA. This phenotype was represented in 10% of (WLAxC57BL/6)F2 mice, while all the remaining individuals died either early with low parasitaemia, in some cases with clear signs of ECM (17.4%), or later due to HP (72.6%). This constitutes the first experimental model of spontaneous cure to P. berghei ANKA infection.

The novel phenotype of complete parasitaemia clearance, cure and improved response to reinfection in the F2-resistant mice is the result of combinatorial factors derived from the two parental strains. Most likely, the novel phenotype represents an immunological phenotype, implicating genes with immune-related function as likely candidates genes for one or several of the loci involved.

Two major loci, on distal chromosomes 1 and 9, and a third suggested locus on chromosome 4 were identified as controlling the segregation of the observed phenotypes. The WLA allele at the locus on chromosome 1 appears to increase the resistance to early death, as significant under-representation of this allele was found among the mice that died early. This locus maps to a chromosomal region reported to harbour the Berr1 locus, previously found to control susceptibility/resistance to severe malaria after P. berghei ANKA infection.12 At Berr1, the WLA allele was identified as protecting from ECM in a (WLAxC56BL/6)xC57BL/6 backcrossed cohort. These results suggest that the locus on chromosome 1 described here could represent the same genetic factor as Berr1. Interestingly, several QTLs associated with resistance/susceptibility to intracellular pathogens have been mapped to the Berr1 region, suggesting that a common genetic factor that determines the host response to different pathogens is located in this chromosomal region. Candidate genes in the region that may be of particular interest from this perspective include the Toll-like receptor 5 (Tlr5), the transforming growth factor beta 2 and the tumour necrosis factor receptor-associated factor 5.

The C57BL/6 allele at the Berr3 locus on chromosome 9 increases the probability of survival and cure following infection, as demonstrated by its over-representation among the surviving mice. The localization of the Berr3 locus close to the loci Char1 and Pymr, described to be associated with resistance to P. chabaudi and P. yoelli, respectively, emphasizes the importance of this region in resistance to infection by different Plasmodium strains. This suggests that a common genetic factor underlies these observations. The chromosomal region harbours several candidate genes encoding chemokine receptors (Ccr1–5, Cxcr6, Ccr9), of which particular interest has been spurred by recent reports indicating that deficiency in Ccr5 reduces susceptibility to ECM induced by P. berghei ANKA.20 The adaptor protein Myd88 is another candidate gene in the region that has previously been associated with malaria pathogenesis. This protein is recruited by TLRs to initiate signalling pathways and was demonstrated to be a necessary factor in the development of liver injury induced by P. berghei Nk65 infection in mice.21

The locus identified on chromosome 4 (Berr4) was found to be associated with prolonged survival after infection among the mice with high parasitaemia levels, with death occurring several weeks later due to HP. Similar to the Berr1 locus, the WLA allele at Berr4 appears to confer prolonged resistance to death due to HP, with individuals homozygous for WLA at Berr4 developing severe HP significantly later. This phenomenon could be the result of an efficient mechanism of host defense against parasite infection, such as improved clearance of infected erythrocytes. The attractive genes in this region include one member of the toll-like receptors (Tlr4), a granulocyte colony-stimulating factor receptor and the complement component 8 beta subunit.

Together, these results demonstrate the strength of combining the functional dissection of pathogenesis with a genetic mapping approach, thereby taking advantage of the genetic diversity among wild-derived inbred mouse strains. Using this approach, two previously reported loci associated with malaria resistance/susceptibility have been confirmed and one novel locus has been suggested. More importantly, however, by associating each of these loci with defined biological functions, including the unique phenotype of long-term survival and cure from infection, further analysis aimed at positional cloning of the candidate genes can be approached with greater accuracy. This will be of particular importance in the continued analysis of congenic mouse strains currently under analysis, which will provide a critical region narrowing candidate genes, and in the efforts aimed at enhancing our current understanding of human malaria pathology.

Materials and methods

Mice

The C57Bl/6/J mice were purchased from Elevage Janvier (Le Genest St Isle, France). WLA/Pas mice (Mus musculus domesticus) and the (WLAxC56BL/6)F1 and (WLAxC56BL/6)F2 were bred at the facilities of Pasteur Institute, France. The French Committee for Animal Welfare ‘Ministère de l'Agriculture et de la Pêche’ no A 75485 gave prior approval to all experiments reported.

Parasite and infection

P. berghei ANKA clone 1.49L, kindly provided by Dr Walliker (Institute of Genetics, Edinburgh, UK), was maintained by passage on C57Bl/6J mice. This clone was selected for its lethality and ability to induce ECM. Mice at 8–12 weeks of age were infected with 106 parasitized erythrocytes by intraperitoneal injection. Neurological symptoms and survival were recorded every day. ECM was diagnosed by clinical signs including ataxia, trembling, paralysis (mono, hemi, para or tetraplegia), deviation of the head, convulsions and coma, followed by death. Parasitaemia progression was determined on day 3, day 5 and every day until day 30 postinfection using flow cytometry analysis as described elsewhere.22

Genetic analysis

Genomic DNA from 219 F2 mice was genotyped for 113 microsatellite markers obtained from the Whitehead MIT Center for Genome Research (www.broad.mit.edu/cgi-bin/mouse/index). Cosegregation analysis was performed by using the Pearson's χ2 test of association and the Wilks likelihood ratio test. Evidence for significant genetic linkage was considered for P-values less than 0.00005 (a value proposed for an intercross).13 QTL were analysed using the R/QTL software.14 The parametric two-part and the nonparametric models were used since the analysed phenotypes did not follow a normal distribution.14, 15 The LOD thresholds were estimated by performing 1000 permutation tests. Evidence for significant linkage in the two-part model was considered for LOD scores above 4.1. For the nonparametric model significant linkage was considered for LOD scores above 3.1.

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Acknowledgements

We are very grateful to Isabelle Lanctin and Danièle Voegtlé for technical assistance. We are most indebted to António Coutinho, Maria Mota, Sarah Auburn and Taane Clark for continuous support during the development of this work and for critical reading of the manuscript. This work was supported by the Fundação para a Ciência e Tecnologia, Portugal (36392/99), and by the CNRS LEA ‘Génétique et dévelopement de la tolérance naturelle’. It was also supported by the Fundação Calouste Gulbenkian, ICCTI, French Embassy in Lisbon, by the ‘Programme de recherche fondamentale en microbiologie, maladies infectieuses et parasitaires’ of the French Ministry of Research, by INSERM U511 and by the Swedish Research Council.

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Correspondence to P-A Cazenave.

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Campino, S., Bagot, S., Bergman, ML. et al. Genetic control of parasite clearance leads to resistance to Plasmodium berghei ANKA infection and confers immunity. Genes Immun 6, 416–421 (2005). https://doi.org/10.1038/sj.gene.6364219

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Keywords

  • Plasmodium berghei
  • experimental cerebral malaria
  • hyperparasitaemia
  • inbred wild-derived mouse strains
  • quantitative trait loci
  • nonparametric analysis

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