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Rodent malaria models: insights into human disease and parasite biology
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ScienceDirect Rodent malaria models: insights into human disease and parasite biology Mariana De Niz1,2 and Volker T Heussler2 The use of rodents as model organisms to study human disease is based on the genetic and physiological similarities between the species. Successful molecular methods to generate transgenic reporter or humanized rodents has rendered rodents as powerful tools for understanding biological processes and host-pathogen interactions relevant to humans. In malaria research, rodent models have been pivotal for the study of liver stages, syndromes arising from blood stages of infection, and malaria transmission to and from the mammalian host. Importantly, many in vivo findings are comparable to pathology observed in humans only when adequate combinations of rodent strains and Plasmodium parasites are used. Addresses 1 Wellcome Centre for Molecular Parasitology, Glasgow, G12 8TA, UK 2 Institute for Cell Biology, University of Bern, CH-3012, Switzerland Corresponding author: Heussler, Volker T ([email protected])
Current Opinion in Microbiology 2018, 46:93–101 This review comes from a themed issue on Host microbe interactions: parasitology Edited by Pascal Maser
https://doi.org/10.1016/j.mib.2018.09.003 1369-5274/ã 2018 Elsevier Ltd. All rights reserved.
Introduction Malaria is responsible for over 400,000 deaths worldwide, most of whom are children under the age of five [1]. Although over 100 species of Plasmodium exist and can infect multiple vertebrates including reptiles, birds, and other mammals, only five species are known to affect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. While the severity of complications caused by infections with each species differs greatly, all five of them cause significant morbidity and/or mortality. Animal models have been invaluable throughout the history of malaria research, including birds, non-human primates, rodent species, and bats. The use of rodents as model organisms to study human biology is largely-based on the biological similarities between the species www.sciencedirect.com
(reviewed in Ref. [2]). These genetic similarities, together with the availability of a plethora of inbred and outbred mouse strains with varied immunological makeup and powerful genetic methods for generating transgenic mice such as CRISPR/Cas9, make rodents an invaluable tool for research. Moreover, the increased use of humanized mouse models and xenografted mice, has further led to successful in vivo investigations of human malaria pathology, host-pathogen dynamics, and antimalarial drug efficacy. The main characteristics of the five most studied rodent strains is presented in Table 1 and in Supplementary Table 1. Four Plasmodium species infecting African rodents, that have been extensively used in in vivo rodent research are Plasmodium berghei, Plasmodium chabaudi, Plasmodium yoelii, and Plasmodium vinckei. Advantages of the rodent malaria models include ease of genetic manipulation through a combination of efficient transfection and various available tools for forward and reverse genetics, which have facilitated the generation of a large number of parasite mutants [3]. In addition, resources such as PlasmoGem [4,5,6], a repository of barcoded vectors, offer the opportunity for large-scale production and genetic screening of parasite mutants, making in vivo studies much more time-efficient while reducing the numbers of mice used.
Pre-erythrocytic stages and link to blood stages Pre-erythrocytic parasite stages have traditionally been studied using the rodent models P. berghei and P. yoelii. Main reasons are that experimental research of liver stages in humans is ethically problematic, and that the general biology of human and rodent Plasmodium species is relatively comparable (Table 2). Although splenectomised chimpanzees can be infected with P. falciparum and P. vivax, and P. cynomolgi serves as a robust non-human primate model, few laboratories in the world can afford liver stage research on monkey Plasmodium parasites. Inhibition of parasite development across stages
Rodent and human parasites share many features in preerythrocytic stages in terms of invasion, development and egress (Table 2). It was recently shown that highlyspecific plasmepsin IX and X inhibitors block P. falciparum egress from, and invasion of erythrocytes [7,8]. However, only by using the rodent P. berghei model it could be shown that plasmepsin IX and X-inhibitor 49c is Current Opinion in Microbiology 2018, 46:93–101
Characteristics of main mouse and rodent Plasmodium models, and their combined pathology outcomes. Mouse strain characteristics
Parasite strain characteristics
P. berghei
P. yoelii
P. chabaudi
P. vinckei
Generally inducing severe pathology. Lines including NK65, ANKA, and K173, differ in pathology.
Widely used for studying receptors for erythrocyte binding. Lines including 17X and YM differ in pathology.
Important model for investigation of drug resistance, immune evasion, sequestration, and antigenic variation. Produces non-lethal, chronic infection.
P. vinckei is the most widely distributed amongst the rodent Plasmodium species. It shows a preference for mature RBCs.
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Balb/c
Inbred strain. Prototypical Th2 response mouse. Increased levels of cytokines and chemokines upon LSP and TLR2-ligand challenges. Increased acute phase proteins.
Lethality: Yes MA: yes SMA: Anaemia with hyperparasitemia. S: yes ALI/ARDS: Pulmonary oedema remains confined. Not severe. PM: yes, lethal to pregnant rodents.
Lethality: Yes (YM) SMA: Anaemia and splenomegaly. Hyperparasitemia. ALI/ARDS: PyXL-infected mice develop interstitial pneumonia and oedema. IMM: 17X results in chronic infection which resolves in 35-40 days. PM: Placental iRBC accumulation.
Lethality: No SMA: PcCB causes severe anaemia unrelated to parasitemia. ALI/ARDS: Limited lung pathology after 20 days of infection. IMM: Model for antigenic switching in vivo. Immunity to homologous challenges. PM: Placental iRBC accumulation.
Lethality: Yes ALI/ARDS: Pulmonary pathology at high parasitemia.
C57BL/6
Inbred strain. Prototypical Th1 response mouse. Activated macrophages produce higher levels of TNFa and IL12.
Lethality: Yes ECM: PbANKA is main model for ECM, but sequestration index of iRBCs is low. ALI/ARDS: Infection with PbNK65 result in severe ARDS with a 90% incidence. Most similar to human ALI/ ARDS. PM: All strains represent good experimental systems to study PM pathogenesis.
Lethality: Yes ALI/ARDS: Limited lung pathology compared to P. berghei infections. PM: Placental iRBC accumulation. IMM: Py17X results in variable outcomes of lethality. Widely used to validate vaccine approaches.
Lethality: No ALI/ARDS: Limited lung pathology compared to P. berghei infections. PM: Females lose embryos half-way through their pregnancy. SMA: Resistant to infection. Develop moderate levels of peak parasitemia, followed by clearance.
Lethality: Yes; Does not develop ECM. Other phenotypes not as widely studied.
DBA/2
Oldest of all inbred strains. Significant genetic disparity with C57BL/6 strain. Have haemolytic complement (C5) deficiency.
Lethality: Yes ALI/ARDS: PbANKA infection considered a model of ALI/ARDS (50% incidence). Recapitulates human syndrome – pulmonary oedema, haemorrhaging, hypoxemia.
Lethality: Yes ALI/ARDS: Limited lung pathology compared to P. berghei infections. Reduced hemozoin.
Lethality: Yes Lethality: No SMA: Resistant to infection. Develop Other phenotypes not as moderate levels of peak parasitemia, widely studied. followed by clearance.
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Table 1
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Table 2 Comparative features of main Plasmodium strains during pre-erythrocytic stages in humans and mice. Human parasites
Exo-erythrocytic stages Sporozoite development in the mosquito Receptor-mediated invasion of hepatocytes Early stage genetically attenuated parasites Duration of development in hepatocytes
Parasite response to restricted diet/ starvation Dormancy Liver schizont size (diameter) Liver stage-derived merozoites
Merosome formation Late stage genetic attenuation of parasites Host susceptibility
P. falciparum
P. vivax
P. berghei
P. yoelii
10-12 days (27 C) CD81, SRBRII
10-14 days (26 C) SRBRII
14-16 days (20 C) CD81
14-16 days (24 C) CD81, SRBRII
Yes 5-7 days
Yes 6-8 days (but forms merozoites that cause relapses) N/A
Yes 2 days
Yes 2 days
Yes
N/A
No 80–100 mm Up to 100 000 per infected hepatocyte
Yes 80–100 mm N/A
No 30–50 mm 10 000–30 000 per infected hepatocyte
Yes No –
Yes No –
No 30–50 mm Up to 10 000–30 000 per infected hepatocyte Yes Yes C57BL/6 mice show higher infection rates over Balb/c mice
Yes
also active against mosquito and liver stage egress of Plasmodium parasites [7]. Since it has recently been shown that late liver stage parasites induce a much more potent immune response than their early stage counterparts [9,10], drug treatment during the transmission season could be a promising strategy to promote superior immune responses. Breakthrough liver stage infections would not be a cause of concern, since these plasmepsin inhibitors also act against RBC invasion and egress. Host dietary restrictions and impact on liver and blood stages
A highly interesting phenomenon affecting parasite development during liver and blood stages, is one induced by host dietary restrictions. In many malariaendemic countries, restricted diet or even starvation is a problem, mainly during dry seasons. In an epidemiological study monitoring parasite growth during wet and dry seasons, it was reported that P. falciparum blood stage parasites can control their replication rate [11]. Evolution has most likely selected for parasites that grow only moderately under starvation conditions to avoid death of the host and to escape immune responses. Using the P. berghei mouse model it was shown that a restricted diet also controls replication of blood stage parasites [12]. Importantly, in both human and rodent parasites, the serine/threonine kinase KIN was identified as a critical www.sciencedirect.com
Rodent parasites
Yes Yes C57BL/6 mice show higher infection rates over Balb/c mice
nutrient-sensing replication regulator (Figure 1). It will be highly interesting to investigate a possible link of these epidemiological studies with the nutrient-sensing kinase KIN or cell cycle regulators like the P. falciparum kinase CRK4 [13]. Conversely to the findings for the blood stages of the parasite, starvation of the host during liver stage development of P. berghei greatly supports parasite development in the liver in terms of growth and survival [14]. Since this phenomenon is strongly associated with starvation-induced host cell autophagy, it will be important to determine whether P. falciparum development in human hepatocytes is also supported by starvation. Biologically and epidemiologically it makes sense that the parasite reacts differently to a restricted diet during blood and liver stage development. The clinically silent liver stage represents an important bottleneck for the parasite, which it must overcome even under starvation conditions. Interestingly, autophagy mechanisms appear to be differentially regulated in rodent and human parasites. For instance, in rodents, autophagy-like cytosolic immune responses are an immediate reaction to invasion, whereas in P. vivax infection of human hepatocytes, similar host cell responses require an additional cytokine-mediated trigger [14,15]. Whether parasite escape mechanisms from host cell cytosolic immune responses, which have been observed for rodent parasites [16,17,18], are also Current Opinion in Microbiology 2018, 46:93–101
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Figure 1
(a)
P.falciparum
P.berghei
EMAP1 EMAP2
? Pf PTEX translocon Pb PTEX translocon
EXP2
EXP2 PTEX150 PTEX150
PTEX88 PTEX88 Hsp101
Hsp101
Trx2
Trx2
KAHRP
Knobs
SBP1 MAHRP1 REX1 KAHRP
SBP1 MAHRP1
?
PfEMP1
IBIS1 CIP1
PfEMP1
Maurer’s cleft-like structures
Maurer’s clefts
Pf Pb
(b)
Replication Virulence KIN
Ad libidum (AL)
Nutrient availability
Caloric restriction (CR) Replication Virulence
KIN Current Opinion in Microbiology
Two research areas where rodent models have provided key insights into malaria pathology. (a) protein export related to sequestration and (b) the link between nutrient sensing and parasite virulence. (a, left) A Plasmodium-infected erythrocyte showing on the left side, the export machinery of the human malaria parasite P. falciparum. Maurer’s clefts are shown in blue, and close-ups of a Maurer cleft show resident proteins SBP1 (brown), MAHRP1 (dark blue) and REX1 (yellow) together with KAHRP (green), and the transported virulence factor PfEMP1. PfEMP1 is accumulated in knobs, which are the result of RBC architectural remodelling induced by the
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What can we learn from rodent malaria models? De Niz and Heussler 97
conserved in human parasites, remains to be determined (Table 3). Genetically attenuated parasites
Limitations of rodent liver stage models became obvious when the knowledge about genetically attenuated parasites (GAPs) for vaccination purposes was transferred to the human system. While fatty acid biosynthesis in the apicoplast is essential for rodent parasite development in the liver (but not in blood or mosquito stages), P. falciparum parasites deficient in fatty acid biosynthesis do not develop beyond the oocyst stage in the mosquito [19]. It is likely that the considerably faster development of human parasites in mosquitoes (10–12 days at 27 C) compared to rodent parasites (14–16 days at 20–24 C) requires more fatty acids in a short time-period and relies on the parasite’s own machinery, whereas rodent parasites can scavenge fatty acids from the mosquito host to generate infectious sporozoites. Despite these limitations, P. berghei and P. yoelii can still be used to pre-select GAP candidates as these species can be easily genetically manipulated and used for high throughput screening [4,5]. In fact, several genes involved in early attenuation in rodent species have been deleted in P. falciparum [20] and successfully used for vaccination trials in human volunteers. However, the information obtained from rodent late liver stage GAPs is more problematic, as already different rodent Plasmodium species vary greatly in their level of attenuation upon deletion of homologue genes [21,22]. In general, for P. falciparum attenuation during late liver stages, the rather long development of this species in hepatocytes needs to be considered, in that it might allow the parasite a longer time frame to scavenge essential components from the host. It is possible that the mechanism or outcome of this scavenging process contrasts to those of the rapidly growing rodent Plasmodium species. A systems biology approach including quantitative modelling offers a solution as it considers the variations in human and model Plasmodium species [23]. Humanized rodent models
The transfer of human hepatocytes to immunocompromised mice has allowed investigating the entire P. falciparum liver stage development, including
parasite egress in merosomes [24]. Despite this great success using humanized mice to study P. falciparum liver stage biology, an important factor to consider is that these mice are severely impaired in many immunological functions. Depending on the degree of manipulation, this immune impairment might have adverse effects on invasion, development, and egress of liver stage parasites. Still there is great hope that the humanized mouse model will help to decipher the molecular basis of dormancy of P. vivax liver stages in vivo [25].
Asexual blood stages Two of the most exciting areas where rodent models provided relevant insights into malaria within a complex living system are sequestration and protein export (Figure 1A), and the link between nutrient sensing and parasite virulence (Figure 1B) discussed in the previous section. Links between Plasmodium protein export, parasite sequestration and virulence in vivo
P. falciparum invests an enormous amount of resources in protein export and host cell remodelling to sequester. While sequestration is thought to be a mechanism for favouring parasite survival, it is also highly linked to pathology. P. falciparum sequestration is mediated by the ligand PfEMP1 and its interaction with multiple host cell vascular receptors (Table 3). The prospect that loss of sequestration could be the solution for diminished lethality was exciting at some point, but without in vivo work, was impossible to prove. Although studies in P. yoelii, P. chabaudi and P. berghei have described phenotypes suggestive of sequestration and cytoadherence [26,27,28 ,29–32], only recently, further research has supported the existence of a conserved machinery for protein export between P. falciparum and rodent malarias (Figure 1A). One of the first findings was the identification of protein PbIBIS1 localizing to membranous structures in the iRBC cytoplasm, reminiscent of Maurer’s clefts in P. falciparuminfected RBCs [33,34]. In 2016, one study identified and functionally characterized Maurer’s clefts resident proteins MAHRP1 and SBP1 homologues in P. berghei [28] (Figure 1). These knockout mutants showed severely attenuated virulence [28]. The successful recovery of virulence upon complementation with the human homologues, demonstrate that the export machinery is
(Figure 1 Legend Continued) parasite. Loss of most Maurer’s cleft resident proteins (red letters) leads to loss of PfEMP1 from the surface of the RBC, and reduced binding to host receptors in vitro. Like-wise, the components of the PTEX translocon at the interface of the parasite and the RBC are essential for export. (a, right) Export machinery of the rodent malaria parasite P. berghei. Maurer’s cleft-like structures are shown in grey. Known proteins of these Maurer’s cleft-like structures include IBIS1 (black), MAHRP1 (dark blue) and SBP1 (brown). Two proteins have been described to reach the surface of the infected RBC, called EMAP1 and EMAP2 but neither was found to be involved in sequestration. Primary figures show immunofluorescence of a P. falciparum infected human RBC (left) and a P. berghei infected mouse RBC (right), proving that P. berghei induces the formation of Maurer’s cleft-like structures (a, lower panel) Effects of nutrient availability on Plasmodium virulence and intraerythrocytic replication. High nutrient levels (e.g. ad libidum, AL) are linked to higher replication rates and higher parasite virulence in vivo. Upon caloric restriction (CR), intraerythrocytic replication and virulence are diminished. The parasite responds to nutrient availability through an AMPK-alpha-related kinase (KIN), which increases upon the parasite facing nutrient deficiency. www.sciencedirect.com
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Table 3 Comparative features of main Plasmodium strains during erythrocytic stages in humans and mice.
P. falciparum Asexual erythrocytic stages Duration 48 h Synchronicity Yes RBC tropism Erythrocytes (unrestricted) Virulence Survival Cerebral malaria Lung pathology
Highest lethality amongst human malarias Yes, low incidence, high mortality rate Yes, low incidence, very high mortality rate
Metabolic acidosis
Yes
Placental malaria Severe anaemia
Yes Yes
Sequestration/ cytoadherence Sequestration Yes, multi-organ phenotype Stages sequestering Trophozoites and schizonts or cytoadhering Ligand identified PfEMP1
P. berghei
P. yoelii
P. chabaudi
22-24h No Reticulocytes (unrestricted)
22-24h No Reticulocytes (unrestricted)
22-24h Yes Erythrocytes
Lethal
Depending on mouse model, 17 YM is lethal No
Chronic
Yes (limited pathology)
Yes (limited pathology)
Yes
Acidosis unknown. Induces hyperlactemia Yes Yes
Yes (PbANKA in C57BL/6 mice) Yes, best model is PbNK65 in DBA/2 mice Yes
Yes Yes (hyperparasitemia)
Yes Yes (hyperparasitemia)
Yes, highest in lungs and adipose tissues Schizonts
Yes
Yes
Schizonts
Schizonts
Unknown
YIR proteins involved in antigenic variation predicted
PIR proteins
No Homologous proteins identified CD36, ICAM1
No Homologous proteins identified ICAM1
Various maturation stages identified
Various maturation stages identified
27h
Variable between strains Mature stages enriched in periphery
Maurer’s cleft presence Knob presence PTEX translocon.
Yes
Yes
Yes Yes
No Yes
Host receptors involved in sequestration
CD36, ICAM1, CSA, PECAM1, VCAM1, NCAM, EPCR, CD9, CD151, TNFR1, TNFR2, EPOR, TSP1
CD36; partly also CSA, ICAM1
Transmission Gametocyte morphology
Clear morphological division into stages I-V
Early and mature gametocytes differentiated. 30h
Duration of maturation Gametocyte distribution
10-12 days Immature stages enriched in the bone marrow and absent from periphery; mature stages enriched in periphery
Immature stages enriched in the bone marrow and spleen; mature stages enriched in periphery
conserved even if the virulence factor protein differs (and remains elusive in rodent parasites). In addition to the Maurer’s clefts, another exciting insight came from studies of the PTEX translocon, involved in general protein export (Figure 1A). The role of five Current Opinion in Microbiology 2018, 46:93–101
No
Mature stages enriched in periphery
predicted
different components of the PTEX translocon in P. berghei were examined in vivo, following the generation of deletion mutants. While 3 of these genes were essential [31,35], deletion of ptex88 and trx2 resulted in attenuated in vivo phenotypes similar to those described for Maurer’s clefts protein mutants [31,32,36]. www.sciencedirect.com
What can we learn from rodent malaria models? De Niz and Heussler 99
Together, there is little doubt that the rodent model offers exciting possibilities to study the effects of key aspects of protein export in vivo.
value of rodent models for the direct study of human malarias, and the significant promise for malaria research.
Conflict of interest statement Nothing declared. Novel rodent models and imaging tools
Humanized and xenografted mouse models promise to overcome some limitations by allowing the direct use of human Plasmodium spp. in vivo [37–39]. Although this will certainly allow the study of sequestration from a mechanical and hemodynamic perspective, it might present limitations in terms of pathology, given the immunocompromised nature of these animals. Altogether, although each individual model displays limitations towards the aim of reproducing the human disease, a combination of reporter, transgenic and xenografted models, holds exciting potential for malaria research. Beyond the molecular advances reached on sequestration in rodent models, various exciting tools have been developed to study Plasmodium interactions in rodents, ranging from intravital microscopy (used to investigate sporozoite recognition of host vasculature in the skin [40], experimental cerebral malaria [41], placental malaria [42], or adipose tissue sequestration [28]) to large-scale genetic approaches for the generation and phenotypic characterization of parasite mutants [5].
Sexual blood stages At each cycle of replication, Plasmodium parasites decide between two fates: an asexual fate via schizogony, which contributes to exponentially increase the parasite burden, or a sexual fate whereby female and male gametocyte are produced and ensure transmission to Anopheles vectors. Important areas where rodent models have contributed insights into gametocyte biology, are sexual commitment, gametocyte production and maturation [43,44] (Table 3). While the transcription factor for sexual commitment, AP2-G, is conserved in human and rodent malaria parasites [45], differences are observed in regulation upstream of AP2-G. While P. falciparum can sense host lyso-phosphotidylcholine levels, and in response to reduced levels of this lipid increase sexual differentiation, P. berghei gametocyte production remains unaffected [43]. It had been previously shown that immature gametocytes preferentially accumulate in protected niches such as the bone marrow extravascular space [46,47]. While this phenomenon had only been shown for P. falciparum in human autopsies, recent work showed that the preferential accumulation of young sexual stages in the bone marrow is also conserved in non-human primates infected with P. vivax [48] and in mice infected with P. berghei [49 ,50]. Most excitingly, the latter studies allowed investigating various aspects of gametocyte dynamics in living mice. P. falciparum gametocyte distribution has also been explored in humanized mice [51], further confirming the www.sciencedirect.com
Acknowledgements We thank Dr. Tobias Spielmann (Bernhard Nocht Institute of Tropical Medicine, Hamburg), Dr. Nisha Philip (Institute of Immunology and Infection Research, University of Edinburgh), Dr Gavin Meehan, Prof. Jim Brewer, and Prof. Matthias Marti (Wellcome Centre for Molecular Parasitology, University of Glasgow) for careful comments and discussions. We thank Dr. Tobias Spielmann and Ashely Vaughn for providing primary images used in this work. MDN is supported by an EMBO Long term postdoctoral Fellowship (EMBO ALTF 1048-2016) and a Swiss National Foundation postdoctoral fellowship (P2BEP3_165396). VTH is supported by the Swiss National Foundation project number: 310030_159519 and by the SystemsX project MalarX, project number 51RTP0_151032.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10. 1016/j.mib.2018.09.003.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
World-Health-Organization: World Malaria Report 2016. 2017.
2.
Perlman RL: Mouse models of human disease: an evolutionary perspective. Evol Med Public Health 2016, 2016:170-176.
3.
de Koning-Ward TF, Gilson PR, Crabb BS: Advances in molecular genetic systems in malaria. Nat Rev Microbiol 2015, 13:373-387.
4.
Gomes AR, Bushell E, Schwach F, Girling G, Anar B, Quail MA, Herd C, Pfander C, Modrzynska K, Rayner JC et al.: A genomescale vector resource enables high-throughput reverse genetic screening in a malaria parasite. Cell Host Microbe 2015, 17:404-413. This paper impressively demonstrates the power of rodent malaria genetics. For the first time it seems to be feasible to knockout all genes ofP. berghei and study their phenotype in blood stage parasites.
5.
Bushell E, Gomes AR, Sanderson T, Anar B, Girling G, Herd C, Metcalf T, Modrzynska K, Schwach F, Martin RE et al.: Functional profiling of a plasmodium genome reveals an abundance of essential genes. Cell 2017, 170:260-272 e268. This paper is the logic consequence of the Gomes paper. It describes the phenotyping of more than 2500 barcodedP. berghei parasites during blood stage development. Surprisingly, it turned out that the parasite requires two-third of its genes for optimal growth in the blood stage. 6.
Schwach F, Bushell E, Gomes AR, Anar B, Girling G, Herd C, Rayner JC, Billker O: PlasmoGEM, a database supporting a community resource for large-scale experimental genetics in malaria parasites. Nucleic Acids Res 2015, 43:D1176-D1182.
7.
Pino P, Caldelari R, Mukherjee B, Vahokoski J, Klages N, Maco B, Collins CR, Blackman MJ, Kursula I, Heussler V et al.: A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress. Science 2017, 358:522-528.
8.
Nasamu AS, Glushakova S, Russo I, Vaupel B, Oksman A, Kim AS, Fremont DH, Tolia N, Beck JR, Meyers MJ et al.: Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion. Science 2017, 358:518-522.
9.
Bijker EM, Bastiaens GJ, Teirlinck AC, van Gemert GJ, Graumans W, van de Vegte-Bolmer M, Siebelink-Stoter R, Arens T, Teelen K, Nahrendorf W et al.: Protection against malaria after Current Opinion in Microbiology 2018, 46:93–101
100 Host microbe interactions: parasitology
immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity. Proc Natl Acad Sci U S A 2013, 110:7862-7867.
Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks. PLoS Comput Biol 2017, 13:e1005397.
10. Mordmuller B, Surat G, Lagler H, Chakravarty S, Ishizuka AS, Lalremruata A, Gmeiner M, Campo JJ, Esen M, Ruben AJ et al.: Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. Nature 2017, 542:445-449.
24. Vaughan AM, Mikolajczak SA, Wilson EM, Grompe M, Kaushansky A, Camargo N, Bial J, Ploss A, Kappe SH: Complete Plasmodium falciparum liver-stage development in liverchimeric mice. J Clin Invest 2012, 122:3618-3628.
11. Portugal S, Moebius J, Skinner J, Doumbo S, Doumtabe D, Kone Y, Dia S, Kanakabandi K, Sturdevant DE, Virtaneva K et al.: Exposure-dependent control of malaria-induced inflammation in children. PLoS Pathog 2014, 10:e1004079.
25. Mikolajczak SA, Vaughan AM, Kangwanrangsan N, Roobsoong W, Fishbaugher M, Yimamnuaychok N, Rezakhani N, Lakshmanan V, Singh N, Kaushansky A et al.: Plasmodium vivax liver stage development and hypnozoite persistence in human liverchimeric mice. Cell Host Microbe 2015, 17:526-535. This paper describes the entire liver stage development ofP. vivax in human liver chimeric mice. Importantly, it could be demonstrated that true hypnozoites (dormant liver stages) are formed and can be reactivated.
12. Mancio-Silva L, Slavic K, Grilo Ruivo MT, Grosso AR, Modrzynska KK, Vera IM, Sales-Dias J, Gomes AR, MacPherson CR, Crozet P et al.: Nutrient sensing modulates malaria parasite virulence. Nature 2017, 547:213-216. This study showed that blood-stage Plasmodium parasites possess a nutrient-sensing mechanism, and that caloric restriction modulates the parasite’s replication rate and virulence. 13. Ganter M, Goldberg JM, Dvorin JD, Paulo JA, King JG, Tripathi AK, Paul AS, Yang J, Coppens I, Jiang RH et al.: Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony. Nat Microbiol 2017, 2:17017. 14. Prado M, Eickel N, De Niz M, Heitmann A, Agop-Nersesian C, Wacker R, Schmuckli-Maurer J, Caldelari R, Janse CJ, Khan SM et al.: Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms. Autophagy 2015, 11:15611579. This paper describes an autophagy-like host cell response against the infection of hepatocytes withP. berghei sporozoites using in vitro but also intravital imaging. It also shows an impressive positive effect of mouse starvation on parasite infection rates of the liver. 15. Boonhok R, Rachaphaew N, Duangmanee A, Chobson P, Pattaradilokrat S, Utaisincharoen P, Sattabongkot J, Ponpuak M: LAP-like process as an immune mechanism downstream of IFN-gamma in control of the human malaria Plasmodium vivax liver stage. Proc Natl Acad Sci U S A 2016, 113:E3519-3528. 16. Agop-Nersesian C, De Niz M, Niklaus L, Prado M, Eickel N, Heussler VT: Shedding of host autophagic proteins from the parasitophorous vacuolar membrane of Plasmodium berghei. Sci Rep 2017, 7:2191. This paper convincingly demonstrates thatP. berghei parasites can escape autophagy-like responses by massive membrane shedding from the PVM.
26. Brugat T, Cunningham D, Sodenkamp J, Coomes S, Wilson M, Spence PJ, Jarra W, Thompson J, Scudamore C, Langhorne J: Sequestration and histopathology in Plasmodium chabaudi malaria are influenced by the immune response in an organspecific manner. Cell Microbiol 2014, 16:687-700. 27. Cunningham DA, Lin JW, Brugat T, Jarra W, Tumwine I, Kushinga G, Ramesar J, Franke-Fayard B, Langhorne J: ICAM-1 is a key receptor mediating cytoadherence and pathology in the Plasmodium chabaudi malaria model. Malar J 2017, 16:185. 28. De Niz M, Ullrich AK, Heiber A, Blancke Soares A, Pick C, Lyck R, Keller D, Kaiser G, Prado M, Flemming S et al.: The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites. Nat Commun 2016, 7:11659. For the first time it is demonstrated thatP. berghei blood stage generates functional Maurer’s clefts that are essential for tissue sequestration of infected red blood cells. 29. Khoury DS, Cromer D, Best SE, James KR, Kim PS, Engwerda CR, Haque A, Davenport MP: Effect of mature blood-stage Plasmodium parasite sequestration on pathogen biomass in mathematical and in vivo models of malaria. Infect Immun 2014, 82:212-220. 30. Lin JW, Sodenkamp J, Cunningham D, Deroost K, Tshitenge TC, McLaughlin S, Lamb TJ, Spencer-Dene B, Hosking C, Ramesar J et al.: Signatures of malaria-associated pathology revealed by high-resolution whole-blood transcriptomics in a rodent model of malaria. Sci Rep 2017, 7:41722.
17. Agop-Nersesian C, Niklaus L, Wacker R, Heussler V: Host cell cytosolic immune response during Plasmodium liver stage development. FEMS Microbiol Rev 2018, 42:324-334.
31. Matz JM, Ingmundson A, Costa Nunes J, Stenzel W, Matuschewski K, Kooij TW: In vivo function of PTEX88 in malaria parasite sequestration and virulence. Eukaryot Cell 2015, 14:528-534.
18. Real E, Rodrigues L, Cabal GG, Enguita FJ, Mancio-Silva L, MelloVieira J, Beatty W, Vera IM, Zuzarte-Luis V, Figueira TN et al.: Plasmodium UIS3 sequesters host LC3 to avoid elimination by autophagy in hepatocytes. Nat Microbiol 2018, 3:17-25.
32. Matz JM, Kooij TW: Towards genome-wide experimental genetics in the in vivo malaria model parasite Plasmodium berghei. Pathog Glob Health 2015, 109:46-60.
19. Cobbold SA, Vaughan AM, Lewis IA, Painter HJ, Camargo N, Perlman DH, Fishbaugher M, Healer J, Cowman AF, Kappe SH et al.: Kinetic flux profiling elucidates two independent acetylCoA biosynthetic pathways in Plasmodium falciparum. J Biol Chem 2013, 288:36338-36350. 20. Kublin JG, Mikolajczak SA, Sack BK, Fishbaugher ME, Seilie A, Shelton L, VonGoedert T, Firat M, Magee S, Fritzen E et al.: Complete attenuation of genetically engineered Plasmodium falciparum sporozoites in human subjects. Sci Transl Med 2017, 9. 21. Nagel A, Prado M, Heitmann A, Tartz S, Jacobs T, Deschermeier C, Helm S, Stanway R, Heussler V: A new approach to generate a safe double-attenuated Plasmodium liver stage vaccine. Int J Parasitol 2013, 43:503-514. 22. Pei Y, Tarun AS, Vaughan AM, Herman RW, Soliman JM, EricksonWayman A, Kappe SH: Plasmodium pyruvate dehydrogenase activity is only essential for the parasite’s progression from liver infection to blood infection. Mol Microbiol 2010, 75:957971. 23. Chiappino-Pepe A, Tymoshenko S, Ataman M, Soldati-Favre D, Hatzimanikatis V: Bioenergetics-based modeling of Current Opinion in Microbiology 2018, 46:93–101
33. Haase S, Hanssen E, Matthews K, Kalanon M, de Koning-Ward TF: The exported protein PbCP1 localises to cleft-like structures in the rodent malaria parasite Plasmodium berghei. PLoS One 2013, 8:e61482. 34. Petersen W, Matuschewski K, Ingmundson A: Trafficking of the signature protein of intra-erythrocytic Plasmodium bergheiinduced structures, IBIS1, to P. falciparum Maurer’s clefts. Mol Biochem Parasitol 2015, 200:25-29. 35. Kalanon M, Bargieri D, Sturm A, Matthews K, Ghosh S, Goodman CD, Thiberge S, Mollard V, McFadden GI, Menard R et al.: The Plasmodium translocon of exported proteins component EXP2 is critical for establishing a patent malaria infection in mice. Cell Microbiol 2016, 18:399-412. 36. Matz JM, Goosmann C, Brinkmann V, Grutzke J, Ingmundson A, Matuschewski K, Kooij TW: The Plasmodium berghei translocon of exported proteins reveals spatiotemporal dynamics of tubular extensions. Sci Rep 2015, 5:12532. 37. Kaushansky A, Mikolajczak SA, Vignali M, Kappe SH: Of men in mice: the success and promise of humanized mouse models for human malaria parasite infections. Cell Microbiol 2014, 16:602-611. www.sciencedirect.com
What can we learn from rodent malaria models? De Niz and Heussler 101
38. Siu E, Ploss A: Modeling malaria in humanized mice: opportunities and challenges. Ann N Y Acad Sci 2015, 1342:2936. 39. Vaughan AM, Kappe SH, Ploss A, Mikolajczak SA: Development of humanized mouse models to study human malaria parasite infection. Future Microbiol 2012, 7:657-665. 40. Hopp CS, Chiou K, Ragheb DR, Salman AM, Khan SM, Liu AJ, Sinnis P: Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition. eLife 2015, 4. 41. Nacer A, Movila A, Sohet F, Girgis NM, Gundra UM, Loke P, Daneman R, Frevert U: Experimental cerebral malaria pathogenesis–hemodynamics at the blood brain barrier. PLoS Pathog 2014, 10:e1004528. 42. de Moraes LV, Tadokoro CE, Gomez-Conde I, Olivieri DN, PenhaGoncalves C: Intravital placenta imaging reveals microcirculatory dynamics impact on sequestration and phagocytosis of Plasmodium-infected erythrocytes. PLoS Pathog 2013, 9:e1003154. 43. Brancucci NMB, Gerdt JP, Wang C, De Niz M, Philip N, Adapa SR, Zhang M, Hitz E, Niederwieser I, Boltryk SD et al.: Lysophosphatidylcholine regulates sexual stage differentiation in the human malaria parasite Plasmodium falciparum. Cell 2017, 171:1532-1544 e1515. 44. Sinha A, Hughes KR, Modrzynska KK, Otto TD, Pfander C, Dickens NJ, Religa AA, Bushell E, Graham AL, Cameron R et al.: A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium. Nature 2014, 507:253-257. 45. Josling GA, Llinas M: Sexual development in Plasmodium parasites: knowing when it’s time to commit. Nat Rev Microbiol 2015, 13:573-587. 46. Aguilar R, Magallon-Tejada A, Achtman AH, Moraleda C, Joice R, Cistero P, Li Wai Suen CS, Nhabomba A, Macete E, Mueller I et al.:
www.sciencedirect.com
Molecular evidence for the localization of Plasmodium falciparum immature gametocytes in bone marrow. Blood 2014, 123:959-966. 47. Joice R, Nilsson SK, Montgomery J, Dankwa S, Egan E, Morahan B, Seydel KB, Bertuccini L, Alano P, Williamson KC et al.: Plasmodium falciparum transmission stages accumulate in the human bone marrow. Sci Transl Med 2014, 6:244re. 48. Obaldia N 3rd, Meibalan E, Sa JM, Ma S, Clark MA, Mejia P, Moraes Barros RR, Otero W, Ferreira MU, Mitchell JR et al.: Bone marrow is a major parasite reservoir in Plasmodium vivax infection. mBio 2018, 9. 49. De Niz M, Meibalan E, Mejia P, Ma S, Brancucci NMB, Agop Nersesian C, Mandt R, Ngotho P, Hughes KR, Waters AP et al.: Plasmodium gametocytes display homing and vascular transmigration in the host bone marrow. Sci Adv 2018, 4: eaat3775. Using the rodent malaria model parasiteP. berghei it was demonstrated that early gametocytes preferentially home to the bone marrow and the spleen of their host to benefit from rich nutrient sources in the reticuloendothelial system. 50. Lee RS, Waters AP, Brewer JM: A cryptic cycle in haematopoietic niches promotes initiation of malaria transmission and evasion of chemotherapy. Nat Commun 2018, 9:1689. This paper demonstrated that a cryptic infection occurs in the erythropoietic organs of mice, where commitment to gametocytogenesis is favoured. This pool alone is capable of replenishing the circulating pool of gametocytes in the periphery. Further, parasites in these niches were found to be less sensitive to anti-malarial treatments. 51. Duffier Y, Lorthiois A, Cistero P, Dupuy F, Jouvion G, Fiette L, Mazier D, Mayor A, Lavazec C, Moreno Sabater A: A humanized mouse model for sequestration of Plasmodium falciparum sexual stages and in vivo evaluation of gametocytidal drugs. Sci Rep 2016, 6:35025.
Current Opinion in Microbiology 2018, 46:93–101
ScienceDirect Rodent malaria models: insights into human disease and parasite biology Mariana De Niz1,2 and Volker T Heussler2 The use of rodents as model organisms to study human disease is based on the genetic and physiological similarities between the species. Successful molecular methods to generate transgenic reporter or humanized rodents has rendered rodents as powerful tools for understanding biological processes and host-pathogen interactions relevant to humans. In malaria research, rodent models have been pivotal for the study of liver stages, syndromes arising from blood stages of infection, and malaria transmission to and from the mammalian host. Importantly, many in vivo findings are comparable to pathology observed in humans only when adequate combinations of rodent strains and Plasmodium parasites are used. Addresses 1 Wellcome Centre for Molecular Parasitology, Glasgow, G12 8TA, UK 2 Institute for Cell Biology, University of Bern, CH-3012, Switzerland Corresponding author: Heussler, Volker T ([email protected])
Current Opinion in Microbiology 2018, 46:93–101 This review comes from a themed issue on Host microbe interactions: parasitology Edited by Pascal Maser
https://doi.org/10.1016/j.mib.2018.09.003 1369-5274/ã 2018 Elsevier Ltd. All rights reserved.
Introduction Malaria is responsible for over 400,000 deaths worldwide, most of whom are children under the age of five [1]. Although over 100 species of Plasmodium exist and can infect multiple vertebrates including reptiles, birds, and other mammals, only five species are known to affect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, and Plasmodium knowlesi. While the severity of complications caused by infections with each species differs greatly, all five of them cause significant morbidity and/or mortality. Animal models have been invaluable throughout the history of malaria research, including birds, non-human primates, rodent species, and bats. The use of rodents as model organisms to study human biology is largely-based on the biological similarities between the species www.sciencedirect.com
(reviewed in Ref. [2]). These genetic similarities, together with the availability of a plethora of inbred and outbred mouse strains with varied immunological makeup and powerful genetic methods for generating transgenic mice such as CRISPR/Cas9, make rodents an invaluable tool for research. Moreover, the increased use of humanized mouse models and xenografted mice, has further led to successful in vivo investigations of human malaria pathology, host-pathogen dynamics, and antimalarial drug efficacy. The main characteristics of the five most studied rodent strains is presented in Table 1 and in Supplementary Table 1. Four Plasmodium species infecting African rodents, that have been extensively used in in vivo rodent research are Plasmodium berghei, Plasmodium chabaudi, Plasmodium yoelii, and Plasmodium vinckei. Advantages of the rodent malaria models include ease of genetic manipulation through a combination of efficient transfection and various available tools for forward and reverse genetics, which have facilitated the generation of a large number of parasite mutants [3]. In addition, resources such as PlasmoGem [4,5,6], a repository of barcoded vectors, offer the opportunity for large-scale production and genetic screening of parasite mutants, making in vivo studies much more time-efficient while reducing the numbers of mice used.
Pre-erythrocytic stages and link to blood stages Pre-erythrocytic parasite stages have traditionally been studied using the rodent models P. berghei and P. yoelii. Main reasons are that experimental research of liver stages in humans is ethically problematic, and that the general biology of human and rodent Plasmodium species is relatively comparable (Table 2). Although splenectomised chimpanzees can be infected with P. falciparum and P. vivax, and P. cynomolgi serves as a robust non-human primate model, few laboratories in the world can afford liver stage research on monkey Plasmodium parasites. Inhibition of parasite development across stages
Rodent and human parasites share many features in preerythrocytic stages in terms of invasion, development and egress (Table 2). It was recently shown that highlyspecific plasmepsin IX and X inhibitors block P. falciparum egress from, and invasion of erythrocytes [7,8]. However, only by using the rodent P. berghei model it could be shown that plasmepsin IX and X-inhibitor 49c is Current Opinion in Microbiology 2018, 46:93–101
Characteristics of main mouse and rodent Plasmodium models, and their combined pathology outcomes. Mouse strain characteristics
Parasite strain characteristics
P. berghei
P. yoelii
P. chabaudi
P. vinckei
Generally inducing severe pathology. Lines including NK65, ANKA, and K173, differ in pathology.
Widely used for studying receptors for erythrocyte binding. Lines including 17X and YM differ in pathology.
Important model for investigation of drug resistance, immune evasion, sequestration, and antigenic variation. Produces non-lethal, chronic infection.
P. vinckei is the most widely distributed amongst the rodent Plasmodium species. It shows a preference for mature RBCs.
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Balb/c
Inbred strain. Prototypical Th2 response mouse. Increased levels of cytokines and chemokines upon LSP and TLR2-ligand challenges. Increased acute phase proteins.
Lethality: Yes MA: yes SMA: Anaemia with hyperparasitemia. S: yes ALI/ARDS: Pulmonary oedema remains confined. Not severe. PM: yes, lethal to pregnant rodents.
Lethality: Yes (YM) SMA: Anaemia and splenomegaly. Hyperparasitemia. ALI/ARDS: PyXL-infected mice develop interstitial pneumonia and oedema. IMM: 17X results in chronic infection which resolves in 35-40 days. PM: Placental iRBC accumulation.
Lethality: No SMA: PcCB causes severe anaemia unrelated to parasitemia. ALI/ARDS: Limited lung pathology after 20 days of infection. IMM: Model for antigenic switching in vivo. Immunity to homologous challenges. PM: Placental iRBC accumulation.
Lethality: Yes ALI/ARDS: Pulmonary pathology at high parasitemia.
C57BL/6
Inbred strain. Prototypical Th1 response mouse. Activated macrophages produce higher levels of TNFa and IL12.
Lethality: Yes ECM: PbANKA is main model for ECM, but sequestration index of iRBCs is low. ALI/ARDS: Infection with PbNK65 result in severe ARDS with a 90% incidence. Most similar to human ALI/ ARDS. PM: All strains represent good experimental systems to study PM pathogenesis.
Lethality: Yes ALI/ARDS: Limited lung pathology compared to P. berghei infections. PM: Placental iRBC accumulation. IMM: Py17X results in variable outcomes of lethality. Widely used to validate vaccine approaches.
Lethality: No ALI/ARDS: Limited lung pathology compared to P. berghei infections. PM: Females lose embryos half-way through their pregnancy. SMA: Resistant to infection. Develop moderate levels of peak parasitemia, followed by clearance.
Lethality: Yes; Does not develop ECM. Other phenotypes not as widely studied.
DBA/2
Oldest of all inbred strains. Significant genetic disparity with C57BL/6 strain. Have haemolytic complement (C5) deficiency.
Lethality: Yes ALI/ARDS: PbANKA infection considered a model of ALI/ARDS (50% incidence). Recapitulates human syndrome – pulmonary oedema, haemorrhaging, hypoxemia.
Lethality: Yes ALI/ARDS: Limited lung pathology compared to P. berghei infections. Reduced hemozoin.
Lethality: Yes Lethality: No SMA: Resistant to infection. Develop Other phenotypes not as moderate levels of peak parasitemia, widely studied. followed by clearance.
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Table 1
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Table 2 Comparative features of main Plasmodium strains during pre-erythrocytic stages in humans and mice. Human parasites
Exo-erythrocytic stages Sporozoite development in the mosquito Receptor-mediated invasion of hepatocytes Early stage genetically attenuated parasites Duration of development in hepatocytes
Parasite response to restricted diet/ starvation Dormancy Liver schizont size (diameter) Liver stage-derived merozoites
Merosome formation Late stage genetic attenuation of parasites Host susceptibility
P. falciparum
P. vivax
P. berghei
P. yoelii
10-12 days (27 C) CD81, SRBRII
10-14 days (26 C) SRBRII
14-16 days (20 C) CD81
14-16 days (24 C) CD81, SRBRII
Yes 5-7 days
Yes 6-8 days (but forms merozoites that cause relapses) N/A
Yes 2 days
Yes 2 days
Yes
N/A
No 80–100 mm Up to 100 000 per infected hepatocyte
Yes 80–100 mm N/A
No 30–50 mm 10 000–30 000 per infected hepatocyte
Yes No –
Yes No –
No 30–50 mm Up to 10 000–30 000 per infected hepatocyte Yes Yes C57BL/6 mice show higher infection rates over Balb/c mice
Yes
also active against mosquito and liver stage egress of Plasmodium parasites [7]. Since it has recently been shown that late liver stage parasites induce a much more potent immune response than their early stage counterparts [9,10], drug treatment during the transmission season could be a promising strategy to promote superior immune responses. Breakthrough liver stage infections would not be a cause of concern, since these plasmepsin inhibitors also act against RBC invasion and egress. Host dietary restrictions and impact on liver and blood stages
A highly interesting phenomenon affecting parasite development during liver and blood stages, is one induced by host dietary restrictions. In many malariaendemic countries, restricted diet or even starvation is a problem, mainly during dry seasons. In an epidemiological study monitoring parasite growth during wet and dry seasons, it was reported that P. falciparum blood stage parasites can control their replication rate [11]. Evolution has most likely selected for parasites that grow only moderately under starvation conditions to avoid death of the host and to escape immune responses. Using the P. berghei mouse model it was shown that a restricted diet also controls replication of blood stage parasites [12]. Importantly, in both human and rodent parasites, the serine/threonine kinase KIN was identified as a critical www.sciencedirect.com
Rodent parasites
Yes Yes C57BL/6 mice show higher infection rates over Balb/c mice
nutrient-sensing replication regulator (Figure 1). It will be highly interesting to investigate a possible link of these epidemiological studies with the nutrient-sensing kinase KIN or cell cycle regulators like the P. falciparum kinase CRK4 [13]. Conversely to the findings for the blood stages of the parasite, starvation of the host during liver stage development of P. berghei greatly supports parasite development in the liver in terms of growth and survival [14]. Since this phenomenon is strongly associated with starvation-induced host cell autophagy, it will be important to determine whether P. falciparum development in human hepatocytes is also supported by starvation. Biologically and epidemiologically it makes sense that the parasite reacts differently to a restricted diet during blood and liver stage development. The clinically silent liver stage represents an important bottleneck for the parasite, which it must overcome even under starvation conditions. Interestingly, autophagy mechanisms appear to be differentially regulated in rodent and human parasites. For instance, in rodents, autophagy-like cytosolic immune responses are an immediate reaction to invasion, whereas in P. vivax infection of human hepatocytes, similar host cell responses require an additional cytokine-mediated trigger [14,15]. Whether parasite escape mechanisms from host cell cytosolic immune responses, which have been observed for rodent parasites [16,17,18], are also Current Opinion in Microbiology 2018, 46:93–101
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Figure 1
(a)
P.falciparum
P.berghei
EMAP1 EMAP2
? Pf PTEX translocon Pb PTEX translocon
EXP2
EXP2 PTEX150 PTEX150
PTEX88 PTEX88 Hsp101
Hsp101
Trx2
Trx2
KAHRP
Knobs
SBP1 MAHRP1 REX1 KAHRP
SBP1 MAHRP1
?
PfEMP1
IBIS1 CIP1
PfEMP1
Maurer’s cleft-like structures
Maurer’s clefts
Pf Pb
(b)
Replication Virulence KIN
Ad libidum (AL)
Nutrient availability
Caloric restriction (CR) Replication Virulence
KIN Current Opinion in Microbiology
Two research areas where rodent models have provided key insights into malaria pathology. (a) protein export related to sequestration and (b) the link between nutrient sensing and parasite virulence. (a, left) A Plasmodium-infected erythrocyte showing on the left side, the export machinery of the human malaria parasite P. falciparum. Maurer’s clefts are shown in blue, and close-ups of a Maurer cleft show resident proteins SBP1 (brown), MAHRP1 (dark blue) and REX1 (yellow) together with KAHRP (green), and the transported virulence factor PfEMP1. PfEMP1 is accumulated in knobs, which are the result of RBC architectural remodelling induced by the
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What can we learn from rodent malaria models? De Niz and Heussler 97
conserved in human parasites, remains to be determined (Table 3). Genetically attenuated parasites
Limitations of rodent liver stage models became obvious when the knowledge about genetically attenuated parasites (GAPs) for vaccination purposes was transferred to the human system. While fatty acid biosynthesis in the apicoplast is essential for rodent parasite development in the liver (but not in blood or mosquito stages), P. falciparum parasites deficient in fatty acid biosynthesis do not develop beyond the oocyst stage in the mosquito [19]. It is likely that the considerably faster development of human parasites in mosquitoes (10–12 days at 27 C) compared to rodent parasites (14–16 days at 20–24 C) requires more fatty acids in a short time-period and relies on the parasite’s own machinery, whereas rodent parasites can scavenge fatty acids from the mosquito host to generate infectious sporozoites. Despite these limitations, P. berghei and P. yoelii can still be used to pre-select GAP candidates as these species can be easily genetically manipulated and used for high throughput screening [4,5]. In fact, several genes involved in early attenuation in rodent species have been deleted in P. falciparum [20] and successfully used for vaccination trials in human volunteers. However, the information obtained from rodent late liver stage GAPs is more problematic, as already different rodent Plasmodium species vary greatly in their level of attenuation upon deletion of homologue genes [21,22]. In general, for P. falciparum attenuation during late liver stages, the rather long development of this species in hepatocytes needs to be considered, in that it might allow the parasite a longer time frame to scavenge essential components from the host. It is possible that the mechanism or outcome of this scavenging process contrasts to those of the rapidly growing rodent Plasmodium species. A systems biology approach including quantitative modelling offers a solution as it considers the variations in human and model Plasmodium species [23]. Humanized rodent models
The transfer of human hepatocytes to immunocompromised mice has allowed investigating the entire P. falciparum liver stage development, including
parasite egress in merosomes [24]. Despite this great success using humanized mice to study P. falciparum liver stage biology, an important factor to consider is that these mice are severely impaired in many immunological functions. Depending on the degree of manipulation, this immune impairment might have adverse effects on invasion, development, and egress of liver stage parasites. Still there is great hope that the humanized mouse model will help to decipher the molecular basis of dormancy of P. vivax liver stages in vivo [25].
Asexual blood stages Two of the most exciting areas where rodent models provided relevant insights into malaria within a complex living system are sequestration and protein export (Figure 1A), and the link between nutrient sensing and parasite virulence (Figure 1B) discussed in the previous section. Links between Plasmodium protein export, parasite sequestration and virulence in vivo
P. falciparum invests an enormous amount of resources in protein export and host cell remodelling to sequester. While sequestration is thought to be a mechanism for favouring parasite survival, it is also highly linked to pathology. P. falciparum sequestration is mediated by the ligand PfEMP1 and its interaction with multiple host cell vascular receptors (Table 3). The prospect that loss of sequestration could be the solution for diminished lethality was exciting at some point, but without in vivo work, was impossible to prove. Although studies in P. yoelii, P. chabaudi and P. berghei have described phenotypes suggestive of sequestration and cytoadherence [26,27,28 ,29–32], only recently, further research has supported the existence of a conserved machinery for protein export between P. falciparum and rodent malarias (Figure 1A). One of the first findings was the identification of protein PbIBIS1 localizing to membranous structures in the iRBC cytoplasm, reminiscent of Maurer’s clefts in P. falciparuminfected RBCs [33,34]. In 2016, one study identified and functionally characterized Maurer’s clefts resident proteins MAHRP1 and SBP1 homologues in P. berghei [28] (Figure 1). These knockout mutants showed severely attenuated virulence [28]. The successful recovery of virulence upon complementation with the human homologues, demonstrate that the export machinery is
(Figure 1 Legend Continued) parasite. Loss of most Maurer’s cleft resident proteins (red letters) leads to loss of PfEMP1 from the surface of the RBC, and reduced binding to host receptors in vitro. Like-wise, the components of the PTEX translocon at the interface of the parasite and the RBC are essential for export. (a, right) Export machinery of the rodent malaria parasite P. berghei. Maurer’s cleft-like structures are shown in grey. Known proteins of these Maurer’s cleft-like structures include IBIS1 (black), MAHRP1 (dark blue) and SBP1 (brown). Two proteins have been described to reach the surface of the infected RBC, called EMAP1 and EMAP2 but neither was found to be involved in sequestration. Primary figures show immunofluorescence of a P. falciparum infected human RBC (left) and a P. berghei infected mouse RBC (right), proving that P. berghei induces the formation of Maurer’s cleft-like structures (a, lower panel) Effects of nutrient availability on Plasmodium virulence and intraerythrocytic replication. High nutrient levels (e.g. ad libidum, AL) are linked to higher replication rates and higher parasite virulence in vivo. Upon caloric restriction (CR), intraerythrocytic replication and virulence are diminished. The parasite responds to nutrient availability through an AMPK-alpha-related kinase (KIN), which increases upon the parasite facing nutrient deficiency. www.sciencedirect.com
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Table 3 Comparative features of main Plasmodium strains during erythrocytic stages in humans and mice.
P. falciparum Asexual erythrocytic stages Duration 48 h Synchronicity Yes RBC tropism Erythrocytes (unrestricted) Virulence Survival Cerebral malaria Lung pathology
Highest lethality amongst human malarias Yes, low incidence, high mortality rate Yes, low incidence, very high mortality rate
Metabolic acidosis
Yes
Placental malaria Severe anaemia
Yes Yes
Sequestration/ cytoadherence Sequestration Yes, multi-organ phenotype Stages sequestering Trophozoites and schizonts or cytoadhering Ligand identified PfEMP1
P. berghei
P. yoelii
P. chabaudi
22-24h No Reticulocytes (unrestricted)
22-24h No Reticulocytes (unrestricted)
22-24h Yes Erythrocytes
Lethal
Depending on mouse model, 17 YM is lethal No
Chronic
Yes (limited pathology)
Yes (limited pathology)
Yes
Acidosis unknown. Induces hyperlactemia Yes Yes
Yes (PbANKA in C57BL/6 mice) Yes, best model is PbNK65 in DBA/2 mice Yes
Yes Yes (hyperparasitemia)
Yes Yes (hyperparasitemia)
Yes, highest in lungs and adipose tissues Schizonts
Yes
Yes
Schizonts
Schizonts
Unknown
YIR proteins involved in antigenic variation predicted
PIR proteins
No Homologous proteins identified CD36, ICAM1
No Homologous proteins identified ICAM1
Various maturation stages identified
Various maturation stages identified
27h
Variable between strains Mature stages enriched in periphery
Maurer’s cleft presence Knob presence PTEX translocon.
Yes
Yes
Yes Yes
No Yes
Host receptors involved in sequestration
CD36, ICAM1, CSA, PECAM1, VCAM1, NCAM, EPCR, CD9, CD151, TNFR1, TNFR2, EPOR, TSP1
CD36; partly also CSA, ICAM1
Transmission Gametocyte morphology
Clear morphological division into stages I-V
Early and mature gametocytes differentiated. 30h
Duration of maturation Gametocyte distribution
10-12 days Immature stages enriched in the bone marrow and absent from periphery; mature stages enriched in periphery
Immature stages enriched in the bone marrow and spleen; mature stages enriched in periphery
conserved even if the virulence factor protein differs (and remains elusive in rodent parasites). In addition to the Maurer’s clefts, another exciting insight came from studies of the PTEX translocon, involved in general protein export (Figure 1A). The role of five Current Opinion in Microbiology 2018, 46:93–101
No
Mature stages enriched in periphery
predicted
different components of the PTEX translocon in P. berghei were examined in vivo, following the generation of deletion mutants. While 3 of these genes were essential [31,35], deletion of ptex88 and trx2 resulted in attenuated in vivo phenotypes similar to those described for Maurer’s clefts protein mutants [31,32,36]. www.sciencedirect.com
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Together, there is little doubt that the rodent model offers exciting possibilities to study the effects of key aspects of protein export in vivo.
value of rodent models for the direct study of human malarias, and the significant promise for malaria research.
Conflict of interest statement Nothing declared. Novel rodent models and imaging tools
Humanized and xenografted mouse models promise to overcome some limitations by allowing the direct use of human Plasmodium spp. in vivo [37–39]. Although this will certainly allow the study of sequestration from a mechanical and hemodynamic perspective, it might present limitations in terms of pathology, given the immunocompromised nature of these animals. Altogether, although each individual model displays limitations towards the aim of reproducing the human disease, a combination of reporter, transgenic and xenografted models, holds exciting potential for malaria research. Beyond the molecular advances reached on sequestration in rodent models, various exciting tools have been developed to study Plasmodium interactions in rodents, ranging from intravital microscopy (used to investigate sporozoite recognition of host vasculature in the skin [40], experimental cerebral malaria [41], placental malaria [42], or adipose tissue sequestration [28]) to large-scale genetic approaches for the generation and phenotypic characterization of parasite mutants [5].
Sexual blood stages At each cycle of replication, Plasmodium parasites decide between two fates: an asexual fate via schizogony, which contributes to exponentially increase the parasite burden, or a sexual fate whereby female and male gametocyte are produced and ensure transmission to Anopheles vectors. Important areas where rodent models have contributed insights into gametocyte biology, are sexual commitment, gametocyte production and maturation [43,44] (Table 3). While the transcription factor for sexual commitment, AP2-G, is conserved in human and rodent malaria parasites [45], differences are observed in regulation upstream of AP2-G. While P. falciparum can sense host lyso-phosphotidylcholine levels, and in response to reduced levels of this lipid increase sexual differentiation, P. berghei gametocyte production remains unaffected [43]. It had been previously shown that immature gametocytes preferentially accumulate in protected niches such as the bone marrow extravascular space [46,47]. While this phenomenon had only been shown for P. falciparum in human autopsies, recent work showed that the preferential accumulation of young sexual stages in the bone marrow is also conserved in non-human primates infected with P. vivax [48] and in mice infected with P. berghei [49 ,50]. Most excitingly, the latter studies allowed investigating various aspects of gametocyte dynamics in living mice. P. falciparum gametocyte distribution has also been explored in humanized mice [51], further confirming the www.sciencedirect.com
Acknowledgements We thank Dr. Tobias Spielmann (Bernhard Nocht Institute of Tropical Medicine, Hamburg), Dr. Nisha Philip (Institute of Immunology and Infection Research, University of Edinburgh), Dr Gavin Meehan, Prof. Jim Brewer, and Prof. Matthias Marti (Wellcome Centre for Molecular Parasitology, University of Glasgow) for careful comments and discussions. We thank Dr. Tobias Spielmann and Ashely Vaughn for providing primary images used in this work. MDN is supported by an EMBO Long term postdoctoral Fellowship (EMBO ALTF 1048-2016) and a Swiss National Foundation postdoctoral fellowship (P2BEP3_165396). VTH is supported by the Swiss National Foundation project number: 310030_159519 and by the SystemsX project MalarX, project number 51RTP0_151032.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10. 1016/j.mib.2018.09.003.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
World-Health-Organization: World Malaria Report 2016. 2017.
2.
Perlman RL: Mouse models of human disease: an evolutionary perspective. Evol Med Public Health 2016, 2016:170-176.
3.
de Koning-Ward TF, Gilson PR, Crabb BS: Advances in molecular genetic systems in malaria. Nat Rev Microbiol 2015, 13:373-387.
4.
Gomes AR, Bushell E, Schwach F, Girling G, Anar B, Quail MA, Herd C, Pfander C, Modrzynska K, Rayner JC et al.: A genomescale vector resource enables high-throughput reverse genetic screening in a malaria parasite. Cell Host Microbe 2015, 17:404-413. This paper impressively demonstrates the power of rodent malaria genetics. For the first time it seems to be feasible to knockout all genes ofP. berghei and study their phenotype in blood stage parasites.
5.
Bushell E, Gomes AR, Sanderson T, Anar B, Girling G, Herd C, Metcalf T, Modrzynska K, Schwach F, Martin RE et al.: Functional profiling of a plasmodium genome reveals an abundance of essential genes. Cell 2017, 170:260-272 e268. This paper is the logic consequence of the Gomes paper. It describes the phenotyping of more than 2500 barcodedP. berghei parasites during blood stage development. Surprisingly, it turned out that the parasite requires two-third of its genes for optimal growth in the blood stage. 6.
Schwach F, Bushell E, Gomes AR, Anar B, Girling G, Herd C, Rayner JC, Billker O: PlasmoGEM, a database supporting a community resource for large-scale experimental genetics in malaria parasites. Nucleic Acids Res 2015, 43:D1176-D1182.
7.
Pino P, Caldelari R, Mukherjee B, Vahokoski J, Klages N, Maco B, Collins CR, Blackman MJ, Kursula I, Heussler V et al.: A multistage antimalarial targets the plasmepsins IX and X essential for invasion and egress. Science 2017, 358:522-528.
8.
Nasamu AS, Glushakova S, Russo I, Vaupel B, Oksman A, Kim AS, Fremont DH, Tolia N, Beck JR, Meyers MJ et al.: Plasmepsins IX and X are essential and druggable mediators of malaria parasite egress and invasion. Science 2017, 358:518-522.
9.
Bijker EM, Bastiaens GJ, Teirlinck AC, van Gemert GJ, Graumans W, van de Vegte-Bolmer M, Siebelink-Stoter R, Arens T, Teelen K, Nahrendorf W et al.: Protection against malaria after Current Opinion in Microbiology 2018, 46:93–101
100 Host microbe interactions: parasitology
immunization by chloroquine prophylaxis and sporozoites is mediated by preerythrocytic immunity. Proc Natl Acad Sci U S A 2013, 110:7862-7867.
Plasmodium falciparum metabolism reveals its essential genes, nutritional requirements, and thermodynamic bottlenecks. PLoS Comput Biol 2017, 13:e1005397.
10. Mordmuller B, Surat G, Lagler H, Chakravarty S, Ishizuka AS, Lalremruata A, Gmeiner M, Campo JJ, Esen M, Ruben AJ et al.: Sterile protection against human malaria by chemoattenuated PfSPZ vaccine. Nature 2017, 542:445-449.
24. Vaughan AM, Mikolajczak SA, Wilson EM, Grompe M, Kaushansky A, Camargo N, Bial J, Ploss A, Kappe SH: Complete Plasmodium falciparum liver-stage development in liverchimeric mice. J Clin Invest 2012, 122:3618-3628.
11. Portugal S, Moebius J, Skinner J, Doumbo S, Doumtabe D, Kone Y, Dia S, Kanakabandi K, Sturdevant DE, Virtaneva K et al.: Exposure-dependent control of malaria-induced inflammation in children. PLoS Pathog 2014, 10:e1004079.
25. Mikolajczak SA, Vaughan AM, Kangwanrangsan N, Roobsoong W, Fishbaugher M, Yimamnuaychok N, Rezakhani N, Lakshmanan V, Singh N, Kaushansky A et al.: Plasmodium vivax liver stage development and hypnozoite persistence in human liverchimeric mice. Cell Host Microbe 2015, 17:526-535. This paper describes the entire liver stage development ofP. vivax in human liver chimeric mice. Importantly, it could be demonstrated that true hypnozoites (dormant liver stages) are formed and can be reactivated.
12. Mancio-Silva L, Slavic K, Grilo Ruivo MT, Grosso AR, Modrzynska KK, Vera IM, Sales-Dias J, Gomes AR, MacPherson CR, Crozet P et al.: Nutrient sensing modulates malaria parasite virulence. Nature 2017, 547:213-216. This study showed that blood-stage Plasmodium parasites possess a nutrient-sensing mechanism, and that caloric restriction modulates the parasite’s replication rate and virulence. 13. Ganter M, Goldberg JM, Dvorin JD, Paulo JA, King JG, Tripathi AK, Paul AS, Yang J, Coppens I, Jiang RH et al.: Plasmodium falciparum CRK4 directs continuous rounds of DNA replication during schizogony. Nat Microbiol 2017, 2:17017. 14. Prado M, Eickel N, De Niz M, Heitmann A, Agop-Nersesian C, Wacker R, Schmuckli-Maurer J, Caldelari R, Janse CJ, Khan SM et al.: Long-term live imaging reveals cytosolic immune responses of host hepatocytes against Plasmodium infection and parasite escape mechanisms. Autophagy 2015, 11:15611579. This paper describes an autophagy-like host cell response against the infection of hepatocytes withP. berghei sporozoites using in vitro but also intravital imaging. It also shows an impressive positive effect of mouse starvation on parasite infection rates of the liver. 15. Boonhok R, Rachaphaew N, Duangmanee A, Chobson P, Pattaradilokrat S, Utaisincharoen P, Sattabongkot J, Ponpuak M: LAP-like process as an immune mechanism downstream of IFN-gamma in control of the human malaria Plasmodium vivax liver stage. Proc Natl Acad Sci U S A 2016, 113:E3519-3528. 16. Agop-Nersesian C, De Niz M, Niklaus L, Prado M, Eickel N, Heussler VT: Shedding of host autophagic proteins from the parasitophorous vacuolar membrane of Plasmodium berghei. Sci Rep 2017, 7:2191. This paper convincingly demonstrates thatP. berghei parasites can escape autophagy-like responses by massive membrane shedding from the PVM.
26. Brugat T, Cunningham D, Sodenkamp J, Coomes S, Wilson M, Spence PJ, Jarra W, Thompson J, Scudamore C, Langhorne J: Sequestration and histopathology in Plasmodium chabaudi malaria are influenced by the immune response in an organspecific manner. Cell Microbiol 2014, 16:687-700. 27. Cunningham DA, Lin JW, Brugat T, Jarra W, Tumwine I, Kushinga G, Ramesar J, Franke-Fayard B, Langhorne J: ICAM-1 is a key receptor mediating cytoadherence and pathology in the Plasmodium chabaudi malaria model. Malar J 2017, 16:185. 28. De Niz M, Ullrich AK, Heiber A, Blancke Soares A, Pick C, Lyck R, Keller D, Kaiser G, Prado M, Flemming S et al.: The machinery underlying malaria parasite virulence is conserved between rodent and human malaria parasites. Nat Commun 2016, 7:11659. For the first time it is demonstrated thatP. berghei blood stage generates functional Maurer’s clefts that are essential for tissue sequestration of infected red blood cells. 29. Khoury DS, Cromer D, Best SE, James KR, Kim PS, Engwerda CR, Haque A, Davenport MP: Effect of mature blood-stage Plasmodium parasite sequestration on pathogen biomass in mathematical and in vivo models of malaria. Infect Immun 2014, 82:212-220. 30. Lin JW, Sodenkamp J, Cunningham D, Deroost K, Tshitenge TC, McLaughlin S, Lamb TJ, Spencer-Dene B, Hosking C, Ramesar J et al.: Signatures of malaria-associated pathology revealed by high-resolution whole-blood transcriptomics in a rodent model of malaria. Sci Rep 2017, 7:41722.
17. Agop-Nersesian C, Niklaus L, Wacker R, Heussler V: Host cell cytosolic immune response during Plasmodium liver stage development. FEMS Microbiol Rev 2018, 42:324-334.
31. Matz JM, Ingmundson A, Costa Nunes J, Stenzel W, Matuschewski K, Kooij TW: In vivo function of PTEX88 in malaria parasite sequestration and virulence. Eukaryot Cell 2015, 14:528-534.
18. Real E, Rodrigues L, Cabal GG, Enguita FJ, Mancio-Silva L, MelloVieira J, Beatty W, Vera IM, Zuzarte-Luis V, Figueira TN et al.: Plasmodium UIS3 sequesters host LC3 to avoid elimination by autophagy in hepatocytes. Nat Microbiol 2018, 3:17-25.
32. Matz JM, Kooij TW: Towards genome-wide experimental genetics in the in vivo malaria model parasite Plasmodium berghei. Pathog Glob Health 2015, 109:46-60.
19. Cobbold SA, Vaughan AM, Lewis IA, Painter HJ, Camargo N, Perlman DH, Fishbaugher M, Healer J, Cowman AF, Kappe SH et al.: Kinetic flux profiling elucidates two independent acetylCoA biosynthetic pathways in Plasmodium falciparum. J Biol Chem 2013, 288:36338-36350. 20. Kublin JG, Mikolajczak SA, Sack BK, Fishbaugher ME, Seilie A, Shelton L, VonGoedert T, Firat M, Magee S, Fritzen E et al.: Complete attenuation of genetically engineered Plasmodium falciparum sporozoites in human subjects. Sci Transl Med 2017, 9. 21. Nagel A, Prado M, Heitmann A, Tartz S, Jacobs T, Deschermeier C, Helm S, Stanway R, Heussler V: A new approach to generate a safe double-attenuated Plasmodium liver stage vaccine. Int J Parasitol 2013, 43:503-514. 22. Pei Y, Tarun AS, Vaughan AM, Herman RW, Soliman JM, EricksonWayman A, Kappe SH: Plasmodium pyruvate dehydrogenase activity is only essential for the parasite’s progression from liver infection to blood infection. Mol Microbiol 2010, 75:957971. 23. Chiappino-Pepe A, Tymoshenko S, Ataman M, Soldati-Favre D, Hatzimanikatis V: Bioenergetics-based modeling of Current Opinion in Microbiology 2018, 46:93–101
33. Haase S, Hanssen E, Matthews K, Kalanon M, de Koning-Ward TF: The exported protein PbCP1 localises to cleft-like structures in the rodent malaria parasite Plasmodium berghei. PLoS One 2013, 8:e61482. 34. Petersen W, Matuschewski K, Ingmundson A: Trafficking of the signature protein of intra-erythrocytic Plasmodium bergheiinduced structures, IBIS1, to P. falciparum Maurer’s clefts. Mol Biochem Parasitol 2015, 200:25-29. 35. Kalanon M, Bargieri D, Sturm A, Matthews K, Ghosh S, Goodman CD, Thiberge S, Mollard V, McFadden GI, Menard R et al.: The Plasmodium translocon of exported proteins component EXP2 is critical for establishing a patent malaria infection in mice. Cell Microbiol 2016, 18:399-412. 36. Matz JM, Goosmann C, Brinkmann V, Grutzke J, Ingmundson A, Matuschewski K, Kooij TW: The Plasmodium berghei translocon of exported proteins reveals spatiotemporal dynamics of tubular extensions. Sci Rep 2015, 5:12532. 37. Kaushansky A, Mikolajczak SA, Vignali M, Kappe SH: Of men in mice: the success and promise of humanized mouse models for human malaria parasite infections. Cell Microbiol 2014, 16:602-611. www.sciencedirect.com
What can we learn from rodent malaria models? De Niz and Heussler 101
38. Siu E, Ploss A: Modeling malaria in humanized mice: opportunities and challenges. Ann N Y Acad Sci 2015, 1342:2936. 39. Vaughan AM, Kappe SH, Ploss A, Mikolajczak SA: Development of humanized mouse models to study human malaria parasite infection. Future Microbiol 2012, 7:657-665. 40. Hopp CS, Chiou K, Ragheb DR, Salman AM, Khan SM, Liu AJ, Sinnis P: Longitudinal analysis of Plasmodium sporozoite motility in the dermis reveals component of blood vessel recognition. eLife 2015, 4. 41. Nacer A, Movila A, Sohet F, Girgis NM, Gundra UM, Loke P, Daneman R, Frevert U: Experimental cerebral malaria pathogenesis–hemodynamics at the blood brain barrier. PLoS Pathog 2014, 10:e1004528. 42. de Moraes LV, Tadokoro CE, Gomez-Conde I, Olivieri DN, PenhaGoncalves C: Intravital placenta imaging reveals microcirculatory dynamics impact on sequestration and phagocytosis of Plasmodium-infected erythrocytes. PLoS Pathog 2013, 9:e1003154. 43. Brancucci NMB, Gerdt JP, Wang C, De Niz M, Philip N, Adapa SR, Zhang M, Hitz E, Niederwieser I, Boltryk SD et al.: Lysophosphatidylcholine regulates sexual stage differentiation in the human malaria parasite Plasmodium falciparum. Cell 2017, 171:1532-1544 e1515. 44. Sinha A, Hughes KR, Modrzynska KK, Otto TD, Pfander C, Dickens NJ, Religa AA, Bushell E, Graham AL, Cameron R et al.: A cascade of DNA-binding proteins for sexual commitment and development in Plasmodium. Nature 2014, 507:253-257. 45. Josling GA, Llinas M: Sexual development in Plasmodium parasites: knowing when it’s time to commit. Nat Rev Microbiol 2015, 13:573-587. 46. Aguilar R, Magallon-Tejada A, Achtman AH, Moraleda C, Joice R, Cistero P, Li Wai Suen CS, Nhabomba A, Macete E, Mueller I et al.:
www.sciencedirect.com
Molecular evidence for the localization of Plasmodium falciparum immature gametocytes in bone marrow. Blood 2014, 123:959-966. 47. Joice R, Nilsson SK, Montgomery J, Dankwa S, Egan E, Morahan B, Seydel KB, Bertuccini L, Alano P, Williamson KC et al.: Plasmodium falciparum transmission stages accumulate in the human bone marrow. Sci Transl Med 2014, 6:244re. 48. Obaldia N 3rd, Meibalan E, Sa JM, Ma S, Clark MA, Mejia P, Moraes Barros RR, Otero W, Ferreira MU, Mitchell JR et al.: Bone marrow is a major parasite reservoir in Plasmodium vivax infection. mBio 2018, 9. 49. De Niz M, Meibalan E, Mejia P, Ma S, Brancucci NMB, Agop Nersesian C, Mandt R, Ngotho P, Hughes KR, Waters AP et al.: Plasmodium gametocytes display homing and vascular transmigration in the host bone marrow. Sci Adv 2018, 4: eaat3775. Using the rodent malaria model parasiteP. berghei it was demonstrated that early gametocytes preferentially home to the bone marrow and the spleen of their host to benefit from rich nutrient sources in the reticuloendothelial system. 50. Lee RS, Waters AP, Brewer JM: A cryptic cycle in haematopoietic niches promotes initiation of malaria transmission and evasion of chemotherapy. Nat Commun 2018, 9:1689. This paper demonstrated that a cryptic infection occurs in the erythropoietic organs of mice, where commitment to gametocytogenesis is favoured. This pool alone is capable of replenishing the circulating pool of gametocytes in the periphery. Further, parasites in these niches were found to be less sensitive to anti-malarial treatments. 51. Duffier Y, Lorthiois A, Cistero P, Dupuy F, Jouvion G, Fiette L, Mazier D, Mayor A, Lavazec C, Moreno Sabater A: A humanized mouse model for sequestration of Plasmodium falciparum sexual stages and in vivo evaluation of gametocytidal drugs. Sci Rep 2016, 6:35025.
Current Opinion in Microbiology 2018, 46:93–101