Heat shock responses: systemic and essential ways of malaria parasite survival

Abstract

Fever is a part of the human innate immune response that contributes to limiting microbial growth and development in many infectious diseases. For the parasite Plasmodium falciparum survival of febrile temperatures is crucial for its successful propagation in human populations as well as a fundamental aspect of malaria pathogenesis. This review discusses recent insights into the biological complexity of the malaria parasite’s heat shock response, which involves many cellular compartments and essential metabolic processes to alleviate oxidative stress and accumulation of damaged and unfolded proteins. We highlight the overlap between heat shock and artemisinin resistance responses, while also explaining how the malaria parasite adapts its fever response to fight artemisinin treatment. Additionally, we discuss how this systemic and essential fight for survival can also contribute to parasite transmission to mosquitoes.

Introduction

The human response to blood-stage infection with malaria parasites involves periodic fever episodes, the classical diagnostic feature of clinical malaria [1,2]. Fever is part of the human TLR-mediated innate immune response trigged by LPS-like metabolic products of an infecting microbe, reducing the burden of infection [3]. In malignant tertian malaria, fever episodes are associated with the parasite’s asexual intra-erythrocytic development via the rupture of red blood cells at the end of the parasite schizont stages, which releases glycolipid debris along with the egress of merozoites. This terminal event of asexual development induces the periodic episodes of malaria fever, or paroxysm, and marks the beginning of a new cycle of blood-stage development [4,5]. Survival of febrile temperatures during the early developmental stages (ring stages) is a fundamental biological property crucial for the parasite’s successful propagation in the human host and beyond. Here, we discuss the modulation of parasite-specific molecular factors that play a role in tolerance to febrile temperatures and highlight the parasite biology necessary for adaptation and survival.

The heat shock response

Heat shock responses in microbial pathogens are triggered by increased temperatures, such as fever conditions. The innate physiology of the host increases body temperature to combat external stressors like infectious agents as well non biological triggers, such as heavy metals, ethanol, and other toxic substances [6,7] (reviewed at [8]). The first consequence of heat shock in the infecting microbe is typically a global perturbation of the conformational structures of proteins resulting in the growing accumulation of unfolded proteins, perturbing function and causing protein aggregation. This imbalance of the protein homeostasis provokes changes to nuclear metabolism [9,10], with the upregulation of the hallmark chaperone genes [11,12], such as the conserved heat shock protein 90 (HSP 90)[13,14] and heat shock protein 70 (HSP 70) [15,16], to prevent the detrimental aggregation of unfolded proteins. HSPs (heat-shock proteins) protect cells exposed to stress conditions, though not necessarily only heat shock conditions. The synthesis of HSPs is a universal phenomenon, occurring in all plant, bacterial, archaean, and animal species, including humans [17].

Heat shock may also adversely impact subcellular organization, such as disturbances to the cytoskeleton [18], endoplasmic reticulum (ER), and Golgi complex (revised by [19]). Increased reactive oxygen species (ROS) in the mitochondrion [20] is connected to a dramatic drop in ATP levels during heat stress (reviewed by [21]). Changes in membrane morphology have been associated with changes in the ratio of protein to lipids and a higher fluidity of the membranes [22–25].

Adaptation of heat shock to artemisinin responses

Studies on Plasmodium have revealed a similar complexity of responses, involving several essential cellular and metabolic processes, after exposure to febrile temperatures (Fig 1). Recently, phenotype-genotype screens, with a large-scale P. falciparum piggyBac mutant library [26], captured the parasite’s heat shock response by highlighting the broad nature of the essential molecular pathways, including many not directly regulated by AP2-HS [27] (reviewed at [28]). Notably, hundreds of genes were annotated as essential for the parasite’s response to heat shock, including the widely described chaperone gene HSP90 (PF3D7_0708400) (Fig 1B).

Figure 1.

Functional assignment of heat-shock and artemisinin resistance response. A). Schematic cell shows the overlap between heat-shock response (Zhang et al 2021) and artemisinin resistance markers (Zhu et al 2022). Figure created with BioRender.com. B). Mutagenesis Index Score (MIS) (Zhang et al 2018) of the main pathways related to heat-shock response and artemisinin resistance, along with gametocytogenesis and AP2-G genes. The plot was generated using R.

As reported in the most recent Tracking Resistance to Artemisinin Collaboration (TRACII) [29] an array of genes implicated in artemisinin resistance overlapped with pathways linked to the parasite’s responses to heat shock [26]. These shared adaptive responses broadly included altered expression of genes for unfolding protein response, exported proteins, and lipid metabolism (Fig 1). These similarities were partly characterized in the small-scaled piggyBac heat shock screen [26], wherein parallel in vitro phenotyping of a 128-mutant library revealed a significant correlation between parasite pathways underlying the responses to febrile temperatures, those altering sensitivity to artemisinin, and altered gene expression profiles defined in the earlier TRACI study [30–32]. Interestingly, a key K13-associated parasite endocytosis pathway, the ubiquitinating components E2/E3 ligases, and K13, have all been linked to artemisinin resistance [33,34] and were also all found to be downregulated in response to heat shock [26], which may increase the parasite’s tolerance of damaged and misfolded proteins.

Parasite processes essential for survival are attractive as potential drug targets, given the decreased likelihood of deleterious off-target effects to the host, and may help limit emerging resistance to artemisinin. Potential druggable targets may lie the intersection of the parasite’s heat shock responses and artemisinin resistance, since for both responses the parasite’s fight for survival relies on the defensive ability to tolerate a toxic accumulation of damaged proteins. Evidence of this adaptation are noted for some of the P. falciparum essential pathways [35], such as redox homeostasis, lipid metabolism, cellular transport, and metabolic processes associated with endosymbiont-derived organelles, that are differentially regulated both in heat shock [26] and in artemisinin resistance [29] responses (Fig 1). These overlaps and discrepancies between the heat shock and artemisinin resistances responses raises the quintessential question: how do the parasites evolve and adapt?

Evolutionary implications and drug discovery

The large-scale heat shock screen of piggyBac mutants identified the vital importance of the isoprenoid biosynthesis of the apicoplast for survival of febrile temperatures [26]. One of the critical processes of the survival mechanism is prenylation of parasite proteins, especially farnesylation of HSP40 that is needed for HSP70 function [36]. Importantly, these processes come from the DOXP mevalonate independent pathway (MEP) present in chloroplasts, algae, some parasitic protozoa, and bacterial pathogens, but absent in all metazoans [37–39]. The essential nature of these processes in parasite survival and absence in humans makes MEP enzymatic processes attractive novel targets for intervention against human pathogens [40]. The parasite’s utilization of the MEP pathway in the innate fever-response mechanisms is likely to extend beyond only generation of farnesylated HSP40 [26,36]. Perhaps most importantly is the role of protein prenylation of the Rab family GTPases [41,42], which is known to be associated with K13-linked artemisinin resistance [43]. Experimental analysis has demonstrated that non-prenylation causes the dysfunction of Rab5 trafficking and disrupts digestive vacuole-morphology [44], showing a similar phenotype to artemisinin mechanism of action [45,46]. Altogether these data strongly support the value of these P. falciparum MEP plant-ortholog gene products as targets for drug development. In particular, the 1-deoxy-D-xylulose-5-phosphate synthase would be an ideal target as it mediates the first step in the DOXP pathway and is the rate-limiting step of MEP [47].

Energy requirements

Increased energy production is another inherent characteristic in the parasite’s responses to the stress responses induced by febrile temperature [26,27] and artemisinin treatment [48], achieved in part by redirecting its own internal biosynthetic pathways to produce glucose (i.e., induction of gluconeogenesis). However, specific factors involved in redox functionalities [27, 48,49] are differently regulated in heat shock responses [26] and artemisinin resistant lines [27] (Fig 1). Lipid and fatty acid metabolism genes [50–52] and ATP production genes linked to metabolism of pyruvate and glutamate in mitochondrial functions [1, 27, 47] are generally up-regulated in heat shock and downregulated in ART-resistant lines. The parallel symmetry of these changes powering the energy production under these adverse conditions suggests that the parasite quiescent state in response ART may have originated by metabolic repurposing of the survival response to heat shock.

Host-cell remodeling

Modulation of the export of proteins, including several members of the FIKK and PHIST gene families, is required to catalyze changes at the host-cell membrane [26,49–52]. The host-cell remodeling could affect hemoglobin uptake through the K13-mediated endocytosis pathway [33,34,53], which reduces artemisinin activation and, thereby, artemisinin toxicity, contributing to ART resistance. The remarkable similarities occurring in genes linked to emerging artemisinin resistance and the genes linked to altered sensitivity to febrile temperature provides another suggestive link that the origins of artemisinin resistance evolve from P. falciparum’s innate febrile stress response mechanisms.

Transcriptomic reprogramming

The changes in the cascade of global transcriptome are tightly controlled by complex transcriptional and post-transcriptional regulatory processes [29,54–56]. The transcription factor identified to have a key role in the heat shock response is AP2-HS (PF3D7_1342900) [27,57]. Two heat shock responses are distinguished one dependent on AP2-HS, activating both the HSP-70-1 and HSP-90 chaperone genes, and another independent of AP2-HS [57]. The AP2-HS independent heat shock response activates other chaperone genes and then enhances processes involved in heat shock responses [26] and artemisinin responses [29], such as energy pathways (lipid and fatty-acid metabolism), apicoplast pathways, and host-interactions (Maurer’s clefts, antigenic variation and cytoadherence, and host-cell surface receptor binding) [27].

Together, the parasite’s fight for survival is a complex, multi-factorial systemic response that cannot be attributed to a single genetic factor. While induction of specific transcription factors can play critical roles in many processes, these alone are not sufficient to regulate the entire complex set of interactions that underlie resistance and heat shock/stress phenotypes. Genome-scale approaches offer an opportunity to deconstruct the intricate web of similar and different processes driving this pathogen’s complex physiologic responses necessary to survive different stress conditions.

Fight for survival, pathway to transmission

The complex life cycle of P. falciparum first involves the fight for survival of gametocytes in its human host and then in the mosquito where sexual reproduction is completed. Relatively few sporozoites, the product of the sexual reproduction, complete the passage from the salivary gland through the new host’s skin to complete their ~10-day development in the liver to initiate a new blood-stage infection. Subsequent repeated rounds of the ~48-hour intraerythrocytic asexual cycle results in exponential parasite growth, potentially giving rise to clinical malaria, including a periodic tertian fever [5,58]. During each round of asexual replication cycle, a small subset of parasites commits to differentiation into non-replicative sexual forms, the gametocytes. Gametocytes are the only stages capable of mediating transmission to a mosquito vector, representing another population bottleneck in the life cycle and a promising target for therapeutic intervention [59–61].

There is growing evidence that the rate of sexual conversion can be affected by artemisinin-based treatment in naturally infected malaria patients [62] and in in vitro infection [63]. More interestingly, febrile temperatures inhibit parasite growth, mostly trophozoites and schizonts [58,64,65] and induce conversion to sexual forms [63]. Indeed, stress responses can influence the sexual fate of the parasite.

Based on previous genome-scale studies, about eight genes involved in heat shock [26] and artemisinin resistance responses [29] were identified to overlap with transcriptomic signatures of gametocyte development [66]. Among them is the glutaminyl-peptide cyclotransferase, putative (PF3D7_1446900), linked to mitochondrial processes/redox, suggesting that altered redox balance redirects the parasite towards sexual commitment. These redox imbalances [29,55,67] can also alter lipid metabolism [68] enabling a shift to energy biosynthesis, which consequently drives the parasites to gametocytes development [69]. Additionally, isoprenoid metabolism, which plays a role in heat shock response [26], could indirectly influence sexual commitment by Ras-related protein Rab-5C (PF3D7_0106800) regulation [66](Fig 2). Not surprisingly, genes involved in translation regulation (tRNA m (1)G methyltransferase, putative, PF3D7_1119100 and 40S ribosomal protein S27, PF3D7_1308300) and DNA replication, (specifically DNA replication initiation via origin recognition complex subunit 1, PF3D7_1203000, and ribonuclease H2 complex subunit A, putative, PF3D7_0623900) are essential for the heat shock [26] and artemisinin survival responses [29], may be potentially involved in sexual commitment as well [66](Fig 2).

Figure 2.

A) Overlap between heat-shock (Zhang et al 2021), artemisinin resistance responses (Zhu et al 2022) and gametogenesis genes (van Biljon et al 2019). Venn diagram shows the number of genes overlapped among three databases: GAM_ART_HS (Zhang et al 2021; Zhu et al 2022; Van Biljon et al 2019). B) Overlapped genes’ representative Gene Ontology terms (GO term), Gene Ontology ID (GO ID) and their associated p values. C) GAM_ART (van Biljon et al 2019 and Zhu et al 2022) and D) GAM_HS (van Biljon et al 2019 and Zhang et al 2021). GO analyses for each overlapped set of genes were performed using the GO enrichment tool in PlasmoDB with a cutoff of p <0.05 (https://plasmodb.org/plasmo/app/).

Notably, processes such as co-translational protein targeting to membrane, protein targeting to endoplasmic reticulum, cell communication, mismatch DNA repair, DNA integrity checkpoint signaling, and RNA metabolism are shared between artemisinin resistance and gametocytogenesis (Fig 2). The master regulator of sexual commitment, PfAP2-G [70], dispensable for blood-stage development [35], is epigenetically activated by the limiting levels of the lipid lysophosphatidyl-choline (LysoPC). Indeed, LysoPC depletion drives upregulation of alternative metabolic pathways for phosphatidylcholine biosynthesis to induce the expression of AP2-G transcription factor [69]. The choline pathway, (GO:0004105- choline-phosphate cytidylyltransferase activity) [63] and splicing regulation (GO:0045292 - mRNA cis splicing, via spliceosome) are shared between sexual conversion and artemisinin resistance, while stimulation of sexual conversion in heat shock responses involves epigenetic processes (GO:0034968 - histone lysine methylation), translation (GO:1901962 S-adenosyl-L-methionine transmembrane transport) and gene expression (GO:0001139-RNA polymerase II complex recruiting activity).

These shared commonalities among different microenvironmental conditions raises an intriguing research question of how the different signals for heat shock response and resistance to artemisinin enhance the malaria parasite’s commitment to sexual development (Fig 3).

Figure 3.

Schematic of Plasmodium falciparum physiological nexus highlighting different directions based on the microenvironmental conditions, inherent strain properties, drug treatment and host immune status. Pathways central to these overlapping responses steer the parasite either towards death or sexual development and transmission into the mosquito vector. Figure created with BioRender.com.

Conclusion

Despite sequencing the genome of P. falciparum over two decades ago, many Plasmodium falciparum genes still lack meaningful functional annotation [71]. Nevertheless, we expect that conserved Plasmodium proteins of unknown function critical for parasite survival in vivo, such as for stress responses, represent opportune targets for development of new intervention therapies. Here, we highlight shared mechanisms between three essential stress responses: heat shock response, artemisinin response, and gametocytogenesis (Figs 3). Three conserved Plasmodium proteins of unknown function link gametocytogenesis with artemisinin and heat shock responses (Fig 2). From the total 960 conserved Plasmodium proteins of unknown function in the P. falciparum genome (https://plasmodb.org/plasmo/app/)[72], 62 proteins are markers of artemisinin resistance [29], and 40 proteins are involved in heat shock responses [26]; with the majority in both sets essential for blood-stage survival [35]. Previous studies on gene co-expression demonstrated the efficiency with which one can identify and characterize the function of those hypothetical proteins [35,73,74]. Genome-wide studies provide a platform to annotate genes with unknown function that lack detectable homology to other organisms. Analysis of shared genes from parallel phenotypic screens, using selective environment and conditions that the parasite is exposed to during its life cycle, such as heat shock, artemisinin exposure, oxidative stress, hemoglobinopathies and gametocyte development, unravel unexpected and essential parasite biology, which expedite the identification of high-value antimalarial targets.