Effect of febrile temperature on the survival of P. falciparum parasites.
During the course of an acute P. falciparum
infection, elevated temperatures as high as 41°C that last between 2 and 6 h are experienced in children and nonimmune adults. The malaria paroxysm is generally known to occur between the cycles of schizont rupture and persists for several hours. The rupture of malaria schizonts is known to release toxins, such as hemozoin pigments and glycophosphoinositol anchor moieties. These toxins activate the host monocytes to release tumor necrosis factor alpha, a major fever-inducing cytokine during malaria infection (29
We studied the effect of febrile temperature on the survival of synchronous and asynchronous asexual erythrocytic-stage cultures of P. falciparum
parasites by comparing the survival rates of parasites grown at 37°C and 41°C over the period of 48 h. Our results show that elevated temperature had a deleterious effect on parasite survival in both synchronous and asynchronous cultures. In synchronous ring-stage cultures, in relation to survival at 37°C, following 2, 8, 16, and 24 h of cultivation at 41°C, parasite survival was reduced by 25%, 60%, 95%, and 88%, respectively (Fig. ). Thirty-two hours of cultivation at 41°C caused elimination of 100% of cultured P. falciparum
parasites. In asynchronous cultures, following 2 and 8 h of cultivation at 41°C, parasite survival was reduced by 23% and 66%, respectively, and 16 h of cultivation at febrile temperature resulted in the death of 100% of cultured parasites (Fig. ). The prolonged survival of synchronous cultures can be attributed to the fact that the starting cultures were solely comprised of ring-stage parasites that have been shown to be less susceptible to cultivation at elevated temperature than the mature forms (29
). To further explore the effect of febrile temperatures on stage-specific killing of blood-form parasites, we examined the Giemsa-stained P. falciparum
blood films for the presence of pyknotic “crisis forms” that give the appearance of parasites undergoing death. Morphological analysis of different developmental stages treated at 41°C by light microscopy revealed the presence of distinct “crisis-form” trophozoites and schizonts, while rings appear to be immune to heat-induced destruction. However, the number and morphological appearance of “crisis forms” was significantly more evident following 4 h of treatment at 41°C (data not shown). Previously, “crisis forms” of trophozoites and schizonts had been described in P. falciparum
cultures undergoing death induced by treatment with antimalaria drugs and other experimentally induced forms of stress (7
). It is important to note that the presence of “crisis forms” has been ascribed as a marker of apoptotic cell death in malaria parasites (7
). These results are in agreement with an earlier report showing an inhibitory effect of temperatures characteristic of the malaria paroxysm on in vitro parasite growth (29
) and suggest that the malaria paroxysm plays a significant role in limiting the exponential growth of parasites in a nonimmune host.
FIG. 1. Effect of febrile temperature on the survival of asexual erythrocytic-stage synchronous and asynchronous cultures of Plasmodium falciparum. Parasite survival rate, defined as the number of parasites at 41°C divided by the number of parasites at (more ...)
To understand the mechanism of febrile temperature-induced death in P. falciparum
parasites, we performed the in situ TUNEL assay in segmented P. falciparum
schizonts cultivated at 37°C and following a 2-h heat shock at 41°C. The TUNEL assay is widely used as a marker for apoptotic cell death in eukaryotic cells. Our results show a strong TUNEL activity in parasites cultured at 41°C (Fig. ). By counting the number of fluorescence-positive cells, we find that approximately 60% of all infected red cells were TUNEL positive. In comparison, barely detectable reactivity was observed in parasites cultured at 37°C (Fig. ). In fact, the intensity of fluorescence signal in parasites cultured at 41°C almost reached the level seen in DNase-treated cells that serve as positive control. The existence of TUNEL-positive reaction in liver forms and mid-gut stages is well documented (16
). While our study clearly demonstrates TUNEL-positive assay results, the existence of TUNEL reactivity in blood forms of malaria parasites has been a subject of controversy. In a recent review article, Deponte and Becker have reported TUNEL activity in P. falciparum
blood-stage schizonts treated with antimalaria drugs and H2
). Other studies have failed to detect TUNEL-positive assay results in P. falciparum
parasites treated in vitro with known antimalarial drugs (38
). Taken together, the presence of “crisis forms” and TUNEL-positive parasites suggests that febrile temperature-induced parasite killing is mediated by the mechanism of apoptotic cell death. However, further studies demonstrating the presence of additional markers of apoptotic cell death in heat-shocked parasites will be needed to firmly establish this conclusion.
FIG. 2. Evidence of in situ DNA fragmentation as monitored by TUNEL assay in P. falciparum blood-form schizonts following the exposure to febrile temperature. (A) P. falciparum parasites cultured at 41°C for 2 h, (B) P. falciparum parasites cultured at (more ...) Measuring febrile temperature-induced alterations in the expression profile of P. falciparum.
The molecular factors and biologic pathways triggered in response to febrile illness during a malaria infection are not known. We compared the global gene expression profiles in P. falciparum parasites cultivated at 37°C and after heat induction at 41°C for 2 h. A 2-h heat induction period was selected for the following reasons. First, during a primary malaria infection, the duration of febrile illness in patients typically lasts between 2 and 6 h. Second, in our studies, we found that a 2-h heat exposure had a minimal effect on the parasite growth and morphology and allowed for the preparation of high-quality RNA. Asynchronous P. falciparum parasites at approximately 3% parasitemia were incubated at 37°C and 41°C for 2 h, and parasite RNA samples were prepared. To measure temperature-induced differential global gene transcription, Cy3- and Cy5-labeled cDNA probes were prepared by reverse transcription of the isolated total RNA samples. The labeled probes were hybridized to a P. falciparum oligonucleotide microarray representing 6,168 open reading frames. To ascertain that the transcription levels determined were the true measure of gene expression and not artifacts introduced by experimental variations, we performed microarray hybridizations with RNA samples isolated in five independent experiments. An altered expression response was defined as an increase of more than twofold (upregulation) or a twofold decrease (downregulation) in the individual gene expression measured in response to heat induction and a cutoff P value of <0.05 by two-tailed Student t test. Our input data were from five arrays, and gene expression was considered altered only if this criterion was met in at least four of five microarray experiments. By this criterion, in the 6,168-oligonucleotide array, 772 genes were excluded for being present in less than four of five arrays, and 4,976 genes were excluded for having an (unaltered) change ratio between 0.5 and 2 in at least two arrays. A total of 46 arrayed sequences were excluded because they did not correspond to any assigned gene in the currently submitted release of the Plasmodium genes in the GenBank database.
We find that, of approximately 5,300 P. falciparum genes analyzed, 336 protein-coding genes consistently show noticeably altered expression patterns in response to elevated temperature, with approximately equal numbers of genes being transcriptionally upregulated (49%) and downregulated (51%) (Table ). Of these 336 genes, 208 genes were annotated as “hypothetical proteins” in the P. falciparum genome database.
Febrile temperature-induced alterations in the P. falciparum genome and assigned biologic function
Six genes with altered expression profiles were randomly selected for additional analysis by real-time PCR to verify that the changes in mRNA abundance observed by microarray were true measures of febrile temperature-induced alterations of expression and not experimental artifacts of microarray chip analysis. Measurements of changes in mRNA abundance by real-time PCR for 70-kDa heat shock protein (7.42), protein with DnaJ domain (6.05), rifin (2.13), acyl carrier protein (−2.80), ribosomal protein L20 (−1.69), and UDP-galactose transporter (−2.69) are in general accordance with our microarray results for 70-kDa heat shock protein (5.29), protein with DnaJ domain (9.51), rifin (4.38), acyl carrier protein (−2.96), ribosomal protein L20 (−2.9), and UDP-galactose transporter (−4.34).
To determine whether there was a relationship between the individual mRNA levels and protein expression, we compared the levels of P. falciparum heat shock protein 70 (PfHSP-70) and P. falciparum chitinase in asynchronous P. falciparum parasites cultivated at 37°C and 41°C. Protein levels were measured as the intensities of specific-antibody reactive bands in enhanced chemiluminescence (ECL)-based semiquantitative assays. By immunoprecipitation assay, integrated optical density (IOD) values for PfHSP-70 at 37°C and 41°C of 507 and 1,894 (a 3.7-fold change) were obtained (see Fig. S1A in the supplemental material); the corresponding change in RNA transcription by microarray was 7.4-fold. By Western blotting, the IOD values for chitinase at 37°C and 41°C were 371 and 660 (a 1.8-fold change) (see Fig. S1B in the supplemental material), while a 3.1-fold change in RNA level was observed by microarray. These results demonstrate a close concordance between the febrile temperature-induced alterations in mRNA levels and the resultant protein expression.
Annotation of febrile temperature-regulated hypothetical malarial genes.
To better understand the functional role of these genes during febrile illness and to improve the quality of annotations in the malaria genome database, we analyzed these genes by using sequence analysis techniques (see Materials and Methods for details). As a result, we were able to detect conserved protein domains in 101 of these “hypothetical proteins,” annotate them, and consequently make new functional predictions at differing levels of detail (see Table S1 in the supplemental material). Of the remaining set, 76 showed conserved regions that were restricted to other Plasmodium species or other apicomplexans like Theileria. Another 28 proteins of the remaining set seemed to be entirely composed of low-complexity regions and seemed not to have any significant hits to other proteins in the nonredundant protein database. A list of the newly annotated genes and their assigned functional/structural predictions is available (see Table S1 in the supplemental material).
Biologic characteristics of the febrile temperature-regulated genes.
To obtain a specific representation of the cellular systems that might be altered in response to temperature stress, we systematically analyzed all the proteins encoded by the responding genes and classified them into specific biologic functional classes on the basis of the presence of conserved motifs and the pathways to which their orthologs belong (Tables and ). Several striking changes were seen across different functional classes, and we discuss the transcriptional changes in terms of these specific biologic classes below.
Biologic functions in a subset of genes in our data set regulated by febrile temperature
About 47% of the genes that show altered transcription are predicted to be either transmembrane or secreted proteins, suggesting that a major component of the transcriptional response to temperature is directed at altering the cell surface and/or interactions with the host. About 22% (75 proteins) of the transcriptionally altered genes are predicted to contain the recently described Pexel motif or host target signal (consensus R/KXLXE/Q) (20
). The Pexel motif has been demonstrated to serve as a key signal for protein export into the erythrocytes, and such exported proteins are known to reside in either the host cytoplasm or host membrane. In P. falciparum
, 400 proteins (8% of the genome) are predicted to contain the putative Pexel motif. Of these, 225 proteins are identified as virulence proteins, and 160 are thought to be involved in the remodeling of the host erythrocyte (33
). Pexel motifs are fairly reliably detected, especially if constrained with the condition requiring them to be closely associated with a signal peptide, and show a more extended general amino acid compositional similarity around the motif. Furthermore, for several of the proteins with confidently identified Pexel motifs, e.g., the rifins, Pfemp1, Psurf 4.2, some R45-like kinases and RESA-like DnaJ domain proteins, there is prior evidence for host targeting, supporting the predictive value of this motif (20
). Nonetheless, further experimental evidence presented by additional molecules containing the Pexel motifs should fully authenticate the validity of the “Pexel motif rule.”
In our studies, 72% of the proteins (54 of 75) with reliably predicted Pexel motifs encoded by the temperature-affected genes are upregulated, suggesting that there is a major extrusion of proteins into the host cytoplasm or membrane upon temperature elevation. The most prominent group of genes encoding Pexel motif-containing proteins in our data set are the rifins. Several of the uncharacterized Pexel motif-containing proteins that show altered expression levels are Plasmodium-specific predicted membrane proteins with large, low-complexity segments and might be involved in remodeling the erythrocyte and mediating interactions with the host, such as cytoadherence-mediated immune evasion. These results suggest that febrile illness conditions result in the en masse upregulation of proteins that might contribute toward parasite-host interactions and cause necessary modifications in the host cell membrane to facilitate parasite sequestration.
Febrile illness and cerebral malaria: role of EMP-1.
The effect of febrile illness on malaria pathogenesis is not well understood. A generalized upregulation in the expression of genes that are identified as virulence factors and potential erythrocyte remodeling proteins strongly suggest that febrile illness directly affects malaria pathogenesis. In this regard, we paid special attention to EMP-1, the most-studied virulence protein of P. falciparum
. We find that in four of five microarray experiments, there was a consistent upregulation in the expression of five var
genes (average change, 2.8-fold; range, 2.6- to 3.0-fold). Among these, four var
transcripts encode full-length Var proteins, and one of the transcripts is a truncated transcript (specifying only 88 amino acids of the Var protein) and could have a regulatory role. How elevated temperature upregulated the expression of multiple var
genes is not known. While simultaneous transcription of multiple var
genes in a parasite isolate culture has been described earlier (5
), of the 60 var
genes present in the P. falciparum
genome, in a single parasite at a given time, only one var
gene is expressed. The regulation of the expression of var
family genes is thought to be controlled by several factors. One recently identified factor is a transcriptional regulatory protein, P. falciparum
Sir2 (PfSir2), a molecule that has been shown to maintain the subtelomeric var
genes in a silent state by deacetylating the histones that are bound to their promoter (14
). Interestingly, we find that following heat shock, there is an average 2.4-fold increase in the level of PfSir2 expression.
Some malaria researchers believe that a permutation of events, such as frequent recombinations, deletions, and gene conversions, give rise to a limitless var
repertoire for antigenic variation and thus make it impossible to attain sterilizing immunity against blood-form parasites (9
). It is reasonable to assume that in an area where malaria is endemic, clinically immune adults possess immunity against a multitude of var
alleles. How febrile illness influences var
-mediated malaria pathogenesis is not known. In sub-Saharan Africa, the regulation of var
gene expression in young children, who are the most susceptible to cerebral malaria, has not been studied. Nonetheless, on the basis of our results, it is tempting to hypothesize that malaria-induced fever causes enhanced expression of multiple Var proteins leading to enhanced cytoadherence in vivo, thereby modulating the pathogenesis of disease in a susceptible host. Similarly, fever-induced expression of multiple Var proteins may accelerate the development of immunity against the disease that prevents cytoadherence-mediated pathogenesis in adults living in areas where malaria is endemic.
We next wanted to determine whether febrile temperatures increase the amount of EMP-1 present at the IRBC surface. We used flow cytometry to examine the reactivity of unfixed (live) parasitized erythrocytes to a mouse polyclonal antibody specific for the EMP-1 variant expressed by the mature trophozoite stage of P. falciparum
line 3D7.41. We found that the median fluorescence intensities (MFI) of parasitized erythrocyte populations incubated for 2 h at 41°C were slightly lower than those incubated for 2 h at 37°C (ratio of MFI at 41°C/MFI at 37°C [mean ± standard deviation], 0.93 ± 0.03, P
= 0.0002, one sample t
test of the mean). Similar results (0.95 ± 0.03, P
= 0.04) were obtained after incubation for an additional 2 h at 37°C to enable sufficient time for translation and subsequent transport of EMP-1 to the erythrocyte surface (Fig. ). These data suggest that heat shock does not increase the amount of EMP-1 expressed at the cell surface. These results differ somewhat from those reported earlier by Udomsangpetch et al. who detected EMP-1 expression on the surfaces of ring and mature trophozoite IRBCs incubated at 40°C but not at 37°C (48
). Among the possible reasons for disagreement observed in the level of EMP-1 expressed on the surfaces of RBCs following treatment at febrile temperatures may include the different P. falciparum
parasites used in the study and differences in assay sensitivity. It is important to note that we were able to detect EMP-1 expression on the surfaces of IRBC incubated at 37°C. Nonetheless, further studies will be needed to determine how febrile illness influences EMP-1-mediated cytoadherence. It is feasible that elevated temperature could influence the conformational folding of adhesion moieties on EMP-1 (e.g., CSA, CD36, and ICAM) or alter its distribution on IRBCs, making it more accessible for binding to endothelial cells. Precedence exists for such a possibility. In West African children, the presence of the homozygous hemoglobin CC genotype is associated with an increased protection against P. falciparum
). P. falciparum
-infected CC erythrocytes display an abnormal cell surface distribution of EMP-1 and consequently have a reduced binding affinity to endothelial cells expressing CD36 and ICAM-1 (11
FIG. 3. Flow cytometric analysis of heat-induced P. falciparum EMP-1 expression on erythrocytes parasitized with the mature trophozoite stage of P. falciparum parasites. Parasitized erythrocytes cultured at 37°C for 2 h (A), 41°C for 2 h (B), (more ...)
These in vitro studies suggest that in an infected host, febrile illness could have both protective and deleterious effects. While febrile temperature could directly kill in vivo parasites by causing physiologic stress, it can also simultaneously prevent parasite immunologic clearance by allowing enhanced sequestration within the deep venules of the host tissues that could contribute toward the pathogenesis of cerebral malaria.
Secreted and cell surface molecules.
This class is basically defined as those parasite-encoded polypeptides that are secreted outside the parasite cell or anchor themselves in the parasite membrane or host cell or membrane. The most striking molecules that showed a strong tendency for overexpression are the Rif and Var proteins that are predicted to localize to the erythrocyte membrane. Proteins of the Rif and Var families are known to be involved in the binding of malaria parasites to receptors on the host cells, causing rosetting and sequestration, two phenomena that are associated with malaria pathogenesis. Two other surface molecules encoded by subtelomerically located genes are also upregulated, namely, PFA0135w, a homolog of the P. falciparum
merozoite-associated tryptophan-rich antigen, and Plasmodium yoelii
), a homolog of Psurf 4.2, a P. falciparum
protein related to P. vivax
Vir proteins (52
). Along with these proteins, other surface molecules that are overexpressed include an ortholog of the ookinete-expressed protein SOAP of Plasmodium berghei
(murine malaria), the so-called glycophorin-binding related antigen, a surface molecule with the anthrax-protective antigen domain (46
), a protein with the membrane attack complex-perforin domain that has been implicated in invasion (3
), the merozoite surface protein 7, and the erythrocyte-binding protein 3, a paralog of MAEBL. The elevated expression of the P. falciparum
SOAP at febrile temperatures is of interest because this molecule is expressed in the micronemes of the ookinete in P. berghei
malaria and plays a role in adhesion to the mosquito basement layer (8
). If this temperature-induced increase in expression of P. falciparum
SOAP also occurs at the level of translation, it could mean that P. falciparum
SOAP may have acquired a different function or that this gene may have an additional function in the blood stages of the vertebrate host that was previously unknown. In a similar vein, the P. falciparum
chitinase, a parasite enzyme shown to play an important role in the degradation of the insect peritrophic membrane (50
), was also overexpressed in our study. These data again suggest a second function for this enzyme in modifying the deglycosylation of host molecules. However, direct biochemical studies will be needed to confirm the precise effects of altered expression of individual surface molecules in mediating different interactions with host cells.
In contrast, other membrane proteins, such as at least six distinct small-molecule transporters predicted to be localized to the parasite membrane and two subunits of the vacuolar ATPase, are downregulated. The genome of P. falciparum
possesses an intact pathway for the synthesis of glycosylphosphatidylinositol (GPI) anchors for membrane proteins, and this is consistent with the presence of several GPI-anchored proteins on the parasite membranes (46
). In this study, five key enzymes in the GPI anchor biosynthesis pathway, including GPI transamidase and glycosyltransferase are consistently downregulated. This suggests that in response to elevated temperature, GPI-anchored proteins are likely to be depleted from the parasite membrane. This observation, taken together with the overexpression of proteins released into the host, suggests that some type I membrane proteins on the parasite membrane might possibly be modulated to allow the aforesaid export. Interestingly, we also observed that a predicted secreted/cell surface glycosyltransferase (PF11_0487) is downregulated under febrile conditions. Sequence analysis showed that it contains a glycosyltransferase domain of the O-linked N
-acetylglucosamine transferase family related to the plant Spindly-type proteins (17
). We predicted that this protein might mediate as yet unnoticed glycosylation of serine and threonine residues in host or parasite proteins, which might be shut down or modulated in the febrile response.
Heat shock response and protein stability.
The next functional category in which genes showed dramatic changes in expression were those involved in the heat shock response and protein stability. Not surprisingly, two chaperones, the HSP70 and HSP90 orthologs, which have been implicated in the heat shock response across the phylogenetic spectrum of life, show an increased expression. P. falciparum
, in contrast to other Plasmodium
species and other Apicomplexa, shows a dramatic lineage-specific expansion of a particular family of DnaJ domain proteins (3
). Outside of Apicomplexa, orthologs of these proteins are currently encountered only in plants, further suggesting an ultimate origin from the plastid progenitor (Fig. ). Nine members of this DnaJ expansion show elevated expression in our study. In P. falciparum
, these proteins are characterized by an additional C-terminal domain that is predicted to form a multihelical bundle enriched in charged amino acids that may serve as a surface for mediating interactions with specific protein targets. These DnaJ domain proteins also contain an N-terminal hydrophobic signal and a Pexel motif, suggesting that they are secreted into the host cell wherein they might stabilize certain complexes by acting in conjunction with their usual functional partner, HSP70. In addition to the nine members of this expansion that are expressed under elevated temperature conditions (Fig. ), we observed that there are several other members of the expansion that are not expressed. This observation suggests that after the recent lineage-specific expansion in P. falciparum
, some were adapted for specific roles in the febrile response, whereas other members of the expansion may be deployed under as-yet-unknown conditions. This suggests that the expansion of this family might have a role in terms of multiple specific adaptations of P. falciparum
FIG. 4. Tree of DnaJ family showing lineage-specific expansion. A phylogenetic tree of the DnaJ lineage-specific expansion in Plasmodium falciparum, with other eukaryotes as an outgroup, is shown. The P. falciparum proteins found to be upregulated in this study (more ...)
Most of these P. falciparum-specific RESA-type DnaJ domain proteins were found to contain an additional conserved N-terminal domain. We accordingly named this conserved domain the PRESAN domain for Plasmodium RESA N-terminal domain. Overall, we detected at least 67 proteins in P. falciparum (see supplemental material) with complete copies of the PRESAN domain and several additional fragmentary versions (~5 to 10) of the domain which might represent mispredicted genes or pseudogenes. In the publicly available draft of protein sequences of P. yoelii, P. berghei, and P. vivax in the GenBank database, we detected at least one protein each with a copy of the PRESAN domain. No versions of this domain were detected in other apicomplexan genera, suggesting that the domain was “invented” after the divergence of the lineage leading to genus Plasmodium but underwent a dramatic proliferation only in P. falciparum. A secondary structure prediction based on the amino acid frequency, a hidden Markov model, and a position-specific score matrix derived from the multiple alignment of the PRESAN family revealed that it is composed of an all-α-helical fold (JPred2 program; see Materials and Methods for details). The core domain is predicted to contain six conserved helical segments, which are likely to form a compact bundle. Most of the highly conserved positions seen throughout the family are hydrophobic residues that are likely to form the buried core of the helical bundle. Less conserved regions are enriched in both positively and negatively charged polar residues and likely comprise the exposed surface, which suggests a role for the PRESAN domain in protein-protein interactions. Further iterative searches with the PRESAN domain led to the identification of the conserved extracellular domains within the Vir superfamily of proteins, including the P. falciparum protein PfSURFIN4.2 (see Fig. S2 in the supplemental material). Both of these domains are α-helical and share a similar pattern of secondary structural elements; however, the Vir superfamily contains conserved cysteines that are absent in the PRESAN domains. This suggests that the two domains are likely to have emerged from a common ancestor, with the Vir superfamily specializing in extracellular interactions, whereas the PRESAN superfamily specialized in cytoplasmic interactions.
Paradoxically, 10 different genes for proteins of the ubiquitin metabolism system were observed to be consistently downregulated in this study. These include proteasomal enzymes, different E1 and E3 enzymes, as well as some ubiquitin C-terminal hydrolases. As a validation of our microarray results, we noted that polyubiquitin (PFL0585w), the only ubiquitin pathway gene found to be upregulated in our data set (1.53-fold change), was also upregulated in response to elevated temperature in an earlier published report (22
To establish a relationship between our microarray data and its biologic relevance, we measured total ubiquitination of proteins isolated from parasites incubated at 37°C and 41°C, using a rabbit polyclonal bovine antiubiquitin antibody. Comparison of expression levels obtained by ECL-based semiquantitative Western blot analysis of P. falciparum parasite extracts collected after incubation at 37°C or 41°C for 2 h demonstrated that temperature elevation causes a generalized downregulation in the ubiquitination process. On the basis of its immunoreactivity with antiubiquitin antibody, there is a significant depression in ubiquitination of both high-molecular-mass and low-molecular-mass protein adducts following treatment at 41°C (Fig. ). A quantitative analysis based on intensities of bands measured between the areas marked by asterisks that includes the high- and low-molecular-weight proteins from parasites incubated at 37°C and 41°C gave IOD units of 19,047 and 1,291, respectively, demonstrating a 14.8-fold downregulation in the ubiquitination process.
FIG. 5. Effect of febrile temperature on ubiquitination of parasite proteins. Total ubiquitination of 5 μg protein from P. falciparum parasites was measured by Western blot analysis using an antiubiquitin antibody following incubation at 37°C (more ...)
The significance of this biologic assay is twofold. First, it confirms that changes in expression are occurring at the protein level as well as the mRNA level. Second, while our microarray data capture changes in expression of individual enzymes in the ubiquitin pathway, this assay quantifies total ubiquitination of all parasite proteins. It may seem rather counterintuitive that the ubiquitin pathway is downregulated in response to elevated temperature, which undoubtedly results in the accumulation of misfolded proteins that may become toxic to the cell if not removed. However, depression of the ubiquitin pathway may be a mechanism to increase the half-lives of certain proteins under stressful conditions. A recent study suggested that protein degradation by ubiquitination and HSP-assisted refolding do, in fact, act in concert with one another and may even at times compete for the same substrates (misfolded proteins) (32
). Another plausible explanation for a generalized depression in the ubiquitin pathway could be a parasite strategy to conserve energy at times of duress. It is estimated that approximately 30% of nascent proteins are degraded by the proteasome in unstressed cells (43
); therefore, even a slight decrease in the ubiquitin pathway will result in a considerable increase in energy available for other cellular processes.
Cytoplasmic systems and signal transduction.
We found that four of the five genes encoding conserved cytoskeletal proteins that were recovered in our study were upregulated, including tubulin and a homolog of the Drosophila
actin-binding protein kelch. The only downregulated gene in this category was ADF3, an actin-depolymerizing factor related to gelsolin. A probable explanation for the observed expression pattern may be that the cytoskeleton is strengthened to compensate for the destabilizing effects of elevated temperature. All 10 genes related to cytoplasmic protein trafficking, vacuolar sorting, and secretion that were recovered in this study were found to be consistently downregulated. These included various small GTPases of the vesicular biogenesis and fusion pathway, a potential vesicular cargo-binding protein with the conserved GOLD domain (2
), the microsomal signal peptidase, and one of the luminal disulfide bond isomerases. Similarly, 12 ribosomal protein genes and 2 genes for proteins with ribosome-associated functions were downregulated. This apparent downregulation of several components of the protein synthesis and protein-trafficking apparatus as well as the ubiquitin-dependent protein degradation system (noted above) might indicate a multilevel process to slow down the synthesis and turnover of proteins.
In terms of signal transduction, members of three distinct families of protein kinases are upregulated. Most interesting of these are the protein kinases of the Apicomplexa-specific R45 family. These predicted serine/threonine kinases are thus far found only in Apicomplexa and are characterized by several structural features that distinguish them from all other Ser/Thr kinases that have been characterized thus far. These unique structural features include the peculiar structure of the ATP-binding site in the N-terminal subdomain of the kinase and a conserved extension with a characteristic tryptophan N terminal to the kinase domain. These features suggest that these kinases target a unique set of substrates. Furthermore, they possess a conserved Pexel motif, which has been shown to be required for their translocation to the host cytoplasm and are likely to phosphorylate targets in the host cytoplasm. The R45 family shows a lineage-specific expansion unique to the Plasmodium falciparum species (Fig. ), of which three members were found to be consistently upregulated. The fact that none of the other members of this large lineage-specific expansion in Plasmodium are upregulated suggests that there is again a functional diversification of this recently diversified family, just as in the earlier-mentioned DnaJ proteins, with some members being recruited in the context of the febrile response.
FIG. 6. Tree of R45 protein kinase family showing lineage-specific expansion. A phylogenetic tree of the R45 protein kinase lineage-specific expansion in Plasmodium falciparum, with human kinases with structures as a outgroup, is shown. The P. falciparum proteins (more ...)
In addition to the R45 family, two paralogous kinases of the GCN2 family of kinases, which are involved in regulating translation by phosphorylating components of the translation machinery (51
), are also upregulated. These kinases may also be exported to the host cytoplasm and may thereby interfere with the basic metabolism of the host cell. Two members of the calcium-dependent kinase family with EF-hand domains fused to the kinase domains are also strongly overexpressed. This family shows a lineage-specific expansion in various alveolates and might be widely used by organisms of this lineage in various signaling contexts (32
). In contrast, two genes for predicted calcium-binding proteins with EF-hand domains, and a mitogen-activated protein kinase are downregulated. Beyond this, no conserved signaling genes appear to be under any kind of regulation. This suggests that the transcriptional response to elevated temperature specifically affects only a small set of phosphorylation-dependent signaling pathways.
We found that 17 genes for proteins involved in different aspects of RNA metabolism, particularly splicing, mRNA maturation, and posttranscriptional gene regulation, are overexpressed, compared to only three genes for RNA metabolism proteins that are downregulated. A striking, opposite regulation of two genes for Sm proteins was observed in our study. The classical Sm protein, Sm-G, which is a core component of the U1, U2, U4, and U5 spliceosomal particles, is strongly upregulated, whereas LSM6, which is a component of the U6 spliceosomal particle and decapping-dependent RNA degradation pathway, is downregulated. This pattern might indicate a change in stoichiometry of the spliceosomal components, which might affect the splicing or stability of specific mRNAs. We had earlier reported a family of predicted RNA-binding proteins with multiple Zn-chelating CCCH domains (typified by PFE1245w), which show a lineage-specific expansion in Plasmodium
). Two members of this expansion show a strong overexpression in response to temperature stress and might participate in an Apicomplexa-specific posttranscriptional regulatory mechanism. These observations point to a major potential regulatory input occurring at the level of mRNA stability
and perhaps splicing.
Eight genes for chromatin components are upregulated; in comparison, only two genes are downregulated. The upregulated genes include the histones (H2B and H4) and the NAD-dependent histone deacetylase of the Sir2p family (PfSir2). Several genes of the DNA replication and repair systems, including the RP-A and RF-C are downregulated, whereas a Rad25-like helicase/ATPase and a DNA repair nuclease, Dem1p of the RecB family are upregulated. The exact implication of these changes in the expression patterns of the nuclear proteins is unclear, but it might indicate a tendency for condensation of chromatin and a possible slowdown in replication. A few DNA-binding proteins other than the histones that are associated with chromatin structure maintenance also show upregulation, namely, the BRIGHT domain protein (MAL6P1.39), which is likely to be a component of the SWI2/SNF2-dependent chromatin remodeling complexes, and the histone-type nuclear factor Y homolog (PF14_0374). We observed that the mRNA levels of three predicted specific transcription factors show noticeable changes in response to elevated temperature. Two of these, PFL0455c with two C-terminal C2H2 zinc finger domains, and PFD0200c with the recently identified ApiAP2 DNA-binding domain, are upregulated. In contrast, the third transcription factor, PFE1025c, has a DNA-binding domain related to the plant p24/PBF-2 transcription factors and the ciliate TIF1 transcription factor and is downregulated. In ciliates, the orthologous transcription factor TIF1 is known to be required for the transcription of ribosomal DNA in ribosomal biogenesis (42
). It is likely that the Plasmodium
protein plays a similar role, and its downregulation is consistent with the downregulation of several other ribosomal components (see above).
Another striking observation we made was that about 26% (90 genes) of the genes showing a change in transcription in response to febrile conditions map to the subtelomeric gene arrays that, in addition to members of the rif, var, and DnaJ families, also encode several other proteins. This observation indicates a strong bias in the preferential regulation of genes associated with chromosome ends (<0.001 chance probability of obtaining the observed numbers by the chi test) and points to probable special chromatin-related changes in the subtelomeric regions. In particular, we noticed that at least 70% of subtelomeric genes found in our data set were overexpressed, suggesting there might be an increased accessibility of particular regions of subtelomeric chromatin to allow increased transcription of certain genes.
We did not observe expression patterns suggesting systematic down- or upregulation of entire metabolic pathways; however, expression of genes for specific components of a few metabolic pathways did seem to show alterations. The most striking alterations were seen in the case of lipid metabolism. Plasmodium
possesses multiple paralogs of a fatty acyl coenzyme A synthetase, some of which have been shown to function on long-chain fatty acids. Recently, these proteins have been demonstrated to be exported in specific vesicular structures to the host cell (34
). We observed that three members of this family are strongly or moderately overexpressed under temperature stress. Furthermore, a serine C-palmitoyltransferase (ortholog of yeast Lcb2p), which functions in sphingolipid biosynthesis, is also upregulated, and this protein is predicted on the basis of the Pexel motif to be exported into the host cell. Likewise, two paralogous genes encoding phospholipases that are predicted to convert fatty acid monoglycerides to free fatty acids are also overexpressed. Interestingly, the gene for an enzyme catalyzing the opposite step in the pathway, a membrane-associated lysophosphatidic acyltransferase, is strongly downregulated, implying a two-level modification of the pathway in the same general direction. These patterns suggest potential mechanisms for modification of the lipids of the host and the parasite that might be conducive for the localization of the parasite proteins and also allow the formation and maintenance of the parasitophorous membrane.
PFB0590w encodes a predicted monooxygenase related to the bacteria antibiotic biosynthesis monooxygenases (44
) and is downregulated under febrile conditions. It would be of interest to further investigate whether it might be involved in the modification of as-yet-unknown metabolites in the parasite. The gene for allantoicase, which is involved in purine degradation, is also quite strongly upregulated. This suggests that under heat shock conditions, there might be a shift to utilization of purine breakdown products as a secondary nitrogen source. A Cof-like phosphatase of the HAD superfamily of hydrolases, which belongs to a family of highly conserved hydrolases, is strongly overexpressed in our study. However, the functional implications of this protein remain largely unclear.
We believe that a combination of gene expression data, sequence analysis, and biologic experiments has helped us piece together the potential activities involved in the febrile temperature response in P. falciparum and is depicted in Fig. . We note, particularly, that a large number of polypeptides that are predicted or known to be exported into the host cell or expressed on the host cell surface are overexpressed to various degrees under temperature stress. In particular, the PRESAN domain proteins, such as the DnaJ family, might form specific complexes in the host cytoplasm and modify its properties in response to the temperature elevation. In terms of a general intracellular response, the upregulation of several genes related to mRNA metabolism and splicing appears to suggest a major posttranscriptional regulatory response. In terms of protein stability, trafficking, and protein synthesis itself, a general tendency to slow down synthesis of new proteins and degradation of existing proteins is suggested by our data. On a more pragmatic note, we observe that several Plasmodium- or apicomplexan-specific gene families and other enzymes with no close homologs in humans are overexpressed. If this observation were to be reflected in comparable elevated protein levels, then they might serve as potential targets for therapeutic intervention or as vaccine candidates. In summary, our data present for the first time a comprehensive view of the alterations in gene expression and predicted biochemical pathways in P. falciparum parasites exposed in vitro to temperatures characteristic of febrile illness, independent of confounding factors, such as host genetics and immune status.
FIG. 7. Cell cartoon with subcellular localization of various proteins showing altered mRNA levels under febrile temperatures. The cells and organelles are schematically shown and not drawn to scale. The membranous system depicted on the right side of the parasite (more ...)