Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37°C and 41°C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles.

Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions.

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Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as “hypothetical.” We find that cultivation of P. Falciparum parasites at 41°C leads to parasite death in a time-dependent manner. The presence of the “crisis forms” and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures.

These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures. Febrile illness is the most common and benign feature of malaria pathogenesis. Historically, malaria has been associated with a unique pattern of cyclical fever, later recognized to coincide with schizont rupture, which varies (depending on the length of the erythrocytic-stage cycle) for different Plasmodium species (). The duration of the erythrocytic-stage cycle and, hence, the pattern of cyclical fever for Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale is 48 h, and for Plasmodium malariae, it is 72 h. Febrile illness, defined by an elevated host body temperature, is a common clinical symptom seen in response to distress caused by several pathogens, autoimmune diseases, and many other diseases including cancers (). In malaria, febrile illness is induced via a poorly defined immunologic mechanism activated by malaria toxins and hemoglobin metabolites released from the ruptured, infected red blood cells (IRBCs) ().

It has been argued that malarial febrile illness is an evolutionary adaptation that benefits both the parasite and its host. Cultivation at febrile temperatures has been shown to inhibit in vitro growth of P. Falciparum cultures (). It is possible that during acute malaria infection, elevated host temperature induces a cascade of molecular events that maintain the total parasite burden at a threshold level by limiting its replication rate, allowing host defense mechanisms to activate and mature. Although inhibition of exponential parasite growth caused by febrile temperature may appear to aid only the host, it may also provide the parasite sufficient time to further transmit infection, making it a potential parasite survival strategy. Notwithstanding the possible beneficial effects of malaria-induced febrile illness, a recent study suggests that fever may, in fact, augment the pathogenesis of malaria by enhancing cytoadherence of parasite IRBCs to CD36 and intercellular adhesion molecule 1 (ICAM-1) molecules that serve as host receptors on endothelial cells (). The authors found an increased level of the variant antigen erythrocyte membrane protein 1 (EMP-1) (a parasite ligand that mediates binding to host receptors on endothelial cells) on the surfaces of ring and trophozoite IRBCs when heated to 40°C, leading them to speculate that the enhanced cytoadherence observed could be due to increased trafficking of EMP-1 to the surfaces of IRBCs.

In mammalian cells, an increase in temperature can lead to a number of changes within the cell, including protein denaturation, transient cell cycle arrest, and changes in membrane fluidity (, ). Heat shock proteins (HSPs), the primary mediators of the heat shock response, act as molecular chaperones by preventing aggregation and promoting folding of cellular proteins (). In humans, HSPs appear to be possible regulators of key apoptotic pathways, and targeting HSPs that interact with the cellular apoptotic machinery is emerging as a novel approach for pharmacologic intervention in cancer (). To understand the molecular changes that occur and biochemical pathways altered in P.

Falciparum parasites in response to febrile temperatures, we compared the genome-wide gene expression profiles of parasites cultivated at 37°C and 41°C. We used a combination of gene expression data and computational sequence analyses to reconstruct a detailed picture of the response of the parasite to elevated temperature. The use of sensitive sequence profile analysis methods allowed the detection of conserved domains and sequence motifs that are known to greatly assist in elucidating the biologic role of uncharacterized proteins. Furthermore, various forms of contextual information gleaned from comparative genomics, such as the presence of lineage-specific gene expansions and gene losses, phyletic profiles, and shared gene ontology, also complement the results obtained from direct sequence analysis of the proteins themselves. Accordingly, we carried out in-depth sequence and comparative genomic analyses of all the proteins encoded by the genes showing noticeable changes in response to temperature elevation. We confirmed a subset of the findings, based on transcriptional activation of genes with biologically relevant assays, by measuring the expression of EMP-1 on the surfaces of P.

Falciparum IRBCs and by detecting the ubiquitinization of total parasite proteins using an antiubiquitin antibody. We also investigated whether the febrile temperature-induced in vitro killing of P.

Falciparum parasites (; this study) is mediated by an apoptosis-like cell death. The results of our analyses suggest that a number of parasite pathways are strongly altered at the level of gene expression—particularly those involved with protein stability and folding, RNA metabolism, and a significant component of the secretome that is transported from the parasite into the host cell. We also find a positive terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) activity in the heat-treated parasites, suggesting the existence of an apoptosis-like cell death mechanism in blood-form malaria parasites. We believe that the identification of heat-inducible parasite factors and biochemical pathways that contribute to the regulation of parasite density and that potentially influence their virulence in a nonimmune host could lead to new antimalarial drug and vaccine targets. The 3D7 strain of Plasmodium falciparum was cultured according to the procedures used in our laboratory ().

Briefly, parasites were cultured in modified RPMI 1640 with 25 mM HEPES equilibrated with 5% CO 2, 5% O 2, and 90% N 2 and containing 10% pooled heat-inactivated human type O-positive serum (obtained commercially) and human type O-positive RBCs from blood. The blood was collected in bags containing citrate-phosphate-dextrose-adenine, filtered to remove leukocytes, and handled as a biological hazardous substance. Cultures were maintained in suspension at 2.5% to 5% hematocrit with 0.2% to 5% of the RBCs infected by parasites. For growth inhibition studies, parasites were synchronized using a temperature cycling incubator as described by Haynes and Moch ().

Parasite survival studies. The effect of febrile temperature on parasite survival was evaluated in both synchronous and asynchronous cultures of P. Parasite cultures with an initial parasitemia of 3% and hematocrit of 2.5% were grown at either 37°C or 41°C for 48 h in a final volume of 10 ml. Over the course of the experiment, the number of parasites was counted in Giemsa-stained thin blood smears prepared at 2, 8, 16, 24, 32, 40 and 48 h time points. Experiments were replicated three times for synchronous cultures and two times for asynchronous cultures.

Temperature-dependent survival rate at each time point was defined as follows: number of parasites at 41°C/ number of parasites at 37°C. For monitoring apoptosis, terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling assay (In Situ Cell Death Detection kit; Roche Diagnostic Inc., Indianapolis, IL) was used. Falciparum schizont-stage parasites were cultured at 37°C and 41°C for 2 h. The air-dried blood films were fixed for 1 hour in freshly prepared fixative (4% paraformaldehyde in phosphate-buffered saline [PBS], pH 7.4). The slides were rinsed with PBS and incubated with permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate dihydrate) for 2 min on ice.

The positive-control slides were treated with DNase I (300 U/ml in 50 mM Tris-HCl, pH 7.5, and 2 mg/ml bovine serum albumin) for 10 min. The slides were washed twice with PBS, and 50 μl of TUNEL reaction mixture was added to each slide. The slides were incubated for 60 min at 37°C in a humidified atmosphere in the dark. The slides were rinsed thrice with PBS for 5 min each time and were mounted using VectaShield mounting medium for fluorescence (Vector Laboratories Inc., Burlingame, CA). The images were viewed with a Zeiss Axioplan II imaging microscope.

Images were transferred to the personal computer version of Adobe Photoshop 5.5 for labeling and printing. Heat shock treatment of parasites and RNA preparation. A seed culture of asynchronous P. Falciparum parasites at approximately 3% parasitemia was divided into two flasks, and parasites were grown at either 37°C or 41°C in a final volume of 50 ml. After 2 hours of incubation, total RNA was isolated from parasite cultures by saponin lysis of RBCs, followed by RNA extraction with TRIzol. Abracadabra Flashing Software Download.

RNA quantity was determined by optical densitometry, and its quality was evaluated by agarose gel electrophoresis. Microarray expression profiles were determined from RNA samples isolated in five independent experiments. Real-time PCR. To confirm microarray data, a small subset of genes varying in abundance (upregulation or downregulation) by array was quantified using real-time PCR. Primers (20 to 23 bp) with a melting temperature of 55 to 60°C were designed to yield a 90- to 150-bp product containing an exon/intron boundary. Total RNA (2 μg) was treated with 1 U of RNase-free DNase I (Invitrogen, San Diego, CA), and synthesis of cDNA was performed for 50 min at 42°C using 50 U of reverse transcriptase (Superscript II; Invitrogen), 100 mM of MgCl 2, 40 U of RNaseOUT (Invitrogen), 200 mM of dithiothreitol, 10 mM of deoxynucleoside triphosphate mix, and random hexamer primers.

The reaction mixture was then subjected to RNase H treatment with 2 U of enzyme for 20 min at 37°C. Real-time quantitative PCR was performed in a 20-μl reaction volume containing 10 μl of a dilution of cDNA preparation, 2 μl of FastStart DNA Master SYBR green I (Roche, Nutley, NJ) and 10 μM of gene-specific primers.

Amplification and detection of specific product were performed with the MX4000 LightCycler with the following cycle profile: one cycle at 95°C for 2 min and 40 cycles, with 1 cycle consisting of 30 s of denaturation at 95°C and 1 min of annealing-elongation at 60°C. A standard curve derived from 10-fold serial dilutions of purified PCR products of the target gene was used to determine the absolute concentration of target RNA/DNA. Gene annotation, ontology, and identification of biological pathways. The nonredundant database of protein sequences (National Center for Biotechnology Information, NIH, Bethesda, MD) was searched using the BLASTP program. All large-scale BLAST searches were run using a parallelized version of the BLASTP program running on 1,250 compute nodes of the NIH Biowulf cluster. Profile searches were conducted using the PSI-BLAST program () with either a single sequence or an alignment used as the query, with a default profile inclusion expectation (E) value threshold of 0.01 (unless specified otherwise), and was iterated until convergence. For all searches involving membrane-spanning domains or low-complexity sequences, we used a statistical correction for compositional bias to reduce false-positive results due to the general hydrophobicity of these proteins.

Hidden Markov models (HMMs) were built from alignments using the hmmbuild program, and searches were carried out using the hmmsearch program from the HMMer package (). Multiple alignments were constructed using the T_Coffee program () followed by manual correction based on the PSI-BLAST results. A library of a large set of alignments of conserved protein domains including those from the PFAM database () as well as an additional set of unpublished conserved domains was used for domain searches with the HMMer package (HMMs) or with PSI-BLAST (position-specific score matrices). Signal peptides were predicted using the SignalP program ().

Multiple alignments of the N-terminal regions of proteins were used additionally to verify the presence of a conserved signal peptide, and only those signal peptides that were conserved across orthologous groups of proteins were considered true positives. Transmembrane regions were predicted in individual proteins using the TMPRED, TMHMM2.0, and TOPRED1.0 programs with default parameters (, ). For TOPRED1.0, the organism parameter was set at “eukaryote” (). All large-scale sequence analysis procedures were carried out using the TASS package (S. Anantharaman, and L. Aravind, unpublished data).

The recently reported Plasmodium-specific motif for protein export into the host cells, the Pexel motif, was detected using a HMM, constructed using the alignment of the bona fide Pexel motif-containing proteins. The proteins encoded by the genes affected by elevated temperature were searched with this HMM and constrained by position towards the N-terminal region of the polypeptide and confirmed by further searching for an upstream hydrophobic signal. Secondary structure predictions based on multiple alignments were carried out using the Jpred2 program (). All Plasmodium genes are referred to by the standard gene identifiers that can be retrieved from PlasmoDB. Similarity-based clustering of proteins was carried out using the BLASTCLUST program (). Phylogenetic analysis was carried out using the maximum-likelihood (ML), neighbor-joining, and least-squares methods. Briefly, this process involved the construction of a least-squares tree using the FITCH program or a neighbor-joining tree using the NEIGHBOR program (both from the Phylip package), followed by local rearrangement using the Protml program of the Molphy package to arrive at the ML tree (, ).

The statistical significance of various nodes of this ML tree was assessed using the relative estimate of logarithmic likelihood bootstrap (Protml RELL-BP), with 10,000 replicates. All large-scale sequence and structure analysis procedures were carried out using the TASS package which operates similarly to the SEALS package (S.

Anantharaman, L. Aravind, unpublished data). Text versions of all alignments reported in this study can be downloaded from. Flow cytometry. Mouse polyclonal antiserum (kindly provided by Morris O. Makobongo) was raised against the cysteine-rich interdomain region (CIDR1) of EMP-1 expressed by the 3D7.41 parasite line. Approximately 1 × 10 6 erythrocytes at 1% parasitemia were stained with antiserum at various dilutions (1:200, 1:400, 1:800, and 1:1,600) in PBS containing 2% fetal calf serum for 45 min at 25°C and then washed.

Bound antibody was detected using Alexa Fluor 488-conjugated anti-mouse immunoglobulin G (Molecular Probes, Carlsbad, CA). Parasitized erythrocytes were stained with ethidium bromide (2 μg/ml) for 30 min. A FACSort instrument (Becton-Dickinson, San Jose, CA) and FlowJo software were used to acquire 500,000 events from each sample and to determine the median fluorescence intensity of populations of antibody-reactive, parasitized erythrocytes. Ubiquitin bioassay. Total ubiquitination of parasite proteins was assayed by Western blotting after growth of P.

Falciparum 3D7 parasites (5% initial parasitemia and 5% hematocrit) at 37°C for 2 h or at 41°C for 2 h. After incubation, erythrocytes were lysed with saponin, and parasite protein was then extracted and washed with M-PER mammalian protein extraction reagent (catalog no. 78503; Pierce). Total parasite protein (5 μg) was loaded per well, and ubiquitination of proteins was detected and quantitated using a rabbit antiubiquitin antibody (catalog no.

89899; Pierce) and a commercially obtained chemiluminescence-linked Western blot kit (Western Light Tropix, Bedford, MA). 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 (, ). 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 (). 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 (, ). It is important to note that the presence of “crisis forms” has been ascribed as a marker of apoptotic cell death in malaria parasites (). 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 (, ) and suggest that the malaria paroxysm plays a significant role in limiting the exponential growth of parasites in a nonimmune host. 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. 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 (, ). 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 H 2O 2 (). Other studies have failed to detect TUNEL-positive assay results in P. Falciparum parasites treated in vitro with known antimalarial drugs (, ). 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.

Measuring febrile temperature-induced alterations in the expression profile of P. 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. 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. 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) (, ). 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.

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 (). 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 (,, ). 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.

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 (), 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 (, ).

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 (). 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 (). 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 malaria (). 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 (). 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). 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 pAg-3 (), a homolog of Psurf 4.2, a P. Falciparum protein related to P. Vivax Vir proteins (). 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 (), a protein with the membrane attack complex-perforin domain that has been implicated in invasion (, -), 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 (). 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 (), 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 (). 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 (). 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. Falciparum, in contrast to other Plasmodium species and other Apicomplexa, shows a dramatic lineage-specific expansion of a particular family of DnaJ domain proteins ().

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. 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.

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. Falciparum proteins found to be upregulated in this study.

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. 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.

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. Cms Dvr Software Download Windows.

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 (). 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. 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. 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) (). 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 (); 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 (), 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. 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.

Falciparum proteins. 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 (), 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 (). 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. RNA metabolism. 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 stabilit y and perhaps splicing. Nuclear functions. 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 ().

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 (. General metabolism. 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 (). 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 () 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.

We thank Guojian Jiang and Tim Myers at the NIAID microarray research facility for assistance with the microarray studies and Nancy Shulman for editorial assistance. We also thank Nirbhay Kumar for anti-PfHSP-70, Morris Makobongo for anti-EMP-1, and Sanjay Singh for anti- P. Falciparum chitinase antibodies. Were supported by the intramural research program of the NCBI, NIH. The views and opinions expressed here are those of the authors and should not be construed as the official opinion of the Food and Drug Administration.