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Human malaria is caused by four species of the parasitic protozoan genus Plasmodium . Of these four species, Plasmodium falciparum is responsible for the vast majority of the 300–500 million episodes of malaria worldwide and accounts for 0.7–2.7 million annual deaths. In many endemic countries, malaria is responsible for economic stagnation, lowering the annual economic growth in some regions by up to 1.5% . While isolated efforts to curb malaria with combinations of vector control, education, and drugs have proven successful, a global solution has not been reached. Currently, there are few antimalarial chemotherapeutics available that serve as both prophylaxis and treatment. Compounding this paucity of drugs is a worldwide increase in P. falciparum strains resistant to the mainstays of antimalarial treatment . In addition, the search for a malaria vaccine has thus far been unsuccessful. Given the genetic flexibility and the immunogenic complexity of P. falciparum , a comprehensive understanding of Plasmodium molecular biology will be essential for the development of new chemotherapeutic and vaccine strategies.

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Introduction

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The 22.8 Mb genome of P. falciparum is comprised of 14 linear chromosomes, a circular plastid-like genome, and a linear mitochondrial genome. The malaria genome sequencing consortium estimates that more than 60% of the 5,409 predicted open reading frames (ORFs) lack sequence similarity to genes from any other known organism . Although ascribing putative roles for these ORFs in the absence of sequence similarity remains challenging, their unique nature may be key to identifying Plasmodium -specific pathways as candidates for antimalarial strategies.

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Introduction

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The complete P. falciparum lifecycle encompasses three major developmental stages: the mosquito, liver, and blood stages. It has long been a goal to understand the regulation of gene expression throughout each developmental stage. Previous attempts to apply functional genomics methods to address these questions used various approaches, including DNA microarrays , serial analysis of gene expression , and mass spectrometry on a limited number of samples from different developmental stages. While all of these approaches have provided insight into the biology of this organism, there have been no comprehensive analyses of any single developmental stage. Here we present an examination of the full transcriptome of one of these stages, the asexual intraerythrocytic developmental cycle (IDC), at a 1-h timescale resolution.

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Introduction

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The 48-h P. falciparum IDC initiates with merozoite invasion of red blood cells (RBCs) and is followed by the formation of the parasitophorous vacuole (PV) during the ring stage. The parasite then enters a highly metabolic maturation phase, the trophozoite stage, prior to parasite replication. In the schizont stage, the cell prepares for reinvasion of new RBCs by replicating and dividing to form up to 32 new merozoites. The IDC represents all of the stages in the development of P. falciparum responsible for the symptoms of malaria and is also the target for the vast majority of antimalarial drugs and vaccine strategies.

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Introduction

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Our laboratory has developed a P. falciparum– specific DNA microarray using long (70 nt) oligonucleotides as representative elements for predicted ORFs in the sequenced genome (strain 3D7) . Using this DNA microarray, we have examined expression profiles across 48 individual 1-h timepoints from the IDC of P. falciparum . Our data suggest that not only does P. falciparum express the vast majority of its genes during this lifecycle stage, but also that greater than 75% of these genes are activated only once during the IDC. For genes of known function, we note that this activation correlates well with the timing for the respective protein's biological function, thus illustrating an intimate relationship between transcriptional regulation and the developmental progression of this highly specialized parasitic organism. We also demonstrate the potential of this analysis to elucidate the function of the many unknown gene products as well as for further understanding the general biology of this parasitic organism.

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Introduction

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The genome-wide transcriptome of the P. falciparum IDC was generated by measuring relative mRNA abundance levels in samples collected from a highly synchronized in vitro culture of parasites. The strain used was the well-characterized Honduran chloroquine-sensitive HB3 strain, which was used in the only two experimental crosses carried out thus far with P. falciparum . To obtain sufficient quantities of parasitized RBCs and to ensure the homogeneity of the samples, a large-scale culturing technique was developed using a 4.5 l bioreactor (see Materials and Methods ). Samples were collected for a 48-h period beginning 1 h postinvasion (hpi). Culture synchronization was monitored every hour by Giemsa staining. We observed only the asexual form of the parasite in these stains. The culture was synchronous, with greater than 80% of the parasites invading fresh RBCs within 2 h prior to the harvesting of the first timepoint. Maintenance of synchrony throughout the IDC was demonstrated by sharp transitions between the ring-to-trophozoite and trophozoite-to-schizont stages at the 17- and 29-h timepoints, respectively .

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Expression Profiling of the IDC

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The DNA microarray used in this study consists of 7,462 individual 70mer oligonucleotides representing 4,488 of the 5,409 ORFs manually annotated by the malaria genome sequencing consortium . Of the 4,488 ORFs, 990 are represented by more than one oligonucleotide. Since our oligonucleotide design was based on partially assembled sequences periodically released by the sequencing consortium over the past several years, our set includes additional features representing 1,315 putative ORFs not part of the manually annotated collection. In this group, 394 oligonucleotides are no longer represented in the current assembled sequence. These latter ORFs likely fall into the gaps present in the published assembly available through the Plasmodium genome resource PlasmoDB.org .

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Expression Profiling of the IDC

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To measure the relative abundance of mRNAs throughout the IDC, total RNA from each timepoint was compared to an arbitrary reference pool of total RNA from all timepoints in a standard two-color competitive hybridization . The transcriptional profile of each ORF is represented by the mean-centered series of ratio measurements for the corresponding oligonucleotide(s) . Inspection of the entire dataset revealed a striking nonstochastic periodicity in the majority of expression profiles. The relative abundance of these mRNAs continuously varies throughout the IDC and is marked by a single maximum and a single minimum, as observed for the representative schizont-specific gene, erythrocyte-binding antigen 175 ( eba175 ), and the trophozoite-specific gene, dihydrofolate reductase–thymidylate synthetase ( dhfr-ts ) . However, there is diversity in both the absolute magnitude of relative expression and in the timing of maximal expression (phase). In addition, a minority of genes, such as adenylosuccinate lyase ( asl ) , displayed a relatively constant expression profile. The accuracy of measurements from individual oligonucleotides was further verified by the ORFs that are represented by more than one oligonucleotide feature on the microarray. The calculated average pairwise Pearson correlation ( r ) is greater than 0.90 for 68% (0.75 for 86%) of the transcripts represented by multiple oligonucleotides with detectable expression during the IDC ( Table S1 ). Cases in which data from multiple oligonucleotides representing a single putative ORF disagree may represent incorrect annotation. The internal consistency of expression profile measurements for ORFs represented by more than one oligonucleotide sequence is graphically shown in Figure 1 E for the hypothetical protein MAL6P1.147, the largest predicted ORF in the genome (31 kb), which is represented by 14 oligonucleotide elements spanning the entire length of the coding sequence. The average pairwise correlation ( r ) for these features is 0.98±0.02.

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Expression Profiling of the IDC

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Periodicity in genome-wide gene expression datasets has been used to identify cell-cycle-regulated genes in both yeast and human cells . Owing to the cyclical nature of the P. falciparum IDC dataset, a similar computational approach was taken. We performed simple Fourier analysis, which allowed us to calculate both the apparent phase and frequency of expression for each gene during the IDC (see Materials and Methods ). The fast Fourier transform (FFT) maps a function in a time domain (the expression profile) into a frequency domain such that when the mapped function is plotted (the power spectra), sharp peaks appear at frequencies where there is intrinsic periodicity. The calculated power spectra for each expression profile confirmed the observation that the data are highly periodic. The majority of profiles exhibited an overall expression period of 0.75–1.5 cycles per 48 h.

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12929205

Expression Profiling of the IDC

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We have used the FFT data for the purpose of filtering the expression profiles that are inherently noisy (i.e., that have low signal) or that lack differential expression throughout the IDC. Since the majority of the profiles display a single low-frequency peak in the power spectrum, we have taken advantage of this feature to classify profiles, similar to the application of a low-pass filter in signal processing. By measuring the power present in the peak frequency window (the main component plus two adjacent peaks) relative to the power present at all frequencies of the power spectrum, we were able to define a score (percent power) that we have used to stratify the dataset. The resulting distribution of expression profiles, scored in this way, is shown in Figure 1 F for all oligonucleotides. For reference, the positions of profiles corresponding to eba175 (peak B), dhfr-ts (peak C), and asl (peak D) are indicated. It is striking that 79.5% of the expression profiles have a very high score (greater than 70%). For comparison, we applied our FFT analysis to the Saccharomyces cerevisiae cell cycle data, yielding only 194 profiles (3.8%) above a 70% score . In addition, we randomly permuted the columns of the complete dataset 1,000 times, each time recalculating the FFT, for a total of 5 million profiles . The randomized set exhibits essentially no periodicity: the probability of any random profile scoring above 70% is 1.3 × 10 −5 .

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Expression Profiling of the IDC

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To provide an overview of the IDC transcriptome, we selected all 3,719 microarray elements whose profiles exhibited greater than 70% of the power in the maximum frequency window and that were also in the top 75% of the maximum frequency magnitudes. Although hierarchical clustering is extremely useful for comparing any set of expression data, regardless of the experimental variables, we sought to specifically address temporal order within the dataset. To accomplish this, the FFT phase was used to order the expression profiles to create a phaseogram of the IDC transcriptome of P. falciparum . The overview set represents 2,714 unique ORFs (3,395 oligonucleotides). An additional 324 oligonucleotides represent ORFs that are not currently part of the manually annotated collection.

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P. falciparum Transcriptome Overview

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The IDC phaseogram depicts a cascade of continuous expression lacking clear boundaries or sharp transitions. During the first half of the IDC, a large number of genes involved in general eukaryotic cellular functions are induced with broad expression profiles. This gradual continuum includes the transition from the ring to the early trophozoite stage and the trophozoite to the early schizont stage, encompassing approximately 950 and 1,050 genes, respectively. Next, the mid- and late-schizont stages are marked by a rapid, large amplitude induction of approximately 550 genes, many of which appear to be continually expressed into the early-ring stage. However, owing to the level of synchrony in the culture, the ring-stage signal may be partially attributed to cross-contamination from residual schizonts. In the final hours of the IDC, approximately 300 genes corresponding to the early-ring stage are induced, indicating that reinvasion occurs without obvious interruptions to initiate the next cycle. The expression profiles for developmentally regulated genes in the P. falciparum IDC transcriptome reveal an orderly timing of key cellular functions. As indicated in Figure 2 B–2M, groups of functionally related genes share common expression profiles and demonstrate a programmed cascade of cellular processes that ensure the completion of the P. falciparum IDC.

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P. falciparum Transcriptome Overview

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In the following text, we have grouped the genes according to temporal expression phases based on their association with the common P. falciparum cytological stages.

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Ring and Early-Trophozoite Stage

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Following invasion, approximately 950 ORFs are induced during the ring and early trophozoite stage, including genes associated with the cytoplasmic transcriptional and translational machinery, glycolysis and ribonucleotide biosynthesis . Represented in this group are 23 ORFs involved in transcription, including the four subunits of RNA polymerase I, nine subunits of RNA polymerase II, three subunits of RNA polymerase III, and four transcription factors. The average expression profile for this group is shown in Figure 2 B. (See Table S2 for all functional group details.) Also in this set are three previously identified P. falciparum RNA polymerase genes: the large subunits of P. falciparum RNA polymerase I and RNA polymerase II and RNA polymerase III . The cytoplasmic translation gene group consists of 135 ORFs including homologues for 34 small and 40 large ribosomal subunits, 15 translation initiation factors, five translation elongation factors, 18 aminoacyl-tRNA synthetases, and 23 RNA helicases. In addition to the manually annotated ORFs, the translation gene group contains three ORFs predicted only by automated annotation including two ribosomal proteins (chr5.glm_215, chr5.glm_185) and a homologue of eIF-1A (chr11.glm_489) ( PlasmoDB.org ). In one case, chr5.glm_185 overlaps with the manually annotated ORF PFE0850w, which is found on the opposite strand. Oligonucleotide elements for both of these ORFs are present on the array. The oligonucleotide corresponding to the automated prediction yielded a robust FFT score and a phase consistent with the translation machinery, yet no PFE0850w expression was detected. These results suggest that the automated prediction for chr5.glm_185 most likely represents the correct gene model for this genomic locus and illustrates the use of the IDC expression data for further verification of the P. falciparum genome annotations.

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12929205

Ring and Early-Trophozoite Stage

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Another set of 33 ORFs with homology to components of the translational machinery displayed an entirely distinct expression pattern, being induced during the late-trophozoite and early-schizont stage. This group includes 11 homologues of chloroplast ribosomal proteins, four mitochondrial/chloroplast elongation factors, and six amino acid tRNA synthetases ( Table S2 ). These ORFs also share a common pattern of expression, suggesting that these factors are components of the mitochondrial and/or the plastid translation machinery. This observation is supported by the presence of predicted apicoplast-targeting signals in 18 of these proteins ( PlasmoDB.org ). In addition, one of these factors, ribosomal protein S9, has been experimentally immunolocalized within the plastid . These data suggest that the peak of expression for the cytoplasmic translation machinery occurs in the first half of the IDC, whereas plastid and mitochondrial protein synthesis is synchronized with the maturation of these organelles during the second half of the IDC.

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12929205

Ring and Early-Trophozoite Stage

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In addition to transcription and translation, genes involved in several basic metabolic pathways were also induced during the ring and early-trophozoite stage, including glycolysis and ribonucleotide biosynthesis . Unlike the majority of P. falciparum biochemical processes, most of the enzymes involved in nucleotide metabolism and glycolysis have been identified . The glycolysis group is tightly coregulated throughout the IDC and contains all of the 12 known enzymes. Expression initiates after reinvasion and continues to increase toward maximal expression during the trophozoite stage, when metabolism is at its peak. The glycolytic pathway is very well preserved in P. falciparum and exemplifies how data from this study can complement the homology-based interpretation of the genome. First, the genome contains two putative copies of pyruvate kinase on chromosomes 6 and 10, MAL6P1.160 and PF10_0363, respectively . However, only one of these genes, MAL6P1.160, has a similar expression profile to the other known glycolytic enzymes, suggesting that this enzyme is the main factor of this step in the glycolytic pathway. Interestingly, PF10_0363 contains a putative apicoplast-targeting signal ( PlasmoDB.org ). In another case, the malaria genome sequencing consortium has predicted two homologues of triose phosphate isomerase, PF14_0378 and PFC0381w. The latter is not detected by our analysis, suggesting that this gene is utilized in another developmental stage or may be a nonfunctional, redundant homologue.

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12929205

Ring and Early-Trophozoite Stage

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P. falciparum parasites generate pyrimidines through a de novo synthesis pathway while purines must be acquired by the organism through a salvage pathway . The mRNA levels of 16 enzymes corresponding to members of the pyrimidine ribonucleotide synthesis pathway, beginning with carbamoyl phosphate synthetase and ending with CTP synthetase, were uniformly induced immediately after invasion . The relative abundance of these transcripts peaked at approximately 18–22 hpi and then rapidly declined. Similar expression characteristics were detected for the enzymes of the purine salvage pathway, including the nucleoside conversion enzymes, hypoxanthine–guanine–xanthine phosphoribosyltransferase, and both guanylate and adenylate kinases .

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12929205

Ring and Early-Trophozoite Stage

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The mRNA expression data indicate that ribonucleotide and deoxyribonucleotide production is clearly bifurcated into two distinct temporal classes. While ribonucleotide synthesis is required in the early stages of the IDC, deoxyribonucleotide metabolism is a trophozoite/early-schizont function. mRNA transcripts for enzymes that convert ribonucleotides into deoxyribonucleotides, including DHFR-TS and both subunits of ribonucleotide reductase, were induced approximately at 10 hpi, peaking at approximately 32 hpi . This represents a temporal shift from the induction of ribonucleotide synthesis of approximately 8–10 h. The expression of the deoxyribonucleotide biosynthesis is concomitant with the induction of DNA replication machinery transcripts, reflecting a tight relationship between DNA synthesis and production of precursors for this process.

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12929205

Trophozoite and Early-Schizont Stage

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Thirty-two ORFs with homologies to various eukaryotic DNA replication machinery components are transcribed during the late-trophozoite and early-schizont stage. The timing of their transcription presages cell division. This functional gene group , with peak expression around 32 hpi, contains the previously characterized P. falciparum DNA Polα, DNA Polδ, and proliferating cell nuclear antigen, as well as the vast majority of the DNA replication components predicted by the malaria genome sequencing consortium . These additional components include eight predicted DNA polymerase subunits, two putative origin recognition complex subunits, six minichromosome maintenance proteins, seven endo- and exonucleases, seven replication factor subunits, and two topoiosomerases. Interestingly, a number of proteins typically required for eukaryotic DNA replication, including the majority of the subunits of the origin recognition complex, have not yet been identified by conventional sequence similarity searches of the P. falciparum genome.

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12929205

Trophozoite and Early-Schizont Stage

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All genes necessary for the completion of the tricarboxylic acid (TCA) cycle were detected in the Plasmodium genome , although earlier studies indicate an unconventional function for this metabolic cycle. These studies suggest that the TCA cycle does not play a major role in the oxidation of glycolytic products. Instead, it is essential for the production of several metabolic intermediates, such as succinyl-CoA, a precursor of porphyrin biosynthesis . The peak of expression for all TCA factors was detected during the late-trophozoite and early-schizont stage . Consistent with the model suggesting a disconnection of the TCA cycle from glycolysis during the IDC, no expression was detected for the subunits of the pyruvate dehydrogenase complex, including the α and β chains of pyruvate dehydrogenase and dihydrolipoamide S-acetyl transferase, the typical links between glycolysis and the TCA cycle. On the other hand, expression of TCA cycle genes is well synchronized with the expression of a large number of mitochondrial genes, including the three ORFs of the mitochondrial genome , and several factors of electron transport ( Table S2 ). Although some of the TCA cycle proteins have been localized to the cytoplasm , the expression data suggest an association of this biochemical process with mitochondrial development and possibly with the abbreviated electron transport pathway detected in this organelle.

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12929205

Trophozoite and Early-Schizont Stage

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A transition from early to mid-schizont is marked by the maximal induction of 29 ORFs predicted to encode various subunits of the proteasome . Seven α and six β subunits of the 20S particle and 16 ORFs of the 19S regulatory particle were identified in this gene group. The common expression profile for the subunits of both of the 26S particle complexes suggests the involvement of ubiquitin-dependent protein degradation in the developmental progression of the parasite. The peak of proteasome expression coincides with a transition in the IDC transcriptome from metabolic and generic cellular machinery to specialized parasitic functions in the mid-schizont stage. This suggests an association between transcriptional regulation and protein turnover during this and possibly other transitions during the progression of the P. falciparum IDC.

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Schizont Stage

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In the schizont stage, one of the first specialized processes induced was expression from the plastid genome . The essential extrachromosomal plastid (or apicoplast) genome contains 60 potentially expressed sequences, including ribosomal proteins, RNA polymerase subunits, ribosomal RNAs, tRNAs, and nine putative ORFs, including a ClpC homologue . Very little is known about the regulation of gene expression in the plastid, but it is thought to be polycistronic . In support of this observation, we find that 27 of the 41 plastid-specific elements present on our microarray displayed an identical expression pattern . The remaining elements correspond mainly to tRNAs and failed to detect appreciable signal. The highly coordinated expression of the plastid genome, whose gene products are maximally expressed in the late-schizont stage, is concomitant with the replicative stage of the plastid . Note that not all plastid ORFs are represented on the microarray used in this study, and thus it is a formal possibility that the expression of the missing genes may differ from those shown in Figure 3 C.

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12929205

Schizont Stage

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Offset from the plastid by approximately 6 h, a set of approximately 500 ORFs exhibited peak expression during the late-schizont stage. Merozoite invasion of a new host cell is a complex process during which the parasite must recognize and dock onto the surface of the target erythrocyte, reorient with its apical tip toward the host cell, and internalize itself through invagination of the erythrocytic plasma membrane. The entire sequence of invasion events is facilitated by multiple receptor–ligand interactions with highly specialized plasmodial antigens . The merozoite invasion group contains 58 ORFs, including 26 ORFs encoding antigens previously demonstrated to be important for the invasion process . These include integral membrane proteins delivered to the merozoite surface from the micronemes (AMA1 and EBA175), GPI-anchored proteins of the merozoite membrane (MSP1, MSP4, and MSP5), proteins extrinsically associated with the merozoite surface during their maturation in the PV (MSP3 and MSP6), and soluble proteins secreted to the parasite–host cell interface (RAP1, RAP2, and RAP3). In addition, late-schizont-specific expression was observed for several antigens whose functions are not completely understood, but which have been associated with the invasion process. These ORFs include the merozoite-capping protein (MCP1), erythrocyte-binding-like protein 1 (EBL1), reticulocyte-binding proteins (RBP1 and RBP2), acid basic repeat antigen (ABRA), MSP7, and a homologue of the Plasmodium yoelii merozoite antigen 1. As expected, peak expression of these antigens coincides with the maturation of merozoites and development of several apical organelles, including rhoptries, micronemes, and dense granules. Many of these proteins have been considered as vaccine candidates since antibodies against these antigens were readily detected in the immune sera of both convalescent patients as well as individuals with naturally acquired immunity .

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Schizont Stage

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The sensitivity of invasion to protease and kinase inhibitors indicates an essential role for these activities in merozoite release as well as in the reinvasion process . The merozoite invasion gene group contains three serine proteases, including PfSUB1, PfSUB2, and an additional homologue to plasmodial subtilases , and two aspartyl proteases, plasmepsin (PM) IX and X. Peak expression during the mid-schizont stage was also observed for seven members of the serine repeat antigen (SERA) family, all of which contain putative cysteine protease domains. In addition to the proteases, expression of 12 serine/threonine protein kinases and three phophorylases was tightly synchronized with the genes of the invasion pathway, including six homologues of protein kinase C, three Ca + -dependent and two cAMP-dependent kinases, phosphatases 2A and 2B, and protein phosphatase J.

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12929205

Schizont Stage

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Another functionally related gene group whose expression is sharply induced during the late-schizont stage includes components of actin–myosin motors . As in other apicomplexa, actin and myosin have been implicated in host cell invasion . Schizont-specific expression was observed for three previously described class XIV myosin genes, one associated light chain, two actin homologues, and three additional actin cytoskeletal proteins, including actin-depolymerizing factor/cofilin (two isoforms) and coronin (one isoform). Although the molecular details of plasmodial actin–myosin invasion are not completely understood, the tight transcriptional coregulation of the identified factors indicates that the examination of schizont-specific expression may help to identify additional, possibly unique elements of this pathway.

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Schizont Stage

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The expression data are continuous throughout the invasion process, with no observable abrupt change in the expression program upon successful reinvasion. However, a set of approximately 300 ORFs whose expression is initiated in the late-schizont stage persists throughout the invasion process and peaks during the early-ring stages . It was previously determined that immediately after invasion, a second round of exocytosis is triggered, ensuring successful establishment of the parasite within the host cell . One of the main P. falciparum virulence factors associated with this process is ring-infected surface antigen 1 (RESA1). RESA1 is secreted into the host cell cytoplasm at the final stages of the invasion process, where it binds to erythrocytic spectrin, possibly via its DnaJ-like chaperone domain . The early stages of the IDC contain a variety of putative molecular chaperones in addition to RESA1, including RESA2 and RESAH3, plus five additional proteins carrying DnaJ-like domains. However, the functional roles of these chaperones remain unclear. Despite the cytoplasmic role of RESA1, abundant antibodies specific for RESA1 are present in individuals infected with P. falciparum , indicating that RESA1 is also presented to the host immune system . Several genes encoding additional antigenic factors are found among the early ring gene group, including frequently interspersed repeat antigen (FIRA), octapeptide antigen, MSP8, and sporozoite threonine- and asparagine-rich protein (STARP). Like RESA1, antibodies against these antigens are also found in the sera of infected individuals, suggesting that the final stages of invasion might be a target of the immune response.

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12929205

Early-Ring Stage

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Overall, the genes expressed during the mid- to late-schizont and early-ring stage encode proteins predominantly involved in highly parasite-specific functions facilitating various steps of host cell invasion. The expression profiles of these genes are unique in the IDC because of the large amplitudes and narrow peak widths observed. The sharp induction of a number of parasite-specific functions implies that they are crucial for parasite survival in the mammalian host and hence should serve as excellent targets for both chemotherapeutic and vaccine-based antimalarial strategies.

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12929205

Early-Ring Stage

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Transcriptional regulation of chromosomal gene expression in P. falciparum is thought to be monocistronic, with transcriptional control of gene expression occurring through regulatory sequence elements upstream and downstream of the coding sequence . This is in contrast to several other parasites, such as Leishmania sp. , in which polycistronic mRNA is synthesized from large arrays of coding sequences positioned unidirectionally along the arms of relatively short chromosomes . Recent proteomic analyses failed to detect any continuous chromosomal regions with common stage-specific gene expression in several stages of the P. falciparum lifecycle . However, transcriptional domains have previously been suggested for Chromosome 2 . The availability of the complete P. falciparum genome coupled with the IDC transcriptome allows us to investigate the possibility of chromosomal clustering of gene expression . To systematically explore the possibility of coregulated expression as a function of chromosomal location, we applied a Pearson correlation to identify similarities in expression profiles among adjacent ORFs. The pairwise Pearson correlation was calculated for every ORF pair within each chromosome . Gene groups in which the correlation of 70% of the possible pairs was greater than r = 0.75 were classified as putative transcriptionally coregulated groups. Using these criteria, we identified only 14 coregulation groups consisting of greater than three genes, with the total number of genes being 60 (1.4% of all represented genes) ( Table S3 ). In eight of the 14 groups, the coregulation of a pair of genes may be explained by the fact that they are divergently transcribed from the same promoter. A set of 1,000 randomized permutations of the dataset yielded 2.25 gene groups. Contrary to the nuclear chromosomes, there was a high correlation of gene expression along the plastid DNA element, consistent with polycistronic transcription . The average pairwise Pearson correlation for a sliding window of seven ORFs along the plastid genome is 0.92±0.03.

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12929205

IDC Transcriptional Regulation and Chromosomal Structure

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The largest group demonstrating coregulation on the nuclear chromosomes corresponds to seven genes of the SERA family found on Chromosome 2 . Besides the SERA gene cluster and a group containing three ribosomal protein genes, no additional functional relationship was found among the other chromosomally adjacent, transcriptionally coregulated gene groups. The limited grouping of regional chromosomal expression was independent of strand specificity and, with the exception of the SERA group, did not overlap with the groups of “recently duplicated genes” proposed by the malaria genome sequencing consortium .

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12929205

IDC Transcriptional Regulation and Chromosomal Structure

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Three major surface antigens, the var , rifin , and stevor families, have a high degree of genomic variability and are highly polymorphic between strains and even within a single strain . Expression profiles for only a small subset of these genes were detected in the IDC transcriptome and were typically characterized by low-amplitude profiles. This could be due to two nonmutually exclusive possibilities: first, the HB3 DNA sequence for these genes may be substantially rearranged or completely deleted relative to the reference strain, 3D7; second, only a few of these genes may be selectively expressed, as has been proposed . To identify regions of genomic variability between 3D7 and HB3, we performed microarray-based comparative genomic hybridization (CGH) analysis. Array-based CGH has been performed with human cDNA and bacterial artificial chromosome-based microarrays to characterize DNA copy-number changes associated with tumorigenesis . Using a similar protocol, CGH analysis revealed that the majority of genetic variation between HB3 and 3D7 is confined to the subtelomeric chromosomal regions containing the aforementioned gene families . Only 28.3% of rifin , 47.1% of var , and 51.0% of stevor genes predicted for the 3D7 strain were detected for the HB3 genomic DNA (gDNA) when hybridized to the 3D7-based microarray. Thus, the underrepresentation of these gene families in the HB3 IDC transcriptome is likely due to the high degree of sequence variation present in these genes. Excluding the three surface antigen families in the subtelomeric regions, 97% of the remaining oligonucleotide microarray elements exhibit an equivalent signal in the CGH analysis. However, 144 of the differences detected by CGH reside in internal chromosomal regions and include several previously identified plasmodial antigens: MSP1, MSP2 , S antigen, EBL1, cytoadherence-linked asexual gene 3.1 (CLAG3.1), glutamine-rich protein (GLURP), erythrocyte membrane protein 3 (PfEMP3), knob-associated histidine-rich protein (KAHRP), and gametocyte-specific antigen Pfg377 ( Table S4 ). These results demonstrate a high degree of genetic variation within the genes considered to be crucial for antigenic variation between these two commonly used laboratory strains of P. falciparum .

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12929205

IDC Transcriptional Regulation and Chromosomal Structure

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The majority of the nuclear-encoded proteins targeted to the plastid are of prokaryotic origin, making them excellent drug targets . Moreover, inhibitors of plastid-associated isoprenoid biosynthesis, DNA replication, and translation have been shown to kill the P. falciparum parasite, demonstrating that the plastid is an essential organelle . The plastid has been implicated in various metabolic functions, including fatty acid metabolism, heme biosynthesis, isoprenoid biosynthesis, and iron–sulfur cluster formation . It is clear that, within the plastid, functional ribosomes are assembled to express the ORFs encoded by the plastid genome . However, nuclear-encoded components are required to complete the translational machinery as well as for all other plastid metabolic functions. A bipartite signal sequence is required for efficient transport of these nuclear proteins from the cytoplasm to the plastid via the endoplasmic reticulum . Computational predictions suggest that the P. falciparum genome may contain over 550 nuclear-encoded proteins with putative transit peptides .

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12929205

Implications for Drug Discovery

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Given that over 10% of the ORFs in the P. falciparum genome are predicted to contain an apicoplast-targeting sequence, we sought to use the IDC transcriptome as a means to narrow the search space for candidate apicoplast-targeted genes. As mentioned above, the expression profiles for genes encoded on the plastid genome are tightly coordinated . We reasoned that genes targeted to the plastid would be expressed slightly before or coincidentally with the plastid genome. Therefore, we utilized the FFT phase information to identify ORFs in phase with expression of the plastid genome (see Materials and Methods ) ( Table S5 ). Because the genes of the plastid genome are maximally expressed between 33 and 36 hpi, we searched for all genes in the dataset with an FFT phase in this time window and then cross-referenced the list of predicted apicoplast-targeted sequences ( PlasmoDB.org ), resulting in a list of 124 in-phase apicoplast genes . Within this list are two ORFs that have been directly visualized in the apicoplast, acyl carrier protein and the ribosomal subunit S9 , as well as many ORFs associated with the putative plastid ribosomal machinery, enzymes involved in the nonmevalonate pathway, additional caseineolytic proteases (Clps), the reductant ferredoxin, and replication/transcriptional machinery components. However, this list contains only 14 of the 43 proteins categorized in the Gene Ontology (GO) assignments at PlasmoDB.org as apicoplast proteins by inference from direct assay (IDA). In addition, 30% of the nuclear-encoded translational genes that are not coexpressed with the known cytoplasmic machinery are found within this small group of genes. More importantly, 76 ORFs (62%) are of unknown function, with little or no homology to other genes. This limited subgroup of putative plastid-targeted ORFs are likely excellent candidates for further studies in the ongoing search for malaria-specific functions as putative drug targets.

12929205_p31

12929205

Implications for Drug Discovery

biomedical

Study

en

Similarly, P. falciparum proteases have received much attention, since they are candidates as drug targets and have been shown to play important roles in regulation as well as metabolism throughout the IDC . A temporal ordering of expression profiles for several well-characterized P. falciparum proteases is shown in Figure 4 B, demonstrating the broad significance of these enzymes throughout the IDC. One of the principal proteolytic functions is considered to be the degradation of host cell hemoglobin in the food vacuole (FV) to produce amino acids essential for protein synthesis. This elaborate process is carried out by a series of aspartyl proteases, cysteine proteases, metalloproteases, and aminopeptidases .

12929205_p32

12929205

Implications for Drug Discovery

biomedical

Study

en

A family of ten aspartyl proteases, the plasmepsins (PMs), has been identified in the P. falciparum genome, four of which have been characterized as bona fide hemoglobinases: PM I, II, III (a histo-aspartic protease [HAP]), and IV . Our data reveal that the PMs are expressed at different times throughout the lifecycle, suggesting that they are involved in different processes throughout the IDC. PM I, II, HAP, and PM IV are adjacent to one another on Chromosome 14 and have been localized to the FV. While HAP and PM II are expressed in the mid-trophozoite stage, during peak hemoglobin catabolism, PMI and IV are maximally expressed in the ring stage along with the cysteine protease falcipain-1 (FP-1). FP-1 has recently been implicated in merozoite invasion and has been localized to the interior of the PV . The coincident expression of these proteases implies that the development of the PV and the FV occurs during the very early-ring stage. This observation is corroborated by similar expression profiles for the PV-associated protein RESA1 and the FV protein PGH1. Subsequently, a second group of hemoglobinases, including the m1-family aminopeptidase, FP-2, and falcilysin, is expressed simultaneously with HAP and PM II during the trophozoite stage of the IDC. The expression of PM V and the newly identified FP- 2 homologue during this stage suggests they are also important in the trophozoite stage. The other known falcipain, FP-3, does not show a marked induction in expression throughout the IDC. We fail to detect any transcripts for PM VI, VII, and VIII during the IDC. These genes may have roles in any of the other sexual, liver, or mosquito stages of development.

12929205_p33

12929205

Implications for Drug Discovery

biomedical

Study

en

In addition to the hemoglobinases, P. falciparum contains a variety of proteases involved in cellular processing, including a group of Clps and signal peptidases that are all expressed maximally at the late-trophozoite stage . The timing of these genes may play a key role in protein maturation during trafficking to various compartments, including the plastid. The three Clps contain putative leader peptides and may actually function within the plastid. Finally, a group of proteases are expressed in the schizont stage and include the P. falciparum subtilisin-like proteases PfSUB1 and PfSUB2 as well as PMs IX and X. PfSUB1 and PfSUB2 are believed to be involved in merozoite invasion and have been localized apically in the dense granules. Interestingly, there are two PfSUB1 protease homologues ; PM X parallels the expression of PfSUB1 , suggesting that aspartyl proteases may also be involved in merozoite invasion. In addition, the phase of the PfSUB1 homologue suggests a concomitant role, with PM IX slightly preceding merozoite invasion. In total, we have detected gene expression for over 80 putative proteases throughout the entire IDC ( Table S6 ). This set includes over 65 proteases from a group of recently predicted proteases . The differing temporal expression of these proteases may allow for a multifaceted approach toward identifying protease inhibitors with efficacy at all stages of the IDC.

12929205_p34

12929205

Implications for Drug Discovery

biomedical

Study

en

Merozoite invasion is one of the most promising target areas for antimalarial vaccine development . Many vaccine efforts thus far have focused primarily on a set of plasmodial antigens that facilitate receptor–ligand interaction between the parasite and the host cell during the invasion process . Merozoite invasion antigens are contributing factors to naturally acquired immunity, triggering both humoral and antibody-independent cell-mediated responses . Antibodies against these antigens have been demonstrated to effectively block the merozoite invasion process in vitro and in animal models . Owing to the highly unique character of merozoite surface antigens, homology-based searches have yielded only a limited set of additional invasion factors.

12929205_p35

12929205

Implications for New Vaccine Therapies

biomedical

Study

en

We utilized the IDC transcriptome to predict a set of likely invasion proteins by identifying expression profiles with characteristics similar to previously studied merozoite invasion proteins. The expression profiles for all known invasion factors undergo a sharp induction during the mid- to late-schizont stage and are characterized by large expression amplitudes . Among these proteins are seven of the best-known malaria vaccine candidates, including AMA1, MSP1, MSP3, MSP5, EBA175, RAP1, and RESA1. To identify ORFs with a possible involvement in the merozoite invasion process, we have calculated the similarity, by Euclidian distance, between the expression profiles of these seven vaccine candidates and the rest of the IDC transcriptome. A histogram of the distance values reveals a bimodal distribution with 262 ORFs in the first peak of the distribution . This represents the top 5% of expression profiles when ranked by increasing Euclidian distance ( Table S7 ). In addition to the seven vaccine candidate genes used for the search, essentially all predicted P. falciparum merozoite-associated antigens were identified in this gene set . These include the GPI-anchored MSP4; several integral merozoite membrane proteins, such as EBA140 and EBL1; three RBPs (RBP1, RBP2a, RBP2b); and a previously unknown RBP homologue. In addition, components of two proteins secreted from the rhoptries to the host cell membranes, RhopH1 and RhopH3, or to the PVs RAP1, RAP2, and RAP3 were found in the selected set. Surprisingly, CLAG2 and CLAG9 were also classified into the merozoite invasion group. Although the biological function of these genes is believed to be associated with cytoadherence of the infected erythrocyte to the vascular endothelium, a highly related homologue, CLAG3.1 (RhopH1), was recently detected in the rhoptries, suggesting a possible secondary role for these genes in merozoites .

12929205_p36

12929205

Implications for New Vaccine Therapies

biomedical

Study

en

A number of antigens are presently in various stages of clinical trials and are yielding encouraging results . However, many single-antigen vaccine studies indicate that the most promising approach will require a combination of antigenic determinants from multiple stages of the complex plasmodial lifecycle . Searches for new target antigens in the P. falciparum genome are thus vital to the development of future vaccines, since no fully protective vaccine has been assembled thus far. Of the 262 ORFs whose expression profiles were closest to the profiles of the seven major vaccine candidates, 189 are of unknown function. These ORFs represent a candidate list for new vaccine targets.

12929205_p37

12929205

Implications for New Vaccine Therapies

biomedical

Study

en

The transcriptome of the IDC of P. falciparum constitutes an essential tool and baseline foundation for the analysis of all future gene expression studies in this organism, including response to drugs, growth conditions, environmental perturbations, and genetic alterations. Essentially all experiments involving asexual intraerythrocytic-stage parasites must be interpreted within the context of the ongoing cascade of IDC-regulated genes.

12929205_p38

12929205

Discussion

biomedical

Study

en

In our global analysis of the P. falciparum transcriptome, over 80% of the ORFs revealed changes in transcript abundance during the maturation of the parasite within RBCs. The P. falciparum IDC significantly differs from the cell cycles of the yeast S. cerevisiae and human HeLa cells, during which only 15% of the total genome is periodically regulated. Instead, the P. falciparum IDC resembles the transcriptome of the early stages of Drosophila melanogaster development, which incorporates the expression of over 80% of its genome as well . Unlike the development of multicellular eukaryotes, there is no terminal differentiation and, with the exception of gametocytogenesis, the parasite is locked into a repeating cycle. In this respect, the P. falciparum IDC mirrors a viral-like lifecycle, in which a relatively rigid program of transcriptional regulation governs the progress of the course of infection.

12929205_p39

12929205

Discussion

biomedical

Study

en

The lack of continuous chromosomal domains with common expression characteristics suggests that the genes are regulated individually, presumably via distinct sets of cis - and trans -acting elements. However, the extent and the simple mechanical character of transcriptional control observed in the IDC suggest a fundamentally different mode of regulation than what has been observed in other eukaryotes. It is plausible that a comparatively small number of transcription factors with overlapping binding site specificities could account for the entire cascade. While further experiments are ongoing, it may be the case that P. falciparum gene regulation is streamlined to the extent that it has lost the degree of dynamic flexibility observed in other unicellular organisms, from Escherichia coli to yeast. This observation also implies that disruption of a key transcriptional regulator, as opposed to a metabolic process, may have profound inhibitory properties. While a few putative transcription factors have been identified in the P. falciparum genome, no specific regulatory elements have been defined in basepair-level detail. A further analysis of the upstream regions of genes with similar phases should facilitate the elucidation of regulatory regions and their corresponding regulatory proteins.

12929205_p40

12929205

Discussion

biomedical

Study

en

In general, the timing of mRNA expression for a given gene during the IDC correlates well with the function of the resultant protein. For example, replication of the genome occurs in the early-schizont stage and correlates well with the peak expression of all factors of DNA replication and DNA synthesis. Also, organellar biogenesis of several intracellular compartments such as mitochondria, the plastid, or the apical invasion organelles is concomitant with the maximal induction of mRNAs encoding proteins specific to these organelles. In addition, our data are generally in good agreement with proteomic analyses that have detected intraerythrocytic-stage proteins from the merozoite, trophozoite, and schizont stages. More than 85% of the 1,588 proteins detected in these studies were also expressed in our analysis . However, a more detailed proteomic analysis at different stages of the IDC will be needed to ascertain the temporal changes of these proteins.

12929205_p41

12929205

Discussion

biomedical

Study

en

We initially expected that a high percentage of the genome would be specialized for each lifecycle stage (mosquito, liver, blood), yet this was not observed; the mRNA transcripts for 75% of proteins determined to be gamete-, gametocyte-, or sporozoite-specific by mass spectrometry are also transcribed in the plasmodial IDC. These findings confirm previous studies demonstrating that not only genes used for generic cellular processes are present in multiple developmental stages, but also factors of highly specialized Plasmodium functions . This may indicate that only a small portion of the genome may actually be truly specific to a particular developmental stage and that the majority of the genome is utilized throughout the full lifecycle of this parasite. It is also feasible to speculate that a multilayer regulatory network is employed in the progression of the entire P. falciparum lifecycle. In this model, the same cis - and trans -acting regulatory elements driving the actual mRNA production in IDC are utilized in other developmental stages. These elements are then controlled by an alternate subset of factors determining the status of the lifecycle progression.

12929205_p42

12929205

Discussion

biomedical

Study

en

These findings also outline two contrasting properties of the P. falciparum genome. The Plasmodium parasite devotes 3.9% of its genome to a complex system of antigenic determinants essential for host immune evasion during a single developmental stage . On the other hand, large portions of the genome encode proteins used in multiple stages of the entire lifecycle. Such broad-scope proteins might be excellent targets for both vaccine and chemotherapeutic antimalarial strategies, since they would target several developmental stages simultaneously. While there are certainly proteins specific to these nonerythrocytic stages, a complementary analysis of both proteomic and genomic datasets will facilitate the search.

12929205_p43

12929205

Discussion

biomedical

Study

en

With malaria continuing to be a major worldwide disease, advances toward understanding the basic biology of P. falciparum remain essential. Our analysis of the IDC transcriptome provides a first step toward a comprehensive functional analysis of the genome of P. falciparum . The genome-wide transcriptome will be useful not only for the further annotation of many uncharacterized genes, but also for defining the biological processes utilized by this highly specialized parasitic organism. Importantly, candidate groups of genes can be identified that are both functionally and transcriptionally related and thus provide focused starting points for the further elucidation of genetic and mechanistic aspects of P. falciparum . Such biological characterizations are presently a major objective in the search for novel antimalarial strategies. The public availability of the dataset presented in this study is intended to provide a resource for the entire research community to extend the exploration of P. falciparum beyond the scope of this publication. All data will be freely accessible at two sites: http://plasmodb.org and http://malaria.ucsf.edu .

12929205_p44

12929205

Discussion

biomedical

Study

en

A large-scale culture of P. falciparum (HB3 strain) was grown in a standard 4.5 l microbial bioreactor (Aplikon, Brauwweg, Netherlands) equipped with a Bio Controller unit ADI 1030 (Aplikon, Brauwweg, Netherlands). Cells were initially grown in a 2% suspension of purified human RBCs and RPMI 1640 media supplemented with 0.25% Albumax II (GIBCO, Life Technologies, San Diego, California, United States), 2 g/l sodium bicarbonate, 0.1 mM hypoxanthine, 25 mM HEPES (pH 7.4), and 50 μg/l gentamycin, at 37°C, 5% O 2 , and 6% CO 2 . Cells were synchronized by two consecutive sorbitol treatments for three generations, for a total of six treatments. Large-scale cultures contained 32.5 mM HEPES (pH 7.4). The bioreactor culture was initiated by mixing 25.0 ml of parasitized RBCs (20% late schizonts, approximately 45 hpi) with an additional 115.0 ml of purified RBC in a total of 1.0 l of media (14% hematocrit). Invasion of fresh RBCs occurred during the next 2 h, raising the total parasitemia from an initial 5% to 16%. After this period, the volume of the culture was adjusted to 4.5 l, bringing the final RBC concentration to approximately 3.3% to reduce the invasion of remaining cells. Immediately after the invasion period, greater than 80% of the parasites were in the ring stage. Temperature and gas conditions were managed by the Bio Controller unit. Over the course of 48 h, 3–4 ml of parasitized RBCs was collected every hour, washed with prewarmed PBS, and flash-frozen in liquid nitrogen.

12929205_p45

12929205

Cell culture.

biomedical

Study

en

P. falciparum RNA sample isolation, cDNA synthesis, labeling, and DNA microarray hybridizations were performed as described by Bozdech et al. . Samples for individual timepoints (coupled to Cy5) were hybridized against a reference pool (coupled to Cy3). The reference pool was comprised of RNA samples representing all developmental stages of the parasite. From this pool, sufficient cDNA synthesis reactions, using 12 μg of pooled reference RNA, were performed for all hybridizations. After completing cDNA synthesis, all reference pool cDNAs were combined into one large pool and then split into individual aliquots for subsequent labeling and hybridization. Microarray hybridizations were incubated for 14–18 h.

12929205_p46

12929205

RNA preparation and reference pool.

biomedical

Study

en

In total, 55 DNA microarray hybridizations covering 46 timepoints were performed. Timepoints 1, 7, 11, 14, 18, 20, 27, and 31 were represented by more than one array hybridization. Data were acquired and analyzed by GenePix Pro 3 (Axon Instruments, Union City, California, United States). Array data were stored and normalized using the NOMAD microarray database system ( http://ucsf-nomad.sourceforge.net/ ). In brief, a scalar normalization factor was calculated for each array using unflagged features with median intensities greater than zero for each channel and a pixel regression correlation coefficient greater than or equal to 0.75. Quality spots were retained based on the following criteria. The log 2 (Cy5/Cy3) ratio for array features that were unflagged and had a sum of median intensities greater than the local background plus two times the standard deviation of the background were extracted from the database for further analysis. Subsequently, expression profiles consisting of 43 of 46 timepoints (approximately 95%) were selected. For those timepoints that were represented by multiple arrays, the ratio values were averaged.

12929205_p47

12929205

DNA microarray hybridizations and quality control.

biomedical

Study

en

Fourier analysis was performed on each profile in the quality-controlled set (5,081 oligonucleotides). Profiles were smoothed with missing values imputed using a locally weighted regression algorithm with local weighting restricted to 12% using R ( http://www.R-project.org ). Fourier analysis was performed on each profile using the fft() function of R, padded with zeros to 64 measurements. The power spectrum was calculated using the spectrum() function of R. The power at each frequency ( Power() ), the total power (P tot ), and the frequency of maximum power (F max ) were determined. The periodicity score was defined as Power [(F max−1 ) + (F max ) + (F max+1 )]/P tot . The most frequent value of F max across all profiles was deemed the major frequency (m) and used in determining phase information. The phase of each profile was calculated as atan2\[−(I (m)],R (m)\, where atan2 is R's arctangent function and I and R are the imaginary and real parts of the FFT. Profiles were then ordered in increasing phase from −π to π. The loess smooth profiles were drawn through the raw expression data using the loess() function found in the modern regression library of R (version 1.5.1). The default parameters were used, with the exception that local weighting was reduced to 30%. For the averaged profiles of the functional groups , the loess smooth profiles were calculated for each expression profile individually and subsequently averaged to create the representative profile. These same methods were applied to both the randomized set and the yeast cell cycle dataset .

12929205_p48

12929205

FFT analysis of the expression profiles.

biomedical

Study

en

The raw results files ( Dataset S1 ), the fully assembled raw dataset ( Dataset S2 , the overview dataset ( Dataset S3 , and the quality control dataset ( Dataset S4 ) are available as downloads.

12929205_p49

12929205

FFT analysis of the expression profiles.

biomedical

Other

en

The evaluation of coexpression of genes along chromosomes was carried out as follows. The Pearson correlation coefficient was calculated for each pair of profiles. For ORFs with multiple oligonucleotides, the average profile was calculated. The neighborhood of each ORF profile was defined as a window of between one and ten adjacent ORF profiles. If any window in an ORF profile's neighborhood displayed more than 70% pairwise correlation of greater than 0.75, it was flagged as enriched. The length of the window was then recorded as a region of coexpression. This process was repeated without strand separation of ORFs and with randomly permuted datasets.

12929205_p50

12929205

Evaluation of coexpression along chromosomes.

biomedical

Study

en

P. falciparum strains 3D7 and HB3 were cultured as previously described at a concentration of 10% parasitaemia. Genomic DNA (gDNA) was isolated from a minimum of 500 ml of total culture for each P. falciparum strain, as previously described . Isolated gDNA from each strain was sheared by sonication to an average fragment size of approximately 1–1.5 kb and then was purified and concentrated using a DNA Clean and Concentrator kit (Zymo Research, Orange, California, United States). Amino-allyl-dUTP first was incorporated into the gDNA fragments with a Klenow reaction at 37°C for 6–8 h with random nonamer primers and 3 μg of sheared gDNA. After purification and concentration of the DNA from the Klenow reaction, CyScribe Cy3 and Cy5 dyes (Amersham Biosciences, Buckinghamshire, United Kingdom) were coupled to HB3 DNA and 3D7 DNA, respectively, as previously described . Uncoupled fluorescent dye was removed using a DNA Clean and Concentrator kit. Labeled DNA fragments were hybridized to the oligonucleotide-based DNA microarrays. Fluorescence was detected and analyzed using an Axon Instruments scanner and GenePix Pro 3.0 software. Only features that had median intensities greater than the local background plus two times the standard deviation of the background in each channel were considered for further analysis. For each feature, the percent of the total intensity was determined using the signal in the 3D7 channel as the total amount of intensity for each oligonucleotide; intensity differences less than 50% were considered to be significant for subsequence analysis.

12929205_p51

12929205

Comparative genomic hybridization.

biomedical

Study

en

The range of FFT-based phases for the expression profiles of the plastid genome is between 0.32 and 1.05 (or roughly π/9 −π/3). Using the list of 551 apicoplast-targeted genes available at PlasmoDB.org , we first ordered these genes by phase and then grouped all genes with a phase range between 0.00 and 1.40 (0–4π/9), resulting in 124 genes represented by 128 oligonucleotides on the microarray. This select group represents the in-phase plastid targeted genes (see Table S6 ).

12929205_p52

12929205

Calculation for in-phase plastid-targeted genes.

biomedical

Study

en

To select the expression profiles most related to the AMA1, MSP1, MSP3, MSP5, EBA175, RAP1, and RESA1 vaccine candidates, we calculated the similarity of all expression profiles in the dataset to those of these antigens by Euclidian distance. The minimum Euclidian distance calculated for every profile was then binned into 60 bins and plotted as a histogram. A natural break in the histogram was seen that included the set of 262 ORFs .

12929205_p53

12929205

Calculation for vaccine targets.

biomedical

Study

en

From a clinical perspective, inadequate protection from sunlight has a major impact on human health . In Australia, the lifetime cumulative incidence of skin cancer approaches 50%, yet the oxymoronic “smart tanning” industry continues to grow, and there is controversy over the extent to which different types of melanin can influence susceptibility to ultraviolet (UV) radiation . At the other end of the spectrum, inadequate exposure to sunlight, leading to vitamin D deficiency and rickets, has been mostly cured by nutritional advances made in the early 1900s. In both cases, understanding the genetic architecture of human skin color is likely to provide a greater appreciation of underlying biological mechanisms, much in the same way that mutational hotspots in the gene TP53 have helped to educate society about the risks of tobacco .

14551921_p0

14551921

Why Should We Care?

biomedical

Review

en

From a basic science perspective, variation in human skin color represents an unparalleled opportunity for cell biologists, geneticists, and anthropologists to learn more about the biogenesis and movement of subcellular organelles, to better characterize the relationship between genotypic and phenotypic diversity, to further investigate human origins, and to understand how recent human evolution may have been shaped by natural selection.

14551921_p1

14551921

Why Should We Care?

biomedical

Other

en

Historically, measurement of human skin color is often based on subjective categories, e.g., “moderate brown, rarely burns, tans very easily.” More recently, quantitative methods based on reflectance spectrophotometry have been applied, which allow reddening caused by inflammation and increased hemoglobin to be distinguished from darkening caused by increased melanin . Melanin itself is an organic polymer built from oxidative tyrosine derivatives and comes in two types, a cysteine-rich red–yellow form known as pheomelanin and a less-soluble black--brown form known as eumelanin . Discriminating among pigment types in biological samples requires chemical extraction, but is worth the effort, since the little we do know about common variation in human pigmentation involves pigment type-switching. The characteristic phenotype of fair skin, freckling, and carrot-red hair is associated with large amounts of pheomelanin and small amounts of eumelanin and is caused by loss-of-function alleles in a single gene, the melanocortin 1 receptor (MC1R) However, MC1R variation has a significant effect on pigmentation only in populations where red hair and fair skin are common , and its primary effects—to promote eumelanin synthesis at the expense of pheomelanin synthesis, or vice versa— contribute little to variation of skin reflectance among or between major ethnic groups .

14551921_p2

14551921

The Color Variation Toolbox

biomedical

Study

en

More important than the ratio of melanin types is the total amount of melanin produced. In addition, histological characteristics of different-colored skin provide some clues as to cellular mechanisms that are likely to drive pigmentary variation . For the same body region, light- and dark-skinned individuals have similar numbers of melanocytes (there is considerable variation between different body regions), but pigment-containing organelles, called melanosomes, are larger, more numerous, and more pigmented in dark compared to intermediate compared to light skin, corresponding to individuals whose recent ancestors were from Africa, Asia, or Europe, respectively . From these perspectives, oxidative enzymes like tyrosinase (TYR), which catalyzes the formation of dopaquinone from tyrosine, or melanosomal membrane components like the pink-eyed dilution protein (P) or the membrane-associated transporter protein (MATP), which affect substrate availability and activity of TYR , are logical candidates upon which genetic variation could contribute to the diversity of human skin color.

14551921_p3

14551921

The Color Variation Toolbox

biomedical

Study

en

Of equal importance to what happens inside melanocytes is what happens outside. Each pigment cell actively transfers its melanosomes to about 40 basal keratinocytes; ultimately, skin reflectance is determined by the amount and distribution of pigment granules within keratinocytes rather than melanocytes. In general, melanosomes of African skin are larger and dispersed more widely than in Asian or European skin . Remarkably, keratinocytes from dark skin cocultured with melanocytes from light skin give rise to a melanosome distribution pattern characteristic of dark skin, and vice versa . Thus, at least one component of skin color variation represents a gene or genes whose expression and action affect the pigment cell environment rather than the pigment cell itself.

14551921_p4

14551921

The Color Variation Toolbox

biomedical

Study

en

For any quantitative trait with multiple contributing factors, the most important questions are the overall heritability, the number of genes likely to be involved, and the best strategies for identifying those genes. For skin color, the broad sense heritability (defined as the overall effect of genetic vs. nongenetic factors) is very high , provided one is able to control for the most important nongenetic factor, exposure to sunlight.

14551921_p5

14551921

Genetics of Skin Color

biomedical

Study

en

Statements regarding the number of human skin color genes are attributed to several studies; one of the most complete is by Harrison and Owen . In that study, skin reflectance measurements were obtained from 70 residents of Liverpool whose parents, grandparents, or both were of European (“with a large Irish component”) or West African (“mostly from coastal regions of Ghana and Nigeria”) descent and who were roughly classified into “hybrid” and “backcross” groups on this basis. An attempt to partition and analyze the variance of the backcross groups led to minimal estimates of three to four “effective factors,” in this case, independently segregating genes. Aside from the key word minimal (Harrison and Owen's data could also be explained by 30–40 genes), one of the more interesting findings was that skin reflectance appeared to be mainly additive. In other words, mean skin reflectance of “F1 hybrid” or “backcross hybrid” groups is intermediate between their respective parental groups.

14551921_p6

14551921

Genetics of Skin Color

biomedical

Study

en

An alternative approach for considering the number of potential human pigmentation genes is based on mouse coat color genetics, one of the original models to define and study gene action and interaction, for which nearly 100 different genes have been recognized . Setting aside mouse mutations that cause white spotting or predominant effects outside the pigmentary system, no more than 15 or 20 mutations remain, many of which have been identified and characterized, and most of which have human homologs in which null mutations cause albinism.

14551921_p7

14551921

Genetics of Skin Color

biomedical

Study

en

This brings us to the question of candidate genes for skin color, since, like any quantitative trait, a reasonable place to start is with rare mutations known to cause an extreme phenotype, in this case Mendelian forms of albinism. The underlying assumption is that if a rare null allele causes a complete loss of pigment, then a set of polymorphic, i.e., more frequent, alleles with subtle effects on gene expression will contribute to a spectrum of skin colors. The TYR, P , and MATP genes discussed earlier are well-known causes of albinism whose primary effects are limited to pigment cells ; among these, the P gene is highly polymorphic but the phenotypic consequences of P gene polymorphisms are not yet known.

14551921_p8

14551921

Genetics of Skin Color

biomedical

Study

en

Independent of phenotype, a gene responsible for selection of different skin colors should exhibit a population signature with a large number of alleles and rates of sequence substitution that are greater for nonsynonymous (which change an amino acid in the protein) than synonymous (which do not change any amino acid) alterations. Data have been collected only for MC1R , in which the most notable finding is a dearth of allelic diversity in African samples, which is remarkable given that polymorphism for most genes is greater in Africa than in other geographic regions . Thus, while MC1R sequence variation does not contribute significantly to variation in human skin color around the world, a functional MC1R is probably important for dark skin.

14551921_p9

14551921

Genetics of Skin Color

biomedical

Study

en

Credit for describing the relationship between latitude and skin color in modern humans is usually ascribed to an Italian geographer, Renato Basutti, whose widely reproduced “skin color maps” illustrate the correlation of darker skin with equatorial proximity . More recent studies by physical anthropologists have substantiated and extended these observations; a recent review and analysis of data from more than 100 populations found that skin reflectance is lowest at the equator, then gradually increases, about 8% per 10° of latitude in the Northern Hemisphere and about 4% per 10° of latitude in the Southern Hemisphere. This pattern is inversely correlated with levels of UV irradiation, which are greater in the Southern than in the Northern Hemisphere. An important caveat is that we do not know how patterns of UV irradiation have changed over time; more importantly, we do not know when skin color is likely to have evolved, with multiple migrations out of Africa and extensive genetic interchange over the last 500,000 years .

14551921_p10

14551921

Selection for Skin Color?

biomedical

Review

en

Regardless, most anthropologists accept the notion that differences in UV irradiation have driven selection for dark human skin at the equator and for light human skin at greater latitudes. What remains controversial are the exact mechanisms of selection. The most popular theory posits that protection offered by dark skin from UV irradiation becomes a liability in more polar latitudes due to vitamin D deficiency . UVB (short-wavelength UV) converts 7-dehydrocholesterol into an essential precursor of cholecaliferol (vitamin D 3 ); when not otherwise provided by dietary supplements, deficiency for vitamin D causes rickets, a characteristic pattern of growth abnormalities and bony deformities. An oft-cited anecdote in support of the vitamin D hypothesis is that Arctic populations whose skin is relatively dark given their latitude, such as the Inuit and the Lapp, have had a diet that is historically rich in vitamin D. Sensitivity of modern humans to vitamin D deficiency is evident from the widespread occurrence of rickets in 19th-century industrial Europe, but whether dark-skinned humans migrating to polar latitudes tens or hundreds of thousands of years ago experienced similar problems is open to question. In any case, a risk for vitamin D deficiency can only explain selection for light skin. Among several mechanisms suggested to provide a selective advantage for dark skin in conditions of high UV irradiation , the most tenable are protection from sunburn and skin cancer due to the physical barrier imposed by epidermal melanin.

14551921_p11

14551921

Selection for Skin Color?

biomedical

Review

en

Recent developments in several areas provide a tremendous opportunity to better understand the diversity of human pigmentation. Improved spectrophotometric tools, advances in epidemiology and statistics, a wealth of genome sequences, and efficient techniques for assaying sequence variation offer the chance to replace misunderstanding and myths about skin color with education and scientific insight. The same approaches used to investigate traits such as hypertension and obesity—genetic linkage and association studies—can be applied in a more powerful way to study human pigmentation, since the sources of environmental variation can be controlled and we have a deeper knowledge of the underlying biochemistry and cell biology.

14551921_p12

14551921

Solving the Mystery

biomedical

Review

en

This approach is especially appealing given the dismal success rate in molecular identification of complex genetic diseases. In fact, understanding more about the genetic architecture of skin color may prove helpful in designing studies to investigate other quantitative traits. Current debates in the human genetics community involve strategies for selecting populations and candidate genes to study, the characteristics of sequence polymorphisms worth pursuing as potential disease mutations, and the extent to which common diseases are caused by common (and presumably ancient) alleles. While specific answers will be different for every phenotype, there may be common themes, and some answers are better than none.

14551921_p13

14551921

Solving the Mystery

biomedical

Review

en

Harrison and Owen concluded their 1964 study of human skin color by stating, “The deficiencies in the data in this study are keenly appreciated by the writers, but since there appear at present to be no opportunities for improving the data, it seems justifiable to take the analysis as far as possible.” Nearly 40 years later, opportunities abound, and the mystery of human skin color is ready to be solved.

14551921_p14

14551921

Solving the Mystery

other

Other

en

The heart of PKS function is the synthesis of long chains of carbon atoms by joining (condensing) together small organic acids, such as acetic and malonic acid, by a so-called ketosynthase function. This uses the building units in the form of activated derivatives, called coenzyme A (CoA) esters, so we speak of acetyl-CoA and malonyl-CoA, for example. The special form of condensation that joins them is driven by loss of carbon dioxide. Thus, when acetyl-CoA, with two carbon atoms, joins with malonyl-CoA, with three carbons, one of the latter is lost and a chain of four carbon atoms results . Further rounds of condensation extend the chain by two carbons at each step. If the chain-extender unit, instead of being malonyl-CoA, is methylmalonyl-CoA, which has four carbon atoms, the linear carbon chain is still extended by two carbons, and the ‘extra’ carbon forms a methyl side branch. More complex extender units produce more complex side branches.

14966534_p0

14966534

Molecular Diversity

biomedical

Study

en

Choices of the number and type of the building units are variables in determining polyketide structure. Another concerns the keto groups (C=O) that appear at every alternate carbon atom in the growing chain as a result of the condensation process (accounting for the name polyketide). They may remain intact. Alternatively, some may be modified or removed by a series of three steps , any of which may be omitted. This results in keto groups remaining at some points in the chain; hydroxyl groups (–OH), formed by reduction of a keto group, at others; double bonds between some adjacent carbon atoms, resulting from removal of the hydroxyl by loss of water (dehydration); or full saturation with hydrogen atoms elsewhere, arising by ‘enoyl’ reduction of the double bond . A further variable concerns the stereochemistry of the hydroxyl groups and methyl or other carbon branches, each of which can exist in two possible configurations. Finally, the nascent carbon chain adopts different folding patterns after it leaves the PKS, and ‘tailoring’ enzymes can then add sugars or other chemical groups to it at many alternative positions, enabled by the pattern of chemical reactivity built into the polyketide by the PKS. The challenge has been to understand the programming of the PKS that accounts for this gamut of structural variation.

14966534_p1

14966534

Molecular Diversity

biomedical

Study

en

During the 1990s, the ability to manipulate actinomycete genes, developed over previous decades, mainly using the model species Streptomyces coelicolor , was combined with chemical and biochemical experiments to begin to crack this ‘polyketide code’. The first studies were on organisms making antibiotics of the ‘aromatic’ family, which includes tetracycline and doxorubicin, as well as the model compounds actinorhodin (made by S. coelicolor itself) and tetracenomycin. The main variable in their structure is carbon chain length, with few choices of different building units or keto group modification, so the programming would (in principle) be simple. The DNA sequences responsible for such PKSs revealed sets of genes encoding proteins, including ketosynthases, ketoreductases, and acyl carrier proteins (ACPs) , that would come together to form a multicomponent PKS resembling a typical bacterial fatty acid synthase. In contrast, the DNA sequence of the gene set for the complex polyketide erythromycin, made by a relative of Streptomyces called Saccharopolyspora erythraea , which has more involved programming, revealed multifunctional proteins with the various enzymic functions carried out by active sites on the same polypeptide chain, as in a mammalian fatty acid synthase.

14966534_p2

14966534

Molecular Diversity

biomedical

Study

en

The big surprise, though, was the finding of six sets, or modules, of such active sites, corresponding to the six rounds of condensation needed to build the carbon chain . The modules each contain an acyl transferase (to load the extender unit onto the enzyme), as well as a ketosynthase and an ACP domain, together with exactly those reductive activities needed to generate the required pattern of modification of the chain at each step of elongation. Thus was born an ‘assembly line’ model in which the program for the PKS is hardwired into the DNA and expressed in a linear array of active sites (domains) along the giant protein. This consists of the six chain-building modules, preceded by a short module for loading the starter unit and ending in a domain for releasing the completed carbon chain from the PKS. The carbon chain of the polyketide would be assembled and modified progressively as the molecule moved along the protein, interacting with each domain in turn, which would select extender units, make carbon–carbon bonds, and modify keto groups as appropriate, depending on the presence or absence of domains for the three steps in the reductive cycle.

14966534_p3

14966534

Molecular Diversity

biomedical

Study

en

The model arose from the gene sequence, but was rapidly tested by mutating individual domains or adding or deleting whole modules and by observing predicted changes in the polyketide product. Soon, dozens of engineered compounds had been made, and the field mushroomed with the isolation of more and more clusters of genes for complex polyketides that both proved the generality of the model (with minor variations) and filled the need for spare parts for the engineering of countless new polyketides . Several biotech companies were founded to exploit the potential for drug discovery.

14966534_p4

14966534

Molecular Diversity

biomedical

Review

en

Meanwhile, the programming of the aromatic PKSs was harder to understand. They had been found to contain only a single ketosynthase, which has to operate a specific number of times to build a carbon chain of the correct length, so how is this determined? How does a single reductive enzyme know which keto groups to modify? And how is the starter unit for building the carbon chain selected (the extender units are normally all malonyl-CoA, so no choice is involved)? Considerable progress had been made in constructing novel compounds by mixing and matching PKS subunits, but this was largely based on empirical knowledge about which components to put together . A specific subunit of the PKS, named the chain length factor (CLF), was deduced to have a major influence on carbon chain length , but this conclusion was not universally accepted in the absence of experimental evidence on its mode of action. Two recent publications by the Khosla laboratory at Stanford University describe significant advances in understanding aromatic PKS programming and promise to turn the spotlight back onto engineered members of this class of compounds as potential drug candidates by allowing rational manipulation of the two key variables: carbon chain length and choice of starter unit.

14966534_p5

14966534

Aromatic PKS Programming

biomedical

Review

en

In the first paper , the authors explore the hypothesis that the CLF exerts control over carbon chain length by associating closely with the ketosynthase, a protein with which it shares considerable amino acid sequence similarity, giving rise to a channel of a certain size at the interface between the two proteins. By systematically changing amino acids at four key positions in the CLF, the size of the channel was altered. Thus, large amino acid residues in the CLF of a PKS that makes a 16-carbon chain were replaced by less bulky residues found in one that builds a 20-carbon chain, and the chain length of the product increased as expected. The authors propose that the length of the channel is the main factor in controlling the number of chain-extension steps that can take place to fill it. While protein–protein interactions with other PKS subunits may modulate this chain length control, the work represents a major step in understanding and manipulating the chain length of aromatic polyketides.

14966534_p6

14966534

Aromatic PKS Programming

biomedical

Study

en

What about the choice of starter unit? Most aromatic polyketides start with acetyl-CoA. An important earlier publication by Leadlay and colleagues had shown that this is not loaded directly onto the PKS, as had been assumed, but is derived by loss of carbon dioxide from a molecule of malonyl-CoA previously loaded onto the enzyme. This decarboxylation is catalysed by the CLF as an activity independent of its role in influencing carbon chain length. There are, however, certain aromatic polyketides, including the anticancer drug doxorubicin, an antiparasitic agent called frenolicin, and the estrogen receptor agonist R1128, that have different starters.

14966534_p7

14966534

Aromatic PKS Programming

biomedical

Study

en

What Tang et al. have deduced, as described in this issue of PloS Biology , is that the PKSs for these compounds consist of two modules of active sites. The components of each module are not activities carried on the same protein, as in the PKSs for the complex polyketides, but are all separate proteins. They form functional modules nevertheless. The newly recognized modules in the producers of compounds that start with nonacetate units have a dedicated ACP and a special ketosynthase that carries out a first condensation, joining the unusual starter unit to the first malonyl-CoA extender unit. The starter module then hands the resulting ‘diketide’ on to the second module (first reducing it, if appropriate, using reductive enzymes ‘borrowed’ from fatty acid biosynthesis) for typical extension by successive condensation with malonyl-CoA units to complete the chain. If the starter module is not present, the second module defaults to its typical habit of decarboxylating malonyl-CoA to acetyl-CoA and starts the chain with that.

14966534_p8

14966534

Aromatic PKS Programming

biomedical

Study

en

The excitement of the work for biotechnology is that it offers the prospect of engineering promising drug candidates by making novel combinations of starter and extender modules and perhaps of feeding the starter modules with a whole range of unnatural substrates . It is encouraging that already, in the proof-of-principle studies reported by Tang et al. , some products with improved in vitro antitumor activity were obtained.

14966534_p9

14966534

Aromatic PKS Programming

biomedical

Study

en

Antibiotics have traditionally been plucked from nature's battleground. For billions of years, tiny organisms have engaged in an arms race, hurling toxic molecules at each other in the struggle to prosper. Nearly all of today's antibiotics are versions of weapons long wielded by microbes and fungi. Chemical synthesis of entirely human-created antibiotics has so far yielded only fluoroquinolones, a group of broad-spectrum antibiotics that includes Cipro, which became famously scarce during the 2001 anthrax scare, and linezolid (trade-named Zyvox), which is effective against some resistant strains of Staphylococcus , Streptococcus , and Enterococcus .

14966545_p0

14966545

Tried and True—and Tired?

biomedical

Review

en

The usual way to find a new antibiotic has been laborious screening of immense libraries of compounds, natural and otherwise. Some argue that screening chemical libraries is approaching a deadend. There may be diminishing returns from screening, but it's not quite dead yet: in October, researchers at the University of Wisconsin at Madison reported a new class of bacterial RNA polymerase inhibitors with antibiotic potential. They were found by screening for molecules that prevent Escherichia coli from transcribing RNA.

14966545_p1

14966545

Tried and True—and Tired?

biomedical

Other

en

Christopher T. Walsh of Harvard Medical School says screening's problem may be simply that libraries aren't good enough. Marine organisms have not been studied well, he points out, and 90% of organisms in the biosphere can't be cultured in standard ways. He says, “We're missing 90% of them every time we go and look in nature.”

14966545_p2

14966545

Tried and True—and Tired?

biomedical

Other

en

Walsh is doing his bit to create new libraries. He and his colleagues have recently employed combinatorial biosynthesis to learn how to use part of the machinery for assembling cyclic peptide antibiotics to control their architecture. The result was a small library of natural product analogs, some of which have improved antibiotic activity against common bacterial pathogens. “There are dozens of such enzymatic domains that in principle one could clone, express, and test with other substrates. I view that as the kind of thing we should do,” he says. For example, Walsh suggests, it is a reasonable approach to second-generation improvement of daptomycin, the antibiotic most recently approved for sale in the United States.

14966545_p3

14966545

Tried and True—and Tired?

biomedical

Other

en

Walsh collaborates with Chaitan Khosla of Stanford University on finding ways to make existing antibiotics better. They are studying biosynthesis of rifamycin, an antibiotic that is increasingly less effective against its prime target, tuberculosis (TB) . “In the course of learning about that pathway, we've learned a few interesting things lately about how that molecule is initiated, and we're trying to apply it in other contexts, especially in the context of erythromycin biosynthesis,” Khosla says. The idea would be to make a molecule that might be more effective against bacteria that are becoming resistant to rifamycin—and are already naturally resistant to molecules like erythromycin.

14966545_p4

14966545

Improving on Nature

biomedical

Other

en

“Basically, what we do is to try and figure out new ways to hijack the biosynthesis of antibiotics in nature so as to modify their structures with the goal of improving them,” Khosla explains. He works with an important class of natural antibiotics called polyketides that have generated dozens of drugs, including erythromycin.

14966545_p5

14966545

Improving on Nature

biomedical

Other

en

Polyketides are secondary metabolites (which give their producers a competitive advantage in their environment) produced mostly by bacteria and fungi and made by a complex and structurally diverse family of enzymes called polyketide synthases (see the primer by David Hopwood in this issue of PLoS Biology ). Among them are the anthracyclines, a group of anticancer drugs and antibacterials that includes tetracycline. In this issue of PloS Biology , Khosla and his colleagues report that they can make selective positional modifications in existing anthracycline antibiotics by starting in a different way with a different starting molecule. The molecule came from a natural anthracycline antibiotic, an estrogen receptor antagonist called R1128. R1128 is made via two modules of enzymes that work sequentially; the first module starts the process, and the second completes it. This division of labor permitted the researchers to tack the first R1128 module onto two other enzyme systems, thus engineering completely new anthracyclines. Some were more active in two types of assays than the natural parent molecule. “One setting was an assay on an estrogen-sensitive cancer cell line. Another setting was an assay to probe activity of an enzyme that's of particular interest in Type 2 diabetes, called glucose-6-phosphate translocase.” The work also revealed fundamental mechanistic features of the polyketide synthases, Khosla says.

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14966545

Improving on Nature

biomedical

Study

en

The researchers didn't study the new anthracyclines' effects on bacteria, but Khosla notes that the general principle should apply to other classes of compounds, although the details of how it's implemented will vary from system to system. He says, “The upshot of this paper is that it is now possible to modify a particular methyl group in just about any anthracycline antibiotic.”

14966545_p7

14966545

Improving on Nature

biomedical

Other

en

Instead of searching for new antibiotics by modifying existing ones, some researchers are trying something completely different—first finding the most vulnerable targets in a bacterium and then designing something that hits one or more of them hard. “You have to understand a helluva lot more about how these little cells work. In fact, we think we understand a lot, but I think we can understand almost everything now that we have all the genomes,” says Lucy Shapiro of Stanford University School of Medicine. While having full genome sequences—more than 100 microbe sequences have been completed—is essential, Shapiro believes that knocking outs genes galore to find out which ones are necessary and going after them all is not a sensible strategy. She observes, “People have been doing that for a while with absolutely no success. That's really going after the problem with a Howitzer instead of with an intelligent approach.”

14966545_p8

14966545

Finding New Targets

biomedical

Other

en

So instead of screening libraries of existing compounds, Shapiro prefers using structural information about drug targets or their natural ligands to create new drugs, an approach known as rational drug design. And instead of looking at all essential genes in a bacterium and choosing one to target, she and her colleagues look at genetic circuitry that controls the cell cycle, the pathway that coordinates cell growth and differentiation. They have identified key control points, or nodes, in the circuitry for their favorite study subject, Caulobacter crescentus . Thus, they have found critical genes encoding proteins that control several critical functions in the cell. Their first candidate was an essential enzyme, a methyltransferase called CcrM, that prevents a particular piece of DNA from being expressed in a cell by tagging it with a methyl group.

14966545_p9

14966545

Finding New Targets

biomedical

Study

en

Antibiotic discovery is all chemistry, Shapiro says, which is why she joined with biochemist Stephen J. Benkovic of Pennsylvania State University. They didn't know the structure of CcrM, Benkovic explains, but the literature about other methyltransferases suggested that the adenine molecule, which is the substrate for CcrM within DNA, binds to a specific region of the enzyme.

14966545_p10

14966545

Finding New Targets

biomedical

Other

en

The researchers designed adenine-like molecules that would bind to CcrM and then developed inhibitors. Benkovic says, “We already knew what kind of structure we wanted, and we simply fine-tuned it.” They worked their way through 1,000 inhibitor candidates, ending up with a small subset—no more than about 20—that not only inhibited CcrM, but also killed Caulobacter very quickly.

14966545_p11

14966545

Finding New Targets

biomedical

Other

en

And not only inoffensive Caulobacter . The compounds knock out other gram-negative bacteria, such as the pathogens Brucella abortus and Francisella tularensis . Some even killed off anthrax, a big surprise because it is gram-positive and so has much thicker cell walls than gram-negative bacteria. The researchers undertook an exhaustive series of experiments to identify which gram-positive bacteria would be affected by which compounds. The list of sensitive pathogens now includes multidrug-resistant Streptococcus , Staphylococcus , and Mycobacterium tuberculosis .

14966545_p12

14966545

Finding New Targets

biomedical

Other

en

More recently, Shapiro reports, they have demonstrated efficacy against rats infected with anthrax or multidrug-resistant Staph , although the compounds save only about 60% of the rats at present. She notes, “So we have a long way to go. But this has proven that if you go after something using some rational approach instead of hit-and-miss, you'll probably have more success than by the other method.”

14966545_p13

14966545

Finding New Targets

biomedical

Other

en

Benkovic points out that theirs is an entirely new class of compounds, small molecular weight compounds that can be made in a few steps. He says, “They don't look like the normal antibiotic, so that's why I think they're fairly unique.” The basic research was done under a grant from the Defense Advanced Research Projects Agency (DARPA), the United States Department of Defense's (DOD) central research and development organization, and once the researchers realized they wanted to develop drugs against three agents that have been considered bioterrorism threats — Brucella , tularensis, and anthrax — they established a separate operation, Anacor Pharmaceuticals, which is developing them with DOD funding and without Shapiro. In her Stanford lab, she continues her fundamental research to define the complete genetic circuitry of Caulobacter , hoping to identify additional nodes in the circuit. She says, “I am not doing it to develop antibiotics; that's what comes out of the work. My goal is to understand how the cell works. I think a lot of studies in pathogenesis should not be just to understand pathogenic organisms, but to understand the complete network of regulatory mechanisms that controls the bacterial cell.”

14966545_p14

14966545

Finding New Targets

biomedical

Other

en

The most radical approach to new antibiotics may be the resurrection of an old idea: bacteriophage therapy . Late in the 19th century, a researcher noticed that water from some of India's sacred rivers combated cholera. Some years later, the active agents were identified as viruses that infected bacteria. Such viruses are called bacteriophage, or phage for short. There were reports of phage success against dysentery, typhoid, and plague, and bacteriophage therapy had a brief heyday, especially in the 1920s. Results on other diseases were mixed, and with the appearance of antibiotics, phage therapy became unfashionable in the United States, although it has continued in Russia and Eastern Europe.

14966545_p15

14966545

Phage Therapy

biomedical

Other

en

Phage were the model organisms of choice for genetics research in the 1930s and 1940s, but became less fashionable as research tools when investigators moved on to eukaryotes. A few held on, like Ry Young of Texas A&M University, who has made phage-induced cell lysis his life's work. “The cell is basically genetically dead as soon as the phage goes in there, but it will keep living as sort of an infected zombie for as long as the phage wants it to, with virus particles accumulating inside the cell,” he explains. “Only when the phage is ready and has decided that it's the right time will it pull the trigger. And the cell blows up.” The freed phage then spew forth to infect new cells.

14966545_p16

14966545

Phage Therapy

biomedical

Other

en

Antibiotic resistance has led to new interest in phage therapy by several small biotech companies. Young continues basic research at Texas A&M, but has also joined one of them, GangaGen, providing bacteriophage expertise to its labs.

14966545_p17

14966545

Phage Therapy

biomedical

Other

en

Phage do kill pathogenic bacteria effectively, and they do it without penetrating human cells, which they can't even recognize. So what is keeping phage therapy out of the clinic? Problems that some doubt can be overcome.

14966545_p18

14966545

Phage Therapy

biomedical

Other

en

Because bacteria develop resistance to phage rapidly, phage therapy companies will need to direct cocktails against a single pathogen, according to Vincent Fischetti at The Rockefeller University. Phage are also antigenic, and the antibodies they stimulate will neutralize their effects during subsequent treatment, he says. But the chief problem appears to be regulatory—regulatory in the political, rather than the genetic, sense. When bacteriophage package their DNA, they occasionally include varying amounts of their hosts' DNA, too. This miscellany, Fischetti points out, is likely to make the Food and Drug Administration unhappy. “Phage normally are very fragile, their tails break, so lot-to-lot homogeneity could be a problem too,” he adds. “So even though it will work, I think they'll have an uphill battle.” Phage may well enter agricultural or veterinary use, he predicts, but are probably not going to be available to patients in the United States any time soon.

14966545_p19

14966545

Phage Therapy

biomedical

Other

en

Fischetti chose a different approach to phage therapy. It does not rely on phage themselves, but on enzymes that phage produce to smash their way out of their host bacteria so they can infect new hosts. He and his colleagues employ these enzymes externally to kill bacteria. He reports, “We now have enzymes that will kill Strep pyogenes , pneumococci, Strep pneumoniae , Bacillus anthracis , Enterococcus faecalis , and group B Strep . The beauty of these enzymes is that they are targeted killing. You only kill the organism you intend to kill, without destroying or affecting the surrounding organisms that are necessary for health.”

14966545_p20

14966545

Phage Therapy

biomedical

Other

en