Virulence differences in Toxoplasma mediated by amplification of a family of polymorphic pseudokinases.

The population structure of Toxoplasma gondii includes three highly prevalent clonal lineages referred to as types I, II, and III, which differ greatly in virulence in the mouse model. Previous studies have implicated a family of serine/threonine protein kinases found in rhoptries (ROPs) as important in mediating virulence differences between strain types. Here, we explored the genetic basis of differences in virulence between the highly virulent type I lineage and moderately virulent type II based on successful genetic cross between these lineages. Genome-wide association revealed that a single quantitative trait locus controls the dramatic difference in lethality between these strain types. Neither ROP16 nor ROP18, previously implicated in virulence of T. gondii, was found to contribute to differences between types I and II. Instead, the major virulence locus contained a tandem cluster of polymorphic alleles of ROP5, which showed similar protein expression between strains. ROP5 contains a conserved serine/threonine protein kinase domain that includes only part of the catalytic triad, and hence, all members are considered to be pseudokinases. Genetic disruption of the entire ROP5 locus in the type I lineage led to complete attenuation of acute virulence, and complementation with ROP5 restored lethality to WT levels. These findings reveal that a locus of polymorphic pseudokinases plays an important role in pathogenesis of toxoplasmosis in the mouse model.

Linkage maps from multiple genetic crosses and loci linked to growth-related virulent phenotype in Plasmodium yoelii.

Plasmodium yoelii is an excellent model for studying malaria pathogenesis that is often intractable to investigate using human parasites; however, genetic studies of the parasite have been hindered by lack of genome-wide linkage resources. Here, we performed 14 genetic crosses between three pairs of P. yoelii clones/subspecies, isolated 75 independent recombinant progeny from the crosses, and constructed a high-resolution linkage map for this parasite. Microsatellite genotypes from the progeny formed 14 linkage groups belonging to the 14 parasite chromosomes, allowing assignment of sequence contigs to chromosomes. Growth-related virulent phenotypes from 25 progeny of one of the crosses were significantly associated with a major locus on chromosome 13 and with two secondary loci on chromosomes 7 and 10. The chromosome 10 and 13 loci are both linked to day 5 parasitemia, and their effects on parasite growth rate are independent but additive. The locus on chromosome 7 is associated with day 10 parasitemia. The chromosome 13 locus spans ~220 kb of DNA containing 51 predicted genes, including the P. yoelii erythrocyte binding ligand, in which a C741Y substitution in the R6 domain is implicated in the change of growth rate. Similarly, the chromosome 10 locus spans ~234 kb with 71 candidate genes, containing a member of the 235-kDa rhoptry proteins (Py235) that can bind to the erythrocyte surface membrane. Atypical virulent phenotypes among the progeny were also observed. This study provides critical tools and information for genetic investigations of virulence and biology of P. yoelii.

The construction and use of log-odds substitution scores for multiple sequence alignment.

Most pairwise and multiple sequence alignment programs seek alignments with optimal scores. Central to defining such scores is selecting a set of substitution scores for aligned amino acids or nucleotides. For local pairwise alignment, substitution scores are implicitly of log-odds form. We now extend the log-odds formalism to multiple alignments, using Bayesian methods to construct "BILD" ("Bayesian Integral Log-odds") substitution scores from prior distributions describing columns of related letters. This approach has been used previously only to define scores for aligning individual sequences to sequence profiles, but it has much broader applicability. We describe how to calculate BILD scores efficiently, and illustrate their uses in Gibbs sampling optimization procedures, gapped alignment, and the construction of hidden Markov model profiles. BILD scores enable automated selection of optimal motif and domain model widths, and can inform the decision of whether to include a sequence in a multiple alignment, and the selection of insertion and deletion locations. Other applications include the classification of related sequences into subfamilies, and the definition of profile-profile alignment scores. Although a fully realized multiple alignment program must rely upon more than substitution scores, many existing multiple alignment programs can be modified to employ BILD scores. We illustrate how simple BILD score based strategies can enhance the recognition of DNA binding domains, including the Api-AP2 domain in Toxoplasma gondii and Plasmodium falciparum.

Composite genome map and recombination parameters derived from three archetypal lineages of Toxoplasma gondii.

Toxoplasma gondii is a highly successful protozoan parasite in the phylum Apicomplexa, which contains numerous animal and human pathogens. T.gondii is amenable to cellular, biochemical, molecular and genetic studies, making it a model for the biology of this important group of parasites. To facilitate forward genetic analysis, we have developed a high-resolution genetic linkage map for T.gondii. The genetic map was used to assemble the scaffolds from a 10X shotgun whole genome sequence, thus defining 14 chromosomes with markers spaced at approximately 300 kb intervals across the genome. Fourteen chromosomes were identified comprising a total genetic size of approximately 592 cM and an average map unit of approximately 104 kb/cM. Analysis of the genetic parameters in T.gondii revealed a high frequency of closely adjacent, apparent double crossover events that may represent gene conversions. In addition, we detected large regions of genetic homogeneity among the archetypal clonal lineages, reflecting the relatively few genetic outbreeding events that have occurred since their recent origin. Despite these unusual features, linkage analysis proved to be effective in mapping the loci determining several drug resistances. The resulting genome map provides a framework for analysis of complex traits such as virulence and transmission, and for comparative population genetic studies.

Protein database searches using compositionally adjusted substitution matrices.

Almost all protein database search methods use amino acid substitution matrices for scoring, optimizing, and assessing the statistical significance of sequence alignments. Much care and effort has therefore gone into constructing substitution matrices, and the quality of search results can depend strongly upon the choice of the proper matrix. A long-standing problem has been the comparison of sequences with biased amino acid compositions, for which standard substitution matrices are not optimal. To address this problem, we have recently developed a general procedure for transforming a standard matrix into one appropriate for the comparison of two sequences with arbitrary, and possibly differing compositions. Such adjusted matrices yield, on average, improved alignments and alignment scores when applied to the comparison of proteins with markedly biased compositions. Here we review the application of compositionally adjusted matrices and consider whether they may also be applied fruitfully to general purpose protein sequence database searches, in which related sequence pairs do not necessarily have strong compositional biases. Although it is not advisable to apply compositional adjustment indiscriminately, we describe several simple criteria under which invoking such adjustment is on average beneficial. In a typical database search, at least one of these criteria is satisfied by over half the related sequence pairs. Compositional substitution matrix adjustment is now available in NCBI's protein-protein version of blast.

Expression of a recombinant IRP-like Plasmodium falciparum protein that specifically binds putative plasmodial IREs.

Plasmodium falciparum iron regulatory-like protein (PfIRPa, accession AJ012289) has homology to a family of iron-responsive element (IRE)-binding proteins (IRPs) found in different species. We have previously demonstrated that erythrocyte P. falciparum PfIRPa binds a mammalian consensus IRE and that the binding activity is regulated by iron status. In the work we now report, we have cloned a C-terminus histidine-tagged PfIRPa and overexpressed it in a bacterial expression system in soluble form capable of binding IREs. To overexpress PfIRPa, we used the T7 promoter-driven vector, pET28a(+), in conjunction with the Rosetta(DE3)pLysS strain of E. coli, which carries extra copies of tRNA genes usually found in organisms such as P. falciparum whose genome is (A+T)-rich. The histidine-tagged recombinant protein (rPfIRPa) in soluble form was partially purified using His-bind resin. We searched the plasmodial database, plasmoDB, to identify sequences capable of forming IRE loops using a specially developed algorithm, and found three plasmodial sequences matching the search criteria. In gel retardation assays, rPfIRPa bound three 32P-labeled putative plasmodial IREs with affinity exceeding the affinity for the mammalian consensus IRE. The binding was concentration-dependent and was not inhibited by heparin, an inhibitor of non-specific binding. Immunodepletion of rPfIRPa resulted in substantial inhibition of the signal intensity in the gel retardation assays and in Western blot-determinations of rPfIRPa protein levels. Endogenous PfIRPa retained all three putative 32P-IREs at the same position on the gel as the recombinant PfIRPa.

Discovering simple regions in biological sequences associated with scoring schemes.

Let A denote an alphabet consisting of n types of letters. Given a sequence S of length L with v(i) letters of type i on A, to describe the compositional properties and combinatorial structure of S, we propose a new complexity function of S, called the reciprocal complexity of S, as C(S) = (i=1) product operator (n) (L/nv(i))(vi) Based on this complexity measure, an efficient algorithm is developed for classifying and analyzing simple segments of protein and nucleotide sequence databases associated with scoring schemes. The running time of the algorithm is nearly proportional to the sequence length. The program DSR corresponding to the algorithm was written in C++, associated with two parameters (window length and cutoff value) and a scoring matrix. Some examples regarding protein sequences illustrate how the method can be used to find regions. The first application of DSR is the masking of simple sequences for searching databases. Queries masked by DSR returned a manageable set of hits below the E-value cutoff score, which contained all true positive homologues. The second application is to study simple regions detected by the DSR program corresponding to known structural features of proteins. An extensive computational analysis has been made of protein sequences with known, physicochemically defined nonglobular segments. For the SWISS-PROT amino acid sequence database (Release 40.2 of 02-Nov-2001), we determine that the best parameters and the best BLOSUM matrix are, respectively, for automatic segmentation of amino acid sequences into nonglobular and globular regions by the DSR program: Window length k = 35, cutoff value b = 0.46, and the BLOSUM 62.5 matrix. The average "agreement accuracy (sensitivity)" of DSR segmentation for the SWISS-PROT database is 97.3%.

Coordinated progression through two subtranscriptomes underlies the tachyzoite cycle of Toxoplasma gondii.

BACKGROUND: Apicomplexan parasites replicate by varied and unusual processes where the typically eukaryotic expansion of cellular components and chromosome cycle are coordinated with the biosynthesis of parasite-specific structures essential for transmission. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe the global cell cycle transcriptome of the tachyzoite stage of Toxoplasma gondii. In dividing tachyzoites, more than a third of the mRNAs exhibit significant cyclical profiles whose timing correlates with biosynthetic events that unfold during daughter parasite formation. These 2,833 mRNAs have a bimodal organization with peak expression occurring in one of two transcriptional waves that are bounded by the transition into S phase and cell cycle exit following cytokinesis. The G1-subtranscriptome is enriched for genes required for basal biosynthetic and metabolic functions, similar to most eukaryotes, while the S/M-subtranscriptome is characterized by the uniquely apicomplexan requirements of parasite maturation, development of specialized organelles, and egress of infectious daughter cells. Two dozen AP2 transcription factors form a series through the tachyzoite cycle with successive sharp peaks of protein expression in the same timeframes as their mRNA patterns, indicating that the mechanisms responsible for the timing of protein delivery might be mediated by AP2 domains with different promoter recognition specificities. CONCLUSION/SIGNIFICANCE: Underlying each of the major events in apicomplexan cell cycles, and many more subordinate actions, are dynamic changes in parasite gene expression. The mechanisms responsible for cyclical gene expression timing are likely crucial to the efficiency of parasite replication and may provide new avenues for interfering with parasite growth.

Genetic mapping in the human malaria parasite Plasmodium falciparum.

The Plasmodium falciparum genome sequence has boosted hopes for a new era of malaria research and for the application of comprehensive molecular knowledge to disease control, but formidable obstacles remain: approximately 60% of the predicted P. falciparum proteins have no known functions or homologues, and most life cycle stages of this haploid eukaryotic parasite are relatively intractable to cultivation and biochemical manipulation. Genetic mapping based on high-resolution maps saturated with single-nucleotide polymorphisms or microsatellites is now providing effective strategies for discovering candidate genes determining important parasite phenotypes. Here we review classical linkage studies using laboratory crosses and population associations that are now amenable to genome-wide approaches and are revealing multiple candidate genes involved in complex drug responses. Moreover, mapping by linkage disequilibrium is practicable in cases where chromosomal segments flanking drug-selected genes have been preserved in populations during relatively recent P. falciparum evolution. We discuss the advantages and limitations of these various genetic mapping strategies, results from which offer complementary insights to those emerging from gene knockout experiments and/or high-throughput genomic technologies.

The compositional adjustment of amino acid substitution matrices.

Amino acid substitution matrices are central to protein-comparison methods. In most commonly used matrices, the substitution scores take a log-odds form, involving the ratio of "target" to "background" frequencies derived from large, carefully curated sets of protein alignments. However, such matrices often are used to compare protein sequences with amino acid compositions that differ markedly from the background frequencies used for the construction of the matrices. Of course, the target frequencies should be adjusted in such cases, but the lack of an appropriate way to do this has been a long-standing problem. This article shows that if one demands consistency between target and background frequencies, then a log-odds substitution matrix implies a unique set of target and background frequencies as well as a unique scale. Standard substitution matrices therefore are truly appropriate only for the comparison of proteins with standard amino acid composition. Accordingly, we present and evaluate a rationale for transforming the target frequencies implicit in a standard matrix to frequencies appropriate for a nonstandard context. This rationale yields asymmetric matrices for the comparison of proteins with divergent compositions. Earlier approaches are unable to deal with this case in a fully consistent manner. Composition-specific substitution matrix adjustment is shown to be of utility for comparing compositionally biased proteins, including those of organisms with nucleotide-biased, and therefore codon-biased, genomes or isochores.

Genetic diversity and chloroquine selective sweeps in Plasmodium falciparum.

Widespread use of antimalarial agents can profoundly influence the evolution of the human malaria parasite Plasmodium falciparum. Recent selective sweeps for drug-resistant genotypes may have restricted the genetic diversity of this parasite, resembling effects attributed in current debates to a historic population bottleneck. Chloroquine-resistant (CQR) parasites were initially reported about 45 years ago from two foci in southeast Asia and South America, but the number of CQR founder mutations and the impact of chlorquine on parasite genomes worldwide have been difficult to evaluate. Using 342 highly polymorphic microsatellite markers from a genetic map, here we show that the level of genetic diversity varies substantially among different regions of the parasite genome, revealing extensive linkage disequilibrium surrounding the key CQR gene pfcrt and at least four CQR founder events. This disequilibrium and its decay rate in the pfcrt-flanking region are consistent with strong directional selective sweeps occurring over only approximately 20-80 sexual generations, especially a single resistant pfcrt haplotype spreading to very high frequencies throughout most of Asia and Africa. The presence of linkage disequilibrium provides a basis for mapping genes under drug selection in P. falciparum.

Multiple transporters associated with malaria parasite responses to chloroquine and quinine.

Mutations and/or overexpression of various transporters are known to confer drug resistance in a variety of organisms. In the malaria parasite Plasmodium falciparum, a homologue of P-glycoprotein, PfMDR1, has been implicated in responses to chloroquine (CQ), quinine (QN) and other drugs, and a putative transporter, PfCRT, was recently demonstrated to be the key molecule in CQ resistance. However, other unknown molecules are probably involved, as different parasite clones carrying the same pfcrt and pfmdr1 alleles show a wide range of quantitative responses to CQ and QN. Such molecules may contribute to increasing incidences of QN treatment failure, the molecular basis of which is not understood. To identify additional genes involved in parasite CQ and QN responses, we assayed the in vitro susceptibilities of 97 culture-adapted cloned isolates to CQ and QN and searched for single nucleotide polymorphisms (SNPs) in DNA encoding 49 putative transporters (total 113 kb) and in 39 housekeeping genes that acted as negative controls. SNPs in 11 of the putative transporter genes, including pfcrt and pfmdr1, showed significant associations with decreased sensitivity to CQ and/or QN in P. falciparum. Significant linkage disequilibria within and between these genes were also detected, suggesting interactions among the transporter genes. This study provides specific leads for better understanding of complex drug resistances in malaria parasites.