Identification of parasitic genes by computational methods

A number of parasite genome projects are under way, and large amounts of nucleotide sequence data are becoming available for analysis. There is an urgent need for development of theoretical tools to analyze the genome data, including identification of protein-coding sequences. The majority of the methods developed to date require prior information about the genome before accurate predictions can be made. Because such information is not available for many parasites, these methods cannot be directly applied. In this article, Alok Bhattacharya and colleagues describe some of the gene-prediction methods commonly in use, and a new method, GeneScan, that they have developed for the analysis of parasite genomes.     

Clustering of Schistosoma mansoni mRNA sequences and analysis of the most transcribed genes: implications in metabolism and biology of different developmental stages

The study of the Schistosoma mansoni genome, one of the etiologic agents of human schistosomiasis, is essential for a better understanding of the biology and development of this parasite. In order to get an overview of all S. mansoni catalogued gene sequences, we performed a clustering analysis of the parasite mRNA sequences available in public databases. This was made using softwares PHRAP and CAP3. The consensus sequences, generated after the alignment of cluster constituent sequences, allowed the identification by database homology searches of the most expressed genes in the worm. We analyzed these genes and looked for a correlation between their high expression and parasite metabolism and biology. We observed that the majority of these genes is related to the maintenance of basic cell functions, encoding genes whose products are related to the cytoskeleton, intracellular transport and energy metabolism. Evidences are presented here that genes for aerobic energy metabolism are expressed in all the developmental stages analyzed. Some of the most expressed genes could not be identified by homology searches and may have some specific functions in the parasite.     

A tale of three genomes: the kinetoplastids have arrived

July 2005 marked a milestone in kinetoplastid biology research. A tour de force effort led by the Tri-Trypanosomatidae "Tritryp" genome consortium yielded the publication of three prominent kinetoplastid parasite genome sequences: Trypanosoma brucei, Trypanosoma cruzi and Leishmania major. The individual and combined comparative analyses of these three genome sequences, combined with proteomic analyses, have yielded insights into topics ranging from genome evolution and horizontal gene transfer to potential new therapeutic and vaccine targets.     

Assessing the impact of Plasmodium falciparum genome sequencing

With the publication of the complete sequences for chromosomes 2 and 3 and the increasing availability of shotgun sequence covering most of its genome, Plasmodium falciparum biology is entering its post-genomic era. Analysis of the results generated to date has identified higher-order organisation of gene families involved in parasite pathology, provided information regarding the unique biology of this parasite and allowed the identification of potential chemotherapeutic drug targets. Continuing efforts to complete the P. falciparum genome and the availability of sequences from other protozoan parasites will facilitate a broader understanding of their biology, particularly with respect to their pathogenicity.     

Cyclic nucleotide signalling in malaria parasites

Cyclic nucleotides are so-called intracellular second messenger molecules used by all cells to transform environmental signals into an appropriate response. Interest in the cyclic nucleotides cAMP and cGMP in malaria parasites followed early observations that both molecules might be involved in distinct differentiation events within the sexual phase of the life cycle that is required for transmission of parasites to the mosquito vector. Completed genome sequences combined with biochemical and genetic studies have confirmed the presence of the main enzymatic components of cyclic nucleotide signalling in the parasite. Dissection of their functions is underway and is giving initial insights into some of the cellular processes, which are regulated by these signalling pathways. Malaria parasites occupy terminally differentiated red blood cells for a significant proportion of their life cycle, but although there is some evidence of potential roles for the residual host cell signalling machinery in parasite development, details are few. A major gap in our knowledge is the nature of the cell surface receptors, which might trigger cyclic nucleotide signalling in the parasite.     

Evaluation of genome skimming to detect and characterise human and livestock helminths

The identification of gastrointestinal helminth infections of humans and livestock almost exclusively relies on the detection of eggs or larvae in faeces, followed by manual counting and morphological characterisation to differentiate species using microscopy-based techniques. However, molecular approaches based on the detection and quantification of parasite DNA are becoming more prevalent, increasing the sensitivity, specificity and throughput of diagnostic assays. High-throughput sequencing, from single PCR targets through to the analysis of whole genomes, offers significant promise towards providing information-rich data that may add value beyond traditional and conventional molecular approaches; however, thus far, its utility has not been fully explored to detect helminths in faecal samples. In this study, low-depth whole genome sequencing, i.e. genome skimming, has been applied to detect and characterise helminth diversity in a set of helminth-infected human and livestock faecal material. The strengths and limitations of this approach are evaluated using three methods to characterise and differentiate metagenomic sequencing data based on (i) mapping to whole mitochondrial genomes, (ii) whole genome assemblies, and (iii) a comprehensive internal transcribed spacer 2 (ITS2) database, together with validation using quantitative PCR (qPCR). Our analyses suggest that genome skimming can successfully identify most single and multi-species infections reported by qPCR and can provide sufficient coverage within some samples to resolve consensus mitochondrial genomes, thus facilitating phylogenetic analyses of selected genera, e.g. Ascaris spp. Key to this approach is both the availability and integrity of helminth reference genomes, some of which are currently contaminated with bacterial and host sequences. The success of genome skimming of faecal DNA is dependent on the availability of vouchered sequences of helminths spanning both taxonomic and geographic diversity, together with methods to detect or amplify minute quantities of parasite nucleic acids in mixed samples.     

Genome-Wide Stochastic Adaptive DNA Amplification at Direct and Inverted DNA Repeats in the Parasite Leishmania

Gene amplification of specific loci has been described in all kingdoms of life. In the protozoan parasite Leishmania, the product of amplification is usually part of extrachromosomal circular or linear amplicons that are formed at the level of direct or inverted repeated sequences. A bioinformatics screen revealed that repeated sequences are widely distributed in the Leishmania genome and the repeats are chromosome-specific, conserved among species, and generally present in low copy number. Using sensitive PCR assays, we provide evidence that the Leishmania genome is continuously being rearranged at the level of these repeated sequences, which serve as a functional platform for constitutive and stochastic amplification (and deletion) of genomic segments in the population. This process is adaptive as the copy number of advantageous extrachromosomal circular or linear elements increases upon selective pressure and is reversible when selection is removed. We also provide mechanistic insights on the formation of circular and linear amplicons through RAD51 recombinase-dependent and -independent mechanisms, respectively. The whole genome of Leishmania is thus stochastically rearranged at the level of repeated sequences, and the selection of parasite subpopulations with changes in the copy number of specific loci is used as a strategy to respond to a changing environment.     

Adenylyl and guanylyl cyclases from the malaria parasite Plasmodium falciparum

Completion of several malaria parasite genome sequences and advances in Plasmodium gene manipulation technology, will lead to significant advances in our knowledge of the biology of these organisms. Biochemical analysis of the cyclic nucleotide signalling pathways of P. falciparum has provided important information on malaria parasite development. The Plasmodium purine nucleotide cyclase enzymes have extremely unusual structures and the regulatory mechanisms controlling parasite enzyme activity are distinct from those operating on the analogous host molecules. Study of these enzymes could therefore lead to novel strategies for anti-malarial intervention in addition to providing unique insights into the intriguing biology of the parasite.     

Genomic repetitive DNA elements of Trypanosoma cruzi

Repetitive DNA sequences are interspersed throughout the genomes of mammals and other higher eukaryotes, and represent a substantial portion of the genome. Although it has been generally assumed that the redundant DNA is present only in the complex genomes of high order organisms, over the past few years a number of repetitive DNA sequences have been also detected in the protozoan parasite Trypanosoma cruzi. A compilation of the repetitive DNA sequences found in the T. cruzi genome is here presented by Jose Maria Requena, Manuel Carlos López and Carlos Alonso, who also speculate on their possible origin and functional implications regarding retrotransposition and gene regulation.     

Comparative analysis of BAC and whole genome shotgun sequences from an Anopheles gambiae region related to Plasmodium encapsulation

The only natural mechanism of malaria transmission in sub-Saharan Africa is the mosquito, generally Anopheles gambiae. Blocking malaria parasite transmission by stopping the development of Plasmodium in the insect vector would provide a useful alternative to the current methods of malaria control. Toward this end, it is important to understand the molecular basis of the malaria parasite refractory phenotype in An. gambiae mosquito strains. We have selected and sequenced six bacterial artificial chromosome (BAC) clones from the Pen-1 region that is the major quantitative trait locus involved in Plasmodium encapsulation. The sequence and the annotation of five overlapping BAC clones plus one adjacent, but not contiguous clone, totaling 585kb of genomic sequence from the centromeric end of the Pen-1 region of the PEST strain were compared to that of the genome sequence of the same strain produced by the whole genome shotgun technique. This project identified 23 putative mosquito genes plus putative copies of the retrotransposable elements BEL12 and TRANSIBN1_AG in the six BAC clones. Nineteen of the predicted genes are most similar to their Drosophila melanogaster homologs while one is more closely related to vertebrate genes. Comparison of these new BAC sequences plus previously published BAC sequences to the cognate region of the assembled genome sequence identified three retrotransposons present in one sequence version but not the other. One of these elements, Indy, has not been previously described. These observations provide evidence for the recent active transposition of these elements and demonstrate the plasticity of the Anopheles genome. The BAC sequences strongly support the public whole genome shotgun assembly and automatic annotation while also demonstrating the benefit of complementary genome sequences and of human curation. Importantly, the data demonstrate the differences in the genome sequence of an individual mosquito compared to that of a hypothetical, average genome sequence generated by whole genome shotgun assembly.     

Arbitrary-primed PCR for genomic fingerprinting and identification of differentially regulated genes in Indian isolates of Leishmania donovani

The arbitrary-primed PCR (AP-PCR) technique was employed with the twin goals of identifying genetic polymorphisms within the Indian isolates and to identify differentially expressed gene sequences. The parasite isolates from Indian Kala-azar patients could be differentiated from Leishmania donovani isolates from distinct geographic regions. Moreover, differences within the Indian isolates could also be identified. A majority (17/19) of the Indian isolates gave identical AP-PCR pattern, while two isolates gave consistently divergent pattern. The distinctive AP-PCR fragments obtained with Indian isolates were used as probes in Northern blot analysis. Three such fragments were found to represent transcribed sequences that were differentially expressed in the two stages of the parasite. These sequences led to cloning and characterization of Leishmania Centrin gene and a novel gene termed A-1 that is over-expressed in amastigote stage of the parasite. The study demonstrates the utility of random genome sampling methods in genomic fingerprinting and in identifying differentially transcribed sequences that could potentially contribute to parasite virulence.     

Construction of bacterial artificial chromosome libraries from the parasitic nematode Brugia malayi and physical mapping of the genome of its Wolbachia endosymbiont

The parasitic nematode, Brugia malayi, causes lymphatic filariasis in humans, which in severe cases leads to the condition known as elephantiasis. The parasite contains an endosymbiotic alpha-proteobacterium of the genus Wolbachia that is required for normal worm development and fecundity and is also implicated in the pathology associated with infections by these filarial nematodes. Bacterial artificial chromosome libraries were constructed from B. malayi DNA and provide over 11-fold coverage of the nematode genome. Wolbachia genomic fragments were simultaneously cloned into the libraries giving over 5-fold coverage of the 1.1 Mb bacterial genome. A physical framework for the Wolbachia genome was developed by construction of a plasmid library enriched for Wolbachia DNA as a source of sequences to hybridise to high-density bacterial artificial chromosome colony filters. Bacterial artificial chromosome end sequencing provided additional Wolbachia probe sequences to facilitate assembly of a contig that spanned the entire genome. The Wolbachia sequences provided a marker approximately every 10 kb. Four rare-cutting restriction endonucleases were used to restriction map the genome to a resolution of approximately 60 kb and demonstrate concordance between the bacterial artificial chromosome clones and native Wolbachia genomic DNA. Comparison of Wolbachia sequences to public databases using BLAST algorithms under stringent conditions allowed confident prediction of 69 Wolbachia peptide functions and two rRNA genes. Comparison to closely related complete genomes revealed that while most sequences had orthologs in the genome of the Wolbachia endosymbiont from Drosophila melanogaster, there was no evidence for long-range synteny. Rather, there were a few cases of short-range conservation of gene order extending over regions of less than 10 kb. The molecular scaffold produced for the genome of the Wolbachia from B. malayi forms the basis of a genomic sequencing effort for this bacterium, circumventing the difficult challenge of purifying sufficient endosymbiont DNA from a tropical parasite for a whole genome shotgun sequencing strategy.     

Plasmodium falciparum variant surface antigen expression patterns during malaria

The variant surface antigens expressed on Plasmodium falciparum-infected erythrocytes are potentially important targets of immunity to malaria and are encoded, at least in part, by a family of var genes, about 60 of which are present within every parasite genome. Here we use semi-conserved regions within short var gene sequence "tags" to make direct comparisons of var gene expression in 12 clinical parasite isolates from Kenyan children. A total of 1,746 var clones were sequenced from genomic and cDNA and assigned to one of six sequence groups using specific sequence features. The results show the following. (1) The relative numbers of genomic clones falling in each of the sequence groups was similar between parasite isolates and corresponded well with the numbers of genes found in the genome of a single, fully sequenced parasite isolate. In contrast, the relative numbers of cDNA clones falling in each group varied considerably between isolates. (2) Expression of sequences belonging to a relatively conserved group was negatively associated with the repertoire of variant surface antigen antibodies carried by the infected child at the time of disease, whereas expression of sequences belonging to another group was associated with the parasite "rosetting" phenotype, a well established virulence determinant. Our results suggest that information on the state of the host-parasite relationship in vivo can be provided by measurements of the differential expression of different var groups, and need only be defined by short stretches of sequence data.     

For better or worse: genomic consequences of intracellular mutualism and parasitism

Bacteria that replicate within eukaryotic host cells include a variety of pathogenic and mutualistic species. Early genome data for these intracellular associates suggested they experience continual gene loss, little if any gene acquisition, and minimal recombination in small, isolated populations. This view of reductive evolution is itself evolving as new genome sequences clarify mechanisms and outcomes of diverse intracellular associations. Recently sequenced genomes have confirmed a trajectory of gene loss and exceptional genome stability in long-term, nutritional mutualists and certain pathogens. However, new genome data for the Rickettsiales and Chlamydiales indicate more repeated DNA, a greater abundance of mobile DNA elements, and more labile genome dynamics than previously suspected for ancient intracellular lineages. Surprising discoveries of conjugation machinery in the parasite Rickettsia felis and the amoebae symbiont Parachlamydia sp. suggest that DNA transfer might play key roles in some intracellular taxa.     

Assembly of the Genome of the Disease Vector Aedes aegypti onto a Genetic Linkage Map Allows Mapping of Genes Affecting Disease Transmission

The mosquito Aedes aegypti transmits some of the most important human arboviruses, including dengue, yellow fever and chikungunya viruses. It has a large genome containing many repetitive sequences, which has resulted in the genome being poorly assembled — there are 4,758 scaffolds, few of which have been assigned to a chromosome. To allow the mapping of genes affecting disease transmission, we have improved the genome assembly by scoring a large number of SNPs in recombinant progeny from a cross between two strains of Ae. aegypti, and used these to generate a genetic map. This revealed a high rate of misassemblies in the current genome, where, for example, sequences from different chromosomes were found on the same scaffold. Once these were corrected, we were able to assign 60% of the genome sequence to chromosomes and approximately order the scaffolds along the chromosome. We found that there are very large regions of suppressed recombination around the centromeres, which can extend to as much as 47% of the chromosome. To illustrate the utility of this new genome assembly, we mapped a gene that makes Ae. aegypti resistant to the human parasite Brugia malayi, and generated a list of candidate genes that could be affecting the trait.     

Functional and evolutionary analysis of a eukaryotic parasitic genome

The DNA sequences of the 11 linear chromosomes of the approximately 2.9 Mbp genome of Encephalitozoon cuniculi, an obligate intracellular parasite of mammals, include approximately 2000 putative protein-coding genes. The compactness of this genome is associated with the length reduction of various genes. Essential functions are dependent on a minimal set of genes. Phylogenetic analysis supports the hypotheses that microsporidia are related to fungi and have retained a mitochondrion-derived organelle, the mitosome.     

Analysis of sequences from the extremely A + T-rich genome of Plasmodium falciparum

The genome of the human malaria parasite Plasmodium falciparum has an A + T content of about 82%, higher than any other organism whose DNA has been characterized. Computer analysis of 36 kb of available nucleotide sequences from this species showed that the coding regions, with an A + T content of 69.0%, are flanked by more A + T-rich regions of 86.0% A + T. Within the coding sequences, the A/T ratio was 1.68 in the mRNA sense strand, and overall A + T content in the three codon positions increased in the order 1st-2nd-3rd position. Codons with T or especially A in the third position were strongly preferred. Codon usage among individual parasite genes was very similar compared to genes from other species. Dinucleotide frequencies for the parasite DNA were close to those expected for a random sequence with the known base composition, except that the CpG frequency in the coding sequences was low.