Linkage Group Selection to Investigate Genetic Determinants of Complex Traits of Malaria Parasites

Malaria parasites of the species infecting humans and animal hosts exhibit genetic and phenotypic diversity. Some of this diversity, including the responses to anti-malarial drugs, growth rate and virulence and antigenic variability, is medically significant. This is because these phenotypes may determine the existence and survival of the parasites in the host and, in turn, contribute to the clinical outcome of infection. Understanding of the biological characteristics and the genetic basis underlying these complex phenotypes can thus lead to the development of effective control strategies against the disease, such as anti-malarial drugs and vaccines. Genetic studies in rodent malaria parasites have proved useful in providing insights into the genetic determinants of these complex traits and thus can be used to complement the study of human malaria. The present studies aim to investigate genetic determinants underlying two major medically important phenotypes, Strain Specific Protective Immunity (SSPI) and Growth rate, using the newly devised genetic method of Linkage Group Selection (LGS). The results presented here relate to the accomplishment of these aims. LGS analysis of SSPI using a genetic cross between clones AJ and CB-pyr10 of Plasmodium chabaudi chabaudi has identified a single region on chromosome 8 containing the gene for the Merozoite Surface Protein-1 as encoding a major target of SSPI. A similar finding was also obtained in a previous LGS study using a different genetic cross between clones AS-pyr1 and CB of P. c. chabaudi (Martinelli et al., 2005). Hence, the results of two independent studies strongly indicate that a single locus within the parasite genome contains a major target antigen, or antigens, of SSPI against P. c. chabaudi malaria. These results have particular relevance for research on SSPI in human malaria and the choice of candidate antigens for malaria vaccine development. LGS analysis of growth rate conducted upon a genetic cross between a fast-growing line, 17XYM, and a slow-growing line, 33XC, of Plasmodium yoelii yoelii has identified a ~ 1 megabase pair region on P. y. yoelii chromosome 13 as containing a major genetic determinant(s) of growth rate in these malaria parasites. This is consistent with the finding of the classical linkage analysis by Walliker et al., (1976), that growth rate in P. y. yoelii is mainly determined at a single genetic locus. Because the fast-growing line 17XYM arose spontaneously during infection with a mild strain of P. y. yoelii 17X, identification of parasites with a slow growth rate phenotype derived from the same genetic stock as 17XYM can be useful in determining genes underlying growth rate in these malaria parasites. It has been shown here that parasites of the P. y. yoelii lines 17X consist of two completely distinct genotypes. One is represented by the fast-growing line, 17XYM, and a slow-growing line of P. y. yoelii, 17XNIMR. The other is represented by another slow-growing line 17XA. Comparing the region of P. y. yoelii chromosome 13 under strong growth selection between the two congenic lines, 17XYM and 17XNIMR, could lead to the identification of the gene(s) controlling growth rate differences in these two parasite lines. Such findings could be relevant to the location of genetic determinants of growth rate in human malaria

Linkage Group Selection: Towards Identifying Genes Controlling Strain Specific Protective Immunity in Malaria

Protective immunity against blood infections of malaria is partly specific to the genotype, or strain, of the parasites. The target antigens of Strain Specific Protective Immunity are expected, therefore, to be antigenically and genetically distinct in different lines of parasite. Here we describe the use of a genetic approach, Linkage Group Selection, to locate the target(s) of Strain Specific Protective Immunity in the rodent malaria parasite Plasmodium chabaudi chabaudi. In a previous such analysis using the progeny of a genetic cross between P. c. chabaudi lines AS-pyr1 and CB, a location on P. c. chabaudi chromosome 8 containing the gene for merozoite surface protein-1, a known candidate antigen for Strain Specific Protective Immunity, was strongly selected. P. c. chabaudi apical membrane antigen-1, another candidate for Strain Specific Protective Immunity, could not have been evaluated in this cross as AS-pyr1 and CB are identical within the cell surface domain of this protein. Here we use Linkage Group Selection analysis of Strain Specific Protective Immunity in a cross between P. c. chabaudi lines CB-pyr10 and AJ, in which merozoite surface protein-1 and apical membrane antigen-1 are both genetically distinct. In this analysis strain specific immune selection acted strongly on the region of P. c. chabaudi chromosome 8 encoding merozoite surface protein-1 and, less strongly, on the P. c. chabaudi chromosome 9 region encoding apical membrane antigen-1. The evidence from these two independent studies indicates that Strain Specific Protective Immunity in P. c. chabaudi in mice is mainly determined by a narrow region of the P. c. chabaudi genome containing the gene for the P. c. chabaudi merozoite surface protein-1 protein. Other regions, including that containing the gene for P. c. chabaudi apical membrane antigen-1, may be more weakly associated with Strain Specific Protective Immunity in these parasites

Linkage group selection to investigate genetic determinants of complex traits of malaria parasites

Malaria parasites of the species infecting humans and animal hosts exhibit genetic and phenotypic diversity. Some of this diversity, including the responses to anti-malarial drugs, growth rate and virulence and antigenic variability, is medically significant. This is because these phenotypes may determine the existence and survival of the parasites in the host and, in turn, contribute to the clinical outcome of infection. Understanding of the biological characteristics and the genetic basis underlying these complex phenotypes can thus lead to the development of effective control strategies against the disease, such as anti-malarial drugs and vaccines. Genetic studies in rodent malaria parasites have proved useful in providing insights into the genetic determinants of these complex traits and thus can be used to complement the study of human malaria. The present studies aim to investigate genetic determinants underlying two major medically important phenotypes, Strain Specific Protective Immunity (SSPI) and Growth rate, using the newly devised genetic method of Linkage Group Selection (LGS). The results presented here relate to the accomplishment of these aims. LGS analysis of SSPI using a genetic cross between clones AJ and CB-pyr10 of Plasmodium chabaudi chabaudi has identified a single region on chromosome 8 containing the gene for the Merozoite Surface Protein-1 as encoding a major target of SSPI. A similar finding was also obtained in a previous LGS study using a different genetic cross between clones AS-pyr1 and CB of P. c. chabaudi (Martinelli et al., 2005). Hence, the results of two independent studies strongly indicate that a single locus within the parasite genome contains a major target antigen, or antigens, of SSPI against P. c. chabaudi malaria. These results have particular relevance for research on SSPI in human malaria and the choice of candidate antigens for malaria vaccine development. LGS analysis of growth rate conducted upon a genetic cross between a fast-growing line, 17XYM, and a slow-growing line, 33XC, of Plasmodium yoelii yoelii has identified a ~ 1 megabase pair region on P. y. yoelii chromosome 13 as containing a major genetic determinant(s) of growth rate in these malaria parasites. This is consistent with the finding of the classical linkage analysis by Walliker et al., (1976), that growth rate in P. y. yoelii is mainly determined at a single genetic locus. Because the fast-growing line 17XYM arose spontaneously during infection with a mild strain of P. y. yoelii 17X, identification of parasites with a slow growth rate phenotype derived from the same genetic stock as 17XYM can be useful in determining genes underlying growth rate in these malaria parasites. It has been shown here that parasites of the P. y. yoelii lines 17X consist of two completely distinct genotypes. One is represented by the fast-growing line, 17XYM, and a slow-growing line of P. y. yoelii, 17XNIMR. The other is represented by another slow-growing line 17XA. Comparing the region of P. y. yoelii chromosome 13 under strong growth selection between the two congenic lines, 17XYM and 17XNIMR, could lead to the identification of the gene(s) controlling growth rate differences in these two parasite lines. Such findings could be relevant to the location of genetic determinants of growth rate in human malaria.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

MOESM3 of Global sequence diversity of the lactate dehydrogenase gene in Plasmodium falciparum

Additional file 3. Nucleotide sequence IDs of the ldh gene of the malaria parasites of humans and non-human primates

MOESM4 of Global sequence diversity of the lactate dehydrogenase gene in Plasmodium falciparum

Additional file 4. Neighbour Joining tree of 61 allelic sequences of the gene encoding lactate dehydrogenase (ldh) from 12 Plasmodium parasite species. The sequences are named according to parasite species and allelic type. The first two letters indicate parasite species: Pf (Plasmodium falciparum), Pm (Plasmodium malariae), Po (Plasmodium ovale), Pv (Plasmodium vivax), Pp (Plasmodium praefalciparum), Pr (Plasmodium reichenowi), Pbi (Plasmodium billcollinsi), Pbl (Plasmodium blacklocki), Pa (Plasmodium alderi), Pg (Plasmodium gaboni), Pk (Plasmodium knowlesi) and Pc (Plasmodium cynomolgi). Species showed on the right hand site are labelled with color representing parasite host: Homo sapiens (blue), Gorilla gorilla (black), Pan troglodytes (green) and Macaca fascicularis (red). The tree was constructed using the aligned sequences of 768 nucleotides, corresponding to nucleotide position 52–819 after P. falciparum strain 3D7. Bootstrap values are shown next to the nodes. Scale bar shows nucleotide substitution per site

Genetic diversity of the merozoite surface protein-3 gene in Plasmodium falciparum populations in Thailand

Abstract Background An effective malaria vaccine is an urgently needed tool to fight against human malaria, the most deadly parasitic disease of humans. One promising candidate is the merozoite surface protein-3 (MSP-3) of Plasmodium falciparum. This antigenic protein, encoded by the merozoite surface protein (msp-3) gene, is polymorphic and classified according to size into the two allelic types of K1 and 3D7. A recent study revealed that both the K1 and 3D7 alleles co-circulated within P. falciparum populations in Thailand, but the extent of the sequence diversity and variation within each allelic type remains largely unknown. Methods The msp-3 gene was sequenced from 59 P. falciparum samples collected from five endemic areas (Mae Hong Son, Kanchanaburi, Ranong, Trat and Ubon Ratchathani) in Thailand and analysed for nucleotide sequence diversity, haplotype diversity and deduced amino acid sequence diversity. The gene was also subject to population genetic analysis (F st ) and neutrality tests (Tajima’s D, Fu and Li D* and Fu and Li’ F* tests) to determine any signature of selection. Results The sequence analyses revealed eight unique DNA haplotypes and seven amino acid sequence variants, with a haplotype and nucleotide diversity of 0.828 and 0.049, respectively. Neutrality tests indicated that the polymorphism detected in the alanine heptad repeat region of MSP-3 was maintained by positive diversifying selection, suggesting its role as a potential target of protective immune responses and supporting its role as a vaccine candidate. Comparison of MSP-3 variants among parasite populations in Thailand, India and Nigeria also inferred a close genetic relationship between P. falciparum populations in Asia. Conclusion This study revealed the extent of the msp-3 gene diversity in P. falciparum in Thailand, providing the fundamental basis for the better design of future blood stage malaria vaccines against P. falciparum

The Comparative Intensities of 92 AFLP markers of <i>Plasmodium chabaudi chabaudi</i> strain AJ from the progeny of a genetic cross between <i>P. c. chabaudi</i> strains CB-pyr10 and AJ following selection in mice immunised strain AJ (see text).

<p>AJ-specific markers (indicated by black diamonds) were located on a <i>P. c. chabaudi</i> genetic linkage map, generated from a genetic cross between AS and AJ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000857#pone.0000857-Martinelli2" target="_blank">[34]</a>. Numbers after letter ‘C’ and ‘g’ represent <i>P. c. chabaudi</i> chromosome numbers and <i>P. c. chabaudi</i> unassigned linkage groups, respectively, in the genetic linkage map <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000857#pone.0000857-Martinelli2" target="_blank">[34]</a>. Of the six AJ markers which were most reduced under AJ-specific immune selection (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000857#pone-0000857-t002" target="_blank">Table 2</a>), five (indicated by asterisks) could be located to <i>P. c. chabaudi</i> chromosome 8, forming a selection valley with the <i>P. c. chabaudi msp</i>-1 gene at its lowest point (see text). RTQ-PCR values for the proportions of the AJ-immune selected cross progeny carrying the AJ alleles of the Merozoite Surface Protein-1 (<i>msp</i>-1) are indicated by the red triangle and Apical Membrane Antigen-1 (<i>ama</i>-1) by the green triangle in the AJ-immune selected cross progeny. The red line indicates Comparative Intensity of 50%.</p

Physical and genetic locations of AFLP markers of <i>Plasmodium chabaudi chabaudi</i> strain AJ whose Comparative Intensities (CI) were reduced below 50% in the progeny of the genetic cross between CB-pyr10 and AJ following selection in an AJ-immunised mouse (see text).

<p>The six AJ markers with CI of <20% in the AJ-immune selected cross progeny mapped to positions closely linked to the gene encoding the <i>P. c. chabaudi</i> merozoite surface protein-1 (MSP-1) are indicated in bold. ND not determined.</p>*<p>The first and second numbers in brackets represent percentages of parasite DNA carrying the AJ alleles of the indicated gene ( <i>msp</i>-1 or <i>ama</i>-1), respectively in the AJ-immune selected cross progeny and in the non-immune selected cross progeny, as measured by RTQ-PCR (see text)</p>**<p>Numbers after ‘<i>pf</i>’’ indicate the <i>Plasmodium falciparum</i> chromosome number followed by distance along the chromosome in kilo base pairs</p