Project description:Comaprision of P.falciparum clinical isolates showing Uncomplicated disease with that shwoing complicated disease(Cerebral malaria) The experiment was designed to try and identify differences if any, at the genome level between P.falciparum isolates from patients with uncomplicated malaria vs. patients with complicated malaria (Cerebral malaria). The emphasis was to highlight possible amplifications/deletions in different regions of the parasite genome.
Project description:Genomic variation is an inherent phenomena observed among members of same species belonging to different geographical locations. In case of P. falciparum, an apicomplexan protozoan parasite, its 22.8 MB nuclear genome is known to display vast genetic diversity in the subtelomeric compartments having but not exclusively variant gene families like var, rifins and stevors and examples in other elements of the genome have recently been documented. Microarrays, relies solely on the genomic sequence information to capture the relevant transcript abundance and needs to consider these variations into account for revealing true transcriptional variation.Here, we describe the designing strategy of a custom P. falciparum 15K array using Agilent platform to study the transcriptome of Indian field isolates for which genome sequence information is limited. Array contains probes representing genome sequence of two distinct geographical isolates (i.e 3D7 and HB3) and subtelomeric var gene sequence of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts by performing a 244K array experiment representing multiple probes per gene/transcript. Array performance was evaluated and validated using RNA materials from P. falciparum clinical isolates obtained directly from patients with differing clinical conditions due to malaria infection.Due to pre probe screening large percentage (91 %) of the represented transcripts could be detected from Indian P. falciparum isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. Plasmodium falciparum isolates were collected from patients (n=13) with differing clinical conditions. The patients exhibited symptoms categorized as uncomplicated (n=6) or complicated malaria (n=7). Criteria for determination of complicated disease were based on World Health Organization year 2000 guidelines. Microarray array based transcriptional profiling was carried out to evaluate the performance of the array.
Project description:Genomic variation is an inherent phenomena observed among members of same species belonging to different geographical locations. In case of P. falciparum, an apicomplexan protozoan parasite, its 22.8 MB nuclear genome is known to display vast genetic diversity in the subtelomeric compartments having but not exclusively variant gene families like var, rifins and stevors and examples in other elements of the genome have recently been documented. Microarrays, relies solely on the genomic sequence information to capture the relevant transcript abundance and needs to consider these variations into account for revealing true transcriptional variation.Here, we describe the designing strategy of a custom P. falciparum 15K array using Agilent platform to study the transcriptome of Indian field isolates for which genome sequence information is limited. Array contains probes representing genome sequence of two distinct geographical isolates (i.e 3D7 and HB3) and subtelomeric var gene sequence of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts by performing a 244K array experiment representing multiple probes per gene/transcript. Array performance was evaluated and validated using RNA materials from P. falciparum clinical isolates obtained directly from patients with differing clinical conditions due to malaria infection.Due to pre probe screening large percentage (91 %) of the represented transcripts could be detected from Indian P. falciparum isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains.
Project description:Here we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos regions using genome-scanning, a microarray-based technique which delineates the majority of single-base changes, indels and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon is highly structured with a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes indicating mutations arising during self-mating or mitotic replication. The data also reveal that 1 or 2 meioses separate different isolates showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pair-wise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase clindamycin EC50 in strains harboring one of these mutations. This evidence of clindamycin resistant parasites in the Amazon suggests a shift should be made in health policy away from quinine+clindamycin therapy for malaria in pregnant women and infants and that the development of new lincosamide antibiotics for malaria should be reconsidered.
Project description:In our global differential gene expression analyses,ETRAMP14.1, an ETRAMP family member was found to be highly transcribed in vivo in severe Malaria patients from highly endemic Indian region. This study for the first time reports the interaction of ETRAMP14.1 with PfEMP1, EXP2 and Hsp70-1. Therefore, we propose that ETRAMP14.1 facilitate PfEMP1 to cross the PVM via transcolon machinery component EXP2. Peripheral blood samples containing ring forms of the P.falciparum from mild and severe malaria patients were used for RNA extraction, CDNA synthesis and hybridization on Affymetrix microarrays. In vitro ring stage P.falciparum cultures were used for normalization.
Project description:In our global differential gene expression analyses,ETRAMP14.1, an ETRAMP family member was found to be highly transcribed in vivo in severe Malaria patients from highly endemic Indian region. This study for the first time reports the interaction of ETRAMP14.1 with PfEMP1, EXP2 and Hsp70-1. Therefore, we propose that ETRAMP14.1 facilitate PfEMP1 to cross the PVM via transcolon machinery component EXP2. Peripheral blood samples containing ring forms of the P.falciparum from mild and severe malaria patients were used for RNA extraction, CDNA synthesis and hybridization on Affymetrix microarrays. In vitro ring stage P.falciparum cultures were used for normalization.
Project description:In our global differential gene expression analyses,ETRAMP14.1, an ETRAMP family member was found to be highly transcribed in vivo in severe Malaria patients from highly endemic Indian region. This study for the first time reports the interaction of ETRAMP14.1 with PfEMP1, EXP2 and Hsp70-1. Therefore, we propose that ETRAMP14.1 facilitate PfEMP1 to cross the PVM via transcolon machinery component EXP2.
Project description:In our global differential gene expression analyses,ETRAMP14.1, an ETRAMP family member was found to be highly transcribed in vivo in severe Malaria patients from highly endemic Indian region. This study for the first time reports the interaction of ETRAMP14.1 with PfEMP1, EXP2 and Hsp70-1. Therefore, we propose that ETRAMP14.1 facilitate PfEMP1 to cross the PVM via transcolon machinery component EXP2.
Project description:Here we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos regions using genome-scanning, a microarray-based technique which delineates the majority of single-base changes, indels and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon is highly structured with a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes indicating mutations arising during self-mating or mitotic replication. The data also reveal that 1 or 2 meioses separate different isolates showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pair-wise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase clindamycin EC50 in strains harboring one of these mutations. This evidence of clindamycin resistant parasites in the Amazon suggests a shift should be made in health policy away from quinine+clindamycin therapy for malaria in pregnant women and infants and that the development of new lincosamide antibiotics for malaria should be reconsidered. Genome DNA from Peruvian Isolates vs. Reference 3D7
Project description:Further understanding of the molecular mediators of alternative RBC invasion phenotypes in endemic malaria parasites will support malaria blood stage vaccine or drug development. This study investigated the prevalence of SA-dependent and SA-independent RBC invasion pathways in endemic P. falciparum parasites from Cameroon and compared the schizont stage transcriptomes in these two groups with the goal of uncovering the wider repertoire of transcriptional variation associated with the use of alternative RBC invasion pathway phenotypes. Two-colour flow cytometry-based invasion-inhibition assay against RBCs treated with neuraminidase, trypsin and chymotrypsin and deep RNA sequencing of schizont stages harvested in the first ex-vivo replication cycle in culture were employed in this investigation. RBC invasion phenotypes were determined for 63 isolates from asymptomatic children with uncomplicated malaria. Approximately 80% of the isolates invaded neuraminidase-treated but not chymotrypsin-treated RBCs, representing sialic acid (SA)-independent pathways of RBC invasion. The schizont transcriptome profiles of 16 isolates with invasion phenotypes revealed a total of 5136 gene transcripts, with 85% of isolates predicted at schizont stages. Two distinct transcriptome profile clusters belonging to SA-dependent and SA-independent parasites were obtained by data reduction with principal component analysis. Differential analysis of gene expression between the two clusters implicated in addition to the well characterized adhesins, the up-regulation of genes encoding proteins mediating merozoite organelle discharges and several conserved, virulent, merozoite associated and exported proteins. The latter majority of which have been shown to have structural and physiological relevance to RBC surface remodelling and immune evasion in malaria and thus, have potential as anti-invasion targets.