| 1. |
- Ankarklev, Johan, et al.
(författare)
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A novel high-resolution multilocus sequence typing of Giardia intestinalis Assemblage A isolates reveals zoonotic transmission, clonal outbreaks and recombination
- 2018
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Ingår i: Infection, Genetics and Evolution. - : Elsevier BV. - 1567-1348 .- 1567-7257. ; 60, s. 7-16
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Tidskriftsartikel (refereegranskat)abstract
- Molecular epidemiology and genotyping studies of the parasitic protozoan Giardia intestinalis have proven difficult due to multiple factors, such as low discriminatory power in the commonly used genotyping loci, which has hampered molecular analyses of outbreak sources, zoonotic transmission and virulence types. Here we have focused on assemblage A Giardia and developed a high-resolution assemblage-specific multilocus sequence typing (MLST) method. Analyses of sequenced G. intestinalis assemblage A genomes from different sub-assemblages identified a set of six genetic loci with high genetic variability. DNA samples from both humans (n = 44) and animals (n = 18) that harbored Giardia assemblage A infections, were PCR amplified (557-700 bp products) and sequenced at the six novel genetic loci. Bioinformatic analyses showed five to ten-fold higher levels of polymorphic sites than what was previously found among assemblage A samples using the classic genotyping loci. Phylogenetically, a division of two major clusters in assemblage A became apparent, separating samples of human and animal origin. A subset of human samples (n = 9) from a documented Giardia outbreak in a Swedish day-care center, showed full complementarity at nine genetic loci (the six new and the standard BG, TPI and GDH loci), strongly suggesting one source of infection. Furthermore, three samples of human origin displayed MLST profiles that were phylogenetically more closely related to MLST profiles from animal derived samples, suggesting zoonotic transmission. These new genotyping loci enabled us to detect events of recombination between different assemblage A isolates but also between assemblage A and E isolates. In summary, we present a novel and expanded MLST strategy with significantly improved sensitivity for molecular analyses of virulence types, zoonotic potential and source tracking for assemblage A Giardia.
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| 2. |
- Ankarklev, Johan, et al.
(författare)
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Comparative genomic analyses of freshly isolated Giardia intestinalis assemblage A isolates
- 2015
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: The diarrhea-causing protozoan Giardia intestinalis makes up a species complex of eight different assemblages (A-H), where assemblage A and B infect humans. Comparative whole-genome analyses of three of these assemblages have shown that there is significant divergence at the inter-assemblage level, however little is currently known regarding variation at the intra-assemblage level. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic human patients, to study the biological and genetic diversity within assemblage A isolates. Results: Several biological differences between the new and earlier characterized assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 [AS175] and AII-2 [AS98]) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of the Giardia reference isolate WB, an assemblage AI isolate. Our analyses indicate that the divergence between AI and AII is approximately 1 %, represented by similar to 100,000 single nucleotide polymorphisms (SNP) distributed over the chromosomes with enrichment in variable genomic regions containing surface antigens. The level of allelic sequence heterozygosity (ASH) in the two AII isolates was found to be 0.25-0.35 %, which is 25-30 fold higher than in the WB isolate and 10 fold higher than the assemblage AII isolate DH (0.037 %). 35 protein-encoding genes, not found in the WB genome, were identified in the two AII genomes. The large gene families of variant-specific surface proteins (VSPs) and high cysteine membrane proteins (HCMPs) showed isolate-specific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 8 members, localize to the variable regions of the genomes and show high sequence diversity between the assemblage A isolates. One of the families, Bactericidal/ Permeability Increasing-like protein (BPIL), with eight members was characterized further and the proteins were shown to localize to the ER in trophozoites. Conclusions: Giardia genomes are modular with highly conserved core regions mixed up by variable regions containing high levels of ASH, SNPs and variable surface antigens. There are significant genomic variations in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms and these differences mainly localize to the variable regions of the genomes. The large genetic differences within one assemblage of G. intestinalis strengthen the argument that the assemblages represent different Giardia species.
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| 3. |
- Brolund, Alma, et al.
(författare)
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Plasmidome-Analysis of ESBL-Producing Escherichia coli Using Conventional Typing and High-Throughput Sequencing
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:6, s. e65793-
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Tidskriftsartikel (refereegranskat)abstract
- Infections caused by Extended spectrum beta-lactamase (ESBL)-producing E. coli are an emerging global problem, threatening the effectiveness of the extensively used beta-lactam antibiotics. ESBL dissemination is facilitated by plasmids, transposons, and other mobile elements. We have characterized the plasmid content of ESBL-producing E. coli from human urinary tract infections. Ten diverse isolates were selected; they had unrelated pulsed-field gel electrophoresis (PFGE) types (<90% similarity), were from geographically dispersed locations and had diverging antibiotic resistance profiles. Three isolates belonged to the globally disseminated sequence type ST131. ESBL-genes of the CTX-M-1 and CTX-M-9 phylogroups were identified in all ten isolates. The plasmid content (plasmidome) of each strain was analyzed using a combination of molecular methods and high-throughput sequencing. Hidden Markov Model-based analysis of unassembled sequencing reads was used to analyze the genetic diversity of the plasmid samples and to detect resistance genes. Each isolate contained between two and eight distinct plasmids, and at least 22 large plasmids were identified overall. The plasmids were variants of pUTI89, pKF3-70, pEK499, pKF3-140, pKF3-70, p1ESCUM, pEK204, pHK17a, p083CORR, R64, pLF82, pSFO157, and R721. In addition, small cryptic high copy-number plasmids were frequent, containing one to seven open reading frames per plasmid. Three clustered groups of such small cryptic plasmids could be distinguished based on sequence similarity. Extrachromosomal prophages were found in three isolates. Two of them resembled the E. coli P1 phage and one was previously unknown. The present study confirms plasmid multiplicity in multi-resistant E. coli. We conclude that high-throughput sequencing successfully provides information on the extrachromosomal gene content and can be used to generate a genetic fingerprint of possible use in epidemiology. This could be a valuable tool for tracing plasmids in outbreaks.
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| 4. |
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| 5. |
- Chan, Sherwin, et al.
(författare)
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Regulation of PfEMP1-VAR2CSA translation by a Plasmodium translation-enhancing factor
- 2017
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Ingår i: Nature Microbiology. - : Springer Science and Business Media LLC. - 2058-5276. ; 2:7
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Tidskriftsartikel (refereegranskat)abstract
- Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.
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| 6. |
- Franzen, Oscar, et al.
(författare)
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Draft genome sequencing of Giardia intestinalis assemblage B isolate GS : is human giardiasis caused by two different species?
- 2009
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Ingår i: PLoS Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 5:8, s. e1000560-
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Tidskriftsartikel (refereegranskat)abstract
- Giardia intestinalis is a major cause of diarrheal disease worldwide and two major Giardia genotypes, assemblages A and B, infect humans. The genome of assemblage A parasite WB was recently sequenced, and the structurally compact 11.7 Mbp genome contains simplified basic cellular machineries and metabolism. We here performed 454 sequencing to 16 x coverage of the assemblage B isolate GS, the only Giardia isolate successfully used to experimentally infect animals and humans. The two genomes show 77% nucleotide and 78% amino-acid identity in protein coding regions. Comparative analysis identified 28 unique GS and 3 unique WB protein coding genes, and the variable surface protein (VSP) repertoires of the two isolates are completely different. The promoters of several enzymes involved in the synthesis of the cyst-wall lack binding sites for encystation-specific transcription factors in GS. Several synteny-breaks were detected and verified. The tetraploid GS genome shows higher levels of overall allelic sequence polymorphism (0.5 versus <0.01% in WB). The genomic differences between WB and GS may explain some of the observed biological and clinical differences between the two isolates, and it suggests that assemblage A and B Giardia can be two different species.
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| 7. |
- Franzén, Oscar
(författare)
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Genome and transcriptome studies of the protozoan parasites Trypanosoma cruzi and Giardia intestinalis
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Trypanosoma cruzi and Giardia intestinalis are two human pathogens and protozoan parasites responsible for the diseases Chagas disease and giardiasis, respectively. Both diseases cause su ering and illness in several million individuals. The former disease occurs primarily in South America and Central America, and the latter disease occurs worldwide. Current therapeutics are toxic and lack efficacy, and potential vaccines are far from the market. Increased knowledge about the biology of these parasites is essential for drug and vaccine development, and new diagnostic tests. In this thesis, high-throughput sequencing was applied together with extensive bioinformatic analyses to yield insights into the biology and evolution of Trypanosoma cruzi and Giardia intestinalis. Bioinformatics analysis of DNA and RNA sequences was performed to identify features that may be of importance for parasite biology and functional characterization. This thesis is based on five papers (i-v). Paper i and ii describe comparative genome studies of three distinct genotypes of Giardia intestinalis (A, B and E). The genome-wide divergence between A and B was 23% and 13% between A and E. 4557 groups of three-way orthologs were defined across the three genomes. 5 to 38 genotype-specific genes were identified, along with genomic rearrangements. Genes encoding surface antigens, vsps, had undergone extensive diversification in the three genotypes. Several bacterial gene transfers were identified, one of which encoded an acetyltransferase protein in the E genotype. Paper iii describes a genome comparison of the human infecting Trypanosoma cruzi with the bat-restricted subspecies Trypanosoma cruzi marinkellei. The human infecting parasite had an 11% larger genome, and was found to have expanded repertoires of sequences related to surface antigens. The two parasites had a shared 'core' gene complement. One recent horizontal gene transfer was identified in T. c. marinkellei, representing a eukaryoteto-eukaryote transfer from a photosynthesizing organism. Paper iv describes the repertoire of small non-coding RNAs in Trypanosoma cruzi epimastigotes. Sequenced small RNAs were in the size range 16 to 61 nucleotides, and the majority were derived from transfer RNAs and other non-coding RNAs. 92 novel transcribed loci were identified in the genome, 79 of which were without similarity to known RNA classes. One population of small RNAs were derived from protein-coding genes. Paper v describes transcriptome analysis using paired-end RNA-Seq of three distinct genotypes of Giardia intestinalis (A, B and E). Gene expression profiles recapitulated the known phylogeny of the examined genotypes, and 61 to 176 genes were differentially expressed. 49,027 distinct polyadenylation sites were mapped and compared, and the median 30UTR length was 80 nucleotides (A). One 36-nt novel intron was identified and the previously reported introns (5) were confirmed.
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| 8. |
- Franzen, Oscar, et al.
(författare)
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Global analysis of A-to-I RNA editing reveals association with common disease variants
- 2018
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Ingår i: PeerJ. - : PeerJ. - 2167-8359. ; 6
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Tidskriftsartikel (refereegranskat)abstract
- RNA editing modifies transcripts and may alter their regulation or function. In humans, the most common modification is adenosine to inosine (A-to-I). We examined the global characteristics of RNA editing in 4,301 human tissue samples. More than 1.6 million A-to-I edits were identified in 62% of all protein-coding transcripts. mRNA recoding was extremely rare; only 11 novel recoding sites were uncovered. Thirty single nucleotide polymorphisms from genome-wide association studies were associated with RNA editing; one that influences type 2 diabetes (rs2028299) was associated with editing in ARPIN. Twenty-five genes, including LRP11 and PLIN5, had editing sites that were associated with plasma lipid levels. Our findings provide new insights into the genetic regulation of RNA editing and establish a rich catalogue for further exploration of this process.
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| 9. |
- Franzen, Oscar, et al.
(författare)
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The Short Non-Coding Transcriptome of the Protozoan Parasite Trypanosoma cruzi
- 2011
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Ingår i: PLoS Neglected Tropical Diseases. - : Public Library of Science (PLoS). - 1935-2727 .- 1935-2735. ; 5:8, s. e1283-
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Tidskriftsartikel (refereegranskat)abstract
- The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16-61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95-98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3' end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes.
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| 10. |
- Franzén, Oscar, et al.
(författare)
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Transcriptome Profiling of Giardia intestinalis Using Strand-specific RNAseq
- 2013
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Ingår i: PloS Computational Biology. - : Public Library of Science (PLoS). - 1553-734X .- 1553-7358. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Giardia intestinalis is a common cause of diarrheal disease and it consists of eight genetically distinct genotypes or assemblages (A-H). Only assemblages A and B infect humans and are suggested to represent two different Giardia species. Correlations exist between assemblage type and host-specificity and to some extent symptoms. Phenotypical differences have been documented between assemblages and genome sequences are available for A, B and E. We have characterized and compared the polyadenylated transcriptomes of assemblages A, B and E. Four genetically different isolates were studied (WB (AI), AS175 (AII), P15 (E) and GS (B)) using paired-end, strand-specific RNA-seq. Most ofthe genome was transcribed in trophozoites grown in vitro, but at vastly different levels.RNA-seq confirmed many of the present annotations and refined the current genome annotation. Gene expression divergence was found to recapitulate the known phylogeny, and uncovered lineage-specific differences in expression. Polyadenylation sites were mapped for over 70% of the genes and revealed many examples of conserved and unexpectedly long 3' UTRs. 28 open reading frames were found in a non-transcribed gene cluster on chromosome 5 of the WB isolate. Analysis of allele-specific expression revealed a correlation between allele-dosage and allele expression in the GS isolate. Previously reported cis-splicing events were confirmed and global mapping of cis-splicing identified only one novel intron. These observations can possibly explain differences in host-preference and symptoms, and it will be the basis for further studies of Giardia pathogenesis and biology.
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