| 1. |
- Rosendal, Ebba, et al.
(författare)
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Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections
- 2022
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Ingår i: mBio. - : American Society for Microbiology. - 2161-2129 .- 2150-7511. ; 13:3
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Tidskriftsartikel (refereegranskat)abstract
- The coronavirus disease 2019, COVID-19, is a complex disease with a wide range of symptoms from asymptomatic infections to severe acute respiratory syndrome with lethal outcome. Individual factors such as age, sex, and comorbidities increase the risk for severe infections, but other aspects, such as genetic variations, are also likely to affect the susceptibility to SARS-CoV-2 infection and disease severity. Here, we used a human 3D lung cell model based on primary cells derived from multiple donors to identity host factors that regulate SARS-CoV-2 infection. With a transcriptomics-based approach, we found that less susceptible donors show a higher expression level of serine protease inhibitors SERPINA1, SERPINE1, and SERPINE2, identifying variation in cellular serpin levels as restricting host factors for SARS-CoV-2 infection. We pinpoint their antiviral mechanism of action to inhibition of the cellular serine protease, TMPRSS2, thereby preventing cleavage of the viral spike protein and TMPRSS2-mediated entry into the target cells. By means of single-cell RNA sequencing, we further locate the expression of the individual serpins to basal, ciliated, club, and goblet cells. Our results add to the importance of genetic variations as determinants for SARS-CoV-2 susceptibility and suggest that genetic deficiencies of cellular serpins might represent risk factors for severe COVID-19. Our study further highlights TMPRSS2 as a promising target for antiviral intervention and opens the door for the usage of locally administered serpins as a treatment against COVID-19.
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| 2. |
- Ankarklev, Johan, et al.
(författare)
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Common Coinfections of Giardia intestinalis and Helicobacter pylori in Non-Symptomatic Ugandan Children
- 2012
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Ingår i: PLOS Neglected Tropical Diseases. - : Public Library of Science (PLoS). - 1935-2735. ; 6:8, s. e1780-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The protozoan parasite Giardia intestinalis and the pathogenic bacterium Helicobacter pylori are well known for their high prevalences in human hosts worldwide. The prevalence of both organisms is known to peak in densely populated, low resource settings and children are infected early in life. Different Giardia genotypes/assemblages have been associated with different symptoms and H. pylori with induction of cancer. Despite this, not much data are available from sub-Saharan Africa with regards to the prevalence of different G. intestinalis assemblages and their potential association with H. pylori infections.Methodology/Principal Findings: Fecal samples from 427 apparently healthy children, 0-12 years of age, living in urban Kampala, Uganda were analyzed for the presence of H. pylori and G. intestinalis. G. intestinalis was found in 86 (20.1%) out of the children and children age 1<5 years had the highest rates of colonization. H. pylori was found in 189 (44.3%) out of the 427 children and there was a 3-fold higher risk of concomitant G. intestinalis and H. pylori infections compared to non-concomitant G. intestinalis infection, OR = 2.9 (1.7-4.8). No significant association was found in the studied population with regard to the presence of Giardia and gender, type of toilet, source of drinking water or type of housing. A panel of 45 G. intestinalis positive samples was further analyzed using multi-locus genotyping (MLG) on three loci, combined with assemblage-specific analyses. Giardia MLG analysis yielded a total of five assemblage AII, 25 assemblage B, and four mixed assemblage infections. The assemblage B isolates were highly genetically variable but no significant association was found between Giardia assemblage type and H. pylori infection.Conclusions/Significance: This study shows that Giardia assemblage B dominates in children in Kampala, Uganda and that the presence of H. pylori is an associated risk factor for G. intestinalis infection.
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| 3. |
- Hildebrandt, Franziska, 1994-, et al.
(författare)
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scDual-Seq of Toxoplasma gondii-infected mouse BMDCs reveals heterogeneity and differential infection dynamics
- 2023
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Ingår i: Frontiers in Immunology. - : Frontiers Media S.A.. - 1664-3224. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- Dendritic cells and macrophages are integral parts of the innate immune system and gatekeepers against infection. The protozoan pathogen, Toxoplasma gondii, is known to hijack host immune cells and modulate their immune response, making it a compelling model to study host-pathogen interactions. Here we utilize single cell Dual RNA-seq to parse out heterogeneous transcription of mouse bone marrow-derived dendritic cells (BMDCs) infected with two distinct genotypes of T. gondii parasites, over multiple time points post infection. We show that the BMDCs elicit differential responses towards T. gondii infection and that the two parasite lineages distinctly manipulate subpopulations of infected BMDCs. Co-expression networks define host and parasite genes, with implications for modulation of host immunity. Integrative analysis validates previously established immune pathways and additionally, suggests novel candidate genes involved in host-pathogen interactions. Altogether, this study provides a comprehensive resource for characterizing host-pathogen interplay at high-resolution.
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| 4. |
- Mohammed, Mubasher, Msc, et al.
(författare)
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Single-cell transcriptomics reveals transcriptional programs underlying male and female cell fate during Plasmodium falciparum gametocytogenesis
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The Apicomplexa constitute a large phylum of parasitic protozoa with complex life cycles that typically include meiotic sex. The life cycle of the malaria parasite, Plasmodium falciparum, includes obligate transition and stage development between a human and mosquito host. Asexual parasite replication in the human erythrocytes is followed by differentiation which leads to the formation of a precursor gamete stage, referred to as gametocytes. The gametocyte stage is solely responsible for malaria transmission into the mosquito vector where gamete fusion followed by meiosis occurs. How the parasite differentiates into male and female gametocytes in the absence of sex chromosomes largely remains an open question. Here we combine FACS-based cell enrichment of a gametocyte reporter line followed by single-cell RNA-seq, to enable targeted characterization of the entire gametocyte developmental stage. Our data defines differential transcriptional programs during male and female gametocyte development and highlights a bifurcation point for sexual cell fate. We perform prediction analyses of novel candidate driver genes underlying P. falciparum male and female lineage development. Our data indicate that a large panel of genes linked to the inner membrane complex, known to be involved in morphological life cycle changes, appears to be uniquely expressed in the female gametocyte lineage. Additionally, we delineate the timing of expression of members of the ApiAP2 family of transcription factors and predict their specificity in male and female P. falciparum gametocyte development. A motif-driven gene regulatory network analysis indicates a major role for AP2-G5 in downstream gene regulation along the male lineage developmental trajectory. In total, we anticipate that this study provides the malaria community with an important resource for the development of transmission-blocking intervention strategies.
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| 5. |
- Mohammed, Mubasher, Msc, et al.
(författare)
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Single-Cell Transcriptomics To Define Plasmodium falciparum Stage Transition in the Mosquito Midgut
- 2023
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Ingår i: Microbiology Spectrum. - : American Society for Microbiology. - 2165-0497. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- Malaria inflicts the highest rate of morbidity and mortality among the vector-borne diseases. The dramatic bottleneck of parasite numbers that occurs in the gut of the obligatory mosquito vector provides a promising target for novel control strategies. Using single-cell transcriptomics, we analyzed Plasmodium falciparum development in the mosquito gut, from unfertilized female gametes through the first 20 h after blood feeding, including the zygote and ookinete stages. This study revealed the temporal gene expression of the ApiAP2 family of transcription factors and of parasite stress genes in response to the harsh environment of the mosquito midgut. Further, employing structural protein prediction analyses, we found several upregulated genes predicted to encode intrinsically disordered proteins (IDPs), a category of proteins known for their importance in regulation of transcription, translation, and protein-protein interactions. IDPs are known for their antigenic properties and may serve as suitable targets for antibody- or peptide-based transmission suppression strategies. In total, this study uncovers the P. falciparum transcriptome from early to late parasite development in the mosquito midgut, inside its natural vector, which provides an important resource for future malaria transmission-blocking initiatives.
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| 6. |
- 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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| 7. |
- Ankarklev, Johan, 1980-, et al.
(författare)
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Allelic sequence heterozygosity in single Giardia parasites
- 2012
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Ingår i: BMC Microbiology. - : Springer Science and Business Media LLC. - 1471-2180. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: Genetic heterogeneity has become a major inconvenience in the genotyping and molecular epidemiology of the intestinal protozoan parasite Giardia intestinalis, in particular for the major human infecting genotype, assemblage B. Sequence-based genotyping of assemblage B Giardia from patient fecal samples, where one or several of the commonly used genotyping loci (beta-giardin, triosephosphate isomerase and glutamate dehydrogenase) are implemented, is often hampered due to the presence of sequence heterogeneity in the sequencing chromatograms. This can be due to allelic sequence heterozygosity (ASH) and /or co-infections with parasites of different assemblage B sub-genotypes. Thus, two important questions have arisen; i) does ASH occur at the single cell level, and/or ii) do multiple sub-genotype infections commonly occur in patients infected with assemblage B, G. intestinalis isolates? Results: We used micromanipulation in order to isolate single Giardia intestinalis, assemblage B trophozoites (GS isolate) and cysts from human patients. Molecular analysis at the tpi loci of trophozoites from the GS lineage indicated that ASH is present at the single cell level. Analyses of assemblage B Giardia cysts from clinical samples at the bg and tpi loci also indicated ASH at the single cell level. Additionally, alignment of sequence data from several different cysts that originated from the same patient yielded different sequence patterns, thus suggesting the presence of multiple sub-assemblage infections in congruence with ASH within the same patient. Conclusions: Our results conclusively show that ASH does occur at the single cell level in assemblage B Giardia. Furthermore, sequence heterogeneity generated during sequence-based genotyping of assemblage B isolates may possess the complexity of single cell ASH in concurrence with co-infections of different assemblage B sub-genotypes. These findings explain the high abundance of sequence heterogeneity commonly found when performing sequence based genotyping of assemblage B Giardia, and illuminates the necessity of developing new G. intestinalis genotyping tools.
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| 8. |
- Ankarklev, Johan, et al.
(författare)
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Behind the smile : cell biology and disease mechanisms of Giardia species
- 2010
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Ingår i: Nature Reviews Microbiology. - : Springer Science and Business Media LLC. - 1740-1526 .- 1740-1534. ; 8:6, s. 413-422
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Forskningsöversikt (refereegranskat)abstract
- The eukaryotic intestinal parasite Giardia intestinalis was first described in 1681, when Antonie van Leeuwenhoek undertook a microscopic examination of his own diarrhoeal stool. Nowadays, although G. intestinalis is recognized as a major worldwide contributor to diarrhoeal disease in humans and other mammals, the disease mechanisms are still poorly understood. Owing to its reduced complexity and proposed early evolutionary divergence, G. intestinalis is used as a model eukaryotic system for studying many basic cellular processes. In this Review we discuss recent discoveries in the molecular cell biology and pathogenesis of G. intestinalis.
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| 9. |
- 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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| 10. |
- Ankarklev, johan, 1980-
(författare)
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Genomic variation within Giardia intestinalis assemblage A isolates
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Annan publikation (övrigt vetenskapligt/konstnärligt)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. We have performed whole genome sequencing of two sub-assemblage AII isolates, recently axenized from symptomatic patients, to study the genetic diversity within assemblage A and to identify new assemblage A-specific genotyping targets. Results: Several biological differences between the assemblage A isolates were identified, including a difference in growth medium preference. The two AII isolates were of different sub-assemblage types (AII-1 (AS98) and AII-2 (AS175)) and showed size differences in the smallest chromosomes. The amount of genetic diversity was characterized in relation to the genome of an assemblage AI isolate (WB). Our analyses indicate that the divergence between AI and AII is approximately 1%, represented by ~100,000 single nucleotide polymorphisms (SNP). Moreover, SNPs are homogeneously distributed over the chromosomes with an enrichment in regions containing surface antigens and non-coding sequences. The level of allelic sequence heterozygosity (ASH) in the two AII isolates were found to be 0.25-0.35%, which is 25-30-fold higher than in the WB isolate. 37 proteinencoding 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 isolatespecific divergences of the gene repertoires. Certain genes, often in small gene families with 2 to 7 members, showed high sequence diversity between the assemblage A isolates and they could have important roles in hostparasite interactions. A subset of the variable genes was used to develop new genotyping methods for assemblage A isolates. Conclusions: Our results show that there is a significant genomic variation in assemblage A isolates, in terms of chromosome size, gene content, surface protein repertoire and gene polymorphisms. This identified putative virulence genes and generated a new assemblage A-specific genotyping approach.
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