| 1. |
- B. Moreno, Anaísa
(author)
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Evolution and host-specific adaptations of Legionella pneumophila
- 2022
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Doctoral thesis (other academic/artistic)abstract
- How bacteria evolve pathogenic traits is shaped by their communities and environments. Legionella pneumophila is ubiquitous in aquatic habitats, where it persists by replicating within a broad range of protozoan hosts. Using the same mechanisms, L. pneumophila may also accidentally infect humans, causing a severe pneumonia known as Legionnaires’ disease. As hosts, humans are evolutionary dead-ends, resulting in the loss of human-specific adaptations after infection. This thesis aims to identify and characterise these host adaptations.In Paper I, we study the in-patient evolution of L. pneumophila. We collected a large set of strains from sporadic infections and outbreaks, pairing clinical isolates with their respective environmental sources. Using comparative genomic analyses, we identified two genes individually mutated in three independent infections. One gene encoded an outer membrane protein, a homolog from the OmpP1/FadL family, and the other an EAL domain-containing protein. These results suggest that convergent evolution may be at play and that these mutations are potential candidates for human-specific host adaptations.In Paper II, we investigate host adaptation and the selective pressures that drive it using a long-term experimental evolution approach. We passaged L. pneumophila in Acanthamoeba castellanii and U937 macrophages, separately and in alternation, for over 800 generations. We found 49 fixed mutations across the 18 evolved populations: two distinct mutations in RpsL, which confers streptomycin resistance, as well as two additional mutations, each consistently associated with one of the former, in the chaperonin GroES or in RpsD, a known compensatory mutation. Mutations in the lipopolysaccharide synthesis operon were observed only in lineages passaged in A. castellanii, whilst mutations in LerC were fixed in six lineages passaged in U937, making these candidate mutations for host-specific adaptations.In Paper III, we shift focus to A. castellanii, a natural host of L. pneumophila. We describe a novel method for high-efficiency transfection of this amoeba with a cationic polymer. Using a systematic approach to test different parameters, we found that widely available and inexpensive polyethylenimines can be used to transfect A. castellanii at a much greater efficiency than the currently used reagents.In conclusion, these studies suggest that although L. pneumophila can infect humans, it is sub-optimally adapted for it, and offer potential determinants of host-specificity in L. pneumophila.
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| 2. |
- Berggren, Sofia, et al.
(author)
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ProQ-dependent activation of Salmonella virulence genes mediated by post-transcriptional control of PhoP synthesis
- 2024
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In: mSphere. - : American Society for Microbiology. - 2379-5042. ; 9:3
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Journal article (peer-reviewed)abstract
- Gastrointestinal disease caused by Salmonella enterica is associated with the pathogen's ability to replicate within epithelial cells and macrophages. Upon host cell entry, the bacteria express a type-three secretion system encoded within Salmonella pathogenicity island 2, through which host-manipulating effector proteins are secreted to establish a stable intracellular niche. Transcription of this intracellular virulence program is activated by the PhoPQ two-component system that senses the low pH and the reduced magnesium concentration of host cell vacuoles. In addition to transcriptional control, Salmonella commonly employ RNA-binding proteins (RBPs) and small regulatory RNAs (sRNAs) to regulate gene expression at the post-transcriptional level. ProQ is a globally acting RBP in Salmonella that promotes expression of the intracellular virulence program, but its RNA repertoire has previously been characterized only under standard laboratory growth conditions. Here, we provide a high-resolution ProQ interactome during conditions mimicking the environment of the Salmonella-containing vacuole (SCV), revealing hundreds of previously unknown ProQ binding sites in sRNAs and mRNA 3 ' UTRs. ProQ positively affected both the levels and the stability of many sRNA ligands, some of which were previously shown to associate with the well-studied and infection-relevant RBP Hfq. We further show that ProQ activates the expression of PhoP at the post-transcriptional level, which, in turn, leads to upregulation of the intracellular virulence program.
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| 3. |
- Diesend, Jan, et al.
(author)
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Fractional 2 '-O-methylation in the ribosomal RNA of Dictyostelium discoideum supports ribosome heterogeneity in Amoebozoa
- 2022
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In: Scientific Reports. - : Springer Nature. - 2045-2322. ; 12:1
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Journal article (peer-reviewed)abstract
- A hallmark of ribosomal RNA (rRNA) are 2 '-O-methyl groups that are introduced sequence specifically by box C/D small nucleolar RNAs (snoRNAs) in ribonucleoprotein particles. Most data on this chemical modification and its impact on RNA folding and stability are derived from organisms of the Opisthokonta supergroup. Using bioinformatics and RNA-seq data, we identify 30 novel box C/D snoRNAs in Dictyostelium discoideum, many of which are differentially expressed during the multicellular development of the amoeba. By applying RiboMeth-seq, we find 49 positions in the 17S and 26S rRNA 2 '-O-methylated. Several of these nucleotides are substoichiometrically modified, with one displaying dynamic modification levels during development. Using homology-based models for the D. discoideum rRNA secondary structures, we localize many modified nucleotides in the vicinity of the ribosomal A, P and E sites. For most modified positions, a guiding box C/D snoRNA could be identified, allowing to determine idiosyncratic features of the snoRNA/rRNA interactions in the amoeba. Our data from D. discoideum represents the first evidence for ribosome heterogeneity in the Amoebozoa supergroup, allowing to suggest that it is a common feature of all eukaryotes.
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| 4. |
- Edelbroek, Bart, et al.
(author)
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Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity
- 2024
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In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 52:6, s. 3121-3136
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Journal article (peer-reviewed)abstract
- MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.
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| 5. |
- Edelbroek, Bart, et al.
(author)
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Evolution of microRNAs in Amoebozoa and implications for the origin of multicellularity
- 2024
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In: Nucleic Acids Research. - 0305-1048 .- 1362-4962.
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Journal article (peer-reviewed)abstract
- MicroRNAs (miRNAs) are important and ubiquitous regulators of gene expression in both plants and animals. They are thought to have evolved convergently in these lineages and hypothesized to have played a role in the evolution of multicellularity. In line with this hypothesis, miRNAs have so far only been described in few unicellular eukaryotes. Here, we investigate the presence and evolution of miRNAs in Amoebozoa, focusing on species belonging to Acanthamoeba, Physarum and dictyostelid taxonomic groups, representing a range of unicellular and multicellular lifestyles. miRNAs that adhere to both the stringent plant and animal miRNA criteria were identified in all examined amoebae, expanding the total number of protists harbouring miRNAs from 7 to 15. We found conserved miRNAs between closely related species, but the majority of species feature only unique miRNAs. This shows rapid gain and/or loss of miRNAs in Amoebozoa, further illustrated by a detailed comparison between two evolutionary closely related dictyostelids. Additionally, loss of miRNAs in the Dictyostelium discoideum drnB mutant did not seem to affect multicellular development and, hence, demonstrates that the presence of miRNAs does not appear to be a strict requirement for the transition from uni- to multicellular life.
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| 6. |
- Edelbroek, Bart
(author)
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Function and Evolution of Small Regulatory RNAs and their Associated Proteins : A Journey from Genome to Proteome
- 2024
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Doctoral thesis (other academic/artistic)abstract
- Organisms throughout the tree of life have evolved distinct ways to regulate gene expression. Some of these processes involve non-coding RNAs (ncRNAs), which are not translated but functional nonetheless. These ncRNAs are of utmost importance, with dysregulation of some causing severe developmental effects or even being lethal.In order to get a better fundamental understanding of gene regulation, and the ncRNAs that evolved to regulate gene expression, we study this in Amoebozoa. Members of this taxon vary greatly in lifestyle and organismal complexity. Some are strictly unicellular, free-living, whereas others, such as the social amoeba Dictyostelium discoideum can transition between unicellular and multicellular lifestyles. D. discoideum features a variety of small ncRNAs. Among these are the microRNAs. microRNAs have mostly been studied in plants and animals, where they are believed to have evolved convergently, and hypothesized to have played a role when these taxa evolved multicellular lifestyles. At what point the D. discoideum microRNAs evolved, how they function, and if they are involved in its multicellular lifestyle are fundamental questions addressed in this thesis. Here, we studied the evolution and function of microRNAs in a broad set of species belonging to Amoebozoa. We could identify microRNAs in all studied amoebae, and concluded that they are probably not involved in the evolution of multicellularity. To in detail investigate the evolution of microRNAs, we performed comparative genomics using D. discoideum and the close relative Dictyostelium firmibasis. For this, we sequenced, assembled and annotated the genome of the latter. At this point, our findings suggest that the microRNAs evolved several times in Amoebozoa, although we cannot rule out if they have a deep evolutionary history.The Class I RNAs are another type of ncRNAs. These, on the other hand, are only present in the social amoebae. They are hypothesized to regulate the transition from unicellular to multicellular in these species, potentially in a post-transcriptional manner. In order to investigate this, it is essential to understand to what extent the proteome and transcriptome correlate. Hence, we performed paired transcriptomics and proteomics in a time-series during multicellular development. By including a strain in which a specific Class I RNA is knocked out, we have initiated studies of its role during the transition to multicellularity.In conclusion, we were able to answer broad evolutionary and functional questions about gene regulation and ncRNAs by studying Amoebozoa from genome to proteome.
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| 7. |
- Kjellin, Jonas, et al.
(author)
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Abundantly expressed class of noncoding RNAs conserved through the multicellular evolution of dictyostelid social amoebas
- 2021
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In: Genome Research. - : Cold Spring Harbor Laboratory Press (CSHL). - 1088-9051 .- 1549-5469. ; 31:3, s. 436-447
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Journal article (peer-reviewed)abstract
- Aggregative multicellularity has evolved multiple times in diverse groups of eukaryotes, exemplified by the well-studied development of dictyostelid social amoebas, for example, Dictyostelium discoideum. However, it is still poorly understood why multicellularity emerged in these amoebas while the majority of other members of Amoebozoa are unicellular. Previously, a novel type of noncoding RNA, Class I RNAs, was identified in D. discoideum and shown to be important for normal multicellular development. Here, we investigated Class I RNA evolution and its connection to multicellular development. We identified a large number of new Class I RNA genes by constructing a covariance model combined with a scoring system based on conserved upstream sequences. Multiple genes were predicted in representatives of each major group of Dictyostelia and expression analysis confirmed that our search approach identifies expressed Class I RNA genes with high accuracy and sensitivity and that the RNAs are developmentally regulated. Further studies showed that Class I RNAs are ubiquitous in Dictyostelia and share highly conserved structure and sequence motifs. In addition, Class I RNA genes appear to be unique to dictyostelid social amoebas because they could not be identified in outgroup genomes, including their closest known relatives. Our results show that Class I RNA is an ancient class of ncRNAs, likely to have been present in the last common ancestor of Dictyostelia dating back at least 600 million years. Based on previous functional analyses and the presented evolutionary investigation, we hypothesize that Class I RNAs were involved in evolution of multicellularity in Dictyostelia.
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| 8. |
- Kjellin, Jonas, 1986-
(author)
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All Roads Lead to the Non-Coding RNome : Evolution of Multicellularity and Host Response to Bacterial Infection
- 2020
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Doctoral thesis (other academic/artistic)abstract
- The ability to control gene expression is fundamental for all living organisms. Therefore, a large variety of regulatory mechanisms exist in each cell which are essential for e.g. developmental processes and to quickly adapt to different cellular stresses such as infection. Today we know that much of this regulation depends on non-coding (nc)RNAs. However, the function and evolutionary origin of many ncRNAs remains to be understood.The work presented in this thesis revolves around the evolutionary group of Dictyostelia. These social amoebae grow as single cells but initiate a multicellular development program when food runs low. The evolutionary position of Dictyostelia within Amoebozoa together with their multicellular development make these organisms relevant for investigating the evolution of ncRNAs and their association with multicellularity. Furthermore, the dictyostelid Dictyostelium discoideum is one of few organisms besides plants and animals were miRNAs have been identified. It is also an established model organism, well-adapted for laboratory growth and detailed molecular work.In this thesis, we investigate the biogenesis of miRNAs in D. discoideum and show that the Dicer-like protein DrnB is essential for global miRNA maturation. Next, we study the evolution of another ncRNA, Class I RNAs, and show that these are conserved in all dictyostelids and likely emerged in their last common ancestor. Lastly, we utilize the D. discoideum infection model to study the regulation of messenger RNAs and ncRNAs upon infection by Mycobacterium marinum and Legionella pneumophila to improve our understanding of the complex interactions between host and pathogen. We show that the two bacteria induce distinct mRNA regulation in D. discoideum. In addition, we detected high levels of specific tRNA halves generated in the host in response to M. marinum but not L. pneumophila or bacteria utilized as food. Despite the large evolutionary distances, the regulation of both mRNAs and ncRNAs in D. discoideum was, in many aspects, representative for the regulation in macrophages after infection.In conclusion, by using a seemingly simple group of organisms, social amoebae, this thesis work addresses major questions such as the role of ncRNA in multicellular evolution and the intricate host-pathogen interplay during bacterial infection.
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| 9. |
- Rizvanovic, Alisa, et al.
(author)
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Saturation mutagenesis charts the functional landscape of Salmonella ProQ and reveals a gene regulatory function of its C-terminal domain
- 2021
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In: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 49:17, s. 9992-10006
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Journal article (peer-reviewed)abstract
- The global RNA-binding protein ProQ has emerged as a central player in post-transcriptional regulatory networks in bacteria. While the N-terminal domain (NTD) of ProQ harbors the major RNA-binding activity, the role of the ProQ C-terminal domain (CTD) has remained unclear. Here, we have applied saturation mutagenesis coupled to phenotypic sorting and long-read sequencing to chart the regulatory capacity of Salmonella ProQ. Parallel monitoring of thousands of ProQ mutants allowed mapping of critical residues in both the NTD and the CTD, while the linker separating these domains was tolerant to mutations. Single amino acid substitutions in the NTD associated with abolished regulatory capacity strongly align with RNA-binding deficiency. An observed cellular instability of ProQ associated with mutations in the NTD suggests that interaction with RNA protects ProQ from degradation. Mutation of conserved CTD residues led to overstabilization of RNA targets and rendered ProQ inert in regulation, without affecting protein stability in vivo. Furthermore, ProQ lacking the CTD, although binding competent, failed to protect an mRNA target from degradation. Together, our data provide a comprehensive overview of residues important for ProQ-dependent regulation and reveal an essential role for the enigmatic ProQ CTD in gene regulation.
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