| 1. |
- Skurnik, Mikael, et al.
(författare)
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Characterization of the genome, proteome, and structure of yersiniophage φR1-37
- 2012
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Ingår i: Journal of Virology. - 0022-538X. ; 86:23, s. 12625-12642
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Tidskriftsartikel (refereegranskat)abstract
- The bacteriophage vB_YecM-φR1-37 (φR1-37) is a lytic yersiniophage that can propagate naturally in different Yersinia species carrying the correct lipopolysaccharide receptor. This large-tailed phage has deoxyuridine (dU) instead of thymidine in its DNA. In this study, we determined the genomic sequence of phage φR1-37, mapped parts of the phage transcriptome, characterized the phage particle proteome, and characterized the virion structure by cryo-electron microscopy and image reconstruction. The 262,391-bp genome of φR1-37 is one of the largest sequenced phage genomes, and it contains 367 putative open reading frames (ORFs) and 5 tRNA genes. Mass-spectrometric analysis identified 69 phage particle structural proteins with the genes scattered throughout the genome. A total of 269 of the ORFs (73%) lack homologues in sequence databases. Based on terminator and promoter sequences identified from the intergenic regions, the phage genome was predicted to consist of 40 to 60 transcriptional units. Image reconstruction revealed that the φR1-37 capsid consists of hexameric capsomers arranged on a T=27 lattice similar to the bacteriophage φKZ. The tail of φR1-37 has a contractile sheath. We conclude that phage φR1-37 is a representative of a novel phage type that carries the dU-containing genome in a φKZ-like head.
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| 2. |
- van Belkum, Alex, et al.
(författare)
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Host-pathogen adhesion as the basis of innovative diagnostics for emerging pathogens
- 2021
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Ingår i: Diagnostics. - : MDPI AG. - 2075-4418. ; 11:7
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Forskningsöversikt (refereegranskat)abstract
- Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases re-quires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen–surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin–ligand interaction, supported by present high-throughput “omics” technolo-gies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.
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| 3. |
- Skurnik, Mikael, et al.
(författare)
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Bacteriophages fEV-1 and fD1 Infect Yersinia pestis
- 2021
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 13:7
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Tidskriftsartikel (refereegranskat)abstract
- Bacteriophages vB_YpeM_fEV-1 (fEV-1) and vB_YpeM_fD1 (fD1) were isolated from incoming sewage water samples in Turku, Finland, using Yersinia pestis strains EV76 and KIM D27 as enrichment hosts, respectively. Genomic analysis and transmission electron microscopy established that fEV-1 is a novel type of dwarf myovirus, while fD1 is a T4-like myovirus. The genome sizes are 38 and 167 kb, respectively. To date, the morphology and genome sequences of some dwarf myoviruses have been described; however, a proteome characterization such as the one presented here, has currently been lacking for this group of viruses. Notably, fEV-1 is the first dwarf myovirus described for Y. pestis. The host range of fEV-1 was restricted strictly to Y. pestis strains, while that of fD1 also included other members of Enterobacterales such as Escherichia coli and Yersinia pseudotuberculosis. In this study, we present the life cycles, genomes, and proteomes of two Yersinia myoviruses, fEV-1 and fD1.
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| 4. |
- Vaca, Diana J, et al.
(författare)
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Interaction of Bartonella henselae with Fibronectin Represents the Molecular Basis for Adhesion to Host Cells
- 2022
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Ingår i: Microbiology spectrum. - : American Society for Microbiology. - 2165-0497. ; 10:3
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Tidskriftsartikel (refereegranskat)abstract
- Bacterial adhesion to the host is the most decisive step in infections. Trimeric autotransporter adhesins (TAA) are important pathogenicity factors of Gram-negative bacteria. The prototypic TAA Bartonella adhesin A (BadA) from human-pathogenic Bartonella henselae mediates bacterial adherence to endothelial cells (ECs) and extracellular matrix proteins. Here, we determined the interaction between BadA and fibronectin (Fn) to be essential for bacterial host cell adhesion. BadA interactions occur within the heparin-binding domains of Fn. The exact binding sites were revealed by mass spectrometry analysis of chemically cross-linked whole-cell bacteria and Fn. Specific BadA interactions with defined Fn regions represent the molecular basis for bacterial adhesion to ECs and these data were confirmed by BadA-deficient bacteria and CRISPR-Cas knockout Fn host cells. Interactions between TAAs and the extracellular matrix might represent the key step for adherence of human-pathogenic Gram-negative bacteria to the host. IMPORTANCE Deciphering the mechanisms of bacterial host cell adhesion is a clue for preventing infections. We describe the underestimated role that the extracellular matrix protein fibronectin plays in the adhesion of human-pathogenic Bartonella henselae to host cells. Fibronectin-binding is mediated by a trimeric autotransporter adhesin (TAA) also present in many other human-pathogenic Gram-negative bacteria. We demonstrate that both TAA and host-fibronectin contribute significantly to bacterial adhesion, and we present the exact sequence of interacting amino acids from both proteins. Our work shows the domain-specific pattern of interaction between the TAA and fibronectin to adhere to host cells and opens the perspective to fight bacterial infections by inhibiting bacterial adhesion which represents generally the first step in infections.
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| 5. |
- Vaca, Diana J, et al.
(författare)
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Interaction with the host : the role of fibronectin and extracellular matrix proteins in the adhesion of Gram-negative bacteria
- 2020
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Ingår i: Medical microbiology and immunology. - : Springer Science and Business Media LLC. - 0300-8584 .- 1432-1831. ; 209:3, s. 277-299
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Forskningsöversikt (refereegranskat)abstract
- The capacity of pathogenic microorganisms to adhere to host cells and avoid clearance by the host immune system is the initial and most decisive step leading to infections. Bacteria have developed different strategies to attach to diverse host surface structures. One important strategy is the adhesion to extracellular matrix (ECM) proteins (e.g., collagen, fibronectin, laminin) that are highly abundant in connective tissue and basement membranes. Gram-negative bacteria express variable outer membrane proteins (adhesins) to attach to the host and to initiate the process of infection. Understanding the underlying molecular mechanisms of bacterial adhesion is a prerequisite for targeting this interaction by "anti-ligands" to prevent colonization or infection of the host. Future development of such "anti-ligands" (specifically interfering with bacteria-host matrix interactions) might result in the development of a new class of anti-infective drugs for the therapy of infections caused by multidrug-resistant Gram-negative bacteria. This review summarizes our current knowledge about the manifold interactions of adhesins expressed by Gram-negative bacteria with ECM proteins and the use of this information for the generation of novel therapeutic antivirulence strategies.
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| 6. |
- Chernyaeva, Larisa, et al.
(författare)
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Reduced binding of apoE4 to complement factor H promotes amyloid-β oligomerization and neuroinflammation
- 2023
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Ingår i: EMBO Reports. - 1469-221X. ; 24:7
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Tidskriftsartikel (refereegranskat)abstract
- The APOE4 variant of apolipoprotein E (apoE) is the most prevalent genetic risk allele associated with late-onset Alzheimer's disease (AD). ApoE interacts with complement regulator factor H (FH), but the role of this interaction in AD pathogenesis is unknown. Here we elucidate the mechanism by which isoform-specific binding of apoE to FH alters Aβ1-42-mediated neurotoxicity and clearance. Flow cytometry and transcriptomic analysis reveal that apoE and FH reduce binding of Aβ1-42 to complement receptor 3 (CR3) and subsequent phagocytosis by microglia which alters expression of genes involved in AD. Moreover, FH forms complement-resistant oligomers with apoE/Aβ1-42 complexes and the formation of these complexes is isoform specific with apoE2 and apoE3 showing higher affinity to FH than apoE4. These FH/apoE complexes reduce Aβ1-42 oligomerization and toxicity, and colocalize with complement activator C1q deposited on Aβ plaques in the brain. These findings provide an important mechanistic insight into AD pathogenesis and explain how the strongest genetic risk factor for AD predisposes for neuroinflammation in the early stages of the disease pathology.
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| 7. |
- Happonen, Lotta, et al.
(författare)
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Adenosine triphosphatases of thermophilic archaeal double-stranded DNA viruses.
- 2014
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Ingår i: Cell & bioscience. - : Springer Science and Business Media LLC. - 2045-3701. ; 4:Jul 23
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Forskningsöversikt (refereegranskat)abstract
- Adenosine triphosphatases (ATPases) of double-stranded (ds) DNA archaeal viruses are structurally related to the AAA+ hexameric helicases and translocases. These ATPases have been implicated in viral life cycle functions such as DNA entry into the host, and viral genome packaging into preformed procapsids. We summarize bioinformatical analyses of a wide range of archaeal ATPases, and review the biochemical and structural properties of those archaeal ATPases that have measurable ATPase activity. We discuss their potential roles in genome delivery into the host, virus assembly and genome packaging in comparison to hexameric helicases and packaging motors from bacteriophages.
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| 8. |
- Happonen, Lotta J., et al.
(författare)
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Affinity-Purification Combined with Crosslinking Mass Spectrometry for Identification and Structural Modeling of Host-Pathogen Protein-Protein Complexes
- 2023. - 2
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Ingår i: Bacterial Pathogenesis : Book cover Book © 2023 Bacterial Pathogenesis Methods and Protocols - Book cover Book © 2023 Bacterial Pathogenesis Methods and Protocols. - 1940-6029. - 9781071632420 ; , s. 181-200
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Bokkapitel (refereegranskat)abstract
- Host-pathogen protein-protein interactions are highly complex and dynamic and mediate key steps in pathogen adhesion to host, host invasion, and colonization as well as immune evasion. In bacteria, these interactions most often involve specialized virulence factors or effector proteins that specifically target central host proteins. Here, I present a mass spectrometry-based proteomics approach starting with the identification of host-pathogen interactions by affinity-purification followed by mapping the specific host-pathogen protein-protein interaction interfaces by crosslinking mass spectrometry and structural modeling of the complexes.
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| 9. |
- Happonen, Lotta J., et al.
(författare)
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BtuB-dependent infection of the T5-like Yersinia phage φr2-01
- 2021
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Ingår i: Viruses. - : MDPI AG. - 1999-4915. ; 13:11, s. 1-18
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Tidskriftsartikel (refereegranskat)abstract
- Yersinia enterocolitica is a food-borne Gram-negative pathogen responsible for several gastrointestinal disorders. Host-specific lytic bacteriophages have been increasingly used recently as an alternative or complementary treatment to combat bacterial infections, especially when antibiotics fail. Here, we describe the proteogenomic characterization and host receptor identification of the siphovirus vB_YenS_φR2-01 (in short, φR2-01) that infects strains of several Yersinia enterocolitica serotypes. The φR2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The φR2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical φR2-01 proteins. Label-free massspectrometry-based proteomics confirmed the expression of 90 of the φR2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and φR2-01 adsorption experiments identified the outer membrane vitamin B12 receptor BtuB as the host receptor. This study provides a proteogenomic characterization of a T5-type bacteriophage and identifies specific Y. enterocolitica strains sensitive to infection with possible future applications of φR2-01 as a food biocontrol or phage therapy agent.
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| 10. |
- Happonen, Lotta, et al.
(författare)
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The Structure of the NTPase That Powers DNA Packaging into Sulfolobus Turreted Icosahedral Virus 2
- 2013
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Ingår i: Journal of Virology. - 1098-5514. ; 87:15, s. 8388-8398
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Tidskriftsartikel (refereegranskat)abstract
- Biochemical reactions powered by ATP hydrolysis are fundamental for the movement of molecules and cellular structures. One such reaction is the encapsidation of the double-stranded DNA (dsDNA) genome of an icosahedrally symmetric virus into a preformed procapsid with the help of a genome-translocating NTPase. Such NTPases have been characterized in detail from both RNA and tailed DNA viruses. We present four crystal structures and the biochemical activity of a thermophilic NTPase, B204, from the nontailed, membrane-containing, hyperthermoacidophilic archaeal dsDNA virus Sulfolobus turreted icosahedral virus 2. These are the first structures of a genome-packaging NTPase from a nontailed, dsDNA virus with an archaeal host. The four structures highlight the catalytic cycle of B204, pinpointing the molecular movement between substrate-bound (open) and empty (closed) active sites. The protein is shown to bind both single-stranded and double-stranded nucleic acids and to have an optimum activity at 80 C and pH 4.5. The overall fold of B204 places it in the FtsK-HerA superfamily of P-loop ATPases, whose cellular and viral members have been suggested to share a DNA-translocating mechanism.
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| 11. |
- Kelly, John J., et al.
(författare)
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Snapshots of actin and tubulin folding inside the TRiC chaperonin
- 2022
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Ingår i: Nature Structural and Molecular Biology. - : Springer Science and Business Media LLC. - 1545-9993 .- 1545-9985. ; 29:5, s. 420-429
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Tidskriftsartikel (refereegranskat)abstract
- The integrity of a cell’s proteome depends on correct folding of polypeptides by chaperonins. The chaperonin TCP-1 ring complex (TRiC) acts as obligate folder for >10% of cytosolic proteins, including he cytoskeletal proteins actin and tubulin. Although its architecture and how it recognizes folding substrates are emerging from structural studies, the subsequent fate of substrates inside the TRiC chamber is not defined. We trapped endogenous human TRiC with substrates (actin, tubulin) and cochaperone (PhLP2A) at different folding stages, for structure determination by cryo-EM. The already-folded regions of client proteins are anchored at the chamber wall, positioning unstructured regions toward the central space to achieve their native fold. Substrates engage with different sections of the chamber during the folding cycle, coupled to TRiC open-and-close transitions. Further, the cochaperone PhLP2A modulates folding, acting as a molecular strut between substrate and TRiC chamber. Our structural snapshots piece together an emerging model of client protein folding within TRiC.
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