Publications by authors named "Dirk Linke"

95 Publications

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Diagnostics (Basel) 2021 Jul 14;11(7). Epub 2021 Jul 14.

Interfaculty Institute of Biochemistry, University of Tübingen, 72076 Tübingen, Germany.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires 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" technologies, 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/diagnostics11071259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8305138PMC
July 2021

Inward-facing glycine residues create sharp turns in β-barrel membrane proteins.

Biochim Biophys Acta Biomembr 2021 Oct 17;1863(10):183662. Epub 2021 Jun 17.

School of Physics, Georgia Institute of Technology, Atlanta, GA 30313, United States of America. Electronic address:

The transmembrane region of outer-membrane proteins (OMPs) of Gram-negative bacteria are almost exclusively β-barrels composed of between 8 and 26 β-strands. To explore the relationship between β-barrel size and shape, we modeled and simulated engineered variants of the Escherichia coli protein OmpX with 8, 10, 12, 14, and 16 β-strands. We found that while smaller barrels maintained a roughly circular shape, the 16-stranded variant developed a flattened cross section. This flat cross section impeded its ability to conduct ions, in agreement with previous experimental observations. Flattening was determined to arise from the presence of inward-facing glycines at sharp turns in the β-barrel. An analysis of all simulations revealed that glycines, on average, make significantly smaller angles with residues on neighboring strands than all other amino acids, including alanine, and create sharp turns in β-barrel cross sections. This observation was generalized to 119 unique structurally resolved OMPs. We also found that the fraction of glycines in β-barrels decreases as the strand number increases, suggesting an evolutionary role for the addition or removal of glycine in OMP sequences.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbamem.2021.183662DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8380186PMC
October 2021

Dynamic relocalization of cytosolic type III secretion system components prevents premature protein secretion at low external pH.

Nat Commun 2021 03 12;12(1):1625. Epub 2021 Mar 12.

Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

Many bacterial pathogens use a type III secretion system (T3SS) to manipulate host cells. Protein secretion by the T3SS injectisome is activated upon contact to any host cell, and it has been unclear how premature secretion is prevented during infection. Here we report that in the gastrointestinal pathogens Yersinia enterocolitica and Shigella flexneri, cytosolic injectisome components are temporarily released from the proximal interface of the injectisome at low external pH, preventing protein secretion in acidic environments, such as the stomach. We show that in Yersinia enterocolitica, low external pH is detected in the periplasm and leads to a partial dissociation of the inner membrane injectisome component SctD, which in turn causes the dissociation of the cytosolic T3SS components. This effect is reversed upon restoration of neutral pH, allowing a fast activation of the T3SS at the native target regions within the host. These findings indicate that the cytosolic components form an adaptive regulatory interface, which regulates T3SS activity in response to environmental conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-021-21863-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954860PMC
March 2021

BamA and BamD Are Essential for the Secretion of Trimeric Autotransporter Adhesins.

Front Microbiol 2021 23;12:628879. Epub 2021 Feb 23.

Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD, Australia.

The BAM complex in is composed of five proteins, BamA-E. BamA and BamD are essential for cell viability and are required for the assembly of β-barrel outer membrane proteins. Consequently, BamA and BamD are indispensable for secretion via the classical autotransporter pathway (Type 5a secretion). In contrast, BamB, BamC, and BamE are not required for the biogenesis of classical autotransporters. Recently, we demonstrated that TamA, a homologue of BamA, and its partner protein TamB, were required for efficient secretion of proteins via the classical autotransporter pathway. The trimeric autotransporters are a subset of the Type 5-secreted proteins. Unlike the classical autotransporters, they are composed of three identical polypeptide chains which must be assembled together to allow secretion of their cognate passenger domains. In contrast to the classical autotransporters, the role of the Bam and Tam complex components in the biogenesis of the trimeric autotransporters has not been investigated fully. Here, using the trimeric autotransporter SadA and the structurally similar YadA protein of spp., we identify the importance of BamA and BamD in the biogenesis of the trimeric autotransporters and reveal that BamB, BamC, BamE, TamA and TamB are not required for secretion of functional passenger domain on the cell surface.

Importance: The secretion of trimeric autotransporters (TAA's) has yet to be fully understood. Here we show that efficient secretion of TAAs requires the BamA and D proteins, but does not require BamB, C or E. In contrast to classical autotransporter secretion, neither trimeric autotransporter tested required TamA or B proteins to be functionally secreted.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2021.628879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940764PMC
February 2021

Analysis of O-Antigen-Specific Bacteriophage P22 Inactivation by Outer Membrane Vesicles.

Front Microbiol 2020 24;11:510638. Epub 2020 Sep 24.

Physical Biochemistry, Department for Biochemistry and Biology, University of Potsdam, Potsdam, Germany.

Bacteriophages use a large number of different bacterial cell envelope structures as receptors for surface attachment. As a consequence, bacterial surfaces represent a major control point for the defense against phage attack. One strategy for phage population control is the production of outer membrane vesicles (OMVs). In Gram-negative host bacteria, O-antigen-specific bacteriophages address lipopolysaccharide (LPS) to initiate infection, thus relying on an essential outer membrane glycan building block as receptor that is constantly present also in OMVs. In this work, we have analyzed interactions of (.) bacteriophage P22 with OMVs. For this, we isolated OMVs that were formed in large amounts during mechanical cell lysis of the P22 S. Typhimurium host. , these OMVs could efficiently reduce the number of infective phage particles. Fluorescence spectroscopy showed that upon interaction with OMVs, bacteriophage P22 released its DNA into the vesicle lumen. However, only about one third of the phage P22 particles actively ejected their genome. For the larger part, no genome release was observed, albeit the majority of phages in the system had lost infectivity towards their host. With OMVs, P22 ejected its DNA more rapidly and could release more DNA against elevated osmotic pressures compared to DNA release triggered with protein-free LPS aggregates. This emphasizes that OMV composition is a key feature for the regulation of infective bacteriophage particles in the system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2020.510638DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541932PMC
September 2020

CbpA is a collagen binding cell surface protein under c-di-GMP control.

Cell Surf 2019 Dec 23;5:100032. Epub 2019 Aug 23.

Centre for Integrative Microbial Evolution and Section for Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Norway.

Cyclic diguanylate (c-di-GMP) signalling affects several cellular processes in group bacteria including biofilm formation and motility, and CdgF was previously identified as a diguanylate cyclase promoting biofilm formation in C-di-GMP can exert its function as a second messenger via riboswitch binding, and a functional c-di-GMP-responsive riboswitch has been found upstream of in various group strains. Protein signature recognition predicted CbpA to be a cell wall-anchored surface protein with a fibrinogen or collagen binding domain. The aim of this study was to identify the binding ligand of CbpA and the function of CbpA in cellular processes that are part of the group c-di-GMP regulatory network. By global gene expression profiling was found to be down-regulated in a deletion mutant, and exhibited maximum expression in early exponential growth. Contrary to the wild type, a deletion mutant showed no binding to collagen in a cell adhesion assay, while a CbpA overexpression strain exhibited slightly increased collagen binding compared to the control. For both fibrinogen and fibronectin there was however no change in binding activity compared to controls, and CbpA did not appear to contribute to binding to abiotic surfaces (polystyrene, glass, steel). Also, the CbpA overexpression strain appeared to be less motile and showed a decrease in biofilm formation compared to the control. This study provides the first experimental proof that the binding ligand of the c-di-GMP regulated adhesin CbpA is collagen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tcsw.2019.100032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423583PMC
December 2019

Corrigendum: A New Strain Collection for Improved Expression of Outer Membrane Proteins.

Front Cell Infect Microbiol 2020 3;10:220. Epub 2020 Jun 3.

Section for Evolution and Genetics, Department of Biosciences, University of Oslo, Oslo, Norway.

[This corrects the article DOI: 10.3389/fcimb.2017.00464.].
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcimb.2020.00220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7285654PMC
June 2020

The inverse autotransporters of Yersinia ruckeri, YrInv and YrIlm, contribute to biofilm formation and virulence.

Environ Microbiol 2020 07 17;22(7):2939-2955. Epub 2020 May 17.

Department of Biosciences, University of Oslo, 0316, Oslo, Norway.

Yersinia ruckeri causes enteric redmouth disease (ERM) that mainly affects salmonid fishes and leads to significant economic losses in the aquaculture industry. An increasing number of outbreaks and the lack of effective vaccines against some serotypes necessitates novel measures to control ERM. Importantly, Y. ruckeri survives in the environment for long periods, presumably by forming biofilms. How the pathogen forms biofilms and which molecular factors are involved in this process, remains unclear. Yersinia ruckeri produces two surface-exposed adhesins, belonging to the inverse autotransporters (IATs), called Y. ruckeri invasin (YrInv) and Y. ruckeri invasin-like molecule (YrIlm). Here, we investigated whether YrInv and YrIlm play a role in biofilm formation and virulence. Functional assays revealed that YrInv and YrIlm promote biofilm formation on different abiotic substrates. Confocal microscopy revealed that they are involved in microcolony interaction and formation, respectively. The effect of both IATs on biofilm formation correlated with the presence of different biopolymers in the biofilm matrix, including extracellular DNA, RNA and proteins. Moreover, YrInv and YrIlm contributed to virulence in the Galleria mellonella infection model. Taken together, we propose that both IATs are possible targets for the development of novel diagnostic and preventative strategies to control ERM.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/1462-2920.15051DOI Listing
July 2020

BamA is required for autotransporter secretion.

Biochim Biophys Acta Gen Subj 2020 07 27;1864(7):129581. Epub 2020 Feb 27.

School of Physics, Georgia Institute of Technology, Atlanta, GA 30313, United States of America. Electronic address:

Background: In Gram-negative bacteria, type Va and Vc autotransporters are proteins that contain both a secreted virulence factor (the "passenger" domain) and a β-barrel that aids its export. While it is known that the folding and insertion of the β-barrel domain utilize the β-barrel assembly machinery (BAM) complex, how the passenger domain is secreted and folded across the membrane remains to be determined. The hairpin model states that passenger domain secretion occurs independently through the fully-formed and membrane-inserted β-barrel domain via a hairpin folding intermediate. In contrast, the BamA-assisted model states that the passenger domain is secreted through a hybrid of BamA, the essential subunit of the BAM complex, and the β-barrel domain of the autotransporter.

Methods: To ascertain the models' plausibility, we have used molecular dynamics to simulate passenger domain secretion for two autotransporters, EspP and YadA.

Results: We observed that each protein's β-barrel is unable to accommodate the secreting passenger domain in a hairpin configuration without major structural distortions. Additionally, the force required for secretion through EspP's β-barrel is more than that through the BamA β-barrel.

Conclusions: Secretion of autotransporters most likely occurs through an incompletely formed β-barrel domain of the autotransporter in conjunction with BamA.

General Significance: Secretion of virulence factors is a process used by practically all pathogenic Gram-negative bacteria. Understanding this process is a necessary step towards limiting their infectious capacity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbagen.2020.129581DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7196024PMC
July 2020

The trimeric autotransporter adhesin Ata controls key virulence traits of .

Virulence 2019 12;10(1):68-81

Institute for Medical Microbiology and Infection Control, University Hospital, Goethe University, Frankfurt, Germany.

is a Gram-negative pathogen that causes a multitude of nosocomial infections. The trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that is present in 78% of all sequenced isolates but only in 2% of the closely related species and . Employing a markerless deletion mutant of ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/21505594.2018.1558693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363060PMC
December 2019

Innovative training networks: a new way of collaboration-propped PhD training.

Med Microbiol Immunol 2020 Jun 7;209(3):215-216. Epub 2019 Dec 7.

Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, P.O.Box 1066, Blindern, 0316, Oslo, Norway.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00430-019-00647-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248031PMC
June 2020

Immunogenicity of trimeric autotransporter adhesins and their potential as vaccine targets.

Med Microbiol Immunol 2020 Jun 1;209(3):243-263. Epub 2019 Dec 1.

Institute for Medical Microbiology and Infection Control, University Hospital, Goethe-University, Paul-Ehrlich-Str. 40, 60596, Frankfurt am Main, Germany.

The current problem of increasing antibiotic resistance and the resurgence of numerous infections indicate the need for novel vaccination strategies more than ever. In vaccine development, the search for and the selection of adequate vaccine antigens is the first important step. In recent years, bacterial outer membrane proteins have become of major interest, as they are the main proteins interacting with the extracellular environment. Trimeric autotransporter adhesins (TAAs) are important virulence factors in many Gram-negative bacteria, are localised on the bacterial surface, and mediate the first adherence to host cells in the course of infection. One example is the Neisseria adhesin A (NadA), which is currently used as a subunit in a licensed vaccine against Neisseria meningitidis. Other TAAs that seem promising vaccine candidates are the Acinetobacter trimeric autotransporter (Ata), the Haemophilus influenzae adhesin (Hia), and TAAs of the genus Bartonella. Here, we review the suitability of various TAAs as vaccine candidates.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00430-019-00649-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7247748PMC
June 2020

Native display of a huge homotrimeric protein fiber on the cell surface after precise domain deletion.

J Biosci Bioeng 2020 Apr 22;129(4):412-417. Epub 2019 Oct 22.

Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya University, Nagoya, Aichi 464-8603, Japan. Electronic address:

AtaA, a trimeric autotransporter adhesin from Acinetobacter sp. Tol 5, exhibits nonspecific, high adhesiveness to abiotic surfaces. For identification of the functional domains of AtaA, precise design of domain-deletion mutants is necessary so as not to cause undesirable structural distortion. Here, we designed and constructed three types of AtaA mutants from which the same domain, FGG1, was deleted. The first mutant was designed to preserve the periodicity of hydrophobic residues in the coiled-coil segments sandwiching the deleted region. After the deletion, the protein was properly displayed on the cell surface and had the same adhesive function as the wild type. Transmission electron microscopy (TEM) imaging and circular dichroism (CD) spectroscopy showed that its isolated passenger domain had the same fiber structure as in the AtaA wild type. In contrast, a mutant designed to disturb the coiled-coil periodicity at the deletion site failed to reach the cell surface. Although secretion occurred for the mutant designed with a flexible connector between the coiled coils, the cells exhibited a decrease in adhesiveness. Furthermore, TEM imaging of the mutant fibers showed bending at the fiber tip and changes in their CD spectrum indicated a decrease in secondary structure content. Thus, we succeeded to natively display the huge homotrimeric fiber structure of AtaA on the cell surface after precise deletion of a domain, maintaining the proper folding state and adhesive function by preserving its coiled-coil periodicity. This strategy enables us to construct various domain-deletion mutants of AtaA without structural distortion for complete functional mapping.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbiosc.2019.09.022DOI Listing
April 2020

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases.

Nucleic Acids Res 2019 12;47(21):10994-11006

Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, NO-0316 Oslo, Norway.

The widespread occurrence of repetitive stretches of DNA in genomes of organisms across the tree of life imposes fundamental challenges for sequencing, genome assembly, and automated annotation of genes and proteins. This multi-level problem can lead to errors in genome and protein databases that are often not recognized or acknowledged. As a consequence, end users working with sequences with repetitive regions are faced with 'ready-to-use' deposited data whose trustworthiness is difficult to determine, let alone to quantify. Here, we provide a review of the problems associated with tandem repeat sequences that originate from different stages during the sequencing-assembly-annotation-deposition workflow, and that may proliferate in public database repositories affecting all downstream analyses. As a case study, we provide examples of the Atlantic cod genome, whose sequencing and assembly were hindered by a particularly high prevalence of tandem repeats. We complement this case study with examples from other species, where mis-annotations and sequencing errors have propagated into protein databases. With this review, we aim to raise the awareness level within the community of database users, and alert scientists working in the underlying workflow of database creation that the data they omit or improperly assemble may well contain important biological information valuable to others.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868369PMC
December 2019

Overcoming Fish Defences: The Virulence Factors of .

Genes (Basel) 2019 09 11;10(9). Epub 2019 Sep 11.

Department of Biosciences, University of Oslo, 0316 Oslo, Norway.

is the causative agent of enteric redmouth disease, a bacterial infection of marine and freshwater fish. The disease mainly affects salmonids, and outbreaks have significant economic impact on fish farms all over the world. Vaccination routines are in place against the major serotypes of but are not effective in all cases. Despite the economic importance of enteric redmouth disease, a detailed molecular understanding of the disease is lacking. A considerable number of mostly omics-based studies have been performed in recent years to identify genes related to virulence. This review summarizes the knowledge on virulence factors. Understanding the molecular pathogenicity of will aid in developing more efficient vaccines and antimicrobial compounds directed against enteric redmouth disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes10090700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770984PMC
September 2019

The BtaF Adhesin Is Necessary for Full Virulence During Respiratory Infection by and Is a Novel Immunogen for Nasal Vaccination Against Infection.

Front Immunol 2019 26;10:1775. Epub 2019 Jul 26.

Cátedra de Inmunología, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Buenos Aires, Argentina.

enters their hosts mostly through mucosae from where it spreads systemically. Adhesion to extracellular matrix (ECM) components or to host cells is important for the infectious process, and is mediated by several adhesins, including the BtaF trimeric autotransporter. Although Th1 responses and gamma interferon (IFN-γ) are important for protection, antibodies able to block adhesions might also contribute to prevent infection. We evaluated the importance of BtaF for respiratory infection, and characterized the immune response and protection from mucosal challenge induced by nasal vaccination with recombinant BtaF. While lung CFU numbers did not differ at day 1 p.i. between mice intratracheally inoculated with M1330 (wild type) and those receiving a Δ mutant, they were reduced in the latter group at 7 and 30 days p.i. For vaccination studies the BtaF passenger domain was engineered and expressed as a soluble trimeric protein. Mice were immunized by the nasal route with BtaF or saline (control group) plus the mucosal adjuvant c-di-AMP. Specific anti-BtaF antibodies (IgG and IgA) were increased in serum, including a mixed IgG2a/IgG1 response. , these antibodies reduced bacterial adhesion to A549 alveolar epithelial cells. Specific IgA antibodies were also increased in several mucosae. Spleen cells from BtaF immunized mice significantly increased their IL-2, IL-5, IL-17, and IFN-γ secretion upon antigen stimulation. In cervical draining lymph nodes, antigen-experienced CD4+ T cells were maintained mainly as central memory cells. A BtaF-specific delayed-type hypersensitivity response was detected in BtaF immunized mice. Lung cells from the latter produced high levels of IFN-γ upon antigen stimulation. Although nasal immunization with BtaF did not protect mice against respiratory challenge, it conferred significant protection from intragastric challenge; the splenic load of was reduced by 3.28 log CFU in immunized mice. This study shows that nasal vaccination with BtaF+c-di-AMP protects against intragastric challenge with by inducing local and systemic antibody responses, central memory CD4+ T cells and strong Th1 responses. Therefore, although BtaF vaccination did not protect from respiratory infection, this adhesin constitutes a promising immunogen against mucosal infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fimmu.2019.01775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6676368PMC
October 2020

Colicin Z, a structurally and functionally novel colicin type that selectively kills enteroinvasive Escherichia coli and Shigella strains.

Sci Rep 2019 07 31;9(1):11127. Epub 2019 Jul 31.

Department of Biology, Faculty of Medicine, Masaryk University, Kamenice 5, Building A6, 625 00, Brno, Czech Republic.

Colicin production in Escherichia coli (E. coli) strains represents an important trait with regard to microbial survival and competition in the complex intestinal environment. A novel colicin type, colicin Z (26.3 kDa), was described as a product of an original producer, extraintestinal E. coli B1356 strain, isolated from the anorectal abscess of a 17 years-old man. The 4,007 bp plasmid (pColZ) was completely sequenced and colicin Z activity (cza) and colicin Z immunity (czi) genes were identified. The cza and czi genes are transcribed in opposite directions and encode for 237 and 151 amino acid-long proteins, respectively. Colicin Z shows a narrow inhibitory spectrum, being active only against enteroinvasive E. coli (EIEC) and Shigella strains via CjrC receptor recognition and CjrB- and ExbB-, ExbD-mediated colicin translocation. All tested EIEC and Shigella strains isolated between the years 1958-2010 were sensitive to colicin Z. The lethal effect of colicin Z was found to be directed against cell wall peptidoglycan (PG) resulting in PG degradation, as revealed by experiments with Remazol Brilliant Blue-stained purified peptidoglycans and with MALDI-TOF MS analyses of treated PG. Colicin Z represents a new class of colicins that is structurally and functionally distinct from previously studied colicin types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-47488-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6668396PMC
July 2019

A unified model for BAM function that takes into account type Vc secretion and species differences in BAM composition.

AIMS Microbiol 2018 22;4(3):455-468. Epub 2018 Jun 22.

Department of Biosciences, University of Oslo, Blindernveien 31, 0316 Oslo, Norway.

Transmembrane proteins in the outer membrane of Gram-negative bacteria are almost exclusively β-barrels. They are inserted into the outer membrane by a conserved and essential protein complex called the BAM (for β-barrel assembly machinery). In this commentary, we summarize current research into the mechanism of this protein complex and how it relates to type V secretion. Type V secretion systems are autotransporters that all contain a β-barrel transmembrane domain inserted by BAM. In type Vc systems, this domain is a homotrimer. We argue that none of the current models are sufficient to explain BAM function particularly regarding type Vc secretion. We also find that current models based on the well-studied model system mostly ignore the pronounced differences in BAM composition between different bacterial species. We propose a more holistic view on how all OMPs, including autotransporters, are incorporated into the lipid bilayer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3934/microbiol.2018.3.455DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6604945PMC
June 2018

Type V Secretion Systems: An Overview of Passenger Domain Functions.

Front Microbiol 2019 31;10:1163. Epub 2019 May 31.

Department of Biosciences, Section for Genetics and Evolutionary Biology, University of Oslo, Oslo, Norway.

Bacteria secrete proteins for different purposes such as communication, virulence functions, adhesion to surfaces, nutrient acquisition, or growth inhibition of competing bacteria. For secretion of proteins, Gram-negative bacteria have evolved different secretion systems, classified as secretion systems I through IX to date. While some of these systems consist of multiple proteins building a complex spanning the cell envelope, the type V secretion system, the subject of this review, is rather minimal. Proteins of the Type V secretion system are often called autotransporters (ATs). In the simplest case, a type V secretion system consists of only one polypeptide chain with a β-barrel translocator domain in the membrane, and an extracellular passenger or effector region. Depending on the exact domain architecture of the protein, type V secretion systems can be further separated into sub-groups termed type Va through e, and possibly another recently identified subtype termed Vf. While this classification works well when it comes to the architecture of the proteins, this is not the case for the function(s) of the secreted passenger. In this review, we will give an overview of the functions of the passengers of the different AT classes, shedding more light on the variety of functions carried out by type V secretion systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fmicb.2019.01163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555100PMC
May 2019

Assay development for the discovery of small-molecule inhibitors of YadA adhesion to collagen.

Cell Surf 2019 Dec 23;5:100025. Epub 2019 May 23.

Section for Genetics and Evolutionary Biology, Department of Biosciences, University of Oslo, Oslo, Norway.

We set out to develop scalable assays to measure bacterial adhesion to mammalian extracellular matrix proteins, with the aim to perform high-throughput screening for inhibitors. Our model system is the trimeric autotransporter adhesin YadA from that binds to collagen. Using bacterial cells expressing GFP under an inducible promotor, and co-expressing the adhesin of choice, we were able to establish a 384-well plate-based assay that allowed us to screen 28,000 compounds in 8 days (3520 compounds per day). We have collected all parameters that were essential in assay development, and describe how they can be tuned for improved performance. Out of 28,000 compounds, 5 compounds showed significant inhibitory activity, measured as loss of fluorescence compared to control wells. Our assay is easy to scale up, and can be adopted to different ECM component/Adhesin combinations. Alternatively, bacterial pathogens (harboring deletion mutants of adhesins compared to wildtype) could be used directly in the same assay if they express GFP as a reporter at high levels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tcsw.2019.100025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7388967PMC
December 2019

Catching a SPY: Using the SpyCatcher-SpyTag and Related Systems for Labeling and Localizing Bacterial Proteins.

Int J Mol Sci 2019 Apr 30;20(9). Epub 2019 Apr 30.

Bacterial Cell Surface Group, Section for Evolution and Genetics, Department of Biosciences, University of Oslo, 0316 Oslo, Norway.

The SpyCatcher-SpyTag system was developed seven years ago as a method for protein ligation. It is based on a modified domain from a surface protein (SpyCatcher), which recognizes a cognate 13-amino-acid peptide (SpyTag). Upon recognition, the two form a covalent isopeptide bond between the side chains of a lysine in SpyCatcher and an aspartate in SpyTag. This technology has been used, among other applications, to create covalently stabilized multi-protein complexes, for modular vaccine production, and to label proteins (e.g., for microscopy). The SpyTag system is versatile as the tag is a short, unfolded peptide that can be genetically fused to exposed positions in target proteins; similarly, SpyCatcher can be fused to reporter proteins such as GFP, and to epitope or purification tags. Additionally, an orthogonal system called SnoopTag-SnoopCatcher has been developed from an pilin that can be combined with SpyCatcher-SpyTag to produce protein fusions with multiple components. Furthermore, tripartite applications have been produced from both systems allowing the fusion of two peptides by a separate, catalytically active protein unit, SpyLigase or SnoopLigase. Here, we review the current state of the SpyCatcher-SpyTag and related technologies, with a particular emphasis on their use in vaccine development and in determining outer membrane protein localization and topology of surface proteins in bacteria.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms20092129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6539128PMC
April 2019

Insights into the autotransport process of a trimeric autotransporter, Yersinia Adhesin A (YadA).

Mol Microbiol 2019 03 13;111(3):844-862. Epub 2019 Feb 13.

Department of Biosciences, University of Oslo, Blindernveien 31, 0371, Oslo, Norway.

Trimeric autotransporter adhesins (TAAs) are a subset of a larger protein family called the type V secretion systems. They are localized on the cell surface of Gram-negative bacteria, function as mediators of attachment to inorganic surfaces and host cells, and thus include important virulence factors. Yersinia adhesin A (YadA) from Yersinia enterocolitica is a prototypical TAA that is used extensively to study the structure and function of the type Vc secretion system. A solid-state NMR study of the membrane anchor domain of YadA previously revealed a flexible stretch of small residues, termed the ASSA region, that links the membrane anchor to the stalk domain. In this study, we present evidence that single amino acid proline substitutions produce two different conformers of the membrane anchor domain of YadA; one with the N-termini facing the extracellular surface, and a second with the N-termini located in the periplasm. We propose that TAAs adopt a hairpin intermediate during secretion, as has been shown before for other subtypes of the type V secretion system. As the YadA transition state intermediate can be isolated from the outer membrane, future structural studies should be possible to further unravel details of the autotransport process.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/mmi.14195DOI Listing
March 2019

pYR4 From a Norwegian Isolate of Is a Putative Virulence Plasmid Encoding Both a Type IV Pilus and a Type IV Secretion System.

Front Cell Infect Microbiol 2018 30;8:373. Epub 2018 Oct 30.

Department of Biosciences, University of Oslo, Oslo, Norway.

Enteric redmouth disease caused by the pathogen is a significant problem for fish farming around the world. Despite its importance, only a few virulence factors of have been identified and studied in detail. Here, we report and analyze the complete DNA sequence of pYR4, a plasmid from a highly pathogenic Norwegian isolate, sequenced using PacBio SMRT technology. Like the well-known pYV plasmid of human pathogenic , pYR4 is a member of the IncFII family. Thirty-one percent of the pYR4 sequence is unique compared to other plasmids. The unique regions contain, among others genes, a large number of mobile genetic elements and two partitioning systems. The G+C content of pYR4 is higher than that of the NVH_3758 genome, indicating its relatively recent horizontal acquisition. pYR4, as well as the related plasmid pYR3, comprises operons that encode for type IV pili and for a conjugation system (). In contrast to other plasmids, pYR4 cannot be cured at elevated temperatures. Our study highlights the power of PacBio sequencing technology for identifying mis-assembled segments of genomic sequences. Comparative analysis of pYR4 and other plasmids and genomes, which were sequenced by second and the third generation sequencing technologies, showed errors in second generation sequencing assemblies. Specifically, in the 150 and ATCC29473 genome assemblies, we mapped the entire pYR3 plasmid sequence. Placing plasmid sequences on the chromosome can result in erroneous biological conclusions. Thus, PacBio sequencing or similar long-read methods should always be preferred for genome sequencing. As the operons of pYR3, although misplaced on the chromosome during the genome assembly process, were demonstrated to have an effect on virulence, and type IV pili are virulence factors in many bacteria, we suggest that pYR4 directly contributes to virulence.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcimb.2018.00373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6232867PMC
September 2019

A New Strain Collection for Improved Expression of Outer Membrane Proteins.

Front Cell Infect Microbiol 2017 7;7:464. Epub 2017 Nov 7.

Section for Evolution and Genetics, Department of Biosciences, University of Oslo, Oslo, Norway.

Almost all integral membrane proteins found in the outer membranes of Gram-negative bacteria belong to the transmembrane β-barrel family. These proteins are not only important for nutrient uptake and homeostasis, but are also involved in such processes as adhesion, protein secretion, biofilm formation, and virulence. As surface exposed molecules, outer membrane β-barrel proteins are also potential drug and vaccine targets. High production levels of heterologously expressed proteins are desirable for biochemical and especially structural studies, but over-expression and subsequent purification of membrane proteins, including outer membrane proteins, can be challenging. Here, we present a set of deletion mutants derived from BL21 Gold (DE3) designed for the over-expression of recombinant outer membrane proteins. These strains harbor deletions of four genes encoding abundant β-barrel proteins in the outer membrane (OmpA, OmpC, OmpF, and LamB), both single and in all combinations of double, triple, and quadruple knock-outs. The sequences encoding these outer membrane proteins were deleted completely, leaving only a minimal scar sequence, thus preventing the possibility of genetic reversion. Expression tests in the quadruple mutant strain with four test proteins, including a small outer membrane β-barrel protein and variants thereof as well as two virulence-related autotransporters, showed significantly improved expression and better quality of the produced proteins over the parent strain. Differences in growth behavior and aggregation in the presence of high salt were observed, but these phenomena did not negatively influence the expression in the quadruple mutant strain when handled as we recommend. The strains produced in this study can be used for outer membrane protein production and purification, but are also uniquely useful for labeling experiments for biophysical measurements in the native membrane environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3389/fcimb.2017.00464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681912PMC
June 2018

The repeat structure of two paralogous genes, Yersinia ruckeri invasin (yrInv) and a "Y. ruckeri invasin-like molecule", (yrIlm) sheds light on the evolution of adhesive capacities of a fish pathogen.

J Struct Biol 2018 02 6;201(2):171-183. Epub 2017 Sep 6.

Department of Biosciences, University of Oslo, 0316 Oslo, Norway. Electronic address:

Inverse autotransporters comprise the recently identified type Ve secretion system and are exemplified by intimin from enterohaemorrhagic Escherichia coli and invasin from enteropathogenic Yersiniae. These proteins share a common domain architecture and promote bacterial adhesion to host cells. Here, we identified and characterized two putative inverse autotransporter genes in the fish pathogen Yersinia ruckeri NVH_3758, namely yrInv (for Y. ruckeri invasin) and yrIlm (for Y. ruckeri invasin-like molecule). When trying to clone the highly repetitive genes for structural and functional studies, we experienced problems in obtaining PCR products. PCR failures and the highly repetitive nature of inverse autotransporters prompted us to sequence the genome of Y. ruckeri NVH_3758 using PacBio sequencing, which produces some of the longest average read lengths available in the industry at this moment. According to our sequencing data, YrIlm is composed of 2603 amino acids (7812bp) and has a molecular mass of 256.4kDa. Based on the new genome information, we performed PCR analysis on four non-sequenced Y. ruckeri strains as well as the sequenced. Y. ruckeri type strain. We found that the genes are variably present in the strains, and that the length of yrIlm, when present, also varies. In addition, the length of the gene product for all strains, including the type strain, was much longer than expected based on deposited sequences. The internal repeats of the yrInv gene product are highly diverged, but represent the same bacterial immunoglobulin-like domains as in yrIlm. Using qRT-PCR, we found that yrIlm and yrInv are differentially expressed under conditions relevant for pathogenesis. In addition, we compared the genomic context of both genes in the newly sequenced Y. ruckeri strain to all available PacBio-sequenced Y. ruckeri genomes, and found indications of recent events of horizontal gene transfer. Taken together, this study demonstrates and highlights the power of Single Molecule Real-Time technology for sequencing highly repetitive proteins, and sheds light on the genetic events that gave rise to these highly repetitive genes in a commercially important fish pathogen.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jsb.2017.08.008DOI Listing
February 2018

An evolutionarily conserved glycine-tyrosine motif forms a folding core in outer membrane proteins.

PLoS One 2017 3;12(8):e0182016. Epub 2017 Aug 3.

Department of Biosciences, University of Oslo, Oslo, Norway.

An intimate interaction between a pair of amino acids, a tyrosine and glycine on neighboring β-strands, has been previously reported to be important for the structural stability of autotransporters. Here, we show that the conservation of this interacting pair extends to nearly all major families of outer membrane β-barrel proteins, which are thought to have originated through duplication events involving an ancestral ββ hairpin. We analyzed the function of this motif using the prototypical outer membrane protein OmpX. Stopped-flow fluorescence shows that two folding processes occur in the millisecond time regime, the rates of which are reduced in the tyrosine mutant. Folding assays further demonstrate a reduction in the yield of folded protein for the mutant compared to the wild-type, as well as a reduction in thermal stability. Taken together, our data support the idea of an evolutionarily conserved 'folding core' that affects the folding, membrane insertion, and thermal stability of outer membrane protein β-barrels.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0182016PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5542473PMC
October 2017

Vitronectin Binds to a Specific Stretch within the Head Region of Yersinia Adhesin A and Thereby Modulates Yersinia enterocolitica Host Interaction.

J Innate Immun 2017 1;9(1):33-51. Epub 2016 Nov 1.

Institute for Medical Microbiology and Hygiene, University Hospital Tübingen, Tübingen, Germany.

Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the 'uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1159/000449200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6738804PMC
October 2017

Secretion of the Intimin Passenger Domain Is Driven by Protein Folding.

J Biol Chem 2016 09 27;291(38):20096-112. Epub 2016 Jul 27.

From the Evolution and Genetics, Department of Biosciences, University of Oslo, 0316 Oslo, Norway,

Intimin is an essential adhesin of attaching and effacing organisms such as entropathogenic Escherichia coli It is also the prototype of type Ve secretion or inverse autotransport, where the extracellular C-terminal region or passenger is exported with the help of an N-terminal transmembrane β-barrel domain. We recently reported a stalled secretion intermediate of intimin, where the passenger is located in the periplasm but the β-barrel is already inserted into the membrane. Stalling of this mutant is due to the insertion of an epitope tag at the very N terminus of the passenger. Here, we examined how this insertion disrupts autotransport and found that it causes misfolding of the N-terminal immunoglobulin (Ig)-like domain D00. We could also stall the secretion by making an internal deletion in D00, and introducing the epitope tag into the second Ig-like domain, D0, also resulted in reduced passenger secretion. In contrast to many classical autotransporters, where a proximal folding core in the passenger is required for secretion, the D00 domain is dispensable, as the passenger of an intimin mutant lacking D00 entirely is efficiently exported. Furthermore, the D00 domain is slightly less stable than the D0 and D1 domains, unfolding at ∼200 piconewtons (pN) compared with ∼250 pN for D0 and D1 domains as measured by atomic force microscopy. Our results support a model where the secretion of the passenger is driven by sequential folding of the extracellular Ig-like domains, leading to vectorial transport of the passenger domain across the outer membrane in an N to C direction.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M116.731497DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5025694PMC
September 2016

Epitope-Tagged Autotransporters as Single-Cell Reporters for Gene Expression by a Salmonella Typhimurium wbaP Mutant.

PLoS One 2016 5;11(5):e0154828. Epub 2016 May 5.

Institute of Microbiology, Department of Biology, ETH Zürich, Zürich, Switzerland.

Phenotypic diversity is an important trait of bacterial populations and can enhance fitness of the existing genotype in a given environment. To characterize different subpopulations, several studies have analyzed differential gene expression using fluorescent reporters. These studies visualized either single or multiple genes within single cells using different fluorescent proteins. However, variable maturation and folding kinetics of different fluorophores complicate the study of dynamics of gene expression. Here, we present a proof-of-principle study for an alternative gene expression system in a wbaP mutant of Salmonella Typhimurium (S. Tm) lacking the O-sidechain of the lipopolysaccharide. We employed the hemagglutinin (HA)-tagged inverse autotransporter invasin (invAHA) as a transcriptional reporter for the expression of the type three secretion system 1 (T1) in S. Tm. Using a two-reporter approach with GFP and the InvAHA in single cells, we verify that this reporter system can be used for T1 gene expression analysis, at least in strains lacking the O-antigen (wbaP), which are permissive for detection of the surface-exposed HA-epitope. When we placed the two reporters gfp and invAHA under the control of either one or two different promoters of the T1 regulon, we were able to show correlative expression of both reporters. We conclude that the invAHA reporter system is a suitable tool to analyze T1gene expression in S. Tm and propose its applicability as molecular tool for gene expression studies within single cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0154828PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4858243PMC
July 2017
-->