| 1. |
- Fercher, Christian, et al.
(författare)
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VirB8-like protein TraH is crucial for DNA transfer in Enterococcus faecalis
- 2016
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Untreatable bacterial infections caused by a perpetual increase of antibiotic resistant strains represent a serious threat to human healthcare in the 21st century. Conjugative DNA transfer is the most important mechanism for antibiotic resistance and virulence gene dissemination among bacteria and is mediated by a protein complex, known as type IV secretion system (T4SS). The core of the T4SS is a multiprotein complex that spans the bacterial envelope as a channel for macromolecular secretion. We report the NMR structure and functional characterization of the transfer protein TraH encoded by the conjugative Gram-positive broad-host range plasmid pIP501. The structure exhibits a striking similarity to VirB8 proteins of Gram-negative secretion systems where they play an essential role in the scaffold of the secretion machinery. Considering TraM as the first VirB8-like protein discovered in pIP501, TraH represents the second protein affiliated with this family in the respective transfer operon. A markerless traH deletion in pIP501 resulted in a total loss of transfer in Enterococcus faecalis as compared with the pIP501 wild type (wt) plasmid, demonstrating that TraH is essential for pIP501 mediated conjugation. Moreover, oligomerization state and topology of TraH in the native membrane were determined providing insights in molecular organization of a Gram-positive T4SS.
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| 2. |
- Grohmann, Elisabeth, et al.
(författare)
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Acquired resistance from gene transfer
- 2019
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Ingår i: Antibiotic drug resistance. - : John Wiley & Sons. - 9781119282525 - 9781119282549 ; , s. 141-165
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Bokkapitel (refereegranskat)abstract
- The occurrence of multiple antibiotic-resistant pathogens is steadily increasing, and their presence is not limited to clinical settings as they are also encountered in the environment. Horizontal gene transfer is a crucial means of generation and spread of multiresistant pathogenic bacteria. It is subdivided into three different mechanisms: conjugation (conjugative transfer), transformation, and transduction, of which conjugative transfer of plasmids and integrative conjugative elements (ICE) is the most important one. In 2017, the World Health Organization (WHO) published a list of antibiotic-resistant bacterial pathogens for which alternative drugs or treatments need to be urgently developed. Here, we review the spread and transfer mechanisms of these antibiotic resistances and end with current approaches, which could aid in tackling them.
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| 3. |
- Kohler, Verena, 1992-, et al.
(författare)
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Broad-host-range Inc18 plasmids : Occurrence, spread and transfer mechanisms
- 2018
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Ingår i: Plasmid. - : Elsevier. - 0147-619X .- 1095-9890. ; 99, s. 11-21
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Tidskriftsartikel (refereegranskat)abstract
- Conjugative plasmid transfer is one of the major mechanisms responsible for the spread of antibiotic resistance and virulence genes. The incompatibility (Inc) 18 group of plasmids is a family of plasmids replicating by the theta-mechanism, whose members have been detected frequently in enterococci and streptococci. Inc18 plasmids encode a variety of antibiotic resistances, including resistance to vancomycin, chloramphenicol and the macrolide-lincosamide-streptogramine (MLS) group of antibiotics. These plasmids comprising insertions of Tn1546 were demonstrated to be responsible for the transfer of vancomycin resistance encoded by the vanA gene from vancomycin resistant enterococci (VRE) to methicillin resistant Staphylococcus aureus (MRSA). Thereby vancomycin resistant S. aureus (VRSA) were generated, which are serious multi-resistant pathogens challenging the health care system. Inc18 plasmids are widespread in the clinic and frequently have been detected in the environment, especially in domestic animals and wastewater. pIP501 is one of the best-characterized conjugative Inc18 plasmids. It was originally isolated from a clinical Streptococcus agalactiae strain and is, due to its small size and simplicity, a model to study conjugative plasmid transfer in Gram-positive bacteria. Here, we report on the occurrence and spread of Inc18-type plasmids in the clinic and in different environments as well as on the exchange of the plasmids among them. In addition, we discuss molecular details on the transfer mechanism of Inc18 plasmids and its regulation, as exemplified by the model plasmid pIP501. We finish with an outlook on promising approaches on how to reduce the emerging spread of antibiotic resistances.
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| 4. |
- Kohler, Verena, 1992-, et al.
(författare)
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Conjugative type IV secretion in Gram-positive pathogens : TraG, a lytic transglycosylase and endopeptidase, interacts with translocation channel protein TraM
- 2017
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Ingår i: Plasmid. - : Elsevier BV. - 0147-619X .- 1095-9890. ; 91, s. 9-18
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Tidskriftsartikel (refereegranskat)abstract
- Conjugative transfer plays a major role in the transmission of antibiotic resistance in bacteria. pIP501 is a Grampositive conjugative model plasmid with the broadest transfer host-range known so far and is frequently found in Enterococcus faecalis and Enterococcus faecium clinical isolates. The pIP501 type IV secretion system is encoded by 15 transfer genes. In this work, we focus on the VirB1-like protein TraG, a modular peptidoglycan metabolizing enzyme, and the VirB8-homolog TraM, a potential member of the translocation channel. By providing full-length traG in trans, but not with a truncated variant, we achieved full recovery of wild type transfer efficiency in the traG-knockout mutant E. faecalis pIP501AtraG. With peptidoglycan digestion experiments and tandem mass spectrometry we could assign lytic transglycosylase and endopeptidase activity to TraG, with the CHAP domain alone displaying endopeptidase activity. We identified a novel interaction between TraG and TraM in a bacterial 2-hybrid assay. In addition we found that both proteins localize in focal spots at the E. faecalis cell membrane using immunostaining and fluorescence microscopy. Extracellular protease digestion to evaluate protein cell surface exposure revealed that correct membrane localization of TraM requires the transmembrane helix of TraG. Thus, we suggest an essential role for TraG in the assembly of the pIP501 type IV secretion system.
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| 5. |
- Kohler, Verena, 1992-, et al.
(författare)
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Enterococcus adhesin PrgB facilitates type IV secretion by condensation of extracellular DNA
- 2018
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Ingår i: Molecular Microbiology. - : John Wiley & Sons. - 0950-382X .- 1365-2958. ; 109:3, s. 263-267
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Tidskriftsartikel (refereegranskat)abstract
- Conjugative type IV secretion systems (T4SSs) are multi-protein complexes in Gram-negative and Gram-positive (G+) bacteria, responsible for spreading antibiotic resistances and virulence factors among different species. Compared to Gram-negative bacteria, which establish close contacts for conjugative transfer via sex pili, G+ T4SSs are suggested to employ surface adhesins instead. One example is pCF10, an enterococcal conjugative sex-pheromone responsive plasmid with a narrow host range, thus disseminating genetic information only among closely related species. This MicroCommentary is dedicated to the crystal structure of the pCF10-encoded adhesion domain of PrgB presented by Schmitt et al. The authors show in their work that this adhesion domain is responsible for biofilm formation, tight binding and condensation of extracellular DNA (eDNA) and conjugative transfer of pCF10. A sophisticated two-step mechanism for highly efficient conjugative transfer is postulated, including the formation of PrgB-mediated long-range intercellular contacts by binding and establishment of shorter-range contacts via condensation of eDNA. PrgB binding to lipoteichoic acid on the recipient cell surface stabilizes junctions between the mating partners. The major findings by Schmitt et al. will be brought into a broader context and potential medical applications targeting eDNA as essential component in biofilm formation and conjugation will be discussed.
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| 6. |
- Kohler, Verena, 1992-, et al.
(författare)
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Problematic groups of multidrug-resistant bacteria and their resistance mechanisms
- 2019
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Ingår i: Antibacterial drug discovery to combat MDR. - Singapore : Springer Nature. - 9789811398704 - 9789811398735 - 9789811398711 ; , s. 25-69
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Bokkapitel (refereegranskat)abstract
- The occurrence of multidrug-resistant pathogenic bacteria is steadily increasing, not only in medical centers but also in food, animals and the environment, which is of primordial concern for health authorities worldwide. The World Health Organization (WHO) published a global pathogen priority list to encourage international interdisciplinary research initiatives on the occurrence, dissemination, and epidemiology of the most dangerous multiresistant pathogens with the aim to develop effective prevention strategies against the spread of these bugs and new therapeutic approaches to treat infections in agreement with the One Health concept. According to the WHO global pathogen priority list, the most critical resistant pathogens include carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa and carbapenem-resistant as well as third-generation cephalosporin-resistant Enterobacteriaceae. This critical group is followed by pathogens of high priority including vancomycin-resistant Enterococcus faecium, methicillin- and vancomycin-resistant Staphylococcus aureus, and clarithromycin-resistant Helicobacter pylori. Here, we summarize recent data on the occurrence and spread of these and other harmful resistant pathogens, on their resistance mechanisms as well as on the modes of resistance spread, as far as is known. We finish the chapter with an outlook on promising innovative strategies to treat infectious diseases caused by multiresistant pathogens – in combination with antibiotic therapy – as well as on approaches to combat the antibiotic resistance spread.
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| 7. |
- Kohler, Verena, 1992-, et al.
(författare)
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Regulation of gram-positive conjugation
- 2019
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Ingår i: Frontiers in Microbiology. - : Frontiers Media S.A.. - 1664-302X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Type IV Secretion Systems (T4SSs) are membrane-spanning multiprotein complexes dedicated to protein secretion or conjugative DNA transport (conjugation systems) in bacteria. The prototype and best-characterized T4SS is that of the Gram-negative soil bacterium Agrobacterium tumefaciens. For Gram-positive bacteria, only conjugative T4SSs have been characterized in some biochemical, structural, and mechanistic details. These conjugation systems are predominantly encoded by self-transmissible plasmids but are also increasingly detected on integrative and conjugative elements (ICEs) and transposons. Here, we report regulatory details of conjugation systems from Enterococcus model plasmids pIP501 and pCF10, Bacillus plasmid pLS1, Clostridium plasmid pCW3, and staphylococcal plasmid pSK41. In addition, regulation of conjugative processes of ICEs (ICEBs1, ICESt1, ICESt3) by master regulators belonging to diverse repressor families will be discussed. A special focus of this review lies on the comparison of regulatory mechanisms executed by proteins belonging to the RRNPP family. These regulators share a common fold and govern several essential bacterial processes, including conjugative transfer.
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| 8. |
- Kohler, Verena, 1992-, et al.
(författare)
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TraN: A novel repressor of an Enterococcus conjugative type IV secretion system
- 2018
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Ingår i: Nucleic Acids Research. - : Oxford University Press. - 0305-1048 .- 1362-4962. ; 46:17, s. 9201-9219
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Tidskriftsartikel (refereegranskat)abstract
- The dissemination of multi-resistant bacteria represents an enormous burden on modern healthcare. Plasmid-borne conjugative transfer is the most prevalent mechanism, requiring a type IV secretion system that enables bacteria to spread beneficial traits, such as resistance to last-line antibiotics, among different genera. Inc18 plasmids, like the Gram-positive broad host-range plasmid pIP501, are substantially involved in propagation of vancomycin resistance from Enterococci to methicillin-resistant strains of Staphylococcus aureus. Here, we identified the small cytosolic protein TraN as a repressor of the pIP501-encoded conjugative transfer system, since deletion of traN resulted in upregulation of transfer factors, leading to highly enhanced conjugative transfer. Furthermore, we report the complex structure of TraN with DNA and define the exact sequence of its binding motif. Targeting this protein–DNA interaction might represent a novel therapeutic approach against the spreading of antibiotic resistances.
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