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New structural insights provide mechanistic understanding of enterococcal PrgB conjugation, biofilm formation and virulence
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.ORCID iD: 0000-0001-5470-591x
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Identifiers
URN: urn:nbn:se:umu:diva-194147OAI: oai:DiVA.org:umu-194147DiVA, id: diva2:1656947
Available from: 2022-05-09 Created: 2022-05-09 Last updated: 2022-05-10
In thesis
1. Exploring the mechanistic details of Gram-positive Type 4 Secretion Systems
Open this publication in new window or tab >>Exploring the mechanistic details of Gram-positive Type 4 Secretion Systems
2022 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Utforska de mekanistiska detaljerna i Gram-positiva Typ 4 Sekretionssystem
Abstract [en]

Hospital acquired (i.e. nosocomial) infections and antibiotic resistance are large issues in the world today, with about 1.3 million people estimated to have died from antibiotic resistant infections in 2019 alone, and these problems are on the rise. Type 4 Secretion Systems (T4SSs) are complex nanomachineries commonly found on conjugative plasmids. T4SSs are a major route for the translocation of genes encoding for antibiotic resistance and other virulence factors. These systems have primarily been studied in Gram-negative (G-) bacteria even though Gram-positive (G+) bacteria stand for about half of the nosocomial infections. To develop ways to limit the spread of both antibiotic resistance and virulence factors, we need to gain fundamental knowledge of T4SSs in G+ bacteria.

Our work has focused on the conjugative plasmid pCF10 from the G+ bacteria Enterococcus faecalis where all the genes needed for the T4SS are under the regulation of one promoter named PQ. Most G+ T4SSs consist of three groups of proteins, namely the DNA transfer and replication (Dtr) proteins, the channel proteins and the adhesin proteins. In my work, I have focused my attention specifically on i) the regulatory protein PrgU, ii) the Dtr protein PcfF, and iii) the adhesin protein PrgB. These three proteins provide insights into three different parts of the T4SS. PrgU is part of the regulatory process of T4SS expression and has been shown to inhibit cell-toxicity mitigated by PrgB. The Dtr protein PcfF is needed for the formation of the relaxosome complex critical for conjugative transfer of the plasmid, and PrgB is involved in cellular aggregation events and is also a known virulence factor. Interestingly, increased levels of PrgB have been shown to be toxic to the cells. To inhibit PrgB induced cell toxicity, its production needs to be tightly regulated.

The aims of my PhD thesis were to examine conjugation complexes belonging to Type 4 Secretion Systems in Gram-positive bacteria and to determine their function, molecular structures, and regulation. By using a combination of in vivo and in vitro methods we have; i) showed that PrgU binds to the IGR located downstream of the PQ promoter, and that the deletion of prgU in pCF10 containing cells produces increased mRNA levels of the full prgQ transcript, ii) solved the crystal structure of PcfF and identified residues that are important for the interaction with the relaxase and the origin of transfer (oriT) DNA in vitro, and confirmed this by biochemical assays and, iii) solved the entire structure of PrgB using a combination of X-ray crystallography and cryo-EM and performed in vivo assays to confirm its functions.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2022. p. 61
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 2186
Keywords
Antimicrobial resistance, Horizontal gene transfer, Gram-positive bacteria, Type 4 Secretion Systems, Conjugation, Enterococcus faecalis, pCF10, PrgU, PcfF, PrgB
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy) Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-194545 (URN)978-91-7855-815-5 (ISBN)978-91-7855-814-8 (ISBN)
Public defence
2022-06-03, Hörsal UB.A.240 – Lindellhallen 4, Umeå, 09:00 (English)
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Available from: 2022-05-13 Created: 2022-05-10 Last updated: 2023-06-03Bibliographically approved

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Sun, Wei-ShengLassinantti, LenaJärvå, Michael A.Schmitt, Andreaster Beek, JosyBerntsson, Ronnie

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Sun, Wei-ShengLassinantti, LenaJärvå, Michael A.Schmitt, Andreaster Beek, JosyBerntsson, Ronnie
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Department of Medical Biochemistry and BiophysicsWallenberg Centre for Molecular Medicine at Umeå University (WCMM)
Structural Biology

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