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Exploring the mechanistic details of Gram-positive Type 4 Secretion Systems
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.ORCID iD: 0000-0001-5470-591x
2022 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Utforska de mekanistiska detaljerna i Gram-positiva Typ 4 Sekretionssystem (Swedish)
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 [en]
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: urn:nbn:se:umu:diva-194545ISBN: 978-91-7855-815-5 (electronic)ISBN: 978-91-7855-814-8 (print)OAI: oai:DiVA.org:umu-194545DiVA, id: diva2:1657358
Public defence
2022-06-03, Hörsal UB.A.240 – Lindellhallen 4, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2022-05-13 Created: 2022-05-10 Last updated: 2023-06-03Bibliographically approved
List of papers
1. Enterococcal PrgU Provides Additional Regulation of Pheromone-Inducible Conjugative Plasmids
Open this publication in new window or tab >>Enterococcal PrgU Provides Additional Regulation of Pheromone-Inducible Conjugative Plasmids
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2021 (English)In: mSphere, E-ISSN 2379-5042, Vol. 6, no 3, article id e0026421Article in journal (Refereed) Published
Abstract [en]

Efficient horizontal gene transfer of the conjugative plasmid pCF10 from Enterococcus faecalis depends on the expression of its type 4 secretion system (T4SS) genes, controlled by the PQ promoter. Transcription from the PQ promoter is tightly regulated, partially to limit cell toxicity caused by overproduction of PrgB, a T4SS adhesin. PrgU plays an important role in regulating this toxicity by decreasing PrgB levels. PrgU has an RNA-binding fold, prompting us to test whether PrgU exerts its regulatory control through binding of prgQ transcripts. We used a combination of in vivo methods to quantify PrgU effects on prgQ transcripts at both single-cell and population levels. PrgU function requires a specific RNA sequence within an intergenic region (IGR) about 400 bp downstream of PQ. PrgU interaction with the IGR reduces levels of downstream transcripts. Single-cell expression analysis showed that cells expressing prgU decreased transcript levels more rapidly than isogenic prgU-minus cells. PrgU bound RNA in vitro without sequence specificity, suggesting that PrgU requires a specific RNA structure or one or more host factors for selective binding in vivo. PrgU binding to its IGR target might recruit RNase(s) for targeted degradation of downstream transcripts or reduce elongation of nascent transcripts beyond the IGR.

IMPORTANCE: Bacteria utilize type 4 secretion systems (T4SS) to efficiently transfer DNA between donor and recipient cells, thereby spreading genes encoding antibiotic resistance as well as various virulence factors. Regulation of expression of the T4SS proteins and surface adhesins in Gram-positive bacteria is crucial, as some of these are highly toxic to the cell. The significance of our research lies in identifying the novel mechanism by which PrgU performs its delicate fine-tuning of the expression levels. As prgU orthologs are present in various conjugative plasmids and transposons, our results are likely relevant to understanding of diverse clinically important transfer systems.

Place, publisher, year, edition, pages
American Society for Microbiology (ASM), 2021
Keywords
conjugation, regulation, type 4 secretion systems
National Category
Cell Biology
Identifiers
urn:nbn:se:umu:diva-186581 (URN)10.1128/mSphere.00264-21 (DOI)000687868400017 ()34106752 (PubMedID)2-s2.0-85111789262 (Scopus ID)
Funder
Swedish Research Council, 2016-03599Knut and Alice Wallenberg FoundationThe Kempe Foundations, SMK-1869NIH (National Institute of Health), R35GM131892NIH (National Institute of Health), R01GM48476NIH (National Institute of Health), R35GM118079
Available from: 2021-08-12 Created: 2021-08-12 Last updated: 2023-03-24Bibliographically approved
2. Enterococcal PcfF Is a Ribbon-Helix-Helix Protein That Recruits the Relaxase PcfG Through Binding and Bending of the oriT Sequence
Open this publication in new window or tab >>Enterococcal PcfF Is a Ribbon-Helix-Helix Protein That Recruits the Relaxase PcfG Through Binding and Bending of the oriT Sequence
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2019 (English)In: Frontiers in Microbiology, E-ISSN 1664-302X, Vol. 10, article id 958Article in journal (Refereed) Published
Abstract [en]

The conjugative plasmid pCF10 from Enterococcus faecalis encodes a Type 4 Secretion System required for plasmid transfer. The accessory factor PcfF and relaxase PcfG initiate pCF10 transfer by forming the catalytically active relaxosome at the plasmid’s origin-of-transfer (oriT) sequence. Here, we report the crystal structure of the homodimeric PcfF, composed of an N-terminal DNA binding Ribbon-Helix-Helix (RHH) domain and a C-terminal stalk domain. We identified key residues in the RHH domain that are responsible for binding pCF10’s oriT sequence in vitro, and further showed that PcfF bends the DNA upon oriT binding. By mutational analysis and pull-down experiments, we identified residues in the stalk domain that contribute to interaction with PcfG. PcfF variant proteins defective in oriT or PcfG binding attenuated plasmid transfer in vivo, but also suggested that intrinsic or extrinsic factors might modulate relaxosome assembly. We propose that PcfF initiates relaxosome assembly by binding oriT and inducing DNA bending, which serves to recruit PcfG as well as extrinsic factors necessary for optimal plasmid processing and engagement with the pCF10 transfer machine.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
T4SS, accessory factor, conjugation, relaxosome, X-ray crystallography, protein structural and functional analysis
National Category
Structural Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-159103 (URN)10.3389/fmicb.2019.00958 (DOI)000467035600001 ()31134011 (PubMedID)2-s2.0-85072734530 (Scopus ID)
Available from: 2019-05-17 Created: 2019-05-17 Last updated: 2024-01-17Bibliographically approved
3. New structural insights provide mechanistic understanding of enterococcal PrgB conjugation, biofilm formation and virulence
Open this publication in new window or tab >>New structural insights provide mechanistic understanding of enterococcal PrgB conjugation, biofilm formation and virulence
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(English)Manuscript (preprint) (Other academic)
National Category
Structural Biology
Identifiers
urn:nbn:se:umu:diva-194147 (URN)
Available from: 2022-05-09 Created: 2022-05-09 Last updated: 2022-05-10

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Lassinantti, Lena

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