Structural and Biochemical Analysis of OrfG: The VirB8-like Component of the Conjugative Type IV Secretion System of ICESt3 From Streptococcus thermophilus

Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural analysis of VirB8-like proteins and discusses the interplay between tertiary and quaternary structures of these proteins as an essential component of the conjugative transfer.

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Structural and biochemical analysis of OrfG: the VirB8-like component of the integrative and conjugative element ICESt3 from Streptococcus thermophilus

10.1101/2020.11.26.395491 ◽

2020 ◽

Author(s):

Julien Cappele ◽

Abbas Mohamad-Ali ◽

Nathalie Leblond-Bourget ◽

Sandrine Mathiot ◽

Tiphaine Dhalleine ◽

...

Keyword(s):

Antibiotic Resistance ◽

Pathogenic Bacteria ◽

Quaternary Structure ◽

Structural Alignment ◽

Antibiotic Resistance Genes ◽

Streptococcus Thermophilus ◽

Conjugative Transfer ◽

Structural Level ◽

Secretion Systems ◽

Integrative And Conjugative Elements

AbstractConjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural and assembly mode of VirB8-like proteins, a component essential for conjugative transfer and improves our understanding on these under-examined bacterial nanomachines.

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Peptidomimetic Small Molecules Disrupt Type IV Secretion System Activity in Diverse Bacterial Pathogens

mBio ◽

10.1128/mbio.00221-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 22

Author(s):

Carrie L. Shaffer ◽

James A. D. Good ◽

Santosh Kumar ◽

K. Syam Krishnan ◽

Jennifer A. Gaddy ◽

...

Keyword(s):

Antibiotic Resistance ◽

Small Molecules ◽

Bacterial Pathogens ◽

Effector Proteins ◽

Type Iv Secretion ◽

Target Cells ◽

Effector Protein ◽

Secretion Systems ◽

Type Iv ◽

H Pylori

ABSTRACT Bacteria utilize complex type IV secretion systems (T4SSs) to translocate diverse effector proteins or DNA into target cells. Despite the importance of T4SSs in bacterial pathogenesis, the mechanism by which these translocation machineries deliver cargo across the bacterial envelope remains poorly understood, and very few studies have investigated the use of synthetic molecules to disrupt T4SS-mediated transport. Here, we describe two synthetic small molecules (C10 and KSK85) that disrupt T4SS-dependent processes in multiple bacterial pathogens. Helicobacter pylori exploits a pilus appendage associated with the cag T4SS to inject an oncogenic effector protein (CagA) and peptidoglycan into gastric epithelial cells. In H. pylori , KSK85 impedes biogenesis of the pilus appendage associated with the cag T4SS, while C10 disrupts cag T4SS activity without perturbing pilus assembly. In addition to the effects in H. pylori , we demonstrate that these compounds disrupt interbacterial DNA transfer by conjugative T4SSs in Escherichia coli and impede vir T4SS-mediated DNA delivery by Agrobacterium tumefaciens in a plant model of infection. Of note, C10 effectively disarmed dissemination of a derepressed IncF plasmid into a recipient bacterial population, thus demonstrating the potential of these compounds in mitigating the spread of antibiotic resistance determinants driven by conjugation. To our knowledge, this study is the first report of synthetic small molecules that impair delivery of both effector protein and DNA cargos by diverse T4SSs. IMPORTANCE Many human and plant pathogens utilize complex nanomachines called type IV secretion systems (T4SSs) to transport proteins and DNA to target cells. In addition to delivery of harmful effector proteins into target cells, T4SSs can disseminate genetic determinants that confer antibiotic resistance among bacterial populations. In this study, we sought to identify compounds that disrupt T4SS-mediated processes. Using the human gastric pathogen H. pylori as a model system, we identified and characterized two small molecules that prevent transfer of an oncogenic effector protein to host cells. We discovered that these small molecules also prevented the spread of antibiotic resistance plasmids in E. coli populations and diminished the transfer of tumor-inducing DNA from the plant pathogen A. tumefaciens to target cells. Thus, these compounds are versatile molecular tools that can be used to study and disarm these important bacterial machines.

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Export von Gefahrgut: Helicobacter pylori und sein CagA-Protein

BIOspektrum ◽

10.1007/s12268-020-1454-7 ◽

2020 ◽

Vol 26 (6) ◽

pp. 597-599

Author(s):

Clara Lettl ◽

Wolfgang Fischer

Keyword(s):

Helicobacter Pylori ◽

Pathogenic Bacteria ◽

Type Iv Secretion ◽

Host Cells ◽

Bacterial Protein ◽

Secretion Systems ◽

Unusual Structure ◽

Type Iv ◽

Single Protein ◽

Protein Transporters

Abstract Pathogenic bacteria often utilize type IV secretion systems to interact with host cells and to modify their microenvironment in a favourable way. The human pathogen Helicobacter pylori produces such a system to inject only a single protein, CagA, into gastric cells, but this injection represents a major risk factor for gastric cancer development. Here, we discuss the unusual structure of the Cag secretion nanomachine and other features that make it unique among bacterial protein transporters.

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Translocation through the Conjugative Type IV Secretion System Requires Unfolding of Its Protein Substrate

Journal of Bacteriology ◽

10.1128/jb.00615-17 ◽

2018 ◽

Vol 200 (6) ◽

Cited By ~ 10

Author(s):

Martina Trokter ◽

Gabriel Waksman

Keyword(s):

Antibiotic Resistance ◽

Pathogenic Bacteria ◽

Protein Transport ◽

Recipient Cell ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Single Stranded Dna ◽

Protein Substrate ◽

Type Iv

ABSTRACTBacterial conjugation, a mechanism of horizontal gene transfer, is the major means by which antibiotic resistance spreads among bacteria (1, 2). Conjugative plasmids are transferred from one bacterium to another through a type IV secretion system (T4SS) in the form of single-stranded DNA covalently attached to a protein called relaxase. The relaxase is fully functional both in a donor cell (prior to conjugation) and recipient cell (after conjugation). Here, we demonstrate that the protein substrate has to unfold for efficient translocation through the conjugative T4SS. Furthermore, we present various relaxase modifications that preserve the function of the relaxase but block substrate translocation. This study brings us a step closer to deciphering the complete mechanism of T4SS substrate translocation, which is vital for the development of new therapies against multidrug-resistant pathogenic bacteria.IMPORTANCEConjugation is the principal means by which antibiotic resistance genes spread from one bacterium to another (1, 2). During conjugation, a covalent complex of single-stranded DNA and a protein termed relaxase is transported by a type IV secretion system. To date, it is not known whether the relaxase requires unfolding prior to transport. In this report, we use functional assays to monitor the transport of wild-type relaxase and variants containing unfolding-resistant domains and show that these domains reduce conjugation and protein transport dramatically. Mutations that lower the free energy of unfolding in these domains do not block translocation and can even promote it. We thus conclude that the unfolding of the protein substrate is required during transport.

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Protein Transfer through an F Plasmid-Encoded Type IV Secretion System Suppresses the Mating-Induced SOS Response

mBio ◽

10.1128/mbio.01629-21 ◽

2021 ◽

Author(s):

Abu Amar M. Al Mamun ◽

Kouhei Kishida ◽

Peter J. Christie

Keyword(s):

Heavy Metals ◽

Sos Response ◽

Secretion System ◽

Type Iv Secretion ◽

Type Iv Secretion System ◽

Conjugative Transfer ◽

Secretion Systems ◽

Protein Transfer ◽

Type Iv ◽

Genes Encoding

Many bacteria carry plasmids and other mobile genetic elements (MGEs) whose conjugative transfer through encoded type IV secretion systems (T4SSs), or “mating channels,” can lead to a rapid intra- and interspecies proliferation of genes encoding resistance to antibiotics or heavy metals or virulence traits. Here, we show that a model IncF plasmid-encoded T4SS translocates not only DNA but also several proteins intercellularly.

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