Biology subjects

Sun, W.-S.

Publications and source records attributed to Sun, W.-S..

5 recordsLinked to original sources

bioRxiv2024

PrgE: an OB-fold protein from plasmid pCF10 with striking differences to prototypical bacterial SSBs

A major pathway for horizontal gene transfer is the transmission of DNA from donor to recipient cells via plasmid-encoded Type 4 Secretion Systems (T4SS). Many conjugative plasmids encode for a single-stranded DNA-binding protein (SSB) together with their T4SS. Some of these SSBs have been suggested to aid in establishing the plasmid in the recipient cell, but for many their function remains unclear. Here, we characterize PrgE, a proposed SSB from Enterococcus faecalis plasmid pCF10. We show that PrgE is not essential for conjugation. Structurally, it has the characteristic OB-fold of SSBs, but it has very uncharacteristic DNA-binding properties. Our DNA-bound structure shows that PrgE binds ssDNA like beads on a string, and this plasticity of PrgEs oligomerization is further confirmed by in vitro studies. Unlike other SSBs, PrgE binds both double- and single-stranded DNA equally well. This shows that PrgE has a quaternary assembly and DNA-binding properties that are very different from the prototypical bacterial SSB, but also different from the eukaryotic SSBs.

biochemistry↗

bioRxiv2024

Breaking Barriers: pCF10 Type 4 Secretion System relies on a self-regulating muramidase to modulate the cell wall

Conjugative Type 4 Secretion Systems (T4SS) are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. To deliver the DNA substrate to recipient cells, it must cross the cell envelopes of both donor and recipient mating bacteria. In the T4SS from the enterococcal conjugative plasmid pCF10, PrgK is known to be the active cell wall degrading enzyme. It has 3 predicted extracellular hydrolase domains, LytM, SLT and CHAP. Here, we report the structure of the LytM domain, and show that its active site is degenerate and lacks the active site metal. Further, we show that only the predicted SLT domain is functional, and that it unexpectedly has a muramidase instead of a lytic transglycosylase activity. While we did not observe any peptidoglycan hydrolytic activity for the LytM or CHAP domain, we found that these domains downregulated the SLT muramidase activity. The CHAP domain was also found to be involved in PrgK dimer formation. Further, we show that PrgK interacts with PrgL, which likely targets PrgK to the rest of the T4SS. The presented data provides important information towards understanding the function of Gram-positive T4SSs.

biochemistry↗

bioRxiv2022

Structural foundation for the role of enterococcal PrgB in conjugation, biofilm formation and virulence

Type 4 Secretion Systems are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. In Gram-positives, these secretion systems often rely on surface adhesins to enhance cellular aggregation and mating pair formation. One of the best studied adhesins is PrgB from the conjugative plasmid pCF10 of Enterococcus faecalis, which has been shown to play major roles in conjugation, biofilm formation and importantly also in bacterial virulence. Since prgB orthologs exist on a large number of conjugative plasmids in various different species, this makes PrgB a model protein for this widespread virulence factor. After characterizing the polymer adhesin domain of PrgB previously, we here report the structure for almost the entire remainder of PrgB, which reveals that PrgB contains four immunoglobulin-like domains. Based on this new insight, we re-evaluate previously studied variants and present new in vivo data where specific domains or conserved residues have been removed. For the first time, we can show a decoupling of cellular aggregation from biofilm formation and conjugation in prgB mutant phenotypes. Based on the presented data, we propose a new functional model to explain how PrgB mediates its different functions. We hypothesize that the Ig-like domains act as a rigid stalk that presents the polymer adhesin domain at the right distance from the cell wall.

biochemistry↗

bioRxiv2020

Structure of the enterococcal T4SS protein PrgL reveals unique dimerization interface in the VirB8 protein family

Multidrug resistant bacteria are one of the most important current threats to public health and a serious problem in hospital acquired infections (HAIs). Most antibiotic resistance genes are acquired via conjugative gene transfer, in a process that is mediated by a protein machinery called the Type 4 Secretion System (T4SS). The core of the T4SS is a multiprotein complex that spans both the cell wall and cellular membrane(s), serving as a channel for macromolecular secretion. Although the majority of multidrug resistant bacteria responsible for HAIs are of Gram-positive origin, with Enterococci being major contributors, mostly Gram-negative T4SSs have been characterized. Here we describe the structure and organisation of PrgL, one of the seven membrane proteins forming the translocation channel of the T4SS encoded by the pCF10 plasmid from Enterococcus faecalis. We present the structure of the C-terminal domain of PrgL, which displays similarity to VirB8 proteins of Gram-negative secretion systems. PrgL forms dimers and higher order oligomers but does not interact strongly with the other T4SS components. In vitro experiments show that the soluble domain alone is enough to drive both dimerization and dodecamerisation, with a dimerization interface that differs from all other known VirB8-like proteins. Our findings provide insight into the molecular building blocks of Gram-positive T4SS, highlighting similarities but also unique features in PrgL compared to other VirB8-like proteins.

biochemistry↗

bioRxiv2020

Enterococcal PrgA extends far outside the cell and provides surface exclusion to protect against unwanted conjugation

Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via Type 4 Secretion Systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a 2nd copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggest that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.

biochemistry↗