Expression and purification of phage T7 ejection proteins for cryo-EM analysis

Swanson, Nicholas; Lokareddy, Ravi K.; Li, Fenglin; Hou, Chun-Feng David; Pavlenok, Mikhail; Niederweis, Michael; Cingolani, Gino

doi:10.1016/j.xpro.2021.100960

Cited by 6 publications

(8 citation statements)

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“…It has been reported that about 96% of phages are double-stranded DNA genomes with tailed morphology, belonging to the order of Caudovirales [35]. Based on the tail structure, the Caudovirales order can be categorized into three families: Myoviridae (phages with a contractile tail, such as T4-like phages), Siphoviridae (phages with a non-contractile long tail, such as T5-like phages), and Podoviridae (phages with a short tail, such as T7-like phages) [36][37][38].…”

Section: Phages Infect Bacterial Biofilmsmentioning

confidence: 99%

Phages against Pathogenic Bacterial Biofilms and Biofilm-Based Infections: A Review

Liu

Zhang

et al. 2022

Pharmaceutics

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Bacterial biofilms formed by pathogens are known to be hundreds of times more resistant to antimicrobial agents than planktonic cells, making it extremely difficult to cure biofilm-based infections despite the use of antibiotics, which poses a serious threat to human health. Therefore, there is an urgent need to develop promising alternative antimicrobial therapies to reduce the burden of drug-resistant bacterial infections caused by biofilms. As natural enemies of bacteria, bacteriophages (phages) have the advantages of high specificity, safety and non-toxicity, and possess great potential in the defense and removal of pathogenic bacterial biofilms, which are considered to be alternatives to treat bacterial diseases. This work mainly reviews the composition, structure and formation process of bacterial biofilms, briefly discusses the interaction between phages and biofilms, and summarizes several strategies based on phages and their derivatives against biofilms and drug-resistant bacterial infections caused by biofilms, serving the purpose of developing novel, safe and effective treatment methods against biofilm-based infections and promoting the application of phages in maintaining human health.

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Section: Phages Infect Bacterial Biofilmsmentioning

confidence: 99%

Phages against Pathogenic Bacterial Biofilms and Biofilm-Based Infections: A Review

Liu

Zhang

et al. 2022

Pharmaceutics

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“…Gp14 forms a pore in the OM: Gp14, the smallest ejection protein (196 residues), is entirely water-insoluble in vitro, requiring detergents to be extracted from the expression host’s membranes [ 47 , 58 ]. In the pre-ejection conformation, gp14 is solubilized by gp15 as part of the core stack [ 31 ] ( Figure 1 A).…”

Section: Conformational Gymnastics Of T7 Ejection Proteinsmentioning

confidence: 99%

“…A partial structure of the gp14 post-ejection conformation (residues 37–139) revealed a hexameric helical channel [ 31 ]. Accordingly, detergent-solubilized gp14 has pore-forming activity in vitro [ 55 , 58 ]. The limited structural information on gp14 pre- and post-ejection prevent a detailed analysis of the structural changes occurring upon ejection.…”

Section: Conformational Gymnastics Of T7 Ejection Proteinsmentioning

confidence: 99%

“…Three lines of evidence corroborate the hexameric oligomeric state as physiological. First, two independent groups reported this stoichiometry using different purification and assembly procedures [ 55 , 56 , 58 ]. Second, gp15 extends the gp14 pore, which is hexameric after ejection from the virion into the host-cell envelope [ 61 ].…”

Section: Conformational Gymnastics Of T7 Ejection Proteinsmentioning

confidence: 99%

“…Gp16 refolding: T7 gp16 is nearly double the size of gp15 with 1318 residues. It purifies as a soluble monomer [ 50 , 55 , 58 ] despite having two predicted transmembrane helical regions [ 50 ]. Gp16 is largely α-helical in the pre-ejection conformation and can refold from a partially unfolded state as expected for exit from the narrow portal channel [ 19 , 47 , 50 ].…”

Section: Conformational Gymnastics Of T7 Ejection Proteinsmentioning

confidence: 99%

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Viral Ejection Proteins: Mosaically Conserved, Conformational Gymnasts

2022

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Bacterial viruses (or bacteriophages) have developed formidable ways to deliver their genetic information inside bacteria, overcoming the complexity of the bacterial-cell envelope. In short-tailed phages of the Podoviridae superfamily, genome ejection is mediated by a set of mysterious internal virion proteins, also called ejection or pilot proteins, which are required for infectivity. The ejection proteins are challenging to study due to their plastic structures and transient assembly and have remained less characterized than classical components such as the phage coat protein or terminase subunit. However, a spate of recent cryo-EM structures has elucidated key features underscoring these proteins’ assembly and conformational gymnastics that accompany their expulsion from the virion head through the portal protein channel into the host. In this review, we will use a phage-T7-centric approach to critically review the status of the literature on ejection proteins, decipher the conformational changes of T7 ejection proteins in the pre- and post-ejection conformation, and predict the conservation of these proteins in other Podoviridae. The challenge is to relate the structure of the ejection proteins to the mechanisms of genome ejection, which are exceedingly complex and use the host’s machinery.

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