Bacteriophage-derived Protein Research Articles

Exploring the enzymatic activity of depolymerase gp531 from Klebsiella pneumoniae jumbo phage RaK2

Klebsiella pneumoniae poses a major global challenge due to its virulence, multidrug resistance, and nosocomial nature. Thus, bacteriophage-derived proteins are extensively being investigated as a means to combat this bacterium. In this study, we explored the enzymatic specificity of depolymerase gp531, encoded by the jumbo bacteriophage vB_KleM_RaK2 (RaK2). We used two different methods to modify the reducing end of the oligosaccharides released during capsule hydrolysis with gp531. Subsequent acidic cleavage with TFA, followed by TLC and HPLC-MS analyses, revealed that RaK2 gp531 is a β-(1→4)-endoglucosidase. The enzyme specifically recognizes and cleaves the capsular polysaccharide (CPS) of the Klebsiella pneumoniae K54 serotype, releasing K-unit monomers (the main product), dimers, and trimers. Depolymerase gp531 remains active from 10 to 50 °C and in the pH 3–8 range, indicating its stability and versatility. Additionally, we demonstrated that gp531′s activity is not affected by CPS acetylation, which is influenced by the growth conditions of the bacterial culture. Overall, our findings provide valuable insights into the enzymatic activity of the first characterized depolymerase targeting the capsule of the clinically relevant K54 serotype of K. pneumoniae.

Phage-mimicking nanoagents for rapid depolymerase specificity screening against multidrug resistant bacteria

With the rise of drug resistance, bacteriophages and bacteriophage-derived proteins may become an efficient successor to traditional antibiotics. While the enormous natural diversity of the phages allows matching virtually any bacteria, identification of the potentially life-saving phage is currently a tedious and time-consuming challenge that often cannot be performed within a reasonable time. Here we show a rapid 1-min bacteriophage screening assay based on specially constructed phage-mimicking nanoagents and surface plasmon resonance effect. Within the assay, a panel of phage-mimicking gold nanoparticles, possessing the specificity and enzymatic activity of a particular phage, is mixed with a suspension of the bacteria of interest. The spectral behaviour of the assay mix allows measurement of two critical parameters of the nanoagents and the corresponding bacteriophages: 1) direct assessment of their specificity due to convergence of the particles on the cell walls, and more importantly, 2) real-time evaluation of their enzymatic activity for the destruction of the cell capsule via detection of nanoagent detachment from the surface of bacteria. The proposed assay overcomes the current time limitations of the phage-bacteria matching procedures and thereby can facilitate faster development and adoption of phage-based therapies as a much-needed alternative to traditional antibiotics.

微生物シングルセルゲノム解析技術bit-MAP®を用いたエンドリシン創薬技術開発

The emergence of antimicrobial-resistant (AMR) bacteria poses an imminent threat to our society and requires urgent actions. We here propose that a bacteriophage-derived protein called endolysin can be exploited as a new antimicrobial agent to tackle this global public heath issue. Endolysins are hydrolytic enzymes that cleave bacterial cell-walls (peptidoglycan layers), allowing for rapid cell lysis in a matter of minutes. Not only this swift response makes a stark contrast with conventional antibiotics, but the protein nature of endolysins makes them amenable to modifications. For example, modular domains of endolysin can be recombined to create new artificial endolysins. Using in-house high-throughput screening system, we show that we can quickly discover multiple endolysins against methicillin-resistant Staphylococcus aureus (MRSA) with improved activity compared to previously reported ones. We also discuss the prospects for the development of antimicrobial endolysins against any potential pathogens using our proprietary microbial single-cell genome sequencing platform, bit-MAP(R).

The Advantages and Challenges of Using Endolysins in a Clinical Setting.

Antibiotic-resistant pathogens are increasingly more prevalent and problematic. Traditional antibiotics are no longer a viable option for dealing with these multidrug-resistant microbes and so new approaches are needed. Bacteriophage-derived proteins such as endolysins could offer one effective solution. Endolysins are bacteriophage-encoded peptidoglycan hydrolases that act to lyse bacterial cells by targeting their cell’s wall, particularly in Gram-positive bacteria due to their naturally exposed peptidoglycan layer. These lytic enzymes have received much interest from the scientific community in recent years for their specificity, mode of action, potential for engineering, and lack of resistance mechanisms. Over the past decade, a renewed interest in endolysin therapy has led to a number of successful applications. Recombinant endolysins have been shown to be effective against prominent pathogens such as MRSA, Listeria monocytogenes, Staphylococcus strains in biofilm formation, and Pseudomonas aeruginosa. Endolysins have also been studied in combination with other antimicrobials, giving a synergistic effect. Although endolysin therapy comes with some regulatory and logistical hurdles, the future looks promising, with the emergence of engineered “next-generation” lysins. This review will focus on the likelihood that endolysins will become a viable new antimicrobial therapy and the challenges that may have to be overcome along the way.

Can bacteriophage endolysins be nebulised for inhalation delivery against Streptococcus pneumoniae?

Endolysins are bacteriophage-derived protein molecules highly effective for bacterial killing. Cpl-1 and ClyJ-3 are native and chimeric endolysins, respectively, having antimicrobial activity against Streptococcus pneumoniae which causes lung infections. We conducted the first feasibility study on nebulisation of Cpl-1 and ClyJ-3, with a focus on the antimicrobial activity, structural changes of the proteins and aerosol performance. Bacterial colony counts, live cell imaging and Fourier-transform infrared(FTIR) spectroscopy were used to evaluate the proteins before and after jet or vibrating mesh nebulisation. These nebulised aerosols were inhalable with a volume median size of 3.8-4.2µm (span 1.1-2.3) measured by laser diffraction. How-ever, neb-u-li-sa-tion caused al-most com-plete loss in bioac-tiv-ity of ClyJ-3, which were corroborated with the live cell imaging observation and protein structural damage with a large intensity reduction in the amide absorption bands between 1300 and 1700cm-1. In contrast, the bactericidal activity of Cpl-1 showed no significant difference (p≥0.05) before and after mesh nebulisation with 4.9 and 4.6-log10 bacterial count reduction, respectively. However, jet nebulisation reduced the bioactivity of Cpl-1 and the effect was time-dependent showing 1.7, 1.0-log10 bacterial count reduction at 7 and 14 min with complete loss of antimicrobial activity at 21 min after nebulisation, respectively. The results were consistent with time-dependent changes in live cell images and FTIR amide band changes at 1655, 1640, 1632 and 1548cm-1. In conclusion, it is feasible to nebulise endolysins for inhalation delivery but it depends on both the protein and the nebuliser, with the mesh nebuliser being the preferred choice.

A novel flow cytometry assay based on bacteriophage-derived proteins for Staphylococcus detection in blood

Bloodstream infections (BSIs) are considered a major cause of death worldwide. Staphylococcus spp. are one of the most BSIs prevalent bacteria, classified as high priority due to the increasing multidrug resistant strains. Thus, a fast, specific and sensitive method for detection of these pathogens is of extreme importance. In this study, we have designed a novel assay for detection of Staphylococcus in blood culture samples, which combines the advantages of a phage endolysin cell wall binding domain (CBD) as a specific probe with the accuracy and high-throughput of flow cytometry techniques. In order to select the biorecognition molecule, three different truncations of the C-terminus of Staphylococcus phage endolysin E-LM12, namely the amidase (AMI), SH3 and amidase+SH3 (AMI_SH3) were cloned fused with a green fluorescent protein. From these, a higher binding efficiency to Staphylococcus cells was observed for AMI_SH3, indicating that the amidase domain possibly contributes to a more efficient binding of the SH3 domain. The novel phage endolysin-based flow cytometry assay provided highly reliable and specific detection of 1–5 CFU of Staphylococcus in 10 mL of spiked blood, after 16 hours of enrichment culture. Overall, the method developed herein presents advantages over the standard BSIs diagnostic methods, potentially contributing to an early and effective treatment of BSIs.

Broad-spectrum enzymatic inhibition of CRISPR-Cas12a.

Cas12a (Cpf1) is a bacterial RNA-guided nuclease used widely for genome editing and diagnostic applications. In bacteria, Cas12a enzymes can be inhibited by bacteriophage-derived proteins, anti-CRISPRs (Acrs), to thwart clustered regularly interspaced short palindromic repeat (CRISPR) adaptive immune systems. How these inhibitors disable Cas12a by preventing programmed DNA cleavage is unknown. We show that three inhibitors (AcrVA1, AcrVA4 and AcrVA5) block Cas12a activity using functionally distinct mechanisms, including a previously unobserved enzymatic strategy. AcrVA4 and AcrVA5 inhibit double-stranded DNA (dsDNA) recognition with AcrVA4 driving Cas12a dimerization. In contrast, AcrVA1 is a multiple-turnover inhibitor that triggers cleavage of the target recognition sequence of the Cas12a-bound guide RNA to irreversibly inactivate the Cas12a complex. These distinct mechanisms equip bacteriophage with tools to evade CRISPR-Cas12a and support biotechnological applications where multiple-turnover enzymatic inhibition of Cas12a are desirable.

A Bacteriophage Capsid Protein Is an Inhibitor of a Conserved Transcription Terminator of Various Bacterial Pathogens.

Rho is a hexameric molecular motor that functions as a conserved transcription terminator in the majority of bacterial species and is a potential drug target. Psu is a bacteriophage P4 capsid protein that inhibits Escherichia coli Rho by obstructing its ATPase and translocase activities. In this study, we explored the anti-Rho activity of Psu for Rho proteins from different pathogens. Sequence alignment and homology modeling of Rho proteins from pathogenic bacteria revealed the conserved nature of the Psu-interacting regions in all these proteins. We chose Rho proteins from various pathogens, including Mycobacterium smegmatis, Mycobacterium bovis, Mycobacterium tuberculosis, Xanthomonas campestris, Xanthomonas oryzae, Corynebacterium glutamicum, Vibrio cholerae, Salmonella enterica, and Pseudomonas syringae The purified recombinant Rho proteins of these organisms showed variable rates of ATP hydrolysis on poly(rC) as the substrate and were capable of releasing RNA from the E. coli transcription elongation complexes. Psu was capable of inhibiting these two functions of all these Rho proteins. In vivo pulldown assays revealed direct binding of Psu with many of these Rho proteins. In vivo expression of psu induced killing of M. smegmatis, M. bovis, X. campestris, and E. coli expressing S. enterica Rho indicating Psu-induced inhibition of Rho proteins of these strains under physiological conditions. We propose that the "universal" inhibitory function of the Psu protein against the Rho proteins from both Gram-negative and Gram-positive bacteria could be useful for designing peptides with antimicrobial functions and that these peptides could contribute to synergistic antibiotic treatment of the pathogens by compromising the Rho functions.IMPORTANCE Bacteriophage-derived protein factors modulating different bacterial processes could be converted into unique antimicrobial agents. Bacteriophage P4 capsid protein Psu is an inhibitor of the E. coli transcription terminator Rho. Here we show that apart from antagonizing E. coli Rho, Psu is able to inhibit Rho proteins from various phylogenetically unrelated Gram-negative and Gram-positive pathogens. Upon binding to these Rho proteins, Psu inhibited them by affecting their ATPase and RNA release functions. The expression of Psu in vivo kills various pathogens, such as Mycobacterium and Xanthomonas species. Hence, Psu could be useful for identifying peptide sequences with anti-Rho activities and might constitute part of synergistic antibiotic treatment against pathogens.

O456 Deletion analysis of LysK, a bacteriophage-derived protein with anti-MRSA activity

O456 Deletion analysis of LysK, a bacteriophage-derived protein with anti-MRSA activity