Enzybiotics: Endolysins and Bacteriocins

Heselpoth, Ryan D.; Swift, Steven M.; Linden, Sara B.; Mitchell, Michael S.; Nelson, Daniel

doi:10.1007/978-3-319-40598-8_34-1

Cited by 12 publications

(21 citation statements)

References 170 publications

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“…During the phage replicative cycle, lysins degrade the PG of host bacteria to induce hypotonic lysis and phage progeny liberation. The extrinsic application of purified recombinant lysins has been validated for antibacterial efficacy toward several Gram-positive bacterial pathogens as a result of the PG constituting part of the exposed outer structural component of the cell (6). However, expanding the use of these enzymes to target Gram-negative bacteria has, in many cases, been impeded by the outer membrane (OM).…”

mentioning

confidence: 99%

Lysocins: Bioengineered Antimicrobials That Deliver Lysins across the Outer Membrane of Gram-Negative Bacteria

Heselpoth

Euler

Schuch

et al. 2019

Antimicrob Agents Chemother

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The prevalence of multidrug-resistant Pseudomonas aeruginosa has stimulated development of alternative therapeutics. Bacteriophage peptidoglycan hydrolases, termed lysins, represent an emerging antimicrobial option for targeting Gram-positive bacteria. However, lysins against Gram-negatives are generally deterred by the outer membrane and their inability to work in serum. One solution involves exploiting evolved delivery systems used by colicin-like bacteriocins (e.g., S-type pyocins of P. aeruginosa) to translocate through the outer membrane. Following surface receptor binding, colicin-like bacteriocins form Tol-or TonB-dependent translocons to actively import bactericidal domains through outer membrane protein channels. With this understanding, we developed lysocins, which are bioengineered lysin-bacteriocin fusion molecules capable of periplasmic import. In our proof-ofconcept studies, components from the P. aeruginosa bacteriocin pyocin S2 (PyS2) responsible for surface receptor binding and outer membrane translocation were fused to the GN4 lysin to generate the PyS2-GN4 lysocin. PyS2-GN4 delivered the GN4 lysin to the periplasm to induce peptidoglycan cleavage and log-fold killing of P. aeruginosa with minimal endotoxin release. While displaying narrow-spectrum antipseudomonal activity in human serum, PyS2-GN4 also efficiently disrupted biofilms, outperformed standard-of-care antibiotics, exhibited no cytotoxicity toward eukaryotic cells, and protected mice from P. aeruginosa challenge in a bacteremia model. In addition to targeting P. aeruginosa, lysocins can be constructed to target other prominent Gram-negative bacterial pathogens.A ntimicrobial resistance is a threat to global public health. One of the predominant antibiotic-resistant microorganisms responsible for high mortality rates is Pseudomonas aeruginosa. This Gram-negative pathogen is (i) the leading cause of mortality in cystic fibrosis patients, (ii) the main causative agent of burn wound infections, (iii) the most frequent Gram-negative bacterium associated with nosocomial and ventilatoracquired pneumonia, and (iv) the second most common cause of catheter-associated urinary tract infections (1). Additionally, P. aeruginosa is responsible for 3 to 7% of all bloodstream infections (BSIs) and 23 to 26% of Gram-negative bacteremias, translating to mortality rates ranging from 27 to 48% (2). With the antipseudomonal efficacy of standard-of-care (SOC) antibiotics progressively diminishing due to a combination of intrinsic, acquired, and adaptive resistance mechanisms utilized by the bacteria, the lack of therapeutic options has stimulated the World Health Organization to label P. aeruginosa as a critical priority for the research, discovery, and development of new antibiotics (3, 4).

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confidence: 99%

Lysocins: Bioengineered Antimicrobials That Deliver Lysins across the Outer Membrane of Gram-Negative Bacteria

Heselpoth

Euler

Schuch

et al. 2019

Antimicrob Agents Chemother

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“…As such, there exists an engineering potential for these enzymes to be modified to increase their activity, alter host range, or overcome complex extracellular environments [25]. As a growing amount of research focuses on the modular design and crystal structures of endolysins, structure-based rational engineering approaches, such as chimeragenesis and structure-guided mutagenesis, can be used to produce engineered endolysins with desirable antimicrobial properties [26].…”

Section: Discussionmentioning

confidence: 99%

Contributions of Net Charge on the PlyC Endolysin CHAP Domain

Shang

Nelson

2019

Antibiotics

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Bacteriophage endolysins, enzymes that degrade the bacterial peptidoglycan (PG), have gained an increasing interest as alternative antimicrobial agents, due to their ability to kill antibiotic resistant pathogens efficiently when applied externally as purified proteins. Typical endolysins derived from bacteriophage that infect Gram-positive hosts consist of an N-terminal enzymatically-active domain (EAD) that cleaves covalent bonds in the PG, and a C-terminal cell-binding domain (CBD) that recognizes specific ligands on the surface of the PG. Although CBDs are usually essential for the EADs to access the PG substrate, some EADs possess activity in the absence of CBDs, and a few even display better activity profiles or an extended host spectrum than the full-length endolysin. A current hypothesis suggests a net positive charge on the EAD enables it to reach the negatively charged bacterial surface via ionic interactions in the absence of a CBD. Here, we used the PlyC CHAP domain as a model EAD to further test the hypothesis. We mutated negatively charged surface amino acids of the CHAP domain that are not involved in structured regions to neutral or positively charged amino acids in order to increase the net charge from -3 to a range from +1 to +7. The seven mutant candidates were successfully expressed and purified as soluble proteins. Contrary to the current hypothesis, none of the mutants were more active than wild-type CHAP. Analysis of electrostatic surface potential implies that the surface charge distribution may affect the activity of a positively charged EAD. Thus, we suggest that while charge should continue to be considered for future engineering efforts, it should not be the sole focus of such engineering efforts.

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“…Most endolysins, and in particular those from phage that infect Gram-positive bacterial hosts, are comprised of modular domains. An enzymatically active domain (EAD) is generally found in the N-terminal region, while a cell wall-binding domain (CBD) is located in the C-terminal region [6]. As the name implies, the EAD is a catalytic domain that is responsible for cleaving specific bonds in the PG, the nature of which is dependent on the mechanistic class of the EAD.…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin

King

Castro

Bueren-Calabuig

et al. 2021

Preprint

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Endolysins are peptidoglycan (PG) hydrolases that function as part of the bacteriophage (phage) lytic system to release progeny phage at the end of a replication cycle. Notably, endolysins alone can produce lysis without phage infection, which offers an attractive alternative to traditional antibiotics. Endolysins from phage that infect Gram-positive bacterial hosts contain at least one enzymatically active domain (EAD) responsible for hydrolysis of PG bonds and a cell wall binding domain (CBD) that binds a cell wall epitope, such as a surface carbohydrate, providing some degree of specificity for the endolysin. Whilst the EADs typically cluster into conserved mechanistic classes with well-defined active sites, relatively little is known about the nature of the CBDs and only a few binding epitopes for CBDs have been elucidated. The major cell wall components of many streptococci are the polysaccharides that contain the polyrhamnose (pRha) backbone modified with species-specific and serotype-specific glycosyl side chains. In this report, using molecular genetics, microscopy, flow cytometry and lytic activity assays, we demonstrate the interaction of PlyCB, the CBD subunit of the streptococcal PlyC endolysin, with the pRha backbone of the cell wall polysaccharides, Group A Carbohydrate (GAC) and serotype c-specific carbohydrate (SCC) expressed by the Group A Streptococcus and Streptococcus mutans, respectively. Molecular dynamics simulations reveal a previously unidentified binding pocket that is regulated by a gatekeeper residue and uncover that a previously reported inactive PlyC mutant is locked into a 'closed' conformation. Docking studies with the short GAC backbone oligosaccharides expose potential protein-carbohydrate interactions and are consistent with PlyCB binding to the unmodified pRha or pRha decorated with the GAC side chains.

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Enzybiotics: Endolysins and Bacteriocins

Cited by 12 publications

References 170 publications

Lysocins: Bioengineered Antimicrobials That Deliver Lysins across the Outer Membrane of Gram-Negative Bacteria

Lysocins: Bioengineered Antimicrobials That Deliver Lysins across the Outer Membrane of Gram-Negative Bacteria

Contributions of Net Charge on the PlyC Endolysin CHAP Domain

Molecular basis for recognition of the Group A Carbohydrate backbone by the PlyC streptococcal bacteriophage endolysin

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