Lysin

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Lysozyme-like phage lysin
Crystal structure of the modular CPL-1 endolysin complexed with a peptidoglycan analogue.jpg
Crystal structure of the modular CPL-1 endolysin from Streptococcus phage Cp-1 complexed with a peptidoglycan analogue. PDB entry 2j8g . [1]
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EC no. 3.2.1.17
CAS no. 9001-63-2
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Lysins, also known as endolysins or murein hydrolases, are hydrolytic enzymes produced by bacteriophages in order to cleave the host's cell wall during the final stage of the lytic cycle. Lysins are highly evolved enzymes that are able to target one of the five bonds in peptidoglycan (murein), the main component of bacterial cell walls, which allows the release of progeny virions from the lysed cell. Cell-wall-containing Archaea are also lysed by specialized pseudomurein-cleaving lysins, [2] while most archaeal viruses employ alternative mechanisms. [3] Similarly, not all bacteriophages synthesize lysins: some small single-stranded DNA and RNA phages produce membrane proteins that activate the host's autolytic mechanisms such as autolysins. [4]

Contents

Lysins were first used therapeutically in 2001 by the Fischetti lab (see below) and are now being used as antibacterial agents due to their high effectiveness and specificity in comparison with antibiotics, which are susceptible to bacterial resistance. [5] Because lysins are essential for bacteriophage survival, resistance to lysins is an extremely rare event. Over the >20 years of lysin development as therapeutics, resistance has not been observed, even when resistance is forced by mutagenesis experiments.

Structure

Double-stranded DNA phage lysins tend to lie within the 25 to 40 kDa range in terms of size. A notable exception is the streptococcal PlyC endolysin, which is 114 kDa. PlyC is not only the biggest and most potent lysin, but also structurally unique since it is composed of two different gene products, PlyCA and PlyCB, with a ratio of eight PlyCB subunits for each PlyCA in its active conformation. [6]

All other lysins are monomeric and comprise two domains separated by a short linker region. For gram positive bacteria lysins, the N-terminal domain catalyses the hydrolysis of peptidoglycan whereas the C-terminal domain binds to the cell wall substrate.

Catalytic domain

The catalytic domain is responsible for the cleavage of peptidoglycan bonds. Functionally, five types of lysin catalytic domain can be distinguished:

Peptidoglycan consists of cross-linked amino acids and sugars which form alternating amino sugars: N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). Endo-β-N-acetylglucosaminidase lysins cleave NAGs while N-acetylmuramidase lysins (lysozyme-like lysins) cleave NAMs. Endopeptidase lysins cleave any of the peptide bonds between amino acids, whereas N-acetylmuramoyl-l-alanine amidase lysins (or simply amidase lysins) hydrolyze the amide bond between the sugar and the amino acid moieties. Finally, the recently discovered γ-d-glutaminyl-l-lysine endopeptidase lysins cleave the gamma bond between D-glutamine and L-lysine residues. As is the case for autolysins, early confusion around the cleavage specificity of these individual enzymes has led to some misattributions of the name "lysozyme" to proteins without this activity. [7]

Usually, two or more different catalytic domains are linked to a single cell-binding domain. This is typical in many staphylococcal lysins as well as the streptococcal PlyC holoenzyme, which contains two catalytic domains. [6] [8] Catalytic domains are highly conserved in phage lysins of the same class. [5]

Cell-binding domain

The cell-binding domain (CBD) binds to a specific substrate found in the host bacterium's cell wall, usually a carbohydrate. In contrast to the catalytic domain, the cell-binding domain is variable, which allows a great specificity and decreases bacterial resistance. [9] Binding affinity to the cell wall substrate tends to be high, possibly so as to sequester onto cell wall fragments any free enzyme, which could compete with phage progeny from infecting adjacent host bacteria. [10]

Evolution

It has been proposed that the main mechanism of evolution in phage lysins is the exchange of modular units, a process by which different catalytic and cell-binding domains have been swapped between lysins, which would have resulted in new combinations of both bacterial binding and catalytic specificities. [11]

Mode of action

The lysin catalytic domain digests peptidoglycan locally at a high rate, which causes holes in the cell wall. Since the cross-linked peptidoglycan cell wall is the only mechanism that prevents the spontaneous burst of bacterial cells due to the high internal pressure (3 to 5 atmospheres), enzymatic digestion by lysins irreversibly causes hypotonic lysis. Theoretically, due to the catalytic properties of phage lysins, a single enzyme would be sufficient to kill the host bacterium by cleaving the necessary number of bonds, even though this has yet to be proven. [5] The work by Loessner et al suggests that cleavage is typically achieved by the joint action of multiple lysin molecules at a local region of the host's cell wall. [10] The high binding affinity to the cell wall substrate (close to that of IgG for its substrate) of each lysin appear to be reason why multiple molecules are required, since every lysin binds so tightly to the cell wall that it can't break enough bonds to cause lysis by itself. [10]

In order to reach the cell wall, phage lysins have to cross the cell membrane. However, they generally lack a signal peptide that would allow them to do so. In order to solve such a problem, phage viruses synthesize another protein called holin which binds to the cell membrane and makes holes in it (hence its name), allowing lysins to reach the peptidoglycan matrix. The prototypical holin is the lambda phage S protein, which assists the lambda phage R protein (lysin). All holins embed themselves in the cell membrane and contain at least two transmembrane helical domains. The hole making process is thought to be achieved by holin oligomerization at a specific moment when progeny virions are set to be released. [4] [12]

Efficacy

Phage lysins are generally species or subspecies specific, which means that they are only effective against bacteria from which they were produced. While some lysins only act upon the cell walls of several bacterial phylotypes, some broad-spectrum lysins have been found. [13] Similarly, some thermostable lysins are known, which makes them easier to use in biotechnology. [14] Regarding their use as antibacterial agents, lysins have been found effective mainly against Gram-positive bacteria, since Gram-negative bacteria possess an outer membrane that prevents extracellular lysin molecules from digesting peptidoglycan. [5] However, lysins with activity against Gram-negative bacteria, such as OBPgp279, have garnered interest as potential therapeutics. [15]

Immune response

One of the most problematic aspects of the use of phage lysins as antimicrobial agents is the potential immunogenicity of these enzymes. Unlike most antibiotics, proteins are prone to antibody recognition and binding, which means that lysins could be ineffective when treating bacterial infections or even dangerous, potentially leading to a systemic immune response or a cytokine storm. Nonetheless, experimental data from immunologically-rich rabbit serum showed that hyperimmune serum slows down but does not block the activity of pneumococcal lysin Cpl-1. [16]

Antimicrobial use

Phage lysins have been successfully tested in animal models to control pathogenic antibiotic-resistant bacteria found on mucous membranes and in blood. The main advantage of lysins compared to antibiotics is not only the low bacterial resistance but also the high specificity towards the target pathogen, and low activity towards the host's normal bacterial flora. [5]

Lysins were first used therapeutically in animals in 2001, in a publication in which mice orally colonized with Streptococcus pyogenes were decolonized with a single dose of PlyC lysin delivered orally. [17]

See also

Related Research Articles

<span class="mw-page-title-main">Bacteriophage</span> Virus that infects and replicates within bacteria

A bacteriophage, also known informally as a phage, is a virus that infects and replicates within bacteria and archaea. The term was derived from "bacteria" and the Greek φαγεῖν, meaning "to devour". Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome, and may have structures that are either simple or elaborate. Their genomes may encode as few as four genes and as many as hundreds of genes. Phages replicate within the bacterium following the injection of their genome into its cytoplasm.

<span class="mw-page-title-main">Lambda phage</span> Bacteriophage that infects Escherichia coli

Enterobacteria phage λ is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It was discovered by Esther Lederberg in 1950. The wild type of this virus has a temperate life cycle that allows it to either reside within the genome of its host through lysogeny or enter into a lytic phase, during which it kills and lyses the cell to produce offspring. Lambda strains, mutated at specific sites, are unable to lysogenize cells; instead, they grow and enter the lytic cycle after superinfecting an already lysogenized cell.

Autolysins are endogenous lytic enzymes that break down the peptidoglycan components of biological cells which enables the separation of daughter cells following cell division. They are involved in cell growth, cell wall metabolism, cell division and separation, as well as peptidoglycan turnover and have similar functions to lysozymes.

<span class="mw-page-title-main">Lytic cycle</span> Cycle of viral reproduction

The lytic cycle is one of the two cycles of viral reproduction, the other being the lysogenic cycle. The lytic cycle results in the destruction of the infected cell and its membrane. Bacteriophages that only use the lytic cycle are called virulent phages.

<span class="mw-page-title-main">Pseudopeptidoglycan</span>

Pseudopeptidoglycan is a major cell wall component of some Archaea that differs from bacterial peptidoglycan in chemical structure, but resembles bacterial peptidoglycan in function and physical structure. Pseudopeptidoglycan, in general, is only present in a few methanogenic archaea. The basic components are N-acetylglucosamine and N-acetyltalosaminuronic acid, which are linked by β-1,3-glycosidic bonds.

<span class="mw-page-title-main">Penicillin-binding proteins</span> Class of proteins

Penicillin-binding proteins (PBPs) are a group of proteins that are characterized by their affinity for and binding of penicillin. They are a normal constituent of many bacteria; the name just reflects the way by which the protein was discovered. All β-lactam antibiotics bind to PBPs, which are essential for bacterial cell wall synthesis. PBPs are members of a subgroup of enzymes called transpeptidases. Specifically, PBPs are DD-transpeptidases.

Holins are a diverse group of small proteins produced by dsDNA bacteriophages in order to trigger and control the degradation of the host's cell wall at the end of the lytic cycle. Holins form pores in the host's cell membrane, allowing lysins to reach and degrade peptidoglycan, a component of bacterial cell walls. Holins have been shown to regulate the timing of lysis with great precision. Over 50 unrelated gene families encode holins, making them the most diverse group of proteins with common function. Together with lysins, holins are being studied for their potential use as antibacterial agents.

The Phage-ligand technology is a technology to detect, bind and remove bacteria and bacterial toxins by using highly specific bacteriophage derived proteins.

<span class="mw-page-title-main">Enzybiotics</span> Experimental antibacterial therapy

Enzybiotics are an experimental antibacterial therapy. The term is derived from a combination of the words “enzyme” and “antibiotics.” Enzymes have been extensively utilized for their antibacterial and antimicrobial properties. Proteolytic enzymes called endolysins have demonstrated particular effectiveness in combating a range of bacteria and are the basis for enzybiotic research. Endolysins are derived from bacteriophages and are highly efficient at lysing bacterial cells. Enzybiotics are being researched largely to address the issue of antibiotic resistance, which has allowed for the proliferation of drug-resistant pathogens posing great risk to animal and human health across the globe.

In molecular biology, VanY are protein domains found in enzymes named metallopeptidases. They are vital to bacterial cell wall synthesis and antibiotic resistance.

<span class="mw-page-title-main">Epimerox</span> Chemical compound

Epimerox is an experimental broad-spectrum antibiotic compound being developed by scientists at the Rockefeller University and Astex Pharmaceuticals. It is a small molecule inhibitor compound that blocks the activity of the enzyme UDP-N-acetylglucosamine 2-epimerase, an epimerase enzyme that is called 2-epimerase for short.

<span class="mw-page-title-main">OBPgp279</span>

OBPgp279 is an endolysin that hydrolyzes peptidoglycan, a major constituent in bacterial membrane. OBPgp279 is found in Pseudomonas fluorescens phage OBP, which belongs in the Myoviridae family of bacteriophages. Because of its role in hydrolyzing the peptidoglycan layer, OBPgp279 is a key enzyme in the lytic cycle of the OBP bacteriophage; it allows the bacteriophage to lyse its host internally to escape. Unlike other endolysins, OBPgp279 does not rely on holins to perforate the inner bacterial membrane in order to reach the peptidoglycan layer. Although OBPgp279 is not a well-studied enzyme, it has garnered interest as a potential antibacterial protein due to its activity against multidrug-resistant gram-negative bacteria.

The Phi11 Holin Family constitutes the Holin Superfamily I.

The Phage 21 S Family is a member of the Holin Superfamily II.

The T4 Holin Family is a group of putative pore-forming proteins that does not belong to one of the seven holin superfamilies. T-even phage such as T4 use a holin-endolysin system for host cell lysis. Although the endolysin of phage T4 encoded by the e gene was identified in 1961, the holin was not characterized until 2001. A representative list of proteins belonging to the T4 holin family can be found in the Transporter Classification Database.

The Lactococcus lactis Phage r1t Holin Family is a family of putative pore-forming proteins that typically range in size between about 65 and 95 amino acyl residues (aas) in length, although a few r1t holins have been found to be significantly larger. Phage r1t holins exhibit between 2 and 4 transmembrane segments (TMSs), with the 4 TMS proteins resulting from an intragenic duplication of a 2 TMS region. A representative list of the proteins belonging to the r1t holin family can be found in the Transporter Classification Database.

The Bacterophase Dp-1 Holin Family is a family of proteins present in several Gram-positive bacteria and their phage. The genes coding for the lytic system of the pneumococcal phage, Dp-1, has been cloned and characterized. The holin of phage Dp-1 is 74 amino acyl residues (aas) long with two putative transmembrane segments (TMSs). The lytic enzyme of Dp-1 (Pal), an N-acetyl-muramoyl-L-alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophage in which change in the order of the functional domains changes the enzyme specificity. A representative list of proteins belonging to the Dp-1 family can be found in the Transporter Classification Database.

The Actinobacterial Phage Holin (APH) Family is a fairly large family of proteins between 105 and 180 amino acyl residues in length, typically exhibiting a single transmembrane segment (TMS) near the N-terminus. A representative list of proteins belonging to the APH family can be found in the Transporter Classification Database.

Although cell wall carbohydrates are ideal immunotherapeutic targets due to their abundance in bacteria and high level of conservation, their poor immunogenicity compared with protein targets complicates their use for the development of protective antibodies. A lysibody is a chimeric antibody in which the Fab region is the binding domain from a bacteriophage lysin, or the binding domain from an autolysin or bacteriocin, all of which bind to bacterial cell wall carbohydrate epitopes. This is linked to the Fc of Immunoglobulin G (IgG). The chimera forms a stable homodimer held together by hinge-region disulfide bonds. Thus, lysibodies are homodimeric hybrid immunoglobulin G molecules that can bind with high affinity and specificity to a carbohydrate substrate in the bacterial cell wall peptidoglycan. Lysibodies behave like authentic IgG by binding at high affinity to their bacterial wall receptor, fix complement and therefore promote phagocytosis by macrophages and neutrophils, protecting mice from infection in model systems. Since cell wall hydrolases, autolysins and bacteriocins are ubiquitous in nature, production of lysibodies specific for difficult to treat pathogenic bacteria is possible.

Vincent A. Fischetti is a world renowned American microbiologist and immunologist. He is Professor of and Head of the Laboratory of Bacterial Pathogenesis and Immunology at Rockefeller University in New York City. His Laboratory is the oldest continuous laboratory at Rockefeller that started in 1926 and headed by 4 leading scientists over its near 100-year history: Homer Swift, Maclyn McCarty, Emil Gotschlich and now Vincent Fischetti. Keeping with the historical theme of infectious diseases, Fischetti's primary areas of research are bacterial pathogenesis, bacterial genomics, immunology, virology, microbiology, and therapeutics. He was the first scientist to clone and sequence a surface protein on gram-positive bacteria, the M protein from S. pyogenes, and determine its unique coiled-coil structure. He also was the first use phage lysins as a therapeutic and an effective alternative to conventional antibiotics.

References

  1. ↑ Pérez-Dorado I, Campillo NE, Monterroso B, Hesek D, Lee M, Páez JA, García P, Martínez-Ripoll M, García JL, Mobashery S, Menéndez M, Hermoso JA (August 2007). "Elucidation of the molecular recognition of bacterial cell wall by modular pneumococcal phage endolysin CPL-1". J. Biol. Chem. 282 (34): 24990–9. doi: 10.1074/jbc.M704317200 . hdl: 10261/12517 . PMID   17581815.
  2. ↑ Visweswaran GR, Dijkstra BW, Kok J (November 2010). "Two major archaeal pseudomurein endoisopeptidases: PeiW and PeiP". Archaea. 2010: 480492. doi: 10.1155/2010/480492 . PMC   2989375 . PMID   21113291.
  3. ↑ Quemin ER, Quax TE (5 June 2015). "Archaeal viruses at the cell envelope: entry and egress". Frontiers in Microbiology. 6: 552. doi: 10.3389/fmicb.2015.00552 . PMC   4456609 . PMID   26097469.
  4. 1 2 Young R (September 1992). "Bacteriophage lysis: mechanism and regulation". Microbiological Reviews. 56 (3): 430–81. doi:10.1128/mr.56.3.430-481.1992. PMC   372879 . PMID   1406491.
  5. 1 2 3 4 5 Fischetti VA (Oct 2008). "Bacteriophage lysins as effective antibacterials". Current Opinion in Microbiology. 11 (5): 393–400. doi:10.1016/j.mib.2008.09.012. PMC   2597892 . PMID   18824123.
  6. 1 2 McGowan S, Buckle AM, Mitchell MS, Hoopes JT, Gallagher DT, Heselpoth RD, Shen Y, Reboul CF, Law RH, Fischetti VA, Whisstock JC, Nelson DC (Jul 2012). "X-ray crystal structure of the streptococcal specific phage lysin PlyC". Proceedings of the National Academy of Sciences of the United States of America. 109 (31): 12752–7. Bibcode:2012PNAS..10912752M. doi: 10.1073/pnas.1208424109 . PMC   3412044 . PMID   22807482.
  7. ↑ Baker JR, Liu C, Dong S, Pritchard DG (October 2006). "Endopeptidase and glycosidase activities of the bacteriophage B30 lysin". Applied and Environmental Microbiology. 72 (10): 6825–8. doi: 10.1128/AEM.00829-06 . PMC   1610294 . PMID   17021237.
  8. ↑ Navarre WW, Ton-That H, Faull KF, Schneewind O (May 1999). "Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity". The Journal of Biological Chemistry. 274 (22): 15847–56. doi: 10.1074/jbc.274.22.15847 . PMID   10336488.
  9. ↑ García E, García JL, García P, Arrarás A, Sánchez-Puelles JM, López R (Feb 1988). "Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages". Proceedings of the National Academy of Sciences of the United States of America. 85 (3): 914–8. Bibcode:1988PNAS...85..914G. doi: 10.1073/pnas.85.3.914 . JSTOR   31364. PMC   279667 . PMID   3422470.
  10. 1 2 3 Loessner MJ, Kramer K, Ebel F, Scherer S (Apr 2002). "C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates". Molecular Microbiology. 44 (2): 335–49. doi:10.1046/j.1365-2958.2002.02889.x. PMID   11972774.
  11. ↑ García P, García JL, García E, Sánchez-Puelles JM, López R (Jan 1990). "Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages". Gene. 86 (1): 81–8. doi:10.1016/0378-1119(90)90116-9. PMID   2311937.
  12. ↑ Wang IN, Smith DL, Young R (2000). "Holins: the protein clocks of bacteriophage infections". Annual Review of Microbiology. 54: 799–825. doi:10.1146/annurev.micro.54.1.799. PMID   11018145.
  13. ↑ Yoong P, Schuch R, Nelson D, Fischetti VA (Jul 2004). "Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium". Journal of Bacteriology. 186 (14): 4808–12. doi:10.1128/JB.186.14.4808-4812.2004. PMC   438584 . PMID   15231813.
  14. ↑ Plotka M, Kaczorowska AK, Stefanska A, Morzywolek A, Fridjonsson OH, Dunin-Horkawicz S, Kozlowski L, Hreggvidsson GO, Kristjansson JK, Dabrowski S, Bujnicki JM, Kaczorowski T (Feb 2014). "Novel highly thermostable endolysin from Thermus scotoductus MAT2119 bacteriophage Ph2119 with amino acid sequence similarity to eukaryotic peptidoglycan recognition proteins". Applied and Environmental Microbiology. 80 (3): 886–95. doi:10.1128/AEM.03074-13. PMC   3911187 . PMID   24271162.
  15. ↑ Briers Y, Walmagh M, Van Puyenbroeck V, Cornelissen A, Cenens W, Aertsen A, et al. (July 2014). "Engineered endolysin-based "Artilysins" to combat multidrug-resistant gram-negative pathogens". mBio. 5 (4): e01379-14. doi:10.1128/mBio.01379-14. PMC   4161244 . PMID   24987094.
  16. ↑ Loeffler JM, Djurkovic S, Fischetti VA (Nov 2003). "Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia". Infection and Immunity. 71 (11): 6199–204. doi:10.1128/IAI.71.11.6199-6204.2003. PMC   219578 . PMID   14573637.
  17. ↑ Nelson D, Loomis L, Fischetti VA (Mar 2001). "Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme". Proceedings of the National Academy of Sciences of the United States of America. 98 (7): 4107–12. Bibcode:2001PNAS...98.4107N. doi: 10.1073/pnas.061038398 . PMC   31187 . PMID   11259652.
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