View source for Lysin

{{Distinguish|lysine}}
{{Other uses}}
{{infobox enzyme
| Name       = Lysozyme-like phage lysin
| EC_number  = 3.2.1.17
| CAS_number = 9001-63-2
| GO_code    = 0003796
| image      = Crystal structure of the modular CPL-1 endolysin complexed with a peptidoglycan analogue.jpg
| width      = 270
| caption    = Crystal structure of the modular CPL-1 endolysin from Streptococcus phage Cp-1 complexed with a peptidoglycan analogue. [[Protein Data Bank|PDB]] entry {{PDBe|2j8g}}.<ref name="pmid17581815">{{cite journal | vauthors = 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 | title = Elucidation of the molecular recognition of bacterial cell wall by modular pneumococcal phage endolysin CPL-1 | journal = J. Biol. Chem. | volume = 282 | issue = 34 | pages = 24990–9 |date=August 2007 | pmid = 17581815 | doi = 10.1074/jbc.M704317200 | doi-access = free | hdl = 10261/12517 | hdl-access = free }}</ref>
}}
'''Lysins''', also known as '''endolysins''' or '''murein hydrolases''', are [[hydrolase|hydrolytic enzymes]] produced by [[bacteriophage]]s 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,<ref>{{cite journal | vauthors = Visweswaran GR, Dijkstra BW, Kok J | title = Two major archaeal pseudomurein endoisopeptidases: PeiW and PeiP | journal = Archaea | volume = 2010 | pages = 480492 | date = November 2010 | pmid = 21113291 | pmc = 2989375 | doi = 10.1155/2010/480492 | doi-access = free }}</ref> while most archaeal viruses employ alternative mechanisms.<ref>{{cite journal | vauthors = Quemin ER, Quax TE | title = Archaeal viruses at the cell envelope: entry and egress | journal = Frontiers in Microbiology | volume = 6 | pages = 552 | date = 5 June 2015 | pmid = 26097469 | doi = 10.3389/fmicb.2015.00552 | pmc = 4456609 | doi-access = free }}</ref> Similarly, not all bacteriophages synthesize lysins: some small single-stranded DNA and RNA phages produce membrane proteins that activate the host's [[Autolysis (biology)|autolytic]] mechanisms such as [[autolysin]]s.<ref name="Young1">{{cite journal | vauthors = Young R | title = Bacteriophage lysis: mechanism and regulation | journal = Microbiological Reviews | volume = 56 | issue = 3 | pages = 430–81 | date = September 1992 | doi = 10.1128/mr.56.3.430-481.1992 | pmid = 1406491 | pmc = 372879 }}</ref>

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.<ref name="Fisch">{{cite journal | vauthors = Fischetti VA | title = Bacteriophage lysins as effective antibacterials | journal = Current Opinion in Microbiology | volume = 11 | issue = 5 | pages = 393–400 | date = Oct 2008 | pmid = 18824123 | pmc = 2597892 | doi = 10.1016/j.mib.2008.09.012 }}</ref> 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 k[[Atomic mass unit|Da]] 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.<ref name="McGowan">{{cite journal | vauthors = McGowan S, Buckle AM, Mitchell MS, Hoopes JT, Gallagher DT, Heselpoth RD, Shen Y, Reboul CF, Law RH, Fischetti VA, Whisstock JC, Nelson DC | title = X-ray crystal structure of the streptococcal specific phage lysin PlyC | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 31 | pages = 12752–7 | date = Jul 2012 | pmid = 22807482 | pmc = 3412044 | doi = 10.1073/pnas.1208424109 | bibcode = 2012PNAS..10912752M | doi-access = free }}</ref>

All other lysins are monomeric and comprise two [[protein domain|domains]] separated by a short linker region. For gram positive bacteria lysins, the [[N-terminus|N-terminal]] domain catalyses the hydrolysis of peptidoglycan whereas the [[C-terminus|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:
* Endo-β-N-acetylglucosaminidase ([[Endoglycosidase H]], {{EC number|3.2.1.96}})
* N-acetylmuramidase ([[lysozyme]]-like, {{EC number|3.2.1.17}})
* [[Endopeptidase]]
* [[N-acetylmuramoyl-L-alanine amidase]] (T7-like, {{EC number|3.5.1.28}})
* [[γ-D-glutaminyl-L-lysine endopeptidase]] ({{EC number|3.4.14.13}})

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 [[autolysin]]s, early confusion around the cleavage specificity of these individual enzymes has led to some misattributions of the name "lysozyme" to proteins without this activity.<ref>{{cite journal | vauthors = Baker JR, Liu C, Dong S, Pritchard DG | title = Endopeptidase and glycosidase activities of the bacteriophage B30 lysin | journal = Applied and Environmental Microbiology | volume = 72 | issue = 10 | pages = 6825–8 | date = October 2006 | pmid = 17021237 | doi = 10.1128/AEM.00829-06 | doi-access = free | pmc = 1610294 }}</ref>

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.<ref name="McGowan"/><ref name="Navarre">{{cite journal | vauthors = Navarre WW, Ton-That H, Faull KF, Schneewind O | title = Multiple enzymatic activities of the murein hydrolase from staphylococcal phage phi11. Identification of a D-alanyl-glycine endopeptidase activity | journal = The Journal of Biological Chemistry | volume = 274 | issue = 22 | pages = 15847–56 | date = May 1999 | pmid = 10336488 | doi = 10.1074/jbc.274.22.15847 | doi-access = free }}</ref> Catalytic domains are highly conserved in phage lysins of the same class.<ref name="Fisch"/>

=== 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.<ref name="Garcia1">{{cite journal | vauthors = García E, García JL, García P, Arrarás A, Sánchez-Puelles JM, López R | title = Molecular evolution of lytic enzymes of Streptococcus pneumoniae and its bacteriophages | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 85 | issue = 3 | pages = 914–8 | date = Feb 1988 | pmid = 3422470 | pmc = 279667 | doi = 10.1073/pnas.85.3.914 | bibcode = 1988PNAS...85..914G | jstor = 31364 | doi-access = free }}</ref> 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.<ref name="Loessner">{{cite journal | vauthors = Loessner MJ, Kramer K, Ebel F, Scherer S | title = C-terminal domains of Listeria monocytogenes bacteriophage murein hydrolases determine specific recognition and high-affinity binding to bacterial cell wall carbohydrates | journal = Molecular Microbiology | volume = 44 | issue = 2 | pages = 335–49 | date = Apr 2002 | pmid = 11972774 | doi = 10.1046/j.1365-2958.2002.02889.x | doi-access =  }}</ref>

== 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.<ref name="Garcia2">{{cite journal | vauthors = García P, García JL, García E, Sánchez-Puelles JM, López R | title = Modular organization of the lytic enzymes of Streptococcus pneumoniae and its bacteriophages | journal = Gene | volume = 86 | issue = 1 | pages = 81–8 | date = Jan 1990 | pmid = 2311937 | doi = 10.1016/0378-1119(90)90116-9 }}</ref>

== 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.<ref name="Fisch"/> 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.<ref name="Loessner"/> 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.<ref name="Loessner"/>

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.<ref name="Young1"/><ref name="Wang">{{cite journal | vauthors = Wang IN, Smith DL, Young R | title = Holins: the protein clocks of bacteriophage infections | journal = Annual Review of Microbiology | volume = 54 | pages = 799–825 | year = 2000 | pmid = 11018145 | doi = 10.1146/annurev.micro.54.1.799 }}</ref>

== 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.<ref name="Yoong">{{cite journal | vauthors = Yoong P, Schuch R, Nelson D, Fischetti VA | title = Identification of a broadly active phage lytic enzyme with lethal activity against antibiotic-resistant Enterococcus faecalis and Enterococcus faecium | journal = Journal of Bacteriology | volume = 186 | issue = 14 | pages = 4808–12 | date = Jul 2004 | pmid = 15231813 | pmc = 438584 | doi = 10.1128/JB.186.14.4808-4812.2004 }}</ref> Similarly, some thermostable lysins are known, which makes them easier to use in biotechnology.<ref name="pmid24271162">{{cite journal | vauthors = 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 | title = Novel highly thermostable endolysin from Thermus scotoductus MAT2119 bacteriophage Ph2119 with amino acid sequence similarity to eukaryotic peptidoglycan recognition proteins | journal = Applied and Environmental Microbiology | volume = 80 | issue = 3 | pages = 886–95 | date = Feb 2014 | pmid = 24271162 | pmc = 3911187 | doi = 10.1128/AEM.03074-13 }}</ref> Regarding their use as antibacterial agents, lysins have been found effective mainly against [[Gram-positive]] bacteria, since [[Gram-negative]] bacteria possess an [[bacterial outer membrane|outer membrane]] that prevents extracellular lysin molecules from digesting peptidoglycan.<ref name="Fisch"/> However, lysins with activity against Gram-negative bacteria, such as [[OBPgp279]], have garnered interest as potential therapeutics.<ref>{{cite journal | vauthors = Briers Y, Walmagh M, Van Puyenbroeck V, Cornelissen A, Cenens W, Aertsen A, Oliveira H, Azeredo J, Verween G, Pirnay JP, Miller S, Volckaert G, Lavigne R | display-authors = 6 | title = Engineered endolysin-based "Artilysins" to combat multidrug-resistant gram-negative pathogens | journal = mBio | volume = 5 | issue = 4 | pages = e01379-14 | date = July 2014 | pmid = 24987094 | pmc = 4161244 | doi = 10.1128/mBio.01379-14 }}</ref>

== 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.<ref name="Loeffler">{{cite journal | vauthors = Loeffler JM, Djurkovic S, Fischetti VA | title = Phage lytic enzyme Cpl-1 as a novel antimicrobial for pneumococcal bacteremia | journal = Infection and Immunity | volume = 71 | issue = 11 | pages = 6199–204 | date = Nov 2003 | pmid = 14573637 | pmc = 219578 | doi = 10.1128/IAI.71.11.6199-6204.2003 }}</ref>

== Antimicrobial use ==
Phage lysins have been successfully tested in animal models to control pathogenic antibiotic-resistant bacteria found on [[mucous membrane]]s 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]].<ref name="Fisch"/>

Lysins were first used therapeutically in animals in 2001, in a publication in which mice orally colonized with ''[[Streptococcus pyogenes]]'' were [[Decolonization (medicine)|decolonized]] with a single dose of PlyC lysin delivered orally.<ref>{{cite journal | vauthors = Nelson D, Loomis L, Fischetti VA | title = Prevention and elimination of upper respiratory colonization of mice by group A streptococci by using a bacteriophage lytic enzyme | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 98 | issue = 7 | pages = 4107–12 | date = Mar 2001 | pmid = 11259652 | doi = 10.1073/pnas.061038398 | pmc=31187| bibcode = 2001PNAS...98.4107N | doi-access = free }}</ref>

== See also ==
*[[Phage therapy]]
*[[Enzybiotics]]
*[[Lysozyme]]
*[[OBPgp279]]
*[[Lysibody]]

== References ==
{{reflist|35em}}

{{Sugar hydrolases}}
{{Enzymes}}
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[[Category:Viral enzymes]]
[[Category:EC 3.2.1]]