United States of America, As Represented by the Secretary of Agriculture

University System of Maryland

Western University of Health Sciences

David M Donovan

Igor V Abaev

Beatriz Martinez Fernandez

Pilar Garcia Suarez

Steven M Swift

Stephen C Becker

Ana Rodriguez

Lorena Rodriguez Rubio

Stephen R Hughes

Kenneth M Bischoff

Bruce S Seal

Gregory Ross Siragusa

Johnna Kennedy Garrish

Ibn Mustafa A Simmons

Jerel Waters

Jinzhe Mao

Dayana T Rowley

Dwayne R Roach

Brian B Oakley

Piyum A Khatibi

Mathias Schmelcher

The disclosure relates to chimeric recombinant lysins comprising at least one thermophile endolysin catalytic domain and at least one cell wall binding domain. Also disclosed are polynucleotides encoding the chimeric recombinant lysins, host cells expressing the chimeric recombinant lysins, and use of such chimeric recombinant lysins.

Thermophile peptidoglycan hydrolase fusion proteins and uses thereof

can cause food poisoning and is a major agent in necrotic enteritis. As laws banning the use of antibiotics in animal feed become more common, the need for alternatives to antibiotics becomes greater. Peptidoglycan hydrolases that target the cell wall of specific bacteria are one such alternative. Genes for four endolysins, PlyCP10, PlyCP18, PlyCP33, and PlyCP41, were found within clusters of phage associated genes, likely prophages from strains Cp10, Cp18, Cp33, and Cp41. PlyCP18 and PlyCP33 harbor L-alanine amidase catalytic domains, and PlyCP10 and PlyCP41 have glycosyl hydrolase catalytic domains as predicted by BlastP and PFAM searches. All four genes were synthesized withcodon optimization, expressed inexpression vectors with a 6&#xd7;His tag for nickel column purification, and the recombinant proteins purified. The four endolysins were capable of lysing the 66strains tested but not the other bacteria tested.

Characterization of four prophage endolysins specific for clostridium perfringens

Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. Fusion proteins HydH5SH3b and HydH5Lyso comprising full-length peptidoglycan hydrolase HydH5 from thebacteriophage vB_SauS-phi-IPLA88 in combination with the SH3b cell wall-binding domain from lysostaphin or full length lysostaphin, respectively, exhibited high lytic activity against livecells. CHAPSH3b, a HydH5 CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain from truncated HydH5 in combination with the SH3b domain of lysostaphin, exhibited the highest lytic activity against livecells. HydH5 and its derivative fusions lysed bovine and human, methicillin-resistant(MRSA) N315 strain, and humanstrains in zymogram, plate lysis and turbidity reduction assays. HydH5 and its derivative fusions displayed antimicrobial synergy with the endolysin LysH5 (also encoded by vB_SauS-phi-IPLA88) in vitro suggesting the two enzymes have distinct cut sites and thus may be more efficient in combination for elimination of staphylococcal infections.

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Increases in antibiotic resistant strains ofhave elicited efforts to develop novel antimicrobials. One potential treatment includes lytic enzymes produced by staphylococcal bacteriophage during the lytic cycle. The phage Twort endolysin (PlyTW) harbors three domains, a CHAP endopeptidase, an amidase-2 domain, and a SH3b-5 cell wall binding domain. The CHAP domain alone is necessary and sufficient for lysis of live; the amidase-2 domain alone is insufficient. Loss of the SH3b cell wall binding domain results in a 10 fold reduction of enzymatic activity in turbidity reduction and plate lysis assays compared to the full length protein. Deletion of the amidase-2 domain resulted in a protein (PlyTW &#x394;172-373) with lytic activity that exceeded the activity of the full length construct in both assays. Addition of Caenhanced activity in turbidity reduction assays. Chelation by the addition of EDTA or zinc inhibited the activity of all PlyTW constructs.

The routine use of antibiotics to battle Streptococcal pathogens has produced a new class of superbug&#x2014;multi-drug resistant streptococci resulting in a need for new antimicrobials. The LambdaSa2 prophage endolysin gene harbors an amidase-5 (endopeptidase), an amidase-4 (glycosidase) domain and two Cpl-7 cell wall-binding domains. This endolysin can digest the cell walls ofand. Turbidity reduction and plate lysis assays indicate that this peptidoglycan hydrolase also shows strong lytic activity toward, GES, and GGS. Deletion analysis on the His-tagged version of this gene further indicates that the N-terminal endopeptidase domain is minimally active in the absence of a Cpl-7 domain when lysing cells from without; however, with both Cpl-7 domains, it achieves a higher specific activity than the full length protein (on some strains) and shows weak activity against two Coagulase Negative Staphylococci,and

Isolated recombinant cDNA

is notorious for developing resistance to virtually all antibiotics to which it is exposed. Staphylococcal phage 2638A endolysin is a peptidoglycan hydrolase that is lytic forwhen exposed externally, making it a new antimicrobial candidate. It shares a common protein organization with over 40 other staphylococcal peptidoglycan hydrolases: a CHAP endopeptidase domain, a mid-protein amidase 2 domain and a C-terminal SH3b cell wall binding domain. It is the first phage endolysin reported with a cryptic translational start site between the CHAP and amidase domains. Deletion analysis indicates that the amidase domain confers most of the lytic activity and requires the full SH3b domain for maximal activity. It is common for one domain to demonstrate dominant activity over another; however, the phage 2638A endolysin is the first to show high amidase domain activity dominant over the N-terminal CHAP domain, an important finding for targeting novel peptidoglycan bonds.

Ethanol losses due to bacterial contamination in fermentation cultures weakens the economics of biofuel production.species are the predominant contaminant. Bacteriophage lytic enzymes are peptidoglycan hydrolases which degrade Gram positive cell walls when exposed externally and are a novel source of antimicrobials. The streptococcal phage &#x3bb;SA2 endolysin construct demonstrated strong lytic activity towards 17 of 22 strains of lactobacilli, staphylococci or streptococci maintaining optimal specific activity under fermentation conditions towardsubstrates.bacteriophage endolysin constructs LysA, LysA2 and LysgaY showed exolytic activity towards &#x2dc;60% of the lactobacilli tested including fourisolates from fuel ethanol fermentations. Presence of ethanol (&#x2266;5%) did not affect lytic activity. Lysins were able to reduce bothandcontaminants in mock fermentations of corn fiber hydrolysates. Recombinant LysA and &#x3bb;Sa2 expressed in the yeastare functional; LysA was shown to reduce lactobacilli in experimentally infected fermentations.

Peptidoglycan hydrolase antimicrobials for eradicating lactobacilli that contaminate and reduce ethanol yields in biofuel fermentation

Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. Full-length HydH5, a virion-associated peptidoglycan hydrolase from thebacteriophage vB_SauS-phi-IPLA88, and two truncated derivatives, containing only the CHAP domain, exhibited high lytic activity against livecells. Three different fusion proteins were created and showed higher staphylolytic activity than the parental enzyme or its deletion construct. Parental and fusion proteins lysedcells in zymograms, plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and humanMRSA N315 strain, and humanstrains. HydH5 and its derivative fusions proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro suggesting that the two enzymes have distinct cut sites and thus may be more efficient in combination for the elimination of staphylococcal infections.

Methicillin-resistant (MRSA) and multi-drug resistant strains ofare becoming increasingly prevalent in both human and veterinary clinics.-causing bovine mastitis yields high annual losses to the dairy industry. Treatment of mastitis by broad range antibiotics is often not successful and may contribute to development of antibiotic resistance. Bacteriophage endolysins are a promising new source of antimicrobials. The endolysin of prophage &#x3c6;SH2 ofstrain JCSC1435 (&#x3c6;SH2 lysin) shows lytic activity against live staphylococcal cells. Deletion constructs were tested in zymograms and turbidity reduction assays to evaluate the contribution of each functional module to lysis. The CHAP domain exhibited three-fold higher activity than the full length protein. Activity was further enhanced in the presence of bivalent calcium ions. The full length enzyme and the CHAP domain showed activity against multiple staphylococcal strains, including MRSA strains, mastitis isolates, and coagulase negative staphylococcal (CoNS) strains.

The present invention relates to isolatedbacteriophage lytic enzymes from baccteriophages CP26F and CP39O, and uses in controlling

Bacteriophage lytic enzymes as alternative antimicrobials

Peptidoglycan hydrolases are an effective new source of antimicrobials. A chimeric fusion protein of the Ply187 endopeptidase domain and LysK SH3b cell wall binding domain is a potent agent againstin three functional assays.

Enhanced antimicrobial lytic activity of a chimeric Ply187 endolysin

Multi-drug resistant superbugs are a persistent problem in modern health care. LysK is a staphylococcal bacteriophage endolysin from the phage K. It is a peptidoglycan hydrolase enzyme that can lyse many staphylococcal strains and thus is a potent antimicrobial against, including MRSA. Lysostaphin is a bacteriocin secreted byto kill, and has been shown to also be a potent antimicrobial for many antibiotic resistant strains of. This study describes optimal reaction conditions for the recombinant His-tagged LysK protein, compares its MIC and antimicrobial activity to lysostaphin and demonstrates synergy when the two are used in combination against the MRSA USA300.

Lys K endolysin is synergistic with lysostaphin against MRSA

Multi-drug resistant superbugs are a persistent problem in modern health care. This invention provides an antimicrobial endolysin-Lysostaphin triple fusion protein, comprising (1) an endolysin CHAP endopeptidase domain, (2) an endolysin amidase domain, and (3) a Lysostaphin glycyl-glycine endopeptidase domain. The domains are derived from two proteins that show antimicrobial synergy when used in combination. The protein has specificity and exolytic activity for the peptidoglycan cell wall of untreated, livefrom many growth phases i.e. stationary, logarithmic and biofilm growth. The recombinant triple fusion protein comprising the three functional antimicrobial domains is designed to be refractory to resistance development.

Triple acting antimicrobials that are refractory to resistance development

Lysostaphin is a bacteriocin secreted byto kill, and has been shown to also be a potent antimicrobial for many antibiotic-resistant strains of. By adding a &#x2dc;13 amino acid protein transduction domain (PTD) from the HIV-TAT protein to lysostaphin to form lysostaphin-PTD, both extracellular and intracellular forms ofand MRSA are killed in all (multiple) cell types examined.

Fusion of peptidoglycan hydrolase enzymes to a protein transduction domain allows eradication of both extracellular and intracellular gram positive pathogens

Thebacteriophage phi11 endolysin has two peptidoglycan hydrolase domains (endopeptidase and amidase) and a SH3b cell wall-binding domain. In turbidity reduction assays, the purified protein can lyse untreated staphylococcal mastitis-causing pathogens,and coagulase negative staphylococci (, and), making it a strong antimicrobial protein and an effective candidate for treating multidrug-resistant staphylococci. Lytic activity is maintained at the pH (6.7) and the 'free' calcium concentration (3 mM) of milk. Truncated endolysin-derived proteins, containing just the endopeptidase domain, also lyse staphylococci, in the absence of the SH3b-binding domain.

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Baltimore, MD, United States of America

David M Donovan