Title : Lytic activity of secreted LysH 5 endolysin by Lactococcus lactis using the 1 secretion signal sequence of the bacteriocin Lcn 972 2 3 Running Title : Endolysin secretion in Lactococcus lactis 4

19 20 Bacteriophage endolysins have an interesting potential as antimicrobials. The endolysin 21 LysH5, encoded by Staphylococcus aureus phage vB_SauS-phi-IPLA88, was expressed 22 and secreted in Lactococcus lactis using the signal peptide of the bacteriocin lactococcin 23 972 and lactococcal constitutive and inducible promoters. Up to 80 U/mg of 24 extracelullar active endolysin was detected in culture supernatants but most of the 25 protein (up to 323 U/mg) remained in the cell extracts. 26 27 Staphylococcus aureus is a cause of serious concern in clinical settings 28 especially due to the emergence of methicillin-resistant S. aureus (MRSA) and 29 vancomycin-resistant S. aureus (VRSA) (13). Moreover, S. aureus is a threat to food 30 safety (3). In the dairy environment, this pathogen is recognized as a frequent cause of 31 subclinical intramammary infections in dairy cows (28). Bacteriophage endolysins 32 mediate lysis of the host bacteria at the end of the lytic cycle to release phage progeny 33 (39). They have a huge potential as novel therapeutic antibacterial agents (7), as 34 biopreservatives in foods (27), in pathogen detection (38), and as disinfectants of 35 industrial facilities (32). 36 Lactococcus lactis, a GRAS (Generally Regarded As Safe) microorganism with 37 a long history of safe use in food fermentations, has been proven as suitable host for 38 expression and purification of heterologous proteins with food safety applications such 39 as bacteriocins (1, 23) and bacteriophage lytic enzymes against Listeria monocytogenes 40 (8, 36) and Clostridium difficile (24). To facilitate secretion, proteins have been fused to 41 bacteriocin signals (12) or to the signal peptide of Usp45 (SPusp45), the major Sec42 dependent protein secreted by L. lactis (1). The ability of this microorganism for protein 43 secretion can be exploited to improve large-scale production processes and downstream 44 purification steps (19). 45 LysH5 endolysin from S. aureus bacteriophage vB_SauS-phi-IPLA88 has been 46 characterized. LysH5 is a 53.7-kDa protein, encoded by a 1,446 bp gene (10), which 47 lysed a wide range of staphylococci and it also inhibits S. aureus growth in milk (27, 48 11). 49 The aim of this work was cloning and expression of the LysH5 encoding-gene 50 into a L. lactis strain under the control of lactococcal inducible and constitutive 51 promoters to facilitate the potential application of this endolysin as food preservative. 52 For secretion, the signal peptide (SPLcn972) of the bacteriocin lactococcin 972 (Lcn972), 53 which shows a sec-dependent processing signal (22), has been tagged with LysH5 54