A two‐component, multimeric endolysin encoded by a single gene

Abstract

SummaryBacteriophage endolysins are bacterial cell wall degrading enzymes whose potential to fight bacterial infections has been intensively studied. Endolysins from Gram-positive systems are typically described as monomeric and as having a modular structure consisting of one or two N-terminal catalytic domains (CDs) linked to a C-terminal region responsible for cell wall binding (CWB). We show here that expression of the endolysin gene lys170 of the enterococcal phage F170/08 results in two products, the expected full length endolysin (Lys170FL) and a C-terminal fragment corresponding to the CWB domain (CWB170). The latter is produced from an in-frame, alternative translation start site. Both polypeptides interact to form the fully active endolysin. Biochemical data strongly support a model where Lys170 is made of one monomer of Lys170FL associated with up to three CWB170 subunits, which are responsible for efficient endolysin binding to its substrate. Bioinformatics analysis indicates that similar secondary translation start signals may be used to produce and add independent CWB170-like subunits to different enzymatic specificities. The particular configuration of endolysin Lys170 uncovers a new mode of increasing the number of CWB motifs associated to CD modules, as an alternative to the tandem repetition typically found in monomeric cell wall hydrolases.

EC300: a phage-based, bacteriolysin-like protein with enhanced antibacterial activity against Enterococcus faecalis

“…2). The same fragment is also produced during synthesis of the parental Lys170, and we showed recently that it is essential for robust lytic activity of the endolysin (Proença et al 2014). In fact, the C-terminal product (CWB170) results from an in frame, secondary translational start site lying at the beginning of the CWB170 coding sequence; this secondary start site is present both in Lys170 and EC300 sequences (Fig.…”

“…Gene EC300 in pDPEC300 was subjected to site-directed mutagenesis by using the Quick Change II Site directed mutagenesis kit (Stratagene Agilent Technologies) and the primer pair Fw_BD_M170/ Rv_BD_M170, resulting in plasmid pDPmEC300 carrying mEC300 gene. The introduced nucleotide substitutions eliminated the internal translation start site known to drive the independent synthesis of the CWB170 domain (Proença et al 2014, see text also). The pIVEX vectors allow the expression of genes under the control of the phage T7 ϕ10 promoter, and the production of the corresponding proteins C-terminally fused to a hexahistidine tag.…”

“…The standard proteins (Bio-Rad Laboratories) were thyroglobulin (molecular mass = 670 kDa, Stokes radius = 8.6 nm), γ-globulin (158 kDa, 4.8 nm), ovalbumin (44 kDa, 2.73 nm), myoglobin (17 kDa, 2.08 nm), and vitamin B12 (1.35 kDa, 0.85 nm). Proteins Lys170 and CWB170, also used in this work, were produced from pIVEX2.3 derivatives pDP2 ) and pDP4 (Proença et al 2014) and purified as described above.…”

“…Lys170 endolysin was only tested at the maximum amount (10 μg). EC300, mEC300, Lys170, and CWB170, alone or in combination, were also tested on dense lawns of viable E. faecalis strain 1518/05 prepared in agarized protein buffer (0.7 % agar) as described previously (Proença et al 2014). Negative controls were prepared by spotting 10 μl of protein buffer.…”

“…For that, we have assumed the theoretical advantages of VALs and bacteriolysins referred to above and have generated an artificial, bacteriolysin-like enzyme (EC300) by fusing the M23 endopeptidase CD of the VAL Orf73 to the CWB domain of the previously characterized endolysin Lys170 (Proença et al , 2014, both produced by the E. faecalis phage F170/08. The results show the superior lytic activity of EC300 when compared to the endolysin Lys170.…”