Peptidoglycan hydrolase fusions maintain their parental specificities

Abstract

The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiae are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated S. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans. Both fusions display lytic specificity for streptococcal pathogens and S. aureus. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63 degrees C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.

FIG. 1.

Plate lysis assays of B30 endolysin and lysostaphin constructs. Crude E. coli extracts were prepared from cells harboring pET21a-derived expression vectors. Spots of cleared lawn represent lysis of the target organisms. Lysostaphin (Sigma bact lysostaphin) in lysin buffer A was spotted as a control. B30-90 is an inactive truncation and served as a negative control. S. agal, S. agalactiae; S. aur, S. aureus; S. uber, S. uberis; S. dys, S. dysgalactiae.

FIG. 2.

SDS-PAGE and zymogram analysis of lysin constructs. (A) Analysis of endolysin B30-443 and the fusion protein 443-Lyso. Lane M, markers; lanes 1 and 2, SDS-PAGE of E. coli crude extract and purified B30-443, respectively; lane 3, SDS-PAGE of E. coli crude extract of 443-Lyso; lanes 4 and 5, zymogram with S. agalactiae of E. coli crude extract and purified B30-443, respectively; lane 6, zymogram with S. agalactiae and E. coli crude extract of 443-Lyso; lane 7, zymogram with S. aureus and E. coli crude extract of 443-Lyso. The 443-Lyso fusion was not constructed with a His tag and thus was not purified. (B) Analysis of truncated endolysin B30-182 and the fusion protein 182-Lyso. Lane M, markers; lanes 1 and 2, SDS-PAGE of E. coli crude extract and purified B30-182, respectively; lanes 3 and 4, SDS-PAGE of E. coli crude extract and purified 182-Lyso, respectively; lanes 5 and 6, zymograms with S. agalactiae of E. coli crude extract and purified B30-182, respectively; lanes 7 and 8, zymograms with S. agalactiae of E. coli crude extract and purified 182-Lyso, respectively; lanes 9 and 10, zymograms with S. aureus of E. coli crude extract and purified 182-Lyso, respectively.

FIG. 3.

Immunohistochemistry of 182-Lyso expression in CHO cells with lysostaphin antibody. Transiently transfected CHO cells are shown. Blue, DAPI (4′,6′-diamidino-2-phenylindole)-stained nuclei; red, Alexa 594 chromophore.

FIG. 4.

SH3b sequences in B30 endolysin and lysostaphin proteins. (A) Conserved sequences between SH3b domains of lysostaphin and the B30 endolysin. (B) Conserved sequences between the B30 SH3b domain and the intrahydrolytic region of the B30 endolysin. Solid underlining shows the Acm lysozyme domain (aa 145 to 344); dashed underlining shows the B30 SH3b domain (aa 356 to 443).