As a bacteriocin, lysostaphin is evolved for the lysis of S. aureus cell walls. In contrast, LytM as an autolysin should be evolved to have its activity under tight control. We expected this to apply for the full length enzyme, but hoped to bypass this step by the artificial activation that removes the N-terminal domain and the occluding

region. Apparently, this does not suffice, because there are differences at several other levels which reflect the different in vivo roles of lysostaphin and LytM. We conclude that the use of LytM185-316 as an antibacterial agent is a more remote possibility than originally selleck chemical envisaged and that efforts to develop antibacterial peptidoglycan hydrolases should perhaps be concentrated on proteins that act as bacteriocins rather than autolysins. Methods Bacterial cultures Bacteria were grown in CASO broth (Fluka) at 37°C with strong aeration from a 100-fold dilution of overnight cultures. Three strains of S. aureus were used in the studies. The LS-1 is an arthritogenic JAK inhibitor strain originally isolated from swollen mouse joint [39]. The 8325–4 strain is a derivative of NCTC 8325, which has been cured of resident prophages and has low production of coagulase and surface adhesions [40]. The P1 strain was isolated from a rabbit inoculated with ATCC 25923 SB203580 in vitro and has better adherence than 8325–4 to endothelial

cells (a generous gift from prof. T.J. Foster, Trinity College, Dublin, Reverse transcriptase Ireland) [41]. The LS-1 strain was used to develop the eczema model, while the P1 strain was used for a comparison of enzyme efficacies in the eczema model. Strain 8325–4 was used for all in vitro assays (pulldown, lysis, stability). The susceptibilities of the 8325–4 and P1 strains towards LytM and lysostaphin were comparable.

Proteins A fragment of DNA corresponding to the LytM24-105 protein was amplified by PCR from the previously described full length LytM clone [12] inserted into the pET15mod vector and called pET15modLytM24-105. The construct coded for the LytM fragment fused to an amino-terminal histidine tag and could be expressed in soluble form in E. coli strain BL21(DE3). Protein expression was induced during the logarithmic growth phase of the bacteria (OD595 of 0.8) by the addition of 1 mM IPTG and continued for 4 h at 25°C. The recombinant protein was purified by affinity chromatography on a Ni2+ loaded, nitrilo-triacetic acid (NTA) agarose column (Qiagen), followed by gel filtration on a Sephacryl S-200 column (Amersham Bioscience). LytM26-316, LytM99-316, LytM185-316 and all point mutants were expressed and purified as previously described [12]. Lysostaphin (mature form) was purchased from Sigma and used without further purification. Antibodies Polyclonal antibodies against LytM185-316 were raised in rabbit (Pineda Antibody Service, Berlin, Germany).