Bacteriophage endolysins and their derivatives have strong potential as antibacterial agents considering the increasing prevalence of antibiotic resistance in common bacterial pathogens. The peptidoglycan degrading peptidase CHAPk, a truncated derivate of staphylococcal phage K endolysin (LysK), has proven efficacy in preventing and disrupting staphylococcal biofilms. Nevertheless, the concentration of CHAPk required to eliminate populations of stationary-phase cells was previously found to be four-fold higher than that for log-phase cells. Moreover, CHAPk-mediated lysis of stationary-phase cells was observed to be slower than for log-phase cultures. In the present study, we report the fusion of a 165 amino acid fragment containing CHAPk with a 136 amino acid fragment containing the cell-binding domain of the bacteriocin lysostaphin to create a chimeric enzyme designated CHAPk-SH3blys in the vector pET28a. The chimeric protein was employed in concentrations as low as 5 μg/mL, producing a reduction in turbidity in 7-day-old cultures, whereas the original CHAPk required at least 20 μg/mL to achieve this. Where 7-day old liquid cultures were used, the chimeric enzyme exhibited a 16-fold lower MIC than CHAPk. In terms of biofilm prevention, a concentration of 1 μg/mL of the chimeric enzyme was sufficient, whereas for CHAPk, 125 μg/mL was needed. Moreover, the chimeric enzyme exhibited total biofilm disruption when 5 μg/mL was employed in 4-h assays, whereas CHAPk could only partially disrupt the biofilms at this concentration. This study demonstrates that the cell-binding domain from lysostaphin can make the phage endolysin CHAPk more effective against sessile staphylococcal cells.

Actinin‐1 mutations cause dominantly inherited congenital macrothrombocytopenia (CMTP), with mutations in the actin‐binding domain increasing actinin's affinity for F‐actin. In this study, we examined nine CMTP‐causing mutations in the calmodulin‐like and rod domains of actinin‐1. These mutations increase, to varying degrees, actinin's ability to bundle actin filaments in vitro. Mutations within the calmodulin‐like domain decrease its thermal stability slightly but do not dramatically affect calcium binding, with mutant proteins retaining calcium‐dependent regulation of filament bundling in vitro. The G764S and E769K mutations increase cytoskeletal association of actinin in cells, and all mutant proteins colocalize with F‐actin in cultured HeLa cells. Thus, CMTP‐causing actinin‐1 mutations outside the actin‐binding domain also increase actin association, suggesting a common molecular mechanism underlying actinin‐1 related CMTP.