Engineering of the CHAPk Staphylococcal Phage Endolysin to Enhance Antibacterial Activity against Stationary-Phase Cells

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.

Targeting Receptors on Cancer Cells with Protein Toxins

Cancer cells frequently upregulate surface receptors that promote growth and survival. These receptors constitute valid targets for intervention. One strategy involves the delivery of toxic payloads with the goal of killing those cancer cells with high receptor levels. Delivery can be accomplished by attaching a toxic payload to either a receptor-binding antibody or a receptor-binding ligand. Generally, the cell-binding domain of the toxin is replaced with a ligand or antibody that dictates a new binding specificity. The advantage of this “immunotoxin” approach lies in the potency of these chimeric molecules for killing cancer cells. However, receptor expression on normal tissue represents a significant obstacle to therapeutic intervention.

Fibronectin and Its Role in Human Infective Diseases

Fibronectin is a multidomain glycoprotein ubiquitously detected in extracellular fluids and matrices of a variety of animal and human tissues where it functions as a key link between matrices and cells. Fibronectin has also emerged as the target for a large number of microorganisms, particularly bacteria. There are clear indications that the binding of microorganism’ receptors to fibronectin promotes attachment to and infection of host cells. Each bacterium may use different receptors which recognize specific fibronectin domains, mostly the N-terminal domain and the central cell-binding domain. In many cases, fibronectin receptors have actions over and above that of simple adhesion: In fact, adhesion is often the prerequisite for invasion and internalization of microorganisms in the cells of colonized tissues. This review updates the current understanding of fibronectin receptors of several microorganisms with emphasis on their biochemical and structural properties and the role they can play in the onset and progression of host infection diseases. Furthermore, we describe the antigenic profile and discuss the possibility of designing adhesion inhibitors based on the structure of the fibronectin-binding site in the receptor or the receptor-binding site in fibronectin.