Di-enzymatic Chimeric Endolysin

Bacteriophage-derived endolysins have great potential as alternative antimicrobial agents for Gram-positive bacterial infectious diseases, as they are peptidoglycan hydrolases that can destroy susceptible bacteria when applied exogenously. Due to the modular structure of endolysins, engineering methods can be used to improve their properties or change their host range via manipulation of the functional domains. The multimeric endolysin, PlyC, has potent activity on groups A, C, and E streptococci, as well as Streptococcus uberis, but is devoid of activity on other streptococci such as S. agalactiae (i.e. group B strep), S. mutans, or S. pneumoniae. PlyCA, the enzymatically active domain of PlyC, consists of two catalytic domains, GyH, a glycosyl hydrolase, and CHAP, a cysteine, histidine-dependent amidohydrolase/peptidase. Notably, GyH and CHAP have been shown to work synergistically to achieve lytic rates~ I 00-fold higher than comparable single catalytic domain endolysins. In this work, we provide a new design of chimeric endolysins to take advantage of the synergistic effects of PlyCA. ClyX-1 was created by fusing the pneumococcal Cpl-I cell binding domain (CBD) in between the GyH and CHAP catalytic domains of PlyCA. This chimera displayed an~ 100-fold increase in activity in vitro against S. pneumoniae compared to the parental Cpl-I enzyme. ClyX-2 was then created using a similar strategy by fusing the broad host range PlySs2 CBD between the GyH and CHAP catalytic domains. ClyX-2 not only demonstrated wild-type PlyC activities on groups A, C and E streptococci, but now included high levels of activity (i.e. 20-50 fold higher than PlySs2) against S. mutans and S. agalactiae. Moreover, this design format (i.e. CBD in between of two catalytic domains) can also be applied to other enzymes in order to achieve similar synergistic results. CHAP or GH25 catalytic domains were added to the C-terminus of full-length Cpl-I and PlySs2, respectively, and displayed synergistic effects. To date, with the exception of PlyC, two catalytic domains in one endolysin have not shown synergism, even in enzymes that naturally contain two catalytic domains. Our work demonstrates a novel  design  for adopting  the synergy  of two catalytic domains for increased lytic activity.