Dual activities of lysis and capsule degradation enable the bacteriophage protein Lys33795 to effectively disrupt Streptococcus suis biofilms.

Streptococcus suis (S. suis) is an encapsulated zoonotic pathogen that can form recalcitrant biofilms. Bacteriophage-derived endolysins and depolymerases represent promising antibiofilm agents, but currently, no report has described protein which displays both of lytic and capsule degradation activities simultaneously. Here, we mined the porcine gut virome and identified Lys33795, a dual-activity antibacterial protein that degrades both capsule and peptidoglycan of S. suis. Lys33795 consists of an N-terminal GH25_muramidase domain and a C-terminal cell wall binding domain (CBD). However, translational reinitiation at an internal start codon (M217) in the Lys33795 sequence generates an additional CBD that may assembles with the full-length protein to form an oligomeric complex. Interestingly, the GH25 muramidase truncation fragment of Lys33795 not only retains its classical function as a peptidoglycan hydrolase for degrading bacterial cell walls but also exhibits degradation activity towards capsule. This discovery contrasts sharply with the traditional functional understanding of the GH25 family. In addition, the CBD alone has a stable dimer and, as a nonstandard depolymerase without any classical polysaccharide-degrading catalytic domains, retains its degradation activity against S. suis capsule. We elucidated the molecular mechanism of Lys33795 exhibiting dual activity by combining mutagenesis, lysis, and capsule degradation activity assays. In addition, the results of in vitro and in vivo experiments showed that Lys33795 can effectively prevent and clear S. suis biofilms, and is more effective than single active mutants, and has a enhance effect when used in combination with antibiotics. This study describes the first multimeric bacteriophage protein with dual activities of lysis and capsule degradation against S. suis, encoded by a single gene. These findings provide valuable insights for designing potent antibacterial proteins targeting encapsulated pathogens.