Bacteriophage endolysin treatment for systemic infection of Streptococcus iniae in hybrid striped bass.

Streptococcus iniae, a zoonotic Gram-positive pathogen, poses a threat to finfish aquaculture, causing streptococcosis with an annual economic impact exceeding $150 million globally. As aquaculture trends shift towards recirculating systems, the potential for horizontal transmission of S. iniae among fish intensifies. Current vaccine development provides only short-term protection, driving the widespread use of antibiotics like florfenicol. However, this practice raises environmental concerns and potentially contributes to antibiotic resistance. Thus, alternative strategies are urgently needed. Endolysin therapy, derived from bacteriophages, employs hydrolytic endolysin enzymes that target bacterial peptidoglycan cell walls. This study assesses three synthetic endolysins (PlyGBS 90-1, PlyGBS 90-8, and ClyX-2) alongside the antibiotic carbenicillin in treating S. iniae-infected hybrid striped bass (HSB). Results demonstrate that ClyX-2 exhibits remarkable bacteriolytic potency, with lytic activity detected at concentrations as low as ∼15 µg/mL, approximately 8-fold more potent than the PlyGBS derivatives. In therapeutic effectiveness assessments, both carbenicillin and ClyX-2 treatments achieved significantly higher survival rates (85 % and 95 %, respectively) compared to placebo and PlyGBS-based endolysin treatments. Importantly, no statistical differences were observed between ClyX-2 and carbenicillin treatments. This highlights ClyX-2 as a promising alternative for combating S. iniae infections in aquaculture, offering potent bacteriolytic activity and high survival rates.

Functional Analysis of Short Linear Motifs in the Plant Cyclin-Dependent Kinase Inhibitor SIAMESE.

Endoreplication, a modified cell cycle in which DNA is replicated without subsequent cell division, plays an important but poorly understood role in plant growth and in plant responses to biotic and abiotic stress. The Arabidopsis (Arabidopsis thaliana) SIAMESE (SIM) gene encodes the first identified member of the SIAMESE-RELATED (SMR) family of cyclin-dependent kinase inhibitors. SIM controls endoreplication during trichome development, and sim mutant trichomes divide several times instead of endoreplicating their DNA. The SMR family is defined by several short linear amino acid sequence motifs of largely unknown function, and family members have little sequence similarity to any known protein functional domains. Here, we investigated the roles of the conserved motifs in SIM site-directed Arabidopsis mutants using several functional assays. We identified a potential cyclin-dependent kinase (CDK)-binding site, which bears no resemblance to other known CDK interaction motifs. We also identified a potential site of phosphorylation and two redundant nuclear localization sequences. Surprisingly, the only motif with similarity to the other family of plant CDK inhibitors, the INHIBITOR/INTERACTOR OF CDC2 KINASE/KIP-RELATED PROTEIN proteins, is not required for SIM function in vivo. Because even highly divergent members of the SMR family are able to replace SIM function in Arabidopsis trichomes, it is likely that the results obtained here for SIM will apply to other members of this plant-specific family of CDK inhibitors.

Engineered antibodies targeted to bacterial surface integrate effector functions with toxin neutralization to provide superior efficacy against bacterial infections.

Anti-bacterial monoclonal antibody (mAb) therapies either rely on toxin neutralization or opsonophagocytic killing (OPK). Toxin neutralization protects the host from toxin-induced damage, while leaving the organism intact. OPK inducing antibodies clear the bacteria but leave the released toxins unencountered. Infection site targeted anti-toxin antibodies (ISTAbs) that we report here addresses this binary paradigm by combining both functionalities into a single molecule. ISTAbs consist of cell wall targeting (CWT) domains of bacteriophage endolysins fused to toxin neutralizing mAbs (IgG). CWT governs specific binding to the surface of bacteria while the IgG variable domain neutralizes the toxins as they are released. The complex is then cleared by phagocytic cells. As proof of concept, we generated several ISTAb prototypes targeting major toxins from two Gram-positive spore forming pathogens that have a high clinical significance; Clostridium difficile , causative agent of the most common hospital-acquired infection, and Bacillus anthracis , a Category A select agent pathogen. Both groups of ISTAbs exhibited potent toxin neutralization, binding to their respective bacterial cells, and induction of opsonophagocytosis. In mice infected with B. anthracis , ISTAbs exhibit significantly higher efficacy than parental IgG in both pre- and post-challenge models. Furthermore, ISTAbs fully protected against B. anthracis infection in a nonhuman primate (NHP) aerosol challenge model. These findings establish that as a platform technology, ISTAbs are broadly applicable for therapeutic intervention against several toxigenic bacterial pathogens.