Identification and characterization of 40S ribosomal protein S27a as an antimicrobial protein from starfish, Patiria...

Antimicrobial proteins/peptides (AMPs) are promising bioactive molecules with potent and broad-spectrum activity, representing a key alternative to conventional antibiotics. Among these host defense molecules, ribosomal proteins represent a largely unexplored reservoir with potential antimicrobial capacity, as recent studies suggest that certain members possess moonlighting antimicrobial activity. Thus, studies on the antimicrobial activity of ribosomal protein subunits broaden our knowledge of these antimicrobial moonlighting proteins and contribute to better strategies to combat antimicrobial resistance. In this study, we report the antimicrobial capacity of a newly identified 40S ribosomal protein S27a from the starfish Patiria pectinifera (PpRPS27a). We retrieved a 40S ribosomal protein S27a homolog from a transcriptome-derived EST database of P. pectinifera. Subsequent cDNA cloning yielded an 804-bp cDNA comprising a 52-bp 5'-UTR, a 462-bp ORF, and a 290-bp 3'-UTR, revealing that PpRPS27a is expressed together with ubiquitin as a fusion protein. The mature PpRPS27a consists of 77 amino acids with a molecular mass of 9019.74 Da. Quantitative mRNA expression analysis showed that PpRPS27a is highly expressed in tissues associated with immune function or external exposure, including the cardiac stomach, oral haemal ring, and coelomic epithelium. Its expression was found to be upregulated upon immune challenge, suggesting a potential involvement in host defense mechanisms. The recombinant PpRPS27a (rPpRPS27a) was heterologously expressed in Escherichia coli BL21 (DE3) and purified for functional characterization. rPpRPS27a exhibited broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria and a fungus. Notably, rPpRPS27a showed strong activity against fish pathogens such as Aeromonas hydrophila and Vibrio anguillarum, which are major causative agents of diseases in aquaculture. In addition, the antimicrobial activity was enhanced when rPpRPS27a was co-administered with recombinant ubiquitin (rPpUB), its native fusion partner in P. pectinifera. rPpRPS27a increases outer membrane permeability and exhibits DNA-binding activity in vitro, indicating a multifaceted antibacterial mechanism. These results indicate that PpRPS27a is a previously uncharacterized antimicrobial protein with broad-spectrum activity and a potential role in host immune defense. The discovery of PpRPS27a as an antimicrobial effector in innate immunity expands the known roles of invertebrate ribosomal proteins. Moreover, its potent activity against fish pathogens also suggests potential as a molecular scaffold for developing novel antimicrobial agents. To our knowledge, this is the first report of the antimicrobial property of the 40S ribosomal protein S27a from starfish, P. pectinifera.