A novel tail fiber protein triggers phage DNA ejection by recognizing lipopolysaccharides of K54 hypervirulent Klebsiella pneumoniae

Bacteriophage (phage) therapy is a promising alternative antibacterial approach for treating infectious diseases by hv Kp . However, there has been a lack of studies on the host recognition and infection initiation of hv Kp phages. In this work, we found that both capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of strain SCNJ1 are essential for phage infection, and mutations in genes associated with the CPS or LPS biosynthesis pathway conferred phage resistance. These CPS-related mutants, but not LPS-related mutants, showed virulence attenuation and significantly reduced colonization in mice. We demonstrate that CPS serves as the primary receptor for phage adsorption and can be cleaved by phage depolymerase Dep-Y. Phage DNA ejection was specifically triggered by purified LPS, rendering LPS a secondary receptor during the phage infection process. Furthermore, mutant phages capable of infecting the LPS-related mutant strain were isolated, leading to the identification of a novel tail fiber protein named TFP_Y, which can interact with LPS to initiate phage genome ejection. In conclusion, we propose a molecular model for phage infection in the hv Kp strain SCNJ1. In this model, the phage utilizes both the CPS and LPS as receptors to initiate an infection in hv Kp : the phage attaches to the bacterial cell surface; depolymerase Dep-Y degrades the host CPS to expose the LPS; and TFP_Y interacts with LPS, leading to the ejection of phage genome DNA. These findings deepen our understanding of phage-hvKp strain interactions during phage infection, which is crucial for developing phage-based therapeutic strategies. IMPORTANCE Phage infection begins with the adsorption of the phage particle to the bacterial cell surface, followed by the injection of the phage genome into the cell. In the encapsulated hypervirulent K. pneumoniae (hv Kp ), the capsular polysaccharide (CPS) surrounding the bacterium was assumed to be the adsorption receptor of phage infection. Lipopolysaccharide (LPS) is a common phage receptor in many bacteria. Whether and how CPS and LPS act as receptors simultaneously to initiate the phage infection in hv Kp remains unknown. Here, we demonstrated that bacterial CPS and LPS are both critical determinants for phage infection in hv Kp strain SCNJ1, and mutations in either component confer complete resistance. We revealed that CPS is the primary receptor for phage adsorption, whereas LPS is the secondary receptor for phage injection. Furthermore, we identified a novel tail fiber protein TFP_Y that recognizes host LPS to start phage genome DNA release.