Identification of the Annexin A2-interacting domain of pneumococcal PsaA

Streptococcus pneumoniae is a gram-positive bacterium, colonizer of the human nasopharynx capable of causing severe invasive disease. Colonization of the nasopharynx is a prerequisite for the development of invasive disease and depends upon surface-bound bacterial proteins interacting with host cell receptors. Pneumococcal surface adhesin A (PsaA) is a highly conserved lipoprotein involved in the attachment to host airway cells via the host receptor Annexin A2 (ANXA2). However, the specific structural domains of PsaA responsible for ANXA2 recognition and binding remain unknown. Here, we employed a structure-based peptide approach to map the ANXA2-binding domain of PsaA. Five recombinant PsaA peptides were designed and generated based on the crystal structure of PsaA. Far-western blot analysis using ANXA2-overexpressing HEK293T/17 cell lysates revealed that the interaction with ANXA2 was specific to the C-terminal subdomain of PsaA. Cell-binding assays and flow cytometry further confirmed the interaction between the C-terminal subdomain of PsaA and cell surface-expressed ANXA2. Furthermore, polyclonal antibodies against the C-terminal subdomain significantly inhibited the binding of full-length PsaA to ANXA2-overexpressing cells, whereas antibodies against other subdomains did not. Consistently, polyclonal antibodies against the C-terminus of PsaA reduced the binding of S. pneumoniae to A549 lung epithelial cells to a greater extent than other antibodies. Together, these findings establish that the C-terminus of PsaA is crucial for ANXA2 receptor recognition. Targeting this specific subdomain may be a promising strategy for developing next-generation protein-based pneumococcal vaccines that aim at blocking bacterial adherence.IMPORTANCEStreptococcus pneumoniae is a leading cause of millions of deaths worldwide each year due to its ability to transition from an asymptomatic colonizer to an invasive pathogen. Current pneumococcal conjugate and polysaccharide vaccines protect against pneumococcal disease, but overall colonization rates have remained stable. Since pneumococcus is an opportunistic pathogen, decreasing overall colonization rates is essential for preventing progression to disease. The significance of our research lies in mapping functional epitopes within key pneumococcal adhesins that play a critical role in bacterial adherence. Defining these adhesion epitopes is essential for the rational design of next-generation protein-based vaccines capable of blocking colonization and ultimately reducing the global burden of invasive pneumococcal diseases.