Streptococcus pneumoniae is an opportunistic pathogen that asymptomatically colonizes the upper respiratory tracts and frequently shifts from a commensal to a pathogenic condition within the human body. S. pneumoniae is the leading cause of community‐acquired pneumonia, meningitis, and otitis media and can also cause sepsis or sinusitis in humans [1], [2]. Previous reports indicated that the overall mortality rate in patients with invasive pneumococcal disease ranged from 18 % to 40 % [3]. Administration of antibiotics to patients suffering from S. pneumoniae infections is the primary therapeutic option. However, due to the overuse of antibiotics, it prompts the rise of bacterial antimicrobial resistance which has become a major global health issue [4]. The World Health Organization (WHO) listed S. pneumoniae as one of the global priority pathogens with critical and high antibiotic resistance [5]. In order to resolve this issue, it is necessary and urgent to develop alternative strategies against pneumococcal infections, steering favorable outcomes in patients.

Degradation of the extracellular matrix (ECM) barrier is one of the first steps in bacterial infection. Previous studies indicated that S. pneumoniae expresses particular surface proteins to interact with the ECM secreted by host cells [6], [7], [8]. After the pneumococcal surface proteins bind to the ECM, bacterial proteolytic activity occurs, resulting in the cleavage of plasminogen to plasmin, leading to ECM degradation and bacteria transmigration across tissue barriers into deeper tissue sites [7], [9]. Among the pneumococcal surface proteins, alpha-enolase was reported to specifically bind with human plasminogen, implicating pneumococcal pathogenesis [10], [11], [12].

Alpha-enolase (Eno1), also called 2-phospho-D-glycerate hydrolase, is first discovered in eukaryotic cells and responsible for catalyzing the conversion of 2-phosphoglycerate into phosphoenolpyruvate in the glycolytic pathway [13], [14], [15]. In addition to the glycolytic activity, it can also function as a structural receptor protein depending on the localization [16]. Eno1 was later found on the surface and in the cytosol of most Streptococci species and showed greater plasminogen binding affinity than other streptococcal plasminogen binding proteins [17]. Furthermore, a study demonstrated that spEno1 participates in the human innate immune response, which binds with neutrophil and induces neutrophil extracellular traps (NETs) formation and NETosis [18]. During S. pneumoniae infection, a study indicated that the Eno1 from S. pneumoniae (spEno1) was immunogenic against pathogens and played an important role in limiting microbial tissue invasion [12], [19]. Another study revealed that spEno1 interacted with human C4b-binding protein and modulated the human complement system [20].

Given that spEno1 localizes on the bacterial surface, it plays an important role in the infection and pathogenesis of S. pneumoniae, rendering it as a suitable target for developing diagnostic tools and therapeutic drugs. In this study, monoclonal single-chain variable fragment (scFv) antibodies were selected using the phage display technology after chicken immunization with recombinant spEno1 protein. We analyzed the binding activities of the selected scFv antibodies to spEno1 and evaluated the inhibition effect of these antibodies against the interaction between spEno1 and plasminogen. Overall, our result indicated that the spEno1-targeted scFv antibodies will be useful as diagnostic and therapeutic agents for disease progress monitoring and treatments of patients suffering from S. pneumoniae infection in the future.