<i>Streptococcus suis</i> 5’-nucleotidases contribute to adenosine-mediated immune evasion and virulence in a mouse model

<i>Streptococcus suis</i> (<i>S. suis</i>) is an important swine bacterial pathogen and causes human infections, leading to a wide range of diseases. However, the role of 5'-nucleotidases in its virulence remains to be fully elucidated. Herein, we identified four cell wall-anchored 5'-nucleotidases (Snts) within <i>S. suis</i>, named SntA, SntB, SntC, and SntD, each displaying similar domains yet exhibiting low sequence homology. The malachite green reagent and HPLC assays demonstrated that these recombinant enzymes are capable of hydrolysing ATP, ADP, and AMP into adenosine (Ado), with the hierarchy of catalytic efficiency being SntC>SntB>SntA>SntD. Moreover, comprehensive enzymatic activity assays illustrated slight variances in substrate specificity, pH tolerance, and metal ion requirements, yet highlighted a conserved substrate-binding pocket, His-Asp catalytic dyad, metal, and phosphate-binding sites across Snts, with the exception of SntA. Through bactericidal assays and murine infection assays involving in site-mutagenesis strains, it was demonstrated that SntB and SntC collaboratively enhance bacterial survivability within whole blood and polymorphonuclear leukocytes (PMNs) <i>via</i> the Ado-A2aR pathway <i>in vitro</i>, and within murine blood and organs <i>in vivo</i>. This suggests a direct correlation between enzymatic activity and enhancement of bacterial survival and virulence. Collectively, <i>S. suis</i> 5'-nucleotidases additively contribute to the generation of adenosine, influencing susceptibility within blood and PMNs, and enhancing survival within blood and organs <i>in vivo</i>. This elucidation of their integral functions in the pathogenic process of <i>S. suis</i> not only enhances our comprehension of bacterial virulence mechanisms, but also illuminates new avenues for therapeutic intervention aimed at curbing <i>S. suis</i> infections.