commonly colonizes the human upper respiratory tract (1
). However, when the delicate balance between benign harboring of bacterial flora and deleterious microbial outgrowth is disrupted, invasive pneumococcal disease (IPD) can result (2
). IPD is one of the leading causes of opportunistic infections in young children, the elderly and in individuals immunocompromised due to cancer, diabetes or human immunodeficiency virus infection (3–6
). Though favorable outcomes are primarily expected as a result of early detection and prompt antibacterial therapy, severe complications of IPD include bacteremia, meningitis and death (2
Bacterial colonization in the lungs of infected individuals triggers neutrophil recruitment to the site of infection (9
), resulting in extrusion of DNA/protein scaffolds known as neutrophil extracellular traps [NETs (10
)]. These filamentous NETs physically trap bacteria, leaving them vulnerable to phagocytic attack and/or destruction by NET-bound antimicrobial proteins located in the NETs (9–12
). To combat the innate immune response, constitutive expression of membrane-bound DNA-entry nuclease EndA (13
) is instrumental for respiratory track invasion and increased virulence of S. pneumoniae
. EndA localization to the outer face of the bacterial membrane (14
) enables its sequence non-specific nuclease activity to destroy the NETs. EndA, a DNA/RNA endonuclease (16
), is normally associated with DNA degradation and uptake during the biochemical state of competence, further promoting S. pneumoniae
pathogenicity through gene transfer (13
). By helping streptococci to establish genetic diversity, EndA may influence the ability of this organism to adapt to changing environmental conditions, providing a significant advantage during infection.
Nuclease digestion of the NETs’ DNA scaffold is associated with increased bacterial migration from the upper airway to the lungs, and in 20–30% of cases, invasion into the bloodstream (8
). Streptococcus pneumoniae
strains expressing EndA have been shown to evade and destroy NETs, and cause more virulent, invasive forms of pneumonia (18
). The role of EndA as a virulence factor in pneumococcal infection makes it an attractive target for antimicrobial therapeutics. However, despite the central importance of EndA in promoting virulence, the mechanism by which EndA degrades DNA remains enigmatic, due in part to the lack of EndA protein structural information. Furthermore, the toxicity of recombinantly expressed enzymes (15
) and the subsequent inability to purify active nuclease have hampered biochemical and mechanistic studies of EndA.
To define mechanisms by which DNA-entry nucleases bind and degrade DNA substrates, we report the structure and detailed mutagenesis of S. pneumoniae EndA. We engineered an active site glycine substitution mutant, EndA(H160G), which yielded quantifiable imidazole rescue nuclease activity. Structural information, coupled with imidazole rescue, allowed for biochemical characterization of EndA variants surrounding the active site, delineating catalytic activity versus DNA substrate binding. Based on these results, histidine-to-glycine substitution paired with imidazole rescue could be utilized as an all-purpose strategy for expression, purification and biochemical characterization of other ββα-metal finger nucleases.