Abstract
Nontypeable Haemophilus influenzae (NTHi) has been shown to form biofilms, comprised of extracellular DNA (eDNA), in the middle ear and bronchus during clinical infections. Studies in our laboratory have shown that NTHi possesses a homolog of Staphylococcus aureus thermonuclease (staphylococcal thermonuclease), NTHi nuclease (NTHi Nuc, HI_1296). This enzyme had similar size, heat stability, and divalent cation requirements to those of the staphylococcal homolog as determined by light scattering and circular dichroism spectroscopy. Small angle X-ray scattering (SAXS) analysis suggested an overall shape and substrate-binding site comparable to those of staphylococcal nuclease. However, NTHi Nuc was approximately 25-fold more active in fluorescence resonance energy transfer (FRET) activity assay than staphylococcal thermonuclease. Homology modeling implicates shorter NTHi Nuc loops near the active site for this enhanced activity.
Highlights
Nontypeable Haemophilus influenzae (NTHi) are frequently found as a member of the normal upper respiratory tract bacterial flora
Enzyme activity studies were performed with PCR grade staphylococcal thermonuclease (Sigma-Aldrich) and highly purified recombinant NTHi Nuc without signal sequence
NTHi Nuc was a highly efficient nuclease with greater than 1500 fold increased activity compared to bovine pancreatic DNase I and greater than 25 fold increased activity compared to staphylococcal thermonuclease (Table 1)
Summary
Nontypeable Haemophilus influenzae (NTHi) are frequently found as a member of the normal upper respiratory tract bacterial flora. This species can be a frequent cause of airway infections, including otitis media in children, and sinusitis and acute exacerbations of chronic bronchitis in adults [1]. NTHi has been shown to be capable of forming biofilms in vitro and in the upper and lower human respiratory tract during human disease [2,3,4]. Recent studies have shown that Pseudomonas aeruginosa in cystic fibrosis lung disease forms aggregates of microbes containing dead.
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