Amanda Micklo – Health Sciences

Quantitation of DNABII Proteins Within Biofilms Formed by Nontypeable Haemophilus Influenzae

Biofilms are communities of bacteria that contribute to the chronic nature of 80% of persistent bacterial infections due to the fact that they are resistant to antibiotics and clearance by the host immune system. Extracellular DNA and members of the DNABII family of DNA-binding proteins (integration host factor [IHF] and histone-like protein [HU]) are critical to maintaining the structure of bacterial biofilms. We used nontypeable Haemophilus influenzae [NTHI], a bacterium that causes multiple chronic diseases throughout the upper and lower respiratory tracts, including otitis media, bronchitis, sinusitis, conjunctivitis, and exacerbations in cystic fibrosis and chronic obstructive pulmonary disease, as a model organism to determine the concentration of IHF and HU present within biofilms of differing ages in vitro. NTHI was grown on chocolate agar, seeded into 8-well chambered coverglass, and incubated for 3, 6, 16, 24, 36, or 40 hours. The NTHI biofilms were stained with FM1-43X membrane stain, labeled with anti-IHFNTHI, or labeled with anti-HUNTHI, and measured via fluorescence intensity on a plate reader. The unknown protein concentrations of IHF and HU were quantified using a generated standard curve. These data showed a significantly greater concentration of IHF present in younger (3 and 6 hours) biofilms compared to HU, comparable concentrations of both IHF and HU present at the mid-point (16 and 24 hours) of the assay period, and a significantly greater concentration of HU present in older (36 and 40 hours) biofilms compared to IHF. Overall, by quantitating the protein concentrations of both IHF and HU, therapeutic strategies can be tailored to produce the most effective outcomes against diseases with a biofilm component.