Extracellular DNA and Type IV Pilus Expression Regulate the Structure and Kinetics of Biofilm Formation by Nontypeable Haemophilus influenzae

Abstract

Biofilms formed in the middle ear by nontypeable Haemophilus influenzae (NTHI) are central to the chronicity, recurrence, and refractive nature of otitis media (OM). However, mechanisms that underlie the emergence of specific NTHI biofilm structures are unclear. We combined computational analysis tools and in silico modeling rooted in statistical physics with confocal imaging of NTHI biofilms formed in vitro during static culture in order to identify mechanisms that give rise to distinguishing morphological features. Our analysis of confocal images of biofilms formed by NTHI strain 86-028NP using pair correlations of local bacterial densities within sequential planes parallel to the substrate showed the presence of fractal structures of short length scales (≤10 μm). The in silico modeling revealed that extracellular DNA (eDNA) and type IV pilus (Tfp) expression played important roles in giving rise to the fractal structures and allowed us to predict a substantial reduction of these structures for an isogenic mutant (ΔcomE) that was significantly compromised in its ability to release eDNA into the biofilm matrix and had impaired Tfp function. This prediction was confirmed by analysis of confocal images of in vitro ΔcomE strain biofilms. The fractal structures potentially generate niches for NTHI survival in the hostile middle ear microenvironment by dramatically increasing the contact area of the biofilm with the surrounding environment, facilitating nutrient exchange, and by generating spatial positive feedback to quorum signaling.

Autoinducer 2 (AI-2) Production by Nontypeable Haemophilus influenzae 86-028NP Promotes Expression of a Predicted Glycosyltransferase That Is a Determinant of Biofilm Maturation, Prevention of Dispersal, and Persistence In Vivo

“…Based on these findings, we conclude that while the gstA-mediated changes in the biofilm matrix likely promote bacterial persistence, there are other significant determinants of biofilm maturation that are independent of AI-2 signals or this particular glycosyltransferase. Like many bacteria residing within biofilms, NTHi produces extracellular double-stranded DNA, which is also a significant component of the biofilm matrix (43)(44)(45). Recent reports have demonstrated that NTHi also produces an extracellular nuclease, which may degrade extracellular DNA and facilitate bacterial dispersal from biofilms (46,47); there have also been recent reports suggesting that AI-2 quorum signals might promote biofilm dispersal (48).…”

The structure of deterministic mass and surface fractals: theory and methods of analyzing small-angle scattering data

“…and carries information about the relative positions of the scattering unit centers inside the fractal. Equation (21) can be rewritten as with S(0) = N m . The long-range asymptotics [S(q) ≃ 1 for q 2π/l min ] is governed by the diagonal terms in Eq.…”

Otitis media pathogens – A life entrapped in biofilm communities

“…Another study also showed that the EPS matrix of NTHi biofilms is composed of proteins, nucleic acids and b-glucan, with eDNA being an important component of the EPS and essential in biofilm maintenance . NTHi in vitro biofilms visualization combined with mathematical tools demonstrated that eDNA and type IV pili expression are both important for the development of fractal structures in these biofilms, and may help bacterial survival in hostile environments, such as the ME (Das et al 2017).…”