Role of the Nuclease of Nontypeable Haemophilus influenzae in Dispersal of Organisms from Biofilms

Abstract

Nontypeable Haemophilus influenzae (NTHI) forms biofilms in the middle ear during human infection. The biofilm matrix of NTHI contains extracellular DNA. We show that NTHI possesses a potent nuclease, which is a homolog of the thermonuclease of Staphylococcus aureus. Using a biofilm dispersal assay, studies showed a biofilm dispersal pattern in the parent strain, no evidence of dispersal in the nuclease mutant, and a partial return of dispersion in the complemented mutant. Quantitative PCR of mRNA from biofilms from a 24-h continuous flow system demonstrated a significantly increased expression of the nuclease from planktonic organisms compared to those in the biofilm phase of growth (P < 0.042). Microscopic analysis of biofilms grown in vitro showed that in the nuclease mutant the nucleic acid matrix was increased compared to the wild-type and complemented strains. Organisms were typically found in large aggregates, unlike the wild-type and complement biofilms in which the organisms were evenly dispersed throughout the biofilm. At 48 h, the majority of the organisms in the mutant biofilm were dead. The nuclease mutant formed a biofilm in the chinchilla model of otitis media and demonstrated a propensity to also form similar large aggregates of organisms. These studies indicate that NTHI nuclease is involved in biofilm remodeling and organism dispersal.

Biofilm dispersal: multiple elaborate strategies for dissemination of bacteria with unique properties

“…They concluded that satellite biofilm colonies are dispersed in the direction of higher temperatures, and described a new strategy of biofilm dispersal (Kaplan and Fine, 2002). Their method allows the direct visualization of biofilm dispersal and it has also been used to identify a nuclease involved in the biofilm dispersal of H. influenzae (Cho et al, 2015). Confocal laser scanning microscopy imaging can also be used to observe the dispersal phenomenon at a spatiotemporal level by following the dispersed bacteria ( Fig.…”

“…Endogenous nucleases involved in biofilm remodeling and dispersal were also identified in nontypeable Haemophilus influenzae (NTHI) and Neisseria gonorrhoeae strains (Steichen et al, 2011;Cho et al, 2015). In a biofilm dispersal assay, contrary to a NTHI wild-type strain, a NTHI Dnuc nuclease mutant shows no dispersal phenotype suggesting that Nuc is involved in biofilm dispersal (Cho et al, 2015).…”

“…Endogenous nucleases involved in biofilm remodeling and dispersal were also identified in nontypeable Haemophilus influenzae (NTHI) and Neisseria gonorrhoeae strains (Steichen et al, 2011;Cho et al, 2015). In a biofilm dispersal assay, contrary to a NTHI wild-type strain, a NTHI Dnuc nuclease mutant shows no dispersal phenotype suggesting that Nuc is involved in biofilm dispersal (Cho et al, 2015). In N. gonorrhoeae, the deletion of the nuclease-encoding gene results in thicker biofilms that contained more biomass in continuous-flow chambers, and exogenous addition of purified N. gonorrhoeae nuclease to established N. gonorrhoeae biofilm leads to a statistically significant reduction in both the biomass and average thickness of the biofilm (Steichen et al, 2011).…”

Type IV Pilus Expression Is Upregulated in Nontypeable Haemophilus influenzae Biofilms Formed at the Temperature of the Human Nasopharynx

“…Three sets of biofilms were collected at 37°C and 34°C for both time points, and the relative pilA and ompP5 expression levels were assessed using a SuperScript III Platinum SYBR Green One-Step quantitative reverse transcription-PCR (qRT-PCR) kit (Invitrogen), according to the manufacturer's protocol, with the primers listed in Table 1. Expression was normalized to ompP6, which is constitutively expressed in NTHI (24,32). Results were reported as relative gene expression at 34°C, compared to that at 37°C, by the comparative (⌬⌬C T ) method.…”

A Nuclease from Streptococcus mutans Facilitates Biofilm Dispersal and Escape from Killing by Neutrophil Extracellular Traps

“…The nuclease has been proved to be an endogenous mediator of biofilm dispersal of Staphylococcus aureus and a thermonuclease-deficient mutant strain of S. aureus exhibited increased biofilm formation (Mann et al, 2009). A potent nuclease of nontypeable Haemophilus influenzae has also been implicated in biofilm remodeling and organism dispersal (Cho et al, 2015). …”