The Neisseria gonorrhoeae Biofilm Matrix Contains DNA, and an Endogenous Nuclease Controls Its Incorporation

Abstract

Neisseria gonorrhoeae has been shown to produce biofilms both in experimental flow chambers and in the human host. Our laboratory has shown that extracellular DNA is an essential component of the gonococcal matrix. We have also identified a gene in N. gonorrhoeae, which we designated nuc. This gene has homology with the staphylococcus-secreted thermonuclease. Our laboratory has characterized nuc through phenotypic analysis of a nuc deletion mutant. Biofilms grown with this strain are significantly thicker and of greater biomass than the N. gonorrhoeae 1291 parent strain. Confocal microscopy indicates that the increased size of the mutant biofilms appears to be due to elevated amounts of extracellular DNA in the biofilm matrix. Chromosomal complementation of the nuc mutation restored the wild-type biofilm phenotype. In addition, we have cloned and expressed the Nuc protein in Escherichia coli, and our data indicate that it has the ability to digest multiple forms of DNA and is a thermonuclease. The ability of Nuc to digest DNA also extends to its ability to disrupt established gonococcal biofilms through degradation of the DNA in the biofilm matrix. Our studies indicate that the N. gonorrhoeae biofilm contains DNA and that the Nuc protein appears to play a role in biofilm formation and remodeling.

A Thermonuclease ofNeisseria gonorrhoeaeEnhances Bacterial Escape From Killing by Neutrophil Extracellular Traps

“…Neisseria gonorrhoeae encodes a thermonuclease homolog (Nuc) that degrades singleand double-stranded DNA and helps remodel N. gonorrhoeae biofilms [17]. Here, we show that N. gonorrhoeae induces NET formation from human neutrophils; however, Nuc degrades the DNA backbone of NETs over time.…”

“…Parent, Δnuc, and nuc-complement N. gonorrhoeae grew similarly in liquid medium (Supplementary Figure 1A). Piliated, Opa-expressing N. gonorrhoeae of strain 1291 and an isogenic Δnuc mutant were also used [17]; immunoblotting revealed that the strains had identical Opa expression profiles (data not shown).…”

“…An insertiondeletion mutation with a kanamycin resistance cassette was generated in nuc (NGO0969), using plasmid pCTS#43 [17]. Transformants were selected on gonococcal medium base agar (GCB) containing Kellogg supplements I and II [19] with 50 µg/mL kanamycin and were confirmed by DNA sequencing.…”

“…Transformants were selected on gonococcal medium base agar (GCB) containing Kellogg supplements I and II [19] with 50 µg/mL kanamycin and were confirmed by DNA sequencing. Δnuc N. gonorrhoeae was complemented by transformation with pCTS#33 [17], and colonies were selected on supplemented GCB with 50 µg/mL spectinomycin. All N. gonorrhoeae isolates exhibited identical lipooligosaccharide banding patterns on silver-stained polyacrylamide gels.…”

“…Nuc was expressed in Escherichia coli and purified as described by Steichen et al [17]. In some experiments, Nuc was incubated for 30 minutes at 37°C with 0.1 mg/mL thymidine-3,5-bisphosphate disodium salt (Toronto Research Chemicals) [15] prior to addition of neutrophil DNA.…”