Aggregatibacter actinomycetemcomitans NadN contributes to neutrophil extracellular trap degradation

Neutrophils Extracellular Traps (NET) immobilize and kill extracellular pathogens. Aggregatibacter actinomycetemcomitans (Aa), associated with aggressive forms of periodontitis, has been shown to induce NET release. However, it remains unknown whether Aa evades extracellular traps.

NETs were incubated with wild-type Aa (Aa-WT, strain Y4) or Aa-ΔCdtB, lacking a toxin inducing DNA breaks. NET degradation was measured by imaging and fluorometry. Aa survival and growth was measured by biofilm formation. Aa-WT membrane was fractionated on a sucrose gradient, and fractions positive for NET degradation were subjected to LC-MS/MS. The top protein candidate was expressed as a recombinant protein in Lactococcus lactis to confirm its NET-degrading activity.

Aa-WT and Aa-ΔCdtB strains were captured by NETs but degraded the extracellular traps after three hours. Aa survival in the presence of NETs was independent of CdtB expression. NET degrading activity was not release but associated with cells. NAD nucleotidase (NadN) was identified via mass spectrometry from membrane fraction. In vitro, recombinant NadN degraded NETs, and heterologous expression of NadN in L. lactis was sufficient to confer a NET-degrading phenotype.

Aa degrades NETs to evade host immunity. NadN is a primary mediator of the NET-degrading activity of Aggregatibacter actinomycetemcomitans.