Nucleases from Prevotella intermedia can degrade neutrophil extracellular traps

Abstract

Periodontitis is an inflammatory disease caused by periodontal bacteria in subgingival plaque. These bacteria are able to colonize the periodontal region by evading the host immune response. Neutrophils, the host's first line of defense against infection, use various strategies to kill invading pathogens, including neutrophil extracellular traps (NETs). These are extracellular net-like fibers comprising DNA and antimicrobial components such as histones, LL-37, defensins, myeloperoxidase, and neutrophil elastase from neutrophils that disarm and kill bacteria extracellularly. Bacterial nuclease degrades the NETs to escape NET killing. It has now been shown that extracellular nucleases enable bacteria to evade this host antimicrobial mechanism, leading to increased pathogenicity. Here, we compared the DNA degradation activity of major Gram-negative periodontopathogenic bacteria, Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. We found that Pr. intermedia showed the highest DNA degradation activity. A genome search of Pr. intermedia revealed the presence of two genes, nucA and nucD, putatively encoding secreted nucleases, although their enzymatic and biological activities are unknown. We cloned nucA- and nucD-encoding nucleases from Pr. intermedia ATCC 25611 and characterized their gene products. Recombinant NucA and NucD digested DNA and RNA, which required both Mg²⁺ and Ca²⁺ for optimal activity. In addition, NucA and NucD were able to degrade the DNA matrix comprising NETs.

Keywords: nucA; nucD; deoxyribonucleas; neutrophil extracellular trap degradation; periodontal disease.

Figure 1

Bacterial DNA degradation activity detected on nuclease agar plates. The plates were incubated for 2 days, followed by flooding with 1 m HCl to precipitate non‐digested DNA. Clear zones seen around colonies reflect DNA digestion. Bar, 1 cm.

Figure 2

Neutrophil extracellular traps (NET) degradation by crude nuclease. (A) Quantification of NET degradation by staining using the extracellular DNA stain Sytox orange. Data are presented as percent NET levels relative to time‐point zero. Controls were incubated with 50 mm Tris–HCl buffer (pH 7.5) rather than crude nuclease. Error bars represent the standard deviation of three independent experiments. Statistical significance (*P < 0.05) was determined using Student's t‐test relative to the control. (B) Representative images of NET degradation by crude nuclease prepared from bacterial culture supernatant. Human neutrophils were stimulated with PMA to release NETs, which were then exposed to bacterial crude nucleases for 90 min.

Figure 3

SDS–PAGE nuclease assay and biochemical analysis of Prevotella intermedia crude nuclease. (A) Pr. intermedia crude nuclease was detected in SDS–PAGE gels containing 0.2 mg ml⁻¹ salmon sperm DNA. After electrophoresis, proteins were renatured. This was followed by incubation in activation buffer containing 1 mm MgCl₂ and 1 mm CaCl₂ for 3 h at 37°C. To visualize DNA degradation, gels were stained with ethidium bromide and examined under ultraviolet light. Two protein bands were observed to possess nuclease activity. (B) λ DNA was incubated with Pr. intermedia crude nuclease with or without 1 mm MgCl₂ and 1 mm CaCl₂ at 37°C for 10 min. Following electrophoresis, DNA was stained with ethidium bromide and visualized under ultraviolet light.

Figure 4

Schematic presentation of NucA and NucD protein domains. (A) NucA and (B) NucD.

Figure 5

Enzymatic analysis of recombinant NucA and NucA‐E104G (A) λ DNA was incubated with rNucA with or without 1 mm MgCl₂ and 1 mm CaCl₂. (B) The substrate preference of rNucA was examined using M13mp18 (single‐stranded circular DNA), pUC18 (double‐stranded circular DNA), and total RNA purified from murine macrophages. (C) λ DNA was incubated with rNucA with 1 mm MgCl₂ and 1 mm CaCl₂ at different pH. (D) λ DNA was incubated with rNucA‐E104G with or without 1 mm MgCl₂ and 1 mm CaCl₂.

Figure 6

Enzymatic analysis of recombinant NucD and NucD‐N229G. (A) λ DNA was incubated with rNucD with or without 1 mm MgCl₂ and 1 mm CaCl₂. (B) The substrate preference of rNucD was examined using M13mp18 (single‐stranded circular DNA), pUC18 (double‐stranded circular DNA), and total RNA purified from murine macrophages. (C) λ DNA was incubated with rNucD with 1 mm MgCl₂ and 1 mm CaCl₂ at different pH. (D) λ DNA was incubated with rNucD‐N229G with or without 1 mm MgCl₂ and 1 mm CaCl₂.

Figure 7

Determination of human neutrophil extracellular traps (NET) degradation. (A) Immunofluorescence microscopy of neutrophils stimulated with PMA. Nuclear DNA was stained with DAPI (blue) and neutrophil elastase was detected with an anti‐human neutrophil elastase antibody and Cy3‐labeled secondary antibody (orange). PMA‐stimulated human neutrophils were incubated with rNucA, rNucA‐E104G, rNucD, rNucD‐N229G, or Prevotella intermedia crude nuclease. Recombinant bovine pancreas DNase I (BP DNase I) was used as a positive control. (B) Quantification of NET release. Neutrophil elastase released into the reaction buffer in accordance with NET degradation was quantified. Error bars represent the standard deviation of three independent experiments. Statistical significance (*P < 0.05) was determined by Student's t‐test.