Volume 16, Issue 4, Pages 401-407 (February 2006) An Endonuclease Allows Streptococcus pneumoniae to Escape from Neutrophil Extracellular Traps  Katharina Beiter, Florian Wartha, Barbara Albiger, Staffan Normark, Arturo Zychlinsky, Birgitta Henriques-Normark  Current Biology  Volume 16, Issue 4, Pages 401-407 (February 2006) DOI: 10.1016/j.cub.2006.01.056 Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 1 S. pneumoniae Is Trapped but Not Killed by NETs In Vitro (A and B) Neutrophils were stimulated with PMA and infected with FITC-labeled TIGR4 pneumococci (green) at a multiplicity of infection (MOI) of 1 (A) or 100 (B). Five minutes postinfection, the samples were fixed and stained for DNA (blue) and neutrophil elastase (NE, red). Scale bars represent 20 μm. NETs are identified as filamentous structures. The dose-dependent trapping of pneumococci (arrows) in NET structures (MOI 1 versus MOI 100) can be observed. (C) The percentage of bacterial killing by NETs (gray) and by phagocytosis (black), which together make up total killing, is shown for Shigella flexneri (pos. control) and pneumococci [TIGR4, TIGR4Δ(endA)]. Mean and standard error of the mean (SEM) are shown both for total and for phagocytosis killing. Assays were performed at least three independent times for each strain. Pneumococci were killed neither by NETs nor by phagocytosis. Current Biology 2006 16, 401-407DOI: (10.1016/j.cub.2006.01.056) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 2 EndA Is a DNase that Degrades Neutrophil Extracellular Traps (A) Salmon sperm DNA was incubated with TIGR4, TIGR4Δ(endA), and TIGR4Δ(endA)∇(endA) and resolved on an agarose gel. A sample without pneumococci was used as negative control. DNA incubated with TIGR4 or TIGR4Δ(endA)∇(endA) was degraded. No degradation was observed when DNA was incubated with TIGR4Δ(endA), which lacks the DNase gene. (B–E) Neutrophils were activated to make NETs and treated with medium (control) (B), bovine pancreatic DNase (C), TIGR4Δ(endA) pneumococci (D), or TIGR4 pneumococci (E). The samples were stained for DNA (blue) and NE (red). Scale bars represent 20 μm. NETs are degraded in samples treated with bovine DNase or TIGR4 pneumococci. (F) NET-mediated killing of the reporter strain of S. flexneri. The NETs were exposed to RPMI medium (control), bovine pancreatic DNase, TIGR4, or TIGR4Δ(endA) pneumococci. We measured killing of S. flexneri as a reporter of NET antimicrobial activity. Mean and SEM are shown. The assay was performed three times and analyzed with the nonparametric Mann-Whitney test. A p value < 0.05 was considered significant. Shigella flexneri were killed less efficiently after NETs were treated with DNase or DNase-producing pneumococci. (G) NETs were treated with RPMI medium (control), bovine pancreatic DNase, TIGR4, or TIGR4Δ(endA) pneumococci. We measured the NE concentration in the supernatant as a reporter of NET degradation. Mean and SEM are shown. The assay was performed three times and analyzed with the nonparametric Mann-Whitney test. A p value < 0.05 was considered significant. Higher concentrations of NE could be measured in the supernatant of neutrophils cultures after exposure to TIGR4 compared to TIGR4Δ(endA), indicating that the TIGR4-encoded DNase EndA degraded NETs, thereby releasing NE. Current Biology 2006 16, 401-407DOI: (10.1016/j.cub.2006.01.056) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 3 EndA Allows Pneumococci to Escape NETs Neutrophils were activated to make NETs and infected at a MOI of 100 with TIGR4, TIGR4Δ(endA), or TIGR4Δ(endA)∇(endA). Samples were fixed 5 (top panels) or 30 min (bottom panels) after infection and stained for DNA (blue) and NE (red). Pneumococci were labeled with FITC (green) before infection. Scale bars represent 20 μm. Five minutes p.i., NET formation was observed in all samples (A–C), and similar numbers of TIGR4 (A), TIGR4Δ(endA) (B), and TIGRΔ(endA)∇(endA) (C) were initially associated with NETs (arrows). Thirty minutes after infection, only TIGR4Δ(endA) pneumococci were still bound to NETs (E), whereas the EndA-expressing TIGR4 (D) and TIGR4Δ(endA)∇(endA) (F) detached from the strongly degraded NETs and were washed away. Thus, endA-encoded DNase activity liberates bacteria from NETs. Current Biology 2006 16, 401-407DOI: (10.1016/j.cub.2006.01.056) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 4 NETs Are Formed in Murine Pneumococcal Pneumonia and Play a Role in Defense against Pneumococci (A–D) C57BL/6 mice were infected intranasally with either bacterial growth medium (A and C) or TIGR4 pneumococci (B and D). Forty-eight hours postinfection, the lungs were removed and stained for DNA (blue), histone H1 (green), and a neutrophil-specific surface marker (red). Scale bars represent 20 μm ([C] and [D] represent close-ups). Whereas in mock-infected controls (A and C), DNA and histone both are restricted to nuclei, in infected samples (B and D), extracellular DNA and histone can be observed, lining the alveoli. This shows that NETs are formed in murine pneumococcal pneumonia. (E) C57BL/6 mice were infected intranasally with TIGR4, TIGR4Δ(endA), or TIGR4Δ(endA)∇(endA) pneumococci (n = 66). Two independent observers assessed the health status to assign pathology scores as described previously [8]. Mice infected with TIGR4Δ(endA) had a delayed onset of severe disease and a significantly higher survival rate than both TIGR4 and TIGR4Δ(endA)∇(endA) as determined by the Kaplan-Meier-analysis log-rank test (A p value < 0.05 was considered significant). (F–I) C57BL/6 mice were infected intranasally with a 1:1 mix of TIGR4 and TIGR4Δ(endA) (F-G) or a 1:1 mix of TIGR4Δ(endA)∇(endA) and TIGR4Δ(endA) (H–I). Bacterial counts in the upper respiratory tract (URT), lungs, and bloodstream were determined when mice were sacrificed (F and H). Bacterial counts per ml blood were determined 24 hr and 48 hr postinfection (G and I). The competitive index shown is based on the ratio of number of bacteria recovered from the two strains. A competitive index of 1 indicates equal numbers of wild-type and mutant bacteria. Values lower than 1 indicate that the mutant is outcompeted. Each dot represents one mouse. Median values are indicated by horizontal bars. All strains are able to colonize the URT of mice to the same extent. However, TIGR4Δ(endA) is outcompeted by both TIGR4 (F–G) and TIGR4Δ(endA)∇(endA) (H–I) in the lungs and in the bloodstream. This shows that TIGR4Δ(endA) is defective in its invasion ability. Current Biology 2006 16, 401-407DOI: (10.1016/j.cub.2006.01.056) Copyright © 2006 Elsevier Ltd Terms and Conditions

Figure 5 Most Pneumococcal Serotypes Show DNase Activity and Degrade NETs (A) Salmon sperm DNA was incubated with pneumococcal strains of the indicated serotypes and separated on an agarose gel. A sample without pneumococci was used as negative control. Differences in DNase activity of the tested strains can be observed, with especially low DNase activity for the serotype 1 strain. (B) NETs were treated with RPMI medium (control), pneumococcal strains of different serotypes, or bovine pancreatic DNase. The concentration of NE in the supernatant was measured as a reporter of NET degradation. Mean and SEM are shown. The assay was performed three times and analyzed with the nonparametric Mann-Whitney test. A p value < 0.05 was considered significant. Compared to TIGR4 (serotype 4), a significantly lower concentration of NE could be measured in neutrophils exposed to pneumococci of type 1. This is reflecting the type 1's low DNase activity and capability to degrade NETs. Current Biology 2006 16, 401-407DOI: (10.1016/j.cub.2006.01.056) Copyright © 2006 Elsevier Ltd Terms and Conditions