The origin of extracellular DNA in bacterial biofilm infectionsin vivo

Abstract

35 Mobile +45 20 65 98 88 36 fax + 45 35 45 64 12 37 Tbjarnsholt@sund.ku.dk 38 www.sund.ku.dk 39 40 41 42 43 44 45 46 47 48 49 50 51 52 Abstract 53 Extracellular DNA (eDNA) plays an important role in both the aggregation of bacteria54 and in the interaction of the resulting biofilms with polymorphonuclear leukocytes 55 (PMNs) during an inflammatory response. Here, transmission electron and confocal 56 scanning laser microscopy were used to examine the interaction between biofilms of 57 Pseudomonas aeruginosa and PMNs in a murine implant model and in lung tissue from 3 58 chronically infected cystic fibrosis patients. PNA FISH, DNA staining, labeling of 59 PMN DNA with a thymidine analogue, and immunohistochemistry were applied to 60 localize bacteria, eDNA, PMN-derived eDNA, PMN-derived histone H3 (H3), 61 neutrophil elastase (NE), and citrullinated H3 (citH3). Host-derived eDNA was 62 observed surrounding bacterial aggregates but not within the biofilms. H3 localized to 63 the lining of biofilms while NE was found throughout biofilms. CitH3, a marker for 64 neutrophil extracellular traps (NETs) was detected only sporadically indicating that 65 most host-derived eDNA in vivo was not a result of NETosis. Together these 66 observations show that, in these in vivo biofilm infections with P. aeruginosa, the 67 majority of eDNA is found external to the biofilm and derives from the host. 68 69 70 71 Keywords: polymorphonuclear leukocytes/ neutrophil extracellular traps/ NETosis, 72 /Histone/ elastase 73 74 Author summary75 The role of extracellular DNA (eDNA) has been described in vitro to play a major role in 76 biofilm formation and antibiotic tolerance, but never for biofilm infections in vivo. 77 78 Two important characteristics of human chronic bacterial infections are aggregated 79 bacteria and white blood cells (WBC). Bacteria use eDNA to stabilize biofilms and WBC 80 use eDNA to trap bacteria. Given the importance of eDNA for both bacteria and WBC we 81 show here for the first time that bacterial biofilms do not co-localize with either bacterial 82 or WBC-derived eDNA during chronic infections. Our in vivo findings show eDNA is 83 located outside biofilms as opposed to incorporated within the biofilm as commonly 84 observed in vitro. 85 86 We believe that understanding the interplay between biofilms and WBC during chronic 87 infection is essential if we are to elucidate the mechanisms underlying persistent infection 88 and ascertain why WBC fail to eradicate bacteria; this will enable us to develop new 89 treatment strategies. 4 90 91 92 93

Neutrophil extracellular traps are present in the airways of ENaC-overexpressing mice with cystic fibrosis-like lung disease

“…This accumulation correlated with both CXCL2 expression and reduced lung function, suggesting a link between free DNA and leukocyte infiltration in this model, similar to human CF 12 . A more recent study reported that βENaC-Tg mice can be infected with planktonic and biofilm forms of P. aeruginosa 24 much better than wildtype animals, and the bacterium forms aggregates similar to those seen in human CF patients [25,26]. Neutrophil elastase is also found in the airways of βENaC-Tg mice, similar to human CF patients including children [34], and was found to be a major contributor to lung disease pathogenesis [35].…”

“…Studies of these mice (βENaC-Tg) demonstrated that increased airway sodium absorption causes airway surface liquid depletion, reduced mucus transport, and spontaneous CF-like lung disease with airway mucus obstruction, impaired mucociliary clearance, emphysema, and chronic inflammation including airway neutrophilia, similar to human CF lung disease [19,[21][22][23]. P. aeruginosa can infect the airways of βENaC-Tg mice, both in planktonic and biofilm forms, and form bacterial aggregates [23,24], a well-described feature of P. aeruginosa in CF patients with chronic infection [25][26][27][28]. The goal of this work was to characterize NETs and further details of the neutrophil-mediated inflammation in the airways of βENaC-Tg mice, as the best murine CF lung disease model, in the absence of bacterial infections.…”

Treatment with a neutrophil elastase inhibitor and ofloxacin reduces P. aeruginosa burden in a mouse model of chronic suppurative otitis media

“…Given the abundance of neutrophils and the contribution of dead cells to extracellular DNA of bacterial biofilms 17 , 18 , we examined dsDNA levels resulting from CSOM infection in our model. We observed that dsDNA levels in CSOM were significantly greater compared to uninfected mice (mean of 4.5 ng/mL in uninfected control mice c.f.…”

“…Extracellular traps (NETs) are particularly interesting in cases of biofilm-mediated disease as NETs are primarily made of dsDNA (along with various cytosolic and granule proteins) and dsDNA is also a component of the extracellular biofilm matrix. There is evidence to suggest that necrotic neutrophils contribute DNA to the biofilm extracellular matrix as a result of failed attempts to clear infection 17 , 18 . Host biofilms are stronger and more developed in the host versus in a petri dish, and NETs can add to the integrity of the aggregate.…”

Microbial biofilm: A matter of grave concern for human health and food industry

“…Apart from polysaccharides and proteins some amount of nucleic acid is also present within the EPS matrix, generally termed as extracellular DNA or eDNA. The eDNA originates from autolysis of microbes or phage‐induced cell lysis or through the microbial secretory system [48]. The eDNA works as an intercellular connector between cells in biofilm and protects biofilm from degradation [49].…”