The origin of extracellular DNA in bacterial biofilm infections in vivo

Context and background: In 2002, Whitchurch et al established a functional role for extracellular DNA (eDNA) in biofilms by showing that the presence of DNase I prevented Pseudomonas aeruginosa biofilm formation in vitro. Since then, various studies have confirmed the importance of eDNA in bacterial biofilms in vitro [1, 2], but not many studies focus on eDNA in in vivo biofilms. Within in vitro biofilms, eDNA is shown to be a part of the biofilm matrix and thus plays a role in structural establishment as well as maintenance of bacterial biofilms.

Biofilms are a major obstacle in treating chronic infections. One of the key features of chronicity is a marked persistent inflammatory state. The infection site is infiltrated with immune cells, especially polymorphonuclear leukocytes (PMNs). These PMNs fail to eradicate the infection and undergo cell death, releasing various toxins into the cellular environment, which further enhance inflammation.

As a part of their defence machinery, PMNs can form extracellular traps by extruding intracellular materials to release their DNA and bactericidal molecules, thus forming what are known as Neutrophil Extracellular Traps (NETs) (see [3,4] for reviews on NETs and infections). This cell death process is termed NETosis. These NETs are meant to trap bacteria and clear the infection.

Thus, eDNA is an important component both in bacterial biofilms in vitro as well as in NETosis which releases eDNA. This study visualized eDNA from P. aeruginosa biofilms and PMNs in order to understand its role and localization in vivo.

 

Experimental setup: The study made use of a murine implant model as well as explanted lung tissue from chronically infected cystic fibrosis (CF) patients. The explants were precoated with P. aeruginosa and inserted into the peritoneal cavity of the mice. Transmission electron microscopy (TEM) and confocal scanning laser microscopy (CSLM) were used to visualize P. aeruginosa biofilms and PMNs. Immunohistochemistry was used to analyse the localization of various PMN derived factors.

 

Important Results:

High Density Bacterial Aggregates show damaged PMNs in their surroundings – Microscopic analysis of P. aeruginosa and PMN interactions in vitro revealed that PMNs lyse, resulting in release of their DNA. In order to observe their interactions in vivo, TEM of the explants was performed. Early interactions at 6 hours post insertion revealed healthy PMNs, with bacteria within them, showing successful engulfment. These were observed only in areas with no bacterial aggregates. At both 24 and 48-hours post insertion, bacterial biofilms were formed at the lining of the implants and biofilm matrix was also observed in TEM. PMNs were seen to be enlarged with damaged cell membranes. The PMNs were found to be lining the biofilm edge.

PMN-derived eDNA surrounds biofilms, but is not present within the biofilm – In order to visualize eDNA in biofilms from the murine implant model, SYTO9 was used to label DNA. To differentiate between bacterial and eukaryotic DNA, Click-iT DNA labelling technology was used in mice, wherein a modified thymidine analogue (EdU) is incorporated into the DNA during the cell cycle in vivo and is fluorescently labelled ex vivo. Thus, DNA of murine origin will be distinguishable from bacterial DNA (which is just labelled with SYTO9). Staining revealed biofilms surrounded by PMN-derived eDNA, but no PMN derived eDNA within the regions of the biofilms. This indicates that PMN derived eDNA is not incorporated within the biofilm matrix.

Using DAPI and peptide nuclear acid (PNA) FISH, similar observations were made in CF lung tissue. The eDNA was localized outside the biofilm and not within it.

Overall, the SYTO9 staining was also minimal within the biofilm, indicating that eDNA from bacterial origin was also not significantly present in the biofilm.

PMN-derived eDNA is not a result of NETosis – PMN lysis releases various components into the infected microenvironment. They release components like neutrophil elastase (NE), histones as well as DNA that is attached to the histones, and other antibacterial molecules. NE is also an antibacterial, meant to kill bacteria by damaging their membranes. NE can, however, prove fatal to the host at higher concentrations (such as in chronic infections) by degrading various host components and causing tissue damage. One of the key markers for the NETosis process is citrullinated H3 (citH3), a histone modification that occurs as a part of NETosis. To visualize the three components of histones (H3), NE and citH3 within a biofilm, immunolabeling of sections of the murine implant was performed. H3 was found to surround biofilms, but not be present inside, again indicating that PMN-derived eDNA is not found within biofilms. NE was found to be incorporated within the biofilm and citH3 was almost absent. The absence of citH3 indicated that the PMN-derived eDNA was not released as a result of NETosis. The lack of NE-mediated bacterial killing may be attributed to the production of protease inhibitors by bacteria.

In CF lungs, the localization of the three components was similar to what was observed for the murine implants.

 

Interesting aspects of the study: This study brings to light the importance of in vivo studies. The otherwise important eDNA in in vitro studies of biofilms was not observed within in vivo biofilms. The lack of citH3 and presence of H3 indicates that necrosis rather than NETosis might be the source of the eDNA. This study reveals a zone of PMN-deposited components (eDNA and H3) surrounding the biofilm. This could form a protective mesh around the biofilm and thus prevent antibiotics and small molecules from reaching the bacteria and increase resistance (see the figure for a hypothesized model from the authors for eDNA shield formation).

Thus, this study indicates a protective role for host-derived eDNA for the biofilms in chronic infections.

 

 

 

References/Further Reading:

[1] Okshevsky, Mira, and Rikke Louise Meyer. “The role of extracellular DNA in the establishment, maintenance and perpetuation of bacterial biofilms.” Critical reviews in microbiology 41.3 (2015): 341-352.

[2] Das, Theerthankar, Shama Sehar, and Mike Manefield. “The roles of extracellular DNA in the structural integrity of extracellular polymeric substance and bacterial biofilm development.” Environmental microbiology reports 5.6 (2013): 778-786.

[3] Mesa, Miguel Antonio, and Gloria Vasquez. “NETosis.” Autoimmune diseases 2013 (2013).

[4] Branzk, Nora, and Venizelos Papayannopoulos. “Molecular mechanisms regulating NETosis in infection and disease.” Seminars in immunopathology. Vol. 35. No. 4. Springer-Verlag, 2013.

Tags: biofilm, chronic infection, edna, extracellular dna, neutrophils, polymorphonuclear leukocytes, pseudomonas

doi: https://doi.org/10.1242/prelights.12342

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