Abstract
Biofilms are community architectures adopted by bacteria inclusive of a self-formed extracellular matrix that protects resident bacteria from diverse environmental stresses and, in many species, incorporates extracellular DNA (eDNA) and DNABII proteins for structural integrity throughout biofilm development. Here, we present evidence that this eDNA-based architecture relies on the rare Z-form. Z-form DNA accumulates as biofilms mature and, through stabilization by the DNABII proteins, confers structural integrity to the biofilm matrix. Indeed, substances known to drive B-DNA into Z-DNA promoted biofilm formation whereas those that drive Z-DNA into B-DNA disrupted extant biofilms. Importantly, we demonstrated that the universal bacterial DNABII family of proteins stabilizes both bacterial- and host-eDNA in the Z-form in situ. A model is proposed that incorporates the role of Z-DNA in biofilm pathogenesis, innate immune response, and immune evasion.
Highlights
Bacterial biofilms are comprised of a community of cells either aggregated or attached to a surface, which are embedded in a self-produced extracellular polymeric substance (EPS) matrix
We have previously shown that these proteins serve a role extracellularly, wherein they act as linchpin proteins to stabilize the crossed-strand structure of extracellular DNA (eDNA), and these eDNA structures functionally resemble Holliday junctions (HJs) (Devaraj et al, 2019) within the biofilm EPS and are required for the stability of the biofilm matrix (Brockson et al, 2014; Devaraj et al, 2018)
Via use of these highly specific monoclonal antibodies (mAbs) directed against B-DNA (Heegaard et al, 1996) or Z-DNA (Moller et al, 1982), we first assessed whether Z-DNA was present in biofilms formed by several well-known biofilm-forming pathogens: nontypeable Haemophilus influenzae (NTHI), uropathogenic E. coli (UPEC), Klebsiella pneumoniae (Kp), P. aeruginosa, and Streptococcus mutans using IF. Abundant levels of both B-DNA and Z-DNA were present within the EPS of the mature biofilms (40 h) formed by all pathogens tested (Figure 1A). We showed that both Z-DNA and B-DNA increased within the EPS of biofilms formed over time (24 h, 40 h, and 90 h) by NTHI, Kp, and UPEC, after 1 week, eDNA in biofilms formed by NTHI was so strongly skewed to the Z-DNA form that the B-DNA signal was below the level of detection (Figures S3A and S3B)
Summary
Bacterial biofilms are comprised of a community of cells either aggregated or attached to a surface, which are embedded in a self-produced extracellular polymeric substance (EPS) matrix. This EPS contains extracellular DNA (eDNA), proteins, lipids, polysaccharides, biopolymers, and divalent cations and provides a protective barrier against harsh environments, antimicrobials, and host immune effectors (Flemming and Wingender, 2010; Koo et al, 2017). Targeted removal of DNABII proteins with specific antibodies results in rapid, significant biofilm collapse (Devaraj et al, 2015; Gunn et al, 2016; Novotny et al, 2013a; Rocco et al, 2018) with release of resident bacteria that are markedly more susceptible to antibiotics and host immune effectors (Brockson et al, 2014; Gunn et al, 2016; Mokrzan et al, 2020a; Novotny et al, 2013a)
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