Abstract
Biofilm production is a key virulence factor that facilitates bacterial colonization on host surfaces and is regulated by complex pathways, including quorum sensing, that also control pigment production, among others. To limit colonization, epithelial cells, as part of the first line of defense, utilize a variety of antimicrobial peptides (AMPs) including defensins. Pore formation is the best investigated mechanism for the bactericidal activity of AMPs. Considering the induction of human beta-defensin 2 (HBD2) secretion to the epithelial surface in response to bacteria and the importance of biofilm in microbial infection, we hypothesized that HBD2 has biofilm inhibitory activity. We assessed the viability and biofilm formation of a pyorubin-producing Pseudomonas aeruginosa strain in the presence and absence of HBD2 in comparison to the highly bactericidal HBD3. At nanomolar concentrations, HBD2 – independent of its chiral state – significantly reduced biofilm formation but not metabolic activity, unlike HBD3, which reduced biofilm and metabolic activity to the same degree. A similar discrepancy between biofilm inhibition and maintenance of metabolic activity was also observed in HBD2 treated Acinetobacter baumannii, another Gram-negative bacterium. There was no evidence for HBD2 interference with the regulation of biofilm production. The expression of biofilm-related genes and the extracellular accumulation of pyorubin pigment, another quorum sensing controlled product, did not differ significantly between HBD2 treated and control bacteria, and in silico modeling did not support direct binding of HBD2 to quorum sensing molecules. However, alterations in the outer membrane protein profile accompanied by surface topology changes, documented by atomic force microscopy, was observed after HBD2 treatment. This suggests that HBD2 induces structural changes that interfere with the transport of biofilm precursors into the extracellular space. Taken together, these data support a novel mechanism of biofilm inhibition by nanomolar concentrations of HBD2 that is independent of biofilm regulatory pathways.
Highlights
Biofilms are composed of microbial communities encased in a protective layer of self-produced, extracellular polymers
The resazurin reduction assay showed that both human beta-defensin 2 (HBD2) and HBD3 reduced metabolic activity in a dose-dependent manner, with HBD3 being more effective on a per molar basis, producing around a 30% reduction at 0.5 μM compared to the 4 μM needed by HBD2 at 18 h for the same effect (Figure 1)
At concentrations of 0.25 and 0.5 μM, HBD2 reduced P. aeruginosa biofilm to ∼75% of the control without significantly reducing the metabolic activity (Figure 2A). At these concentrations, HBD3 reduced the formation of P. aeruginosa biofilm in a dose dependent manner that was directly proportional to the cumulative effect on metabolic activity and further reduced both biofilm and resorufin production to nearly undetectable levels at a concentration of 1 μM (Figure 2B) consistent with direct microbicidal activity
Summary
Biofilms are composed of microbial communities encased in a protective layer of self-produced, extracellular polymers. Biofilms are formed on both abiotic and biotic surfaces and play a significant role in a variety of settings such as aquaculture [1], the food industry, and the clinical field as a factor for antimicrobial drug resistance. The biofilm matrix is primarily composed of exopolysaccharide, proteins, and extracellular DNA and has been well studied in Pseudomonas aeruginosa, a ubiquitous, opportunistic, Gramnegative bacterium. Extracellular DNA (eDNA), which is released via cell lysis [10], plays an important role in priming surfaces for the initial adhesion of the bacteria as well as in maintaining the structural integrity of the polysaccharide fibers [3, 6, 11,12,13,14]
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