Abstract
Outer Membrane Vesicles (OMVs) are ubiquitous in bacterial environments and enable interactions within and between species. OMVs are observed in lab-grown and environmental biofilms, but our understanding of their function comes primarily from planktonic studies. Planktonic OMVs assist in toxin delivery, cell-cell communication, horizontal gene transfer, small RNA trafficking, and immune system evasion. Previous studies reported differences in size and proteomic cargo between planktonic and agar plate biofilm OMVs, suggesting possible differences in function between OMV types. In Pseudomonas aeruginosa interstitial biofilms, extracellular vesicles were reported to arise through cell lysis, in contrast to planktonic OMV biogenesis that involves the Pseudomonas Quinolone Signal (PQS) without appreciable autolysis. Differences in biogenesis mechanism could provide a rationale for observed differences in OMV characteristics between systems. Using nanoparticle tracking, we found that P. aeruginosa PAO1 planktonic and biofilm OMVs had similar characteristics. However, P. aeruginosa PA14 OMVs were smaller, with planktonic OMVs also being smaller than their biofilm counterparts. Large differences in Staphylococcus killing ability were measured between OMVs from different strains, and a smaller within-strain difference was recorded between PA14 planktonic and biofilm OMVs. Across all conditions, the predatory ability of OMVs negatively correlated with their size. To address biogenesis mechanism, we analyzed vesicles from wild type and pqsA mutant biofilms. This showed that PQS is required for physiological-scale production of biofilm OMVs, and time-course analysis confirmed that PQS production precedes OMV production as it does in planktonic cultures. However, a small sub-population of vesicles was detected in pqsA mutant biofilms whose size distribution more resembled sonicated cell debris than wild type OMVs. These results support the idea that, while a small and unique population of vesicles in P. aeruginosa biofilms may result from cell lysis, the PQS-induced mechanism is required to generate the majority of OMVs produced by wild type communities.
Highlights
The biofilm mode of growth predominates in natural [1] and disease [2] environments, with estimates of greater than 65 percent of infections being biofilm-related [3]
In contrast to results previously reported using electron microscopy [10], we found that differences in Outer Membrane Vesicles (OMVs) size between biofilms and planktonic cultures were minimal for PAO1 when measured using Nanoparticle Tracking Analysis (NTA) (Fig 1A, 1C and 1D)
Since biofilm OMV production was observed to be dependent on the presence of pqsA, we investigated whether Pseudomonas Quinolone Signal (PQS) concentration and OMV concentration increase as the number of cells in the population increases, as they do in planktonic cultures [19,46,70]
Summary
The biofilm mode of growth predominates in natural [1] and disease [2] environments, with estimates of greater than 65 percent of infections being biofilm-related [3]. Planktonic bacteria transition to a biofilm lifestyle by first attaching to a surface [4,5]. Afterwards, they produce an extracellular polymeric substance (EPS) that encases the bacteria, and protects them from the environment [6]. The EPS is composed of polysaccharides [7], extracellular DNA [7,8], proteins [7,9], and lipids [9] that can be found in the form of OMVs [10]. Recent literature suggests that OMV production may contribute to biofilm formation in Helicobacter pylori [11], Vibrio cholerae [12], and Pseudomonas putida [13], and inhibit biofilm formation in Xylella fastidiosa [14]. Together with the known ubiquity of OMV production among Gram-negative species [15], this suggests that OMVs may carry out important functions for organisms living the biofilm lifestyle
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