Quorum Sensing Signaling Alters Virulence Potential and Population Dynamics in Complex Microbiome-Host Interactomes

Abstract

Despite the discovery of the first N-acyl homoserine lactone (AHL) based quorum sensing (QS) in the marine environment, relatively little is known about the abundance, nature and diversity of AHL QS systems in this diverse ecosystem. Establishing the prevalence and diversity of AHL QS systems and how they may influence population dynamics within the marine ecosystem, may give a greater insight into the evolution of AHLs as signaling molecules in this important and largely unexplored niche. Microbiome profiling of Stelletta normani and BD1268 sponge samples identified several potential QS active genera. Subsequent biosensor-based screening of a library of 650 marine sponge bacterial isolates identified 10 isolates that could activate at least one of three AHL biosensor strains. Each was further validated and profiled by Ultra-High Performance Liquid Chromatography Mass Spectrometry, with AHLs being detected in 8 out of 10 isolate extracts. Co-culture of QS active isolates with S. normani marine sponge samples led to the isolation of genera such as Pseudomonas and Paenibacillus, both of which were low abundance in the S. normani microbiome. Surprisingly however, addition of AHLs to isolates harvested following co-culture did not measurably affect either growth or biofilm of these strains. Addition of supernatants from QS active strains did however impact significantly on biofilm formation of the marine Bacillus sp. CH8a sporeforming strain suggesting a role for QS systems in moderating the microbe-microbe interaction in marine sponges. Genome sequencing and phylogenetic analysis of a QS positive Psychrobacter isolate identified several QS associated systems, although no classical QS synthase gene was identified. The stark contrast between the biodiverse sponge microbiome and the relatively limited diversity that was observed on standard culture media, even in the presence of QS active compounds, serves to underscore the extent of diversity that remains to be brought into culture.

Keywords: acyl homoserine lactone (AHL); cell–cell communication; marine sponge-associated bacteria; microbiome; quorum sensing (QS).

FIGURE 1

Co-culture sponge enrichment assay protocol incorporating QS and QQ samples with appropriate controls. QS or QQ producing isolates are placed in the top chamber with marine both media to facilitate growth. These are separated from the lower chamber containing sponge material by a 0.2 μm filter. QS or QQ signals can transition from the top chamber into the lower chamber by diffusion. Controls are included to ensure no microbial transfer between chambers. Isolation of culturable organisms is achieved on marine agar plates following 3 day incubation. Distinct morphologies are scored and selected for 16S rRNA sequencing.

FIGURE 2

(A) Microbiome 16S DNA bacterial community profiling of sponge DNA. Stacked charts show relative abundance at the genus level. Phylum community profiles are presented in pie-chart format above the corresponding genus-level stacked chart. Comparison with culturable microbiota reveals considerable challenge in activating the “silent” majority of genera present. Two independent samples were taken for each sponge and the average of three independent DNA extraction sequenced replicates is presented for each sample. (B) Diversity and OTU analysis on microbiome data (i) Shannon diversity index (H = –Σp_i ln p_i), (ii) Simpson diversity index, and (iii) OTU abundance.

FIGURE 3

Co-culture sponge enrichment analysis. (A) Proof of concept using QS producing strains. Transfer of the QS active compounds confirmed by validation on Biosensor seeded plates. (B) Outcome of QS-mediated sponge enrichment assays measuring recoverable morphologies which were subsequently 16S rRNA-typed. Each datapoint refers to the number of distinct species recovered after the designated treatment. The species listed represent recoverable isolates identified in the study. Data presented represents at least three independent assays encompassing distinct S. normani sponge preparations. Statistical analysis was performed by Student’s t-test. ^∗∗∗p ≤ 0.001.

FIGURE 4

Addition of purified 3-oxo-C12-HSL did not influence (A) growth of a Pseudomonas sp. isolate or (B) biofilm formation of marine sponge isolates recovered in this study following co-culture.

FIGURE 5

Modulation of Bacillus sp. CH8a biofilm formation by extracts from QS active isolates. Data presented is the average of at least three independent biological replicates. Statistical analysis was performed by one-way ANOVA with post hoc Bonferroni corrective testing (^∗p ≤ 0.05, ^∗∗p ≤ 0.005, ^∗∗∗p ≤ 0.001).

FIGURE 6

Phylogenetic tree of Psychrobacter species including the marine Psychrobacter sp. 230 isolate identified in this study. Species names are listed on the outer edge of each branch and an arbitrary color based system is used to distinguish the various clades in the tree.