Biofilm matrix cloaks bacterial quorum sensing chemoattractants from predator detection

Abstract

Microbes often secrete high levels of quorum sensing (QS) autoinducers into the environment to coordinate gene expression and biofilm formation, but risk detection and subsequent predation by bacterivorous predators. With such prominent signaling molecules acting as chemoattractants that diffuse into the environment at alarmingly high concentrations, it is unclear if bacterial cells can mask their chemical trails from predator detection. Here, we describe a microbial-based anti-detection adaptation, termed as "biofilm cloak", where the biofilm prey produced biofilm matrix exopolysaccharides that "locked" and reduced the leaching of autoinducers into the milieu, thereby concealing their trails to the detection by the bacterivorous Caenorhabditis elegans nematode. The exopolysaccharides act as common good for the non-producers to hide their autoinducers from predator detection. Deficiency in chemosensory gene odr-10 in mutant animals abrogated their ability to detect autoinducers and migrate toward their prey in a directed manner, which led to lower population growth rate of animals. Hence, restriction of bacterial communication activities to the confinements of biofilms is a novel approach for predator evasion, which plays a fundamental role in shaping ecological dynamics of microbial communities and predator-prey interactions.

Fig. 1. C. elegans could not detect secreted metabolites from exopolysaccharide-producing biofilms.

A Experimental design for modified choice assay. B C. elegans preferred biofilm metabolites of ΔpelA, ΔpslBCD, and ΔpelAΔpslBCD mutants over PAO1 control, CI > 0.00. C C. elegans preferred biofilm metabolites of PAO1 control over ΔwspF and PAO1/p_lac-YedQ mutants, CI < 0.00. D C. elegans preferred biofilm metabolites of ΔwspFΔpelA, ΔwspFΔpslBCD, and ΔwspFΔpelAΔpslBCD mutants over ΔwspF control, CI > 0.00. E C. elegans preferred biofilm metabolites of ΔpelAΔpslBCD control over ΔpelAΔpslBCD possessing exogenous Pel and Psl, CI < 0.00. Means and SD from triplicate experiments are shown. ***p < 0.001, one-way ANOVA.

Fig. 2. C. elegans detects bacterial autoinducers.

A C. elegans preferred pure autoinducers over saline control, CI > 0.00. B Pure OdDHL and BHL concentrations in the agar across distances. C BHL concentration in biofilms. D Biofilm-secreted BHL concentrations in agar across distances. E Increasing concentrations of Pel or Psl can trap 10 µM BHL and prevent predator detection. Means and SD from triplicate experiments are shown. F Cellulase degradation of exopolysaccharides results in C. elegans detection of BHL, where C. elegans preferred cellulase-degraded pure exopolysaccharide with released 10 µM BHL over pure exopolysaccharide with trapped 10 µM BHL control, CI > 0.00. *p < 0.05, ***p < 0.001, ns not significant, one-way ANOVA.

Fig. 3. Exopolysaccharide is a public good for trapping autoinducers by non-exopolysaccharide-producers.

A C. elegans preferentially selected PAO1 control strain as compared to ΔlasIΔrhlI mutant, CI < 0.00. B Exogenous addition of Pel or Psl protected ΔlasIΔrhlI mutant from predator detection in the presence of BHL. C Exogenous addition of Pel or Psl protected E. coli OP50 from predator detection. D Exogenous addition of Pel or Psl protected E. coli OP50 from predator detection even in the presence of BHL. Means and SD from triplicate experiments are shown. ***p < 0.001, one-way ANOVA.

Fig. 4. C. elegans requires ODR-10 to sense bacterial autoinducers.

A C. elegans odr-10 mutant could not detect pure autoinducers. B C. elegans odr-10 mutant could not differentiate mutant biofilm mutants from wild-type PAO1. C Experimental design for studying population growth of C. elegans after choice assay. D Higher population growth of wild-type N2 C. elegans which localized on metabolite spot left by ΔpelA, ΔpslBCD, and ΔpelAΔpslBCD mutants relative to odr-10 mutant. Means and SD from triplicate experiments are shown. **p < 0.01, ***p < 0.001, ns not significant, one-way ANOVA.

Fig. 5. Model of biofilms that employ biofilm matrix to hide QS autoinducers from predator (C. elegans) detection.

The exopolysaccharides in the biofilm matrix reduce the leaching of autoinducers into the environment. However, the absence of exopolysaccharides will enable the diffusion of autoinducers into the environment, resulting in the detection by the predator.