Immune-mediated competition benefits protective microbes over pathogens in a novel host species

Abstract

Microbes that protect against infection inhabit hosts across the tree of life. It is unclear whether and how the host immune system may affect the formation of new protective symbioses. We investigated the transcriptomic response of Caenorhabditis elegans following novel interactions with a protective microbe (Enterococcus faecalis) able to defend against infection by pathogenic Staphylococcus aureus. We have previously shown that E. faecalis can directly limit pathogen growth within hosts. In this study, we show that colonisation by protective E. faecalis caused the differential expression of 1,557 genes in pathogen infected hosts, including the upregulation of immune genes such as lysozymes and C-type lectins. The most significantly upregulated host lysozyme gene, lys-7, impacted the competitive abilities of E. faecalis and S. aureus when knocked out. E. faecalis has an increased ability to resist lysozyme activity compared to S. aureus, suggesting that the protective microbe could gain a competitive advantage from this host response. Our finding that protective microbes can benefit from immune-mediated competition after introduction opens up new possibilities for biocontrol design and our understanding of symbiosis evolution. Crosstalk between the host immune response and microbe-mediated protection should favour the continued investment in host immunity and avoid the potentially risky evolution of host dependence.

Fig. 1. Experimental design for testing the effect of protective microbe E. faecalis and pathogen S. aureus on the transcriptional response of C. elegans hosts.

Young adult worms were exposed to bacteria for 12 h to compare host transcriptional response between: (a) S. aureus in the absence vs. presence of E. faecalis; (b) E. coli OP50 (food control) vs. E. faecalis; and (c) E. coli OP50 (food control) vs. S. aureus. We included five replicate populations for each unique treatment (i.e. only a single set of five replicates were completed for S. aureus and E. coli, despite being shown twice in the figure). After 12 h exposure, the RNA from ~1000 worms per sample was sequenced.

Fig. 2. Differential host gene expression and gene ontology analysis under E. faecalis-mediated protection.

a Differentially expressed genes (DEGs) of host C. elegans under single colonisation (orange) by E. faecalis and S. aureus, relative to co-colonisation (green) whereby E. faecalis protects against the S. aureus pathogen. The 46 immune gene families differentially regulated by E. faecalis-mediated protection are detailed in the green box, blue arrows indicate downregulation and red upregulation. See supplementary files 1, 3, 4 and 7 for further detail on DEGs across treatments. b Gene ontology (GO) terms significantly enriched in the list of C. elegans genes differentially regulated under co-colonisation and Enterococcus faecalis-mediated protection. Enrichment analysis performed using g:Profiler with Benjamini-Hochberg FDR correction for multiple comparisons.

Fig. 3. The effect of the lys-7 gene on C. elegans mortality and bacterial colonisation.

Percentage mortality of the lys-7 knock-out mutant and wild-type N2 C.elegans host after single colonisation (orange) by food control E. coli (a), protective E. faecalis (b) and pathogenic S. aureus (c) and after co-colonisation (green) whereby E. faecalis protects against pathogenic S. aureus (d). Within-host bacterial density in colony-forming units (CFUs) of E. faecalis (e), and S. aureus (f) after 24 h of co-colonisation. Each treatment was replicated five independent times and 7–10 worms were collected per replicate for CFU quantification. *P < 0.05, **P < 0.01, ***P < 0.001.