Biofilm development on Caenorhabditis elegans by Yersinia is facilitated by quorum sensing-dependent repression of type III secretion

Abstract

Yersinia pseudotuberculosis forms biofilms on Caenorhabditis elegans which block nematode feeding. This genetically amenable host-pathogen model has important implications for biofilm development on living, motile surfaces. Here we show that Y. pseudotuberculosis biofilm development on C. elegans is governed by N-acylhomoserine lactone (AHL)-mediated quorum sensing (QS) since (i) AHLs are produced in nematode associated biofilms and (ii) Y. pseudotuberculosis strains expressing an AHL-degrading enzyme or in which the AHL synthase (ypsI and ytbI) or response regulator (ypsR and ytbR) genes have been mutated, are attenuated. Although biofilm formation is also attenuated in Y. pseudotuberculosis strains carrying mutations in the QS-controlled motility regulator genes, flhDC and fliA, and the flagellin export gene, flhA, flagella are not required since fliC mutants form normal biofilms. However, in contrast to the parent and fliC mutant, Yop virulon proteins are up-regulated in flhDC, fliA and flhA mutants in a temperature and calcium independent manner. Similar observations were found for the Y. pseudotuberculosis QS mutants, indicating that the Yop virulon is repressed by QS via the master motility regulator, flhDC. By curing the pYV virulence plasmid from the ypsI/ytbI mutant, by growing YpIII under conditions permissive for type III needle formation but not Yop secretion and by mutating the type III secretion apparatus gene, yscJ, we show that biofilm formation can be restored in flhDC and ypsI/ytbI mutants. These data demonstrate that type III secretion blocks biofilm formation and is reciprocally regulated with motility via QS.

Figure 1. Y. pseudotuberculosis QS mutants are attenuated for biofilm formation on C. elegans.

(A) Confocal image showing C. elegans heavily infected with Y. pseudotuberculosis YpIII embedded in a biofilm ECM which surrounds the anterior end of C. elegans and is spreading to other areas of the worm surface. Green, Gfp-labelled Y. pseudotuberculosis red, WGA-R binding to the ECM yellow, red and green overlay. (B) Confocal image of a Y. pseudotuberculosis YpIII biofilm on C. elegans after 48 h in which only the outer surface of the ECM stains with WGA-R which no longer penetrates deep into the biofilm. (C) C. elegans infected with the Y. pseudotuberculosis ypsI/ytbI double mutant.

Figure 2. Y. pseudotuberculosis biofilm ECM on C. elegans contains extracellular DNA.

(A) The ECM fluoresces blue when stained with DAPI consistent with the presence of extracellular DNA. (B), (C) and (D) show the same image labelled with WGA-R (B; red), Gfp-labelled YpIII (C; green) and an overlay image (D) of the three fluorescent labels.

Figure 3. AHLs are produced in Y. pseudotuberculosis YpIII biofilms on C. elegans.

(A) C. violaceum AHL plate assay showing that AHLs are present in a Y. pseudotuberculosis biofilm growing on C. elegans. (i) Y. pseudotuberculosis YpIII biofilm extract harvested from C. elegans; (ii) extract from nematodes grown on E. coli OP50 (iii) cell pellet extract from an overnight liquid culture of Y. pseudotuberculosis and (iv) extract of an overnight liquid culture of Y. pseudotuberculosis YpIII. The AHL levels collected from the biofilm appear to be present at lower levels than in the culture supernatant. (B) Confocal image showing Y. pseudotuberculosis YpIII transformed with the AHL reporter, pJBA89 fluorescing green in response to AHLs in the biofilm. Red and yellow represent WGA-R stain of the ECM and the overlay of red and green respectively.

Figure 4. QS controls Y. pseudotuberculosis biofilm formation on C. elegans.

Biofilm severity as a measurement of biofilm formation by Y. pseudotuberculosis YpIII, transformed with the vector pSU18 or expressing the AHL lactonase AiiA on plasmid pSA236 (A) and for the ypsI/ytbI mutant (B) and complemented ypsI/ytbI mutant (C).

Figure 5. Aberrant translocation of C. elegans on Y. pseudotuberculosis.

(A) E. coli OP50 and (B) Y. pseudotuberculosis YpIII. Worms infected with either the ypsI/ytbI or ypsR/ytbR mutants translocate normally and make tracks in the agar similar to those seen in (A) and only begin to show signs of aberrant movement comparable with (B), 3–4 h post infection.

Figure 6. Y. pseudotuberculosis strains with mutations in flhDC, fliA or flhA but not fliC are attenuated for biofilm formation.

Biofilm severity indices are shown for flhDC and the complemented flhDC mutant (A), flhA and fliA (B) and fliC (C).

Figure 7. SDS-PAGE protein profiles of cell free supernatants prepared from Y. pseudotuberculosis YpIII parent, flhDC, fliA, flhA and fliC mutants grown at 30°C.

The up-regulated proteins YopN, YopM/H, LcrV, KatY and GroEL were identified by MALDI-TOF MS. Molecular masses of the marker proteins are in kDa.

Figure 8. SDS-PAGE protein profiles of the Y. pseudotuberculosis parent and the QS mutants prepared from cell-free supernatants grown at 30°C.

YopN, YopH/M, LcrV, and FliC were identified by MALDI-TOF MS. Molecular masses of the marker proteins are in kDa.

Figure 9. Impact of pYV and type III secretion on biofilm formation by Y. pseudotuberculosis YpIII on C. elegans.

Biofilm severity indices are shown for (A) YpIII and the ypsI/ytbI mutant with or without pYV and (B) YpIII and the ypsI/ytbI mutant compared with the ypsI/ytbI/yscJ triple mutant and the triple mutant complemented with a plasmid borne copy of yscJ.