Campylobacter jejuni biofilms up-regulated in the absence of the stringent response utilize a calcofluor white-reactive polysaccharide

Abstract

The enteric pathogen Campylobacter jejuni is a highly prevalent yet fastidious bacterium. Biofilms and surface polysaccharides participate in stress survival, transmission, and virulence in C. jejuni; thus, the identification and characterization of novel genes involved in each process have important implications for pathogenesis. We found that C. jejuni reacts with calcofluor white (CFW), indicating the presence of surface polysaccharides harboring beta1-3 and/or beta1-4 linkages. CFW reactivity increased with extended growth, under 42 degrees C anaerobic conditions, and in a DeltaspoT mutant defective for the stringent response (SR). Conversely, two newly isolated dim mutants exhibited diminished CFW reactivity as well as growth and serum sensitivity differences from the wild type. Genetic, biochemical, and nuclear magnetic resonance analyses suggested that differences in CFW reactivity between wild-type and DeltaspoT and dim mutant strains were independent of well-characterized lipooligosaccharides, capsular polysaccharides, and N-linked polysaccharides. Targeted deletion of carB downstream of the dim13 mutation also resulted in CFW hyporeactivity, implicating a possible role for carbamoylphosphate synthase in the biosynthesis of this polysaccharide. Correlations between biofilm formation and production of the CFW-reactive polymer were demonstrated by crystal violet staining, scanning electron microscopy, and confocal microscopy, with the C. jejuni DeltaspoT mutant being the first SR mutant in any bacterial species identified as up-regulating biofilms. Together, these results provide new insight into genes and processes important for biofilm formation and polysaccharide production in C. jejuni.

FIG. 1.

CFW fluorescence phenotypes of 81-176 WT and ΔspoT and dim mutants visualized under long-wave UV light. (A and B) CFW reactivity profiles of WT versus the ΔspoT mutant. (A) WT and the ΔspoT mutant were patched onto BHI-CFW plates and grown for 48 h under 37°C microaerobic conditions. (B) Equal OD₆₀₀ equivalents of WT and ΔspoT bacteria were spotted onto BHI-CFW plates and grown for 24 h under 37°C microaerobic conditions and then shifted for 24 h to 42°C anaerobic conditions. (C) Plate growth time course of CFW reactivity and dim mutant profiles compared to those of the WT and the ΔspoT mutant. Equal OD₆₀₀ equivalents of WT and the ΔspoT, dim10, and dim13 mutants were spotted onto BHI-CFW plates and grown for 48, 72, and 96 h under 37°C microaerobic conditions (top three rows) or for 24 h under 37°C microaerobic conditions followed by a 24-h incubation under 42°C anaerobic conditions (48h*). All CFW plate growth and incubation experiments were performed in the dark, and CFW reactivity was visualized by long-wave UV light. All strains were assayed on the same plate, although spot rearrangement was necessary for presentation purposes.

FIG. 2.

Molecular and genetic analyses of dim10 and dim13 mutants. (A to D) Location of dim10 and dim13 Tn inserts and comparison of dim10-related sequences between WT 11168 and 81-176. (A) WT 11168: loci harboring the Cj0967 region (white arrows) and the Cj0500 region (dark gray arrows), based on published sequence data. (B) WT 81-176: two copies of the ∼6-kb Cj0967-Cj0975 locus occur, one downstream of Cj0965c and one between Cj0500 and hemH (Cj0502). Cj0501 is absent in 81-176. A 200-bp 81-176-specific region of intergenic DNA is located upstream of Cj0967 at both loci (black boxes). (C) The dim10 Tn insert (Cm^r flanked by gray boxes) maps within the 200-bp 81-176-specific intergenic region upstream of Cj0967 and downstream of Cj0500. Due to space constraints in panels A to C, two hatched lines (//) were used to represent DNA corresponding to the region between Cj0968 and Cj0975. (D) The dim13 Tn insert (Cm^r flanked by gray boxes) maps upstream of carB (Cj0279) and downstream of Cj0277 in a region conserved between 11168 and 81-176 (hatched gray arrows). (E and F) Forty-eight-hour CFW plate assays on reconstructed dim13 mutant (dim13*) and carB targeted deletion (ΔcarB) strains (37°C microaerobic and 42°C anaerobic analyses) (E) and on dim10 ΔspoT and dim13 ΔspoT double mutants (37°C microaerobic analysis) (F) were performed as described for Fig. 1. Although spot rearrangement for presentation purposes was necessary in panel E, for both panel E and panel F, all appropriate controls were included on the same plates as the test strains.

FIG. 3.

Visual and biochemical investigations into the CFW-reactive polysaccharide by TEM, HR-MAS NMR, Δgne and ΔkpsM CFW assays, and PAGE resolution followed by silver and CFW staining. (A) 81-176 WT and the ΔspoT mutant were grown microaerobically in liquid culture, fixed with glutaraldehyde, negatively stained with uranyl acetate, and visualized using TEM. The arrows point out exaggerated translucent surface material on the ΔspoT mutant and points of agglutination surrounded by the translucent material. (B) HR-MAS NMR spectra of 81-176 WT and ΔspoT, dim10, and dim13 mutants showing the anomeric proton region which highlights the CPS resonances for bacteria grown for 48 h under 37°C microaerobic conditions or for 24 h under 37°C microaerobic conditions and then incubated for 24 h in 42°C anaerobic conditions. (C) CFW reactivity profiles of Δgne and ΔkpsM mutants compared to those for relevant control strains grown on the same plate for 48 h under 37°C microaerobic conditions (as with Fig. 1 and 2, spot rearrangement was necessary for presentation purposes). (D) WT and ΔspoT, dim10, and dim13 mutants were grown under 37°C microaerobic conditions for 48 h, polysaccharides were resolved by 4 to 16% SDS-PAGE (top) and 6 to 16% DOC-PAGE (bottom), and the gels were silver stained. “L” indicates LOS, and “X” indicates an uncharacterized high-MW polysaccharide that migrates just below the stacking gel and near or just above the 250-kDa Bio-Rad Kaleidoscope marker. (E) WT, the ΔspoT, dim10, dim13, Δgne, and ΔkpsM mutants, and a control E. coli DH5α strain were grown as described for panel D, resolved by 4 to 16% (top) or 6 to 16% (middle and bottom) SDS-PAGE, and subjected to CFW staining (top and middle) or silver staining (bottom). Positions of the uncharacterized high-MW polysaccharide (X), running just below the stacking gel, and LOS are shown.

FIG. 4.

Growth and survival of dim mutants in shaking broth culture and in the presence of human serum. (A and B) 81-176 WT (diamonds), the dim10 mutant (triangles), and the dim13 mutant (boxes) were grown in shaking MH broth culture, and OD₆₀₀ (A) and CFU/ml (B) counts were taken at the indicated time points. Error bars are shown but are often too small to see. (C) Survival of WT, the dim10 mutant, and the dim13 mutant in human serum as measured by the ratio of CFU/ml recovered after 60 min in 10% normal human serum versus 10% HK human serum. P < 0.0001 for the dim13 mutant versus the WT. No killing for any strain was observed in HK serum (not shown).

FIG. 5.

CV staining of C. jejuni biofilms. (A) 81-176 WT and ΔspoT, dim10, and dim13 mutants were incubated standing in broth for the indicated times under 37°C microaerobic conditions. Biofilms forming at the air-liquid interface were visualized by staining with 1% CV in 95% ethanol and photographed. (B) Biofilms of strains from panel A, as well as from Δgne and ΔkpsM mutants, were grown in triplicate borosilicate tubes and stained with CV as described for panel A. CV was solubilized in DMSO, intensity was assessed by OD₅₇₀ analyses, and data (with error bars) were graphed.

FIG. 6.

SEM of biofilms formed after 72 h by 81-176 WT and ΔspoT, dim10, and dim13 mutants. 81-176 WT and ΔspoT, dim10, and dim13 mutant biofilms were grown on borosilicate cover glass in standing liquid cultures under 37°C microaerobic conditions for 72 h and visualized by SEM. Bars, 10 μm.

FIG. 7.

CLSM of biofilms formed after 48 h by 81-176 WT and ΔspoT, dim10, and dim13 mutants. 81-176 WT and ΔspoT, dim10, and dim13 mutant biofilms were grown on borosilicate cover glass in standing liquid cultures under 37°C microaerobic conditions for 48 h in the presence of CFW and FM4-64 and visualized by CLSM. As noted at the top of the figure, angled, top-down, and side profiles are shown for each strain.