Oxygen-limiting conditions enrich for fimbriate cells of uropathogenic Proteus mirabilis and Escherichia coli - PubMed (original) (raw)

Oxygen-limiting conditions enrich for fimbriate cells of uropathogenic Proteus mirabilis and Escherichia coli

M Chelsea Lane et al. J Bacteriol. 2009 Mar.

Abstract

MR/P fimbriae of uropathogenic Proteus mirabilis undergo invertible element-mediated phase variation whereby an individual bacterium switches between expressing fimbriae (phase ON) and not expressing fimbriae (phase OFF). Under different conditions, the percentage of fimbriate bacteria within a population varies and could be dictated by either selection (growth advantage of one phase) or signaling (preferentially converting one phase to the other in response to external signals). Expression of MR/P fimbriae increases in a cell-density dependent manner in vitro and in vivo. However, rather than the increased cell density itself, this increase in fimbrial expression is due to an enrichment of fimbriate bacteria under oxygen limitation resulting from increased cell density. Our data also indicate that the persistence of MR/P fimbriate bacteria under oxygen-limiting conditions is a result of both selection (of MR/P fimbrial phase variants) and signaling (via modulation of expression of the MrpI recombinase). Furthermore, the mrpJ transcriptional regulator encoded within the mrp operon contributes to phase switching. Type 1 fimbriae of Escherichia coli, which are likewise subject to phase variation via an invertible element, also increase in expression during reduced oxygenation. These findings provide evidence to support a mechanism for persistence of fimbriate bacteria under oxygen limitation, which is relevant to disease progression within the oxygen-restricted urinary tract.

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Figures

FIG. 1.

FIG. 1.

Phase variation of P. mirabilis MR/P fimbriae. (A) Differential expression of MR/P fimbriae in 5-ml broth cultures of P. mirabilis. All cultures were standardized to the same OD600, and whole-cell lysates were subjected to the IE assay and Western blot analysis with affinity-purified antibodies against MrpA. (B) Electron micrograph showing the phase variation of MR/P fimbrial expression in a broth culture of P. mirabilis. Immunogold labeling of MrpH, the tip adhesin of MR/P fimbriae, was performed as described previously (35). Note that the top left bacterium is gold labeled, while the top right bacterium is unlabeled. Scale bar, 500 nm. (C) Correlation between MR/P fimbrial expression and bacterial colonization in the bladder. Female CBA/J mice were transurethrally challenged with 5 × 107 CFU of the wild-type P. mirabilis strain HI4320. Seven days after challenge, mice were sacrificed. Bacteria in the bladder were both quantitatively cultured and subjected to the IE assay using the technique described previously (36). A positive correlation was found between MR/P fimbrial expression (percentage of IE in the ON orientation; y axis) and bacterial colonization in the bladder (log10 CFU/g tissue; x_-axis): y = 17_x − 30, _r_2 = 0.9, n = 18, P < 0.0001.

FIG. 2.

FIG. 2.

Correlation between MR/P fimbrial expression and oxygenation. (A) As depicted in the diagram, P. mirabilis was incubated in triplicate as 1-ml, 3-ml, or 5-ml cultures in 14-ml tubes or as 10-ml cultures in 110-ml flasks. All cultures were incubated for 24 h at 37°C with constant agitation (200 rpm). Tubes were placed either tilted (45°) or upright in the incubator. Some 1-ml cultures were placed in microaerobic jars or were overlaid with 3-ml mineral oil to further reduce oxygen in the broth. (B) P. mirabilis was cultured in identical 125-ml flasks under atmospheric oxygen (top panel) or 5.0% oxygen (bottom panel). Samples were collected over time and subjected to the IE assay. Nucleotide size markers are shown on the right side of each panel.

FIG. 3.

FIG. 3.

Confirmation of surface expression of MR/P fimbriae by flow cytometry (A and B) and immunofluorescence (C). P. mirabilis was cultured as described in Fig. 2B. Samples were collected over time and processed for flow cytometry or immunofluorescence. (A) Flow cytometry histograms depicting the number of bacterial cells (y axes) and increasing MrpA expression (or log of Alexa Fluor 633 relative fluorescence intensity; x axes). Blue histograms represent bacteria cultured in atmospheric oxygen, while red histograms represent bacteria cultured in 5.0% oxygen. A representative of three experiments is shown. The percentage of MrpA-positive cells that lie within the indicated gated regions for each bacterial population is depicted in the top right corner of each graph. (B) Bars represent the average (n = 3) surface expression of MrpA for each population. Error bars represent the standard errors of the means. *, P < 0.05. WT, wild type. (C) Immunofluorescence images of HI4320 cultured in atmospheric (atm) or 5.0% oxygen. Bacterial DNA is labeled with Syto9 (green), and MR/P fimbriae are labeled with rabbit anti-MrpA antiserum and anti-rabbit IgG conjugated to Alexa Fluor 594 (red).

FIG. 4.

FIG. 4.

(A) Growth curves of the L-OFF and L-ON mutants of P. mirabilis. Bacteria were cultured at 37°C with constant agitation (200 rpm) in atmospheric oxygen (atm) or 5.0% oxygen. The OD600 was measured over time. Data points are the averages and standard errors of the means from three experiments. (B) In vitro growth competition between the L-ON and the L-OFF mutants. The L-ON and the L-OFF mutants of P. mirabilis were combined in specific ratios to obtain mixtures that contained 0, 0.01, 1, 10, 50, or 100% of the L-ON mutant. The wild-type (wt) strain and the mixtures of the mutants were adjusted to an OD600 of 1, diluted 1:100 into LB, and cultured in broth under atmospheric oxygen (middle panel), or in 5.0% oxygen (bottom panel). The inocula (top panel) and cultures were subjected to the IE assay. Nucleotide size markers are shown on the left.

FIG. 5.

FIG. 5.

MR/P fimbrial expression in isogenic mutants of P. mirabilis. Wild-type (WT) and mutant strains of P. mirabilis were cultured in atmospheric oxygen (atm) or 5.0% oxygen. The switch of the IE was measured. Nucleotide size markers are shown on the left.

FIG. 6.

FIG. 6.

The mrpJ and the dsbA mutants of P. mirabilis strain HI4320. Wild-type (WT) and mutant strains of P. mirabilis were cultured in atmospheric or 5.0% oxygen. The orientation of the IE was measured. Nucleotide size markers are shown on the left.

FIG. 7.

FIG. 7.

Wild-type P. mirabilis strain HI4230, cultured under oxygen-limiting conditions, was used to inoculate Luria broth at concentrations of 5 × 106, 5 × 107, 5 × 108, 1 × 109, 1.5 × 109, 2 × 109, and 2.5 × 109 CFU/ml. Cultures were then incubated under oxygen-limiting conditions. Individual cultures were sampled at indicated time points and subjected to the IE assay.

FIG. 8.

FIG. 8.

Transcriptional activities of mrpA and mrpI as measured by the β-galactosidase activities in mrpAΩlacZ and mrpIΩlacZ merodiploid strains, respectively. The mrpAΩlacZ and mrpIΩlacZ merodipoid strains were cultured in atmospheric oxygen (atm) or 5.0% oxygen for 24 h at 37°C. β-Galactosidase activities were assayed in triplicate; the paired one-tailed t test was used to assess significance. Error bars indicate standard errors of the means. Note the different y axes for mrpA and mrpI.

FIG. 9.

FIG. 9.

Effect of oxygen limitation on expression of type 1 fimbriae in uropathogenic E. coli CFT073. Wild-type CFT073 was cultured in atmospheric or 5.0% oxygen. The inocula and cultures were subjected to the IE assay. Nucleotide size markers are shown on the left side of the panel.

References

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