Proteus mirabilis pmrI, an RppA-regulated gene necessary for polymyxin B resistance, biofilm formation, and urothelial cell invasion

Abstract

Proteus mirabilis is naturally resistant to polymyxin B (PB). To investigate the underlying mechanisms, Tn5 mutagenesis was performed, and a mutant exhibiting increased PB susceptibility was isolated. The mutant was found to have Tn5 inserted into the PpmrI (Proteus pmrI) gene, a gene which may encode a UDP-glucuronic acid decarboxylase. In other bacteria, pmrI belongs to the seven-gene pmrF operon, which is involved in lipopolysaccharide (LPS) modification. While the PpmrI knockout mutant had a wild-type LPS profile and produced amounts of LPS similar to those produced by the wild type, LPS of the knockout mutant had higher PB-binding activity than that of the wild type. PB could induce alterations of LPS in the wild type but not in the PpmrI knockout mutant. Moreover, the PpmrI knockout mutant exhibited decreased abilities in biofilm formation and urothelial cell invasion. Complementation of the PpmrI mutant with the full-length PpmrI gene led to restoration of the wild-type phenotypic traits. Previously we identified RppA, a response regulator of the bacterial two-component system, as a regulator of PB susceptibility and virulence factor expression in P. mirabilis. Here we showed that RppA could mediate the induction of PpmrI expression by PB. An electrophoretic mobility shift assay further demonstrated that RppA could bind directly to the putative PpmrI promoter. Together, these results provide a new insight into the regulatory mechanism underlying PB resistance and virulence expression in P. mirabilis.

FIG. 1.

(A) LPS profiles of wild-type P. mirabilis (N2) and the PpmrI knockout mutant (dIp) in the presence or absence of 1 μg/ml PB. Six microliters of LPS purified from the same cell number (OD₆₀₀ × volume [in ml] = 100) of the respective strains was subjected to SDS-PAGE analysis. (B) PB-binding ability of LPS purified from wild-type P. mirabilis (N2) and the PpmrI knockout mutant (dIp). Various amounts of purified LPS were subjected to the PB-binding assay. The unbound PB was then subjected to the E. coli inhibition assay (see Materials and Methods). The data are the averages of results from three independent experiments with standard deviations.

FIG. 2.

(A) Effect of rppA mutation on the expression of PpmrI mRNA in the presence or absence of PB (1 μg/ml). The amounts of PpmrI mRNA for the wild-type, rppA mutant, and RppA-complemented strains were quantified by real-time PCR after a 3.5-h incubation. The value obtained with the wild-type cells in the absence of PB was set at 1. The data represent the averages of results of four independent experiments with standard deviations. (B) The activity of XylE in the PpmrI-xylE reporter plasmid-transformed wild-type, rppA mutant, and RppA-complemented strains in the presence or absence of 1 μg/ml PB after a 3.5-h incubation. The data represent the averages of results of three independent experiments with standard deviations. N2, wild type; dA10, rppA mutant; dA10c, RppA-complemented strain.

FIG. 3.

Electrophoretic mobility shift assay with purified His-tagged RppA and PpmrI promoter DNA. DNA fragments (0.1 μg) containing either the PpmrI promoter region (372 bp) or unrelated DNA (330 bp) were incubated with the indicated concentrations (0, 1, or 2 μM) of the His-tagged RppA protein. After protein-DNA complex formation, the fragments were resolved on a 5% nondenaturing polyacrylamide gel. The supershift assay (lane 7) was performed using 0.3 μl antihistidine antibody. The lower arrow indicates the position of the protein-DNA complex, and the upper arrow indicates the position of the supershifted band. MW, marker; lanes 1 to 3, PpmrI promoter DNA with His-tagged RppA; lanes 4 to 6, negative control DNA with His-tagged RppA.

FIG. 4.

(A) Effect of PpmrI mutation on the biofilm-forming ability of P. mirabilis. Biofilm-forming abilities of the wild-type (N2), the PpmrI knockout mutant (dIp), and the PPmrI-complemented strain (dIpc) of P. mirabilis were determined as described in Materials and Methods. The value obtained with wild-type cells was set at 1. The data represent the averages of results of three independent experiments with standard deviations. A significant difference between the biofilm-forming ability of the wild-type strain and that of the mutant was observed by Student's t-test analysis (*, P < 0.05). (B) Effect of PpmrI mutation on the cell invasion ability of P. mirabilis. Invasion abilities of the wild-type (N2), the PpmrI knockout mutant (dIp), and the PPmrI-complemented strain (dIpc) of P. mirabilis were determined as described in Materials and Methods. The cell invasion ability of wild-type cells was set at 1. The data represent the averages of results from three independent experiments with standard deviations. A significant difference between the invasion ability of the wild-type strain and that of the mutant was observed by Student's t-test analysis (*, P < 0.001).