Requirement of MrpH for mannose-resistant Proteus-like fimbria-mediated hemagglutination by Proteus mirabilis

Abstract

Two new genes, mrpH and mrpJ, were identified downstream of mrpG in the mrp gene cluster encoding mannose-resistant Proteus-like (MR/P) fimbriae of uropathogenic Proteus mirabilis. Since the predicted MrpH has 30% amino acid sequence identity to PapG, the Galalpha(1-4)Gal-binding adhesin of Escherichia coli P fimbriae, we hypothesized that mrpH encodes the functional MR/P hemagglutinin. MR/P fimbriae, expressed in E. coli DH5alpha, conferred on bacteria both the ability to cause mannose-resistant hemagglutination and the ability to aggregate to form pellicles on the broth surface. Both a DeltamrpH mutant expressed in E. coli DH5alpha and an isogenic mrpH::aphA mutant of P. mirabilis were unable to produce normal MR/P fimbriae efficiently, suggesting that MrpH was involved in fimbrial assembly. Amino acid residue substitution of the N-terminal cysteine residues (C66S and C128S) of MrpH abolished the receptor-binding activity (hemagglutinating ability) of MrpH but allowed normal fimbrial assembly, supporting the notion that MrpH was the functional MR/P hemagglutinin. Immunogold electron microscopy of P. mirabilis HI4320 revealed that MrpH was located at the tip of MR/P fimbriae, also consistent with its role in receptor binding. The isogenic mrpH::aphA mutant of HI4320 was less able to colonize the urine, bladder, and kidneys in a mouse model of ascending urinary tract infection (P < 0.01), and therefore MR/P fimbriae contribute significantly to bacterial colonization in mice. While there are similarities between P. mirabilis MR/P and E. coli P fimbriae, there are more notable differences: (i) synthesis of the MrpH adhesin is required to initiate fimbrial assembly, (ii) MR/P fimbriae confer an aggregation phenotype, (iii) site-directed mutation of specific residues can abolish receptor binding but allows fimbrial assembly, and (iv) mutation of the adhesin gene abolishes virulence in a mouse model of ascending urinary tract infection.

FIG. 1

Newly acquired 3′-end sequence of the mrp gene cluster. Predicted polypeptides are translated below the nucleotide sequence. Letters representing amino acid residues are aligned with the first nucleotide of their corresponding codons. Asterisks, aligned with the first nucleotide of the stop codons, mark the end of polypeptides. Gene product designations are given in the right margin. Numbers in the right margin refer to the nucleotide sequence. The downward arrow indicates where the new sequence begins. The pair of inverted arrows and the double line underlie the putative rho-independent terminator.

FIG. 1

Newly acquired 3′-end sequence of the mrp gene cluster. Predicted polypeptides are translated below the nucleotide sequence. Letters representing amino acid residues are aligned with the first nucleotide of their corresponding codons. Asterisks, aligned with the first nucleotide of the stop codons, mark the end of polypeptides. Gene product designations are given in the right margin. Numbers in the right margin refer to the nucleotide sequence. The downward arrow indicates where the new sequence begins. The pair of inverted arrows and the double line underlie the putative rho-independent terminator.

FIG. 2

Alignment of amino acid sequences of putative pilins of the MR/P fimbria. The amino acid sequences of the six putative pilins of the MR/P fimbria were aligned by using the GCG Pileup software. Gaps (.) were introduced to obtain maximal fit. The amino acid residues that are conserved in three or more of the pilins are noted at the bottom of the alignment. The amino acid residues that are conserved in all six pilins are highlighted in black boxes. ∗, the valine residue and the isoleucine residue are both conserved in three of the six pilins. Aligned with the other putative MR/P pilins, MrpH is the only one that contains a distinctive N-terminal domain that could provide a possible basis for the receptor-binding activity.

FIG. 3

Alignment of the amino acid sequence of putative MR/P adhesin with two other adhesins, SmfG of S. marcescens and PapG of E. coli. The amino acid sequences of MrpH, SmfG, and PapG were aligned by using the GCG Pileup software. Gaps (.) were introduced to obtain maximal fit. The amino acid residues that are conserved in two or more of the proteins are noted at the bottom of the alignment. Amino acid residues that are conserved in all three are highlighted in black boxes.

FIG. 4

Southern blot analysis of mrpH::aphA mutants. The predicted restriction map of the wild-type strain and mrpH::aphA mutant is illustrated in the top panel. The arrows represent predicted open reading frames within the defined region. The sizes of the bands that hybridized with the probes in the Southern blots are indicated along the left side.

FIG. 5

Bacterial aggregation and MRHA patterns of the ΔmrpH mutant expressed in E. coli. (A) Pictures of 72-h static cultures of E. coli DH5α containing various constructs grown in 5 ml of Luria-Bertani broth at 37°C. The tubes were handled carefully to avoid disrupting the pellicle. (B) MRHA assay of the bacterial cultures in panel A were performed as described in Materials and Methods.

FIG. 6

Western blot of partially purified fimbrial preparations of various E. coli DH5α strains. Partially purified fimbrial preparations (see Materials and Methods for details) were separated on an SDS–12.5% polyacrylamide gel and subjected to Western blot analysis with polyclonal rabbit antiserum raised against MrpA.

FIG. 7

Immunogold electron micrograph of P. mirabilis HI4320. P. mirabilis HI4320 grown under conditions optimal for fimbrial production (see Materials and Methods) was reacted first with antiserum raised against MrpH and then with a secondary antibody (goat anti-rabbit IgG) conjugated to 30-nm-diameter gold particles. Bar, 200 nm. It is clearly shown that MrpH, targeted by the 30-nm-diameter gold particle, is located at the tip of bacterial fimbriae.

FIG. 8

Immunogold electron micrographs of P. mirabilis HI4320. P. mirabilis HI4320 was reacted first with rabbit antiserum raised against MrpH and then with a secondary antibody (goat anti-rabbit IgG) conjugated to 30-nm-diameter gold particles. (A and B) Bacteria were then reacted with antiserum raised against MR/P fimbriae followed by a secondary antibody (recombinant protein G) conjugated to 5-nm-diameter gold particles. (C and D) Bacteria were then reacted with antiserum raised against MrpA followed by a secondary antibody (recombinant protein G) conjugated to 5-nm-diameter gold particles. Bars, 200 nm. The antiserum raised against MR/P fimbriae, as well as the antiserum raised against MrpA, labeled the shafts of the MR/P fimbriae. While the antiserum raised against MR/P fimbriae resulted in more extensive labeling, the antiserum raised against MrpA gave a cleaner background. The 5-nm-diameter gold particles that labeled the shafts of MR/P fimbriae are difficult to resolve but make the MR/P fimbriae appear thicker. The arrow in panel B indicates MR/P fimbria tips that may have been sheared off during preparation.

FIG. 9

Assessment of the virulence of the mrpH::aphA mutant of HI4320 in the CBA mouse model of ascending UTIs. (A) Independent-challenge experiment. A group of 12 mice and a group of 10 mice were transurethrally challenged with 4.8 × 10⁶ CFU of the wild-type strain per mouse and 5.5 × 10⁶ CFU of the mrpH::aphA mutant per mouse, respectively. (B) Cochallenge experiment. A group of 10 mice were transurethrally challenged with a roughly 1:1 mixture of the wild-type strain and the mrpH::aphA mutant (7.9 × 10⁶ CFU of the wild-type strain and 1.2 × 10⁷ CFU of the mrpH::aphA mutant per each mouse). After 7 days, the mice were sacrificed and the quantitative bacterial counts in the urine, bladders, and kidneys were calculated (see Materials and Methods). Each diamond represents the CFU per milliliter of urine or CFU per gram of tissue from an individual mouse. Horizontal bars represent the geometric means of the colony counts. The range of detection in this assay is 10² to 10⁹ CFU/ml of urine or CFU/g of tissue. Values of ≤10² were set to 10², and values of ≥10⁹ were set to 10⁹. P values (bottom) were derived by using an unpaired, one-tailed Mann-Whitney test. Lf., left; Rt., right. WT, wild-type strain; mrpH, mrpH::aphA mutant.