New aspects of the role of MR/P fimbriae in Proteus mirabilis urinary tract infection

Proteus mirabilis, a common cause of urinary tract infection (UTI), produces a number of different fimbriae including mannose-resistant Proteus-like fimbriae (MR/P). The precise role of different P. mirabilis fimbriae in ascending UTI has not yet been elucidated. In this study, a clinical isolate of P. mirabilis and an isogenic mutant unable to express MR/P were tested using different experimental approaches. They were tested for their ability to cause infection in an ascending co-infection model of UTI and in a haematogenous model in the mouse. In both models, the mutant was less able than the wild-type strain to colonise the lower and upper urinary tracts although infectivity was not abolished. In vitro adherence to uroepithelial cells was also assessed. Significant differences in adherence between both strains were observed at 1 h but not at 15 min post infection. We have also shown that a wild-type strain carries two copies of the mrpA gene. These data reinforce the importance of MR/P fimbriae in P. mirabilis UTI although other virulence factors may be necessary for efficient colonisation and development of infection.     

Proteus mirabilis MR/P fimbriae: molecular cloning, expression, and nucleotide sequence of the major fimbrial subunit gene.

Proteus mirabilis, a cause of serious urinary tract infection and acute pyelonephritis, produces several putative virulence determinants, among them, fimbriae. Principally, two fimbrial types are produced by this species: mannose-resistant/Proteus-like (MR/P) fimbriae and mannose-resistant/Klebsiella-like (MR/K) fimbriae. To isolate MR/P fimbrial gene sequences, a P. mirabilis cosmid library was screened by immunoblotting and by hybridization with an oligonucleotide probe based on the N-terminal amino acid sequence of the isolated fimbrial polypeptide, ADQGHGTVKFVGSIIDAPCS. One clone, pMRP101, reacted strongly with a monoclonal antibody specific for MR/P fimbriae and with the DNA probe. This clone hemagglutinated both tannic acid-treated and untreated chicken erythrocytes with or without 50 mM D-mannose and was shown to be fimbriated by transmission electron microscopy. A 525-bp open reading frame, designated mrpA, predicted a 175-amino-acid polypeptide including a 23-amino-acid hydrophobic leader peptide. The unprocessed and processed polypeptides are predicted to be 17,909 and 15,689 Da, respectively. The N-terminal amino acid sequence of the processed fimbrial subunit exactly matched amino acid residues 24 to 43 predicted by the mrpA nucleotide sequence. The MrpA polypeptide shares 57% amino acid sequence identity with SmfA, the major fimbrial subunit of Serratia marcescens mannose-resistant fimbriae.     

Evaluation of Proteus mirabilis structural fimbrial proteins as antigens against urinary tract infections

Proteus mirabilis is a common cause of urinary tract infection (UTI) and produce several types of different fimbriae, including mannose-resistant/Proteus-like fimbriae, uroepithelial cell adhesin (UCA), and P. mirabilis fimbriae (PMF). Different authors have related these fimbriae with different aspects of P. mirabilis pathogenesis, although the precise role of fimbriae in UTI has not yet been elucidated. In this work we expressed and purified recombinant structural fimbrial proteins of these fimbriae (MrpA, UcaA, and PmfA) and assessed their role as protective antigens using an ascending and a haematogenous model of UTI in the mouse. MrpA protected subcutaneously immunised mice in both models, suggesting that it could be taken into account as a promising vaccine candidate against P. mirabilis UTI. UcaA could also be an interesting subunit to be studied although it only protected mice that were challenged intravenously. All subunits elicited a strong specific serum IgG response but there was no significant correlation between antibody levels and protection. Only PmfA-immunised mice elicited a significant urinary antibody response but this protein was unable to confer protection against P. mirabilis experimental challenges. These results may contribute to the development of vaccines against P. mirabilis, an important cause of complicated UTI.     

Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression.

Proteus mirabilis, an agent of urinary tract infection, expresses at least four fimbrial types. Among these are the MR/P (mannose-resistant/Proteus-like) fimbriae. MrpA, the structural subunit, is optimally expressed at 37 degrees C in Luria broth cultured statically for 48 h by each of seven strains examined. Genes encoding this fimbria were isolated, and the complete nucleotide sequence was determined. The mrp gene cluster encoded by 7,293 bp predicts eight polypeptides: MrpI (22,133 Da), MrpA (17,909 Da), MrpB (19,632 Da), MrpC (96,823 Da), MrpD (27,886 Da), MrpE (19,470 Da), MrpF (17,363 Da), and MrpG (13,169 Da). mrpI is upstream of the gene encoding the major structural subunit gene mrpA and is transcribed in the direction opposite to that of the rest of the operon. All predicted polypeptides share > or = 25% amino acid identity with at least one other enteric fimbrial gene product encoded by the pap, fim, smf, fan, or mrk gene clusters.     

Pathogenesis of Proteus mirabilis urinary tract infection

Proteus mirabilis is a causative agent of cystitis and pyelonephritis primarily in individuals with indwelling catheters or structural abnormalities of the urinary tract. The organism produces a variety of unique virulence factors that contribute to its pathogenicity and persistence in the human host.     

Identity gene expression in Proteus mirabilis

Swarming colonies of independent Proteus mirabilis isolates recognize each other as foreign and do not merge together, whereas apposing swarms of clonal isolates merge with each other. Swarms of mutants with deletions in the ids gene cluster do not merge with their parent. Thus, ids genes are involved in the ability of P. mirabilis to distinguish self from nonself. Here we have characterized expression of the ids genes. We show that idsABCDEF genes are transcribed as an operon, and we define the promoter region upstream of idsA by deletion analysis. Expression of the ids operon increased in late logarithmic and early stationary phases and appeared to be bistable. Approaching swarms of nonself populations led to increased ids expression and increased the abundance of ids-expressing cells in the bimodal population. This information on ids gene expression provides a foundation for further understanding the molecular details of self-nonself discrimination in P. mirabilis.     

Identification of MrpI as the sole recombinase that regulates the phase variation of MR/P fimbria,a bladder colonization factor of uropathogenic Proteus mirabilis

Proteus mirabilis is a common cause of urinary tract infection (UTI) in individuals with structural abnormalities or long-term catheterization. The expression of mannose-resistant/Proteus-like (MR/P) fimbria is phase variable because of the inversion of a 251 bp DNA fragment that carries the promoter for the mrp operon. Previous studies have shown that mrpI, which is transcribed divergently from the mrp operon, encodes a recombinase capable of switching the orientation of this invertible element. In this study, we constructed isogenic mrpI null mutants from a clinical isolate of P. mirabilis, HI4320. A polymerase chain reaction (PCR)-based invertible element assay revealed that the isogenic mrpI null mutants were locked in one phase, either expressing (locked on) MR/P fimbriae or not (locked off), which indicated that MrpI was the sole recombinase that regulated the phase variation of MR/P fimbria. The locked-on and locked-off mutants were evaluated for virulence in the CBA mouse model of ascending UTI by co-challenges with each other and with the wild-type strain. Results from these experiments demonstrated conclusively that the MR/P fimbria was a critical bladder colonization factor of uropathogenic P. mirabilis and also suggested that the ability to switch off the expression of MR/P fimbria might be important for kidney colonization.     

Differential Expression of Nonagglutinating Fimbriae and MR/P Pili in Swarming Colonies of Proteus mirabilis

The expression of nonagglutinating fimbriae (NAF) and mannose-resistant/Proteus-like (MR/P) pili in swarming colonies of Proteus mirabilis was investigated. Elongated swarmer cells do not express pili, and the relative number of bacteria expressing NAF during swarming and early consolidation phases was very low (<5%). Relative expression of NAF in a terrace increased to approximately 30% at 48 h. We also determined the expression of NAF and MR/P pili in two phenotypically distinguishable regions of each terrace. The expression of both NAF and MR/P pili was always higher in the region closer (proximal) to the middle of the colony than in the distal region of the terrace. The relative numbers of bacteria expressing NAF or MR/P pili in the proximal region were between 39.1 and 63% and between 5.9 and 7.7%, respectively. In the distal region, expression levels were between 20.8 and 27.3% and between 3.7 and 5. 6%, respectively. A time course experiment testing NAF expression in both the proximal and distal regions of a terrace indicated that NAF expression in the proximal regions was always higher than in the distal regions and increased to a plateau 40 to 50 h after the start of the swarming phase for any given terrace. These results indicate that expression of NAF or MR/P pili in swarming colonies of P. mirabilis is highly organized, spatially and temporally. The significance of this controlled differentiation remains to be uncovered.     

Native flagellin does not protect mice against an experimental Proteus mirabilis ascending urinary tract infection and neutralizes the protective effect of MrpA fimbrial protein

Proteus mirabilis expresses several virulence factors including MR/P fimbriae and flagella. Bacterial flagellin has frequently shown interesting adjuvant and protective properties in vaccine formulations. However, native P. mirabilis flagellin has not been analyzed so far. Native P. mirabilis flagellin was evaluated as a protective antigen and as an adjuvant in co-immunizations with MrpA (structural subunit of MR/P fimbriae) using an ascending UTI model in the mouse. Four groups of mice were intranasally treated with either MrpA, native flagellin, both proteins and PBS. Urine and blood samples were collected before and after immunization for specific antibodies determination. Cytokine production was assessed in immunized mice splenocytes cultures. Mice were challenged with P. mirabilis, and bacteria quantified in kidneys and bladders. MrpA immunization induced serum and urine specific anti-MrpA antibodies while MrpA coadministered with native flagellin did not. None of the animals developed significant anti-flagellin antibodies. Only MrpA-immunized mice showed a significant decrease of P. mirabilis in bladders and kidneys. Instead, infection levels in MrpA-flagellin or flagellin-treated mice showed no significant differences with the control group. IL-10 was significantly induced in splenocytes of mice that received native flagellin or MrpA-flagellin. Native P. mirabilis flagellin did not protect mice against an ascending UTI. Moreover, it showed an immunomodulatory effect, neutralizing the protective role of MrpA. P. mirabilis flagellin exhibits particular immunological properties compared to other bacterial flagellins.     

Drug resistance and proticinogenic types of Proteus mirabilis isolated from urinary tract infections

Proteus mirabilis strains (88 isolates) from hospitalised patients with urinary tract infection were tested for antibiotic susceptibility, ESBL production and their ability to produce proticin or on their susceptibility to proticin. Antibiotic susceptibility test was performed by standard disc diffusion method according to NCCLS. Proticin typing was made by the standard strain set from the B. W. Senior collection. Most (59%) strains belonged to ESBL producers and were more resistant to antibiotics than ESBL-negative strains. Predominant proticin patterns among the ESBL (+) strains were: P1,2(6)/SO (23%), P1,2/SO (13.5%). Among the ESBL-negative strains more frequent were P6/SO (16.6%), P1,2/SO (13.8%) and P3,6/SO (13.8%) proticin types.     

Effects of the administration of cholera toxin as a mucosal adjuvant on the immune and protective response induced by Proteus mirabilis MrpA fimbrial protein in the urinary tract

Proteus mirabilis is commonly associated with complicated UTI and expresses several virulence factors, including MR/P fimbriae. In the present study mice were immunised nasally with MrpA, the structural subunit of MR/P, with or without CT as a mucosal adjuvant. The animals were then challenged with P. mirabilis and induction of specific serum and urine IgG and IgA, IFN-γ production and bacterial kidney and bladder colonization were assessed. MrpA-immunised mice exhibited significant induction of serum IgA and urine IgA and IgG. MrpA/CT-immunised mice showed both significant serum and urine IgA and IgG production. Only this group showed significant IFN-γ production. Both groups of animals had significant decrease in bacterial colonization of kidneys but not of bladders. No correlation between specific antibody induction in serum and CFU decrease was observed in any group of animals. Our results suggest that a mucosal adjuvant (CT) in the urinary tract enhanced humoral and cytokine response although it did not influence the degree of protection against UTI provided by MrpA. Further studies are necessary to understand immune modulation in the urinary tract.     

Mannose-resistant Proteus-like and P. mirabilis fimbriae have specific and additive roles in P. mirabilis urinary tract infections

Proteus mirabilis is an important uropathogen that can cause complicated urinary tract infections (UTI). It produces several types of fimbriae, including mannose-resistant Proteus-like (MR/P) fimbriae and P. mirabilis fimbriae (PMF). Previously, we determined that these fimbriae affect the ability of P. mirabilis to colonize the urinary tract. The objective of this study was to analyse the effect of the simultaneous lack of P. mirabilis MR/P and PMF fimbriae in UTI pathogenesis. A double mutant lacking both fimbriae was generated by allelic replacement mutagenesis. This mutant was characterized genetically and phenotypically, and tested using an in vitro uroepithelial cell adhesion assay and the ascending UTI murine model. In vitro adhesion to uroepithelial cells by the P. mirabilis pmfA/mrpA-D mutant was reduced when compared with the wild-type, although no significant differences were observed when it was compared with the single mrpA-D and pmfA mutants. However, in vivo assays showed that colonization of kidneys and bladders by the P. mirabilis pmfA/mrpA-D mutant was significantly reduced when compared with the wild-type and both single mutants. These results indicate that, although redundancy can occur, MR/P and PMF fimbriae have specific and additive roles in P. mirabilis UTI.