Construction of an MR/P fimbrial mutant of Proteus mirabilis: role in virulence in a mouse model of ascending urinary tract infection

Abstract

Proteus mirabilis, a cause of acute pyelonephritis, produces at least four types of fimbriae, including MR/P (mannose-resistant/Proteus-like) fimbriae. To investigate the contribution of MR/P fimbriae to colonization of the urinary tract, we constructed an MR/P fimbrial mutant by allelic exchange. A 4.2-kb BamHI fragment carrying the mrpA gene was subcloned into a mobilizable plasmid, pSUP202. A 1.3-kb Kanr cassette was inserted into the mrpA open reading frame, and the construct was transferred to the parent P. mirabilis strain by conjugation. Following passage on nonselective medium, 1 of 500 transconjugants screened was found to have undergone allelic exchange as demonstrated by Southern blot. Colony immunoblot, Western immunoblot, and immunogold labeling with a monoclonal antibody to MR/P fimbriae revealed that MrpA was not expressed. Complementation with cloned mrpA restored MR/P expression as shown by hemagglutination, Western blot, and immunogold electron microscopy. To assess virulence, we challenged 40 CBA mice transurethrally with 10(7) CFU of wild-type or mutant strains. After 1 week, geometric means of log10 CFU per milliliter of urine or per gram of bladder or kidney for the wild-type and mutant strains were as follows: urine, 7.79 (wild type) versus 7.02 (mutant) (P = 0.035); bladder, 6.22 versus 4.78 (P = 0.019); left kidney, 5.02 versus 3.31 (P = 0.009); and right kidney, 5.28 versus 4.46 (P = 0.039). Mice challenged with the wild-type strain showed significantly more severe renal damage than did mice challenged with the MR/P-negative mutant (P = 0.007). We conclude that MR/P fimbriae contribute significantly to colonization of the urinary tract and increase the risk of development of acute pyelonephritis.

Fimbriae of uropathogenicProteus mirabilis

“…The PMF were isolated and their operon nucleotide sequence was determined by Massad & Mobley (1994). The genetic organization of the pmf operon revealed six ORFs with five predicted polypeptides: PmfA (18.9 kDa), the major fimbrial subunit; PmfC (93.1 kDa), the usher; PmfD (28.2 kDa), the chaperone; PmfE (38.9 kDa), the minor fimbrial subunit; and PmfF (19.6 kDa), which is the adhesin.…”

Repression of motility during fimbrial expression: identification of 14 mrpJ gene paralogues in Proteus mirabilis

“…These swarmer cells also exhibit reduced expression of fimbriae (Mobley et al, 1996;Jansen et al, 2003), suggesting possible co-ordinate expression between flagella and fimbriae. Prior to the sequencing of the P. mirabilis genome, expression of five different fimbriae had been documented in P. mirabilis: MR/P fimbriae (Adegbola et al, 1983;Bahrani et al, 1994), uroepithelial cell adhesin (UCA, also known as non-agglutinating fimbriae or NAF) (Wray et al, 1986;Cook et al, 1995), P. mirabilis fimbriae (PMF) (Adegbola et al, 1983;Massad et al, 1994a), ambient temperature fimbriae (ATF) (Massad et al, 1994b) and P. mirabilis P-like pili (PMP) (Bijlsma et al, 1995). The MR/P fimbria is encoded by an operon containing nine genes (Bahrani et al, 1994).…”

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

“…Several potential virulence factors have been suggested for P. mirabilis, including adherence to the uroepithelium mediated by ¢mbriae [3,4], urease production [5], invasion of eucaryotic cells [6], cleavage of IgG and IgA by a proteolytic enzyme [7], haemolysin production [8], and swarming motility dependent on £agella [9].…”

“…Amongst di¡erent types of P. mirabilis ¢mbriae that have been described, the mannose-resistant Proteus-like ¢mbriae (MR/P) have been proposed as important for virulence in UTI [3]. MR/P are expressed in vivo and an in vivo phase variation mechanism has been described [10].…”

“…The genes encoding MR/P ¢mbriae form a complex ¢mbrial operon [11]. P. mirabilis mutants in a number of these genes have been generated and used in infection studies that show that MR/P ¢mbriae play a role in P. mirabilis UTI pathogenesis [3,12,13]. However, the precise roles of the di¡erent ¢mbriae as well as other non-¢mbrial adhesins in P. mirabilis UTI pathogenesis have not yet been elucidated.…”