Proteus, Providencia, and Morganella species produce deaminases that generate ␣-keto acids from amino acids. The ␣-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5␣ for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.Among the most common human infections are those of the urinary tract. After Escherichia coli, Proteus mirabilis is a frequent etiological agent particularly in catheterized patients or individuals with structural abnormalities of the urinary tract (46). P. mirabilis appears to localize in the kidney in higher numbers than other uropathogens (13, 43), and there it can cause serious complications including acute pyelonephritis, bladder and kidney stones, and bacteremia (36, 40). Testing of isogenic mutants in a CBA mouse model of ascending urinary tract infection has demonstrated that urease (18, 19), mannose-resistant/Proteus-like fimbriae (3-5), and P. mirabilis fimbriae (25) contribute significantly to the ability of this organism to establish infection. Other putative virulence determinants include hemolysin (28,32,33,44), flagella associated with swarming motility (6, 31), a uroepithelial cell adhesin (47), ambient temperature fimbriae (24), and an immunoglobulin A-degrading protease (38, 39).The role of iron in infections has been well established (2,7,8,48), and most bacterial species produce phenolate-and/or hydroxamate-type siderophores to compete with the host for iron (7,8,30). Most genera of the family Enterobacteriaceae produce either enterobactin (a phenolate siderophore) or aerobactin (a mixed phenolate-hydroxamate siderophore) (7, 30). These traditional-type siderophores are biosynthesized by the action of a number of enzymes whose genes exist in an operon (30). Despite evidence that the urinary tract is an iron-limited niche (41) and that iron limitation reduces susceptibility of animals to developme...
The mesophilic Aeromonas species are opportunistic pathogens that produce either of the siderophores amonabactin or enterobactin. Acquisition of iron for growth from Fe-transferrin in serum was dependent on the siderophore amonabactin; 50 of 54 amonabactin-producing isolates grew in heat-inactivated serum, whereas none of 30 enterobactin-producing strains were able to grow. Most isolates (regardless of siderophore produced) used haem as a sole source of iron for growth; all of 33 isolates grew with either haematin or haemoglobin and 30 of these used haemoglobin when complexed to human haptoglobin. Mutants unable to synthesize a siderophore used iron from haem, suggesting that this capacity was unrelated to siderophore production. Some members of the mesophilic Aeromonas species have evolved both siderophore-dependent and -independent mechanisms for acquisition of iron from a host.
Thiosulfate-citrate-bile salts-sucrose agar has been routinely used for the isolation of pathogenic vibrios, although its selectivity for Vibrio cholerae and Vibrio vulnificus is inadequate. Therefore, a new plating medium, cellobiose-polymyxin B-colistin agar, was developed for the isolation of these two species. CeMobiosepolymyxin B-colistin agar demonstrated a significant advantage over other media designed for the isolation or differentiation of vibrios: of both the 136 strains representing 19 Vib-rio species and the marine isolates of the genera Pseudomonas, Flavobacterium, and Photobacterium, only V. vulnificus and V. cholerae were able to grow. Furthermore, the fermentation of cellobiose by V. vulnificus aNowed for the easy differentiation of these two species. This medium offers significant potential as a selective and differential medium for these two pathogenic vibrios. Of the 10 human pathogenic vibrios that have been described (4), Vibrio vulnificus and Vibrio cholerae have received the most attention in recent years (11). While V. cholerae has long been of concern as the etiologic agent of cholera, research on its distribution and ecology in the United States has been stimulated by the finding of both 01 and non-O1 strains of this species in coastal waters (3, 5, 18). V. vulnificus in contaminated seafood can cause lifethreatening wound infections and septicemias (1, 16). V. vulnificus also occurs naturally in marine and estuarine environments along much of the United States (7, 12, 14). While thiosulfate-citrate-bile salts-sucrose (TCBS) agar has been routinely used for the isolation of pathogenic vibrios (6, 7, 12, 14, 19), its selectivity for this genus has been questioned (9, 15, 19). While TCBS agar has been judged in several studies to be the best isolation medium currently available for pathogenic Vibrio spp.
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.
Urinary tract infections involving Proteus mirabilis may lead to complications including bladder and kidney stones, acute pyelonephritis, and bacteremia. This bacterium produces a number of fimbriae, two of which, MR/P fimbria and P. mirabilis fimbria, have been shown to contribute to the ability of this pathogen to colonize the bladder and kidney. We have now purified and characterized a previously undescribed fimbria of P. mirabilis, named ambient-temperature fimbria (ATF). Electron microscopy of a pure preparation and immunogold labeling of cells demonstrated that ATF was fimbrial in nature. The major fimbrial subunit of ATF has an apparent molecular weight of 24,000. The N-terminal amino acid sequence, E-X-T-G-T-P-A-P-T-E-V-T-V-D-G-G-T-I-D-F, did not show significant similarity to that of any previously described fimbrial protein. ATF was expressed by all eight P. mirabilis strains examined. Culture conditions affected expression of ATF, with optimal expression observed in static broth cultures at 23 degrees C. This fimbria was not produced by cells grown at 42 degrees C or on solid medium. Expression of ATF did not correlate with mannose-resistant/Proteus-like (MR/P) or mannose-resistant/Klebsiella-like (MR/K) hemagglutination and represents a novel fimbria of P. mirabilis.
Proteus mirabilis, a cause of urinary tract infection and acute pyelonephritis, produces a number of different fimbriae. An isogenic fimbrial mutant of P. mirabilis H14320 was constructed by marker exchange with ApmfA::aphA to determine the role of the P. mirabilis fimbriae (PMF) in hemagglutination and in virulence in the CBA mouse model of ascending urinary tract infection. The pmfA mutant, which did not express the 19,500-Da major subunit of PMF, colonized the bladders of transurethrally challenged CBA mice (n = 20 in each group) in numbers 83-fold lower than those of the wild-type strain (mutant, log1o 4.87 CFU/g; wild-type strain, log1o 6.79 CFU/g; P = 0.023). However, the mutant colonized the kidneys in numbers similar to those of the wild-type strain. Hemagglutination patterns of the mutant ruled out the involvement of PMF in both mannose-resistant, Proteus-like and mannose-resistant, Klebsiella-like hemagglutination. Similarly, PMF does not appear to be involved in adherence to uroepithelial cells (UEC), since the mutant was as adherent as the wild-type strain (mutant, 14.1 ± 11.7 mean bacteria per UEC, 60% of UEC with .10 bacteria; wild-type strain, 18.1 ± 16.2 mean bacteria per UEC, 68% of UEC with .10 bacteria; not significantly different). These data suggest a role for PMF in colonization of the bladder but not in colonization of kidney tissue. PMF appear not to be responsible for mannose-resistant, Proteus-like or mannose-resistant, Klebsiella-like hemagglutination.
Proteus mirabilis, a common cause of urinary tract infection in hospitalized and catheterized patients, produces mannose-resistant/klebsiella-like (MR/K) and mannose-resistant/proteus-like (MR/P) hemagglutinins. The gene encoding the major structural subunit of a fimbria, possibly MR/K, was identified in two strains. A degenerate oligonucleotide probe based on the N terminus of the Proteus uroepithelial cell adhesin and antiserum raised against the denatured polypeptide were used to screen a cosmid gene bank of strain HU1069. A cosmid clone that reacted with the probe and antiserum was identified, and a fimbria-like open reading frame was determined by nucleotide sequencing. The predicted N-terminal amino acid sequence of the processed polypeptide, ENETPAPKVSSTKGEIQLKG (residues 23 to 42), did not match the uroepithelial cell adhesin N terminus but, rather, matched exactly the N-terminal amino acid sequence of a polypeptide with an apparent
Uropathogenic Proteus mirabilis produces at least four types of fimbriae. Amino acid sequences from two peptides, derived by tryptic digestion of the structural subunit of one type of these fimbriae, the ambienttemperature fimbriae, were determined: NVVPGQPSSTQ and LIEGENQLNYNA. PCR primers, based on these sequences and that of the N terminus, were used to amplify a 359-bp fragment. A cosmid clone, isolated from a P. mirabilis genomic library by hybridization with the 359-bp PCR product, was used to determine the nucleotide sequence of the atf gene cluster. A 3,903-bp region encodes three polypeptides: AtfA, the structural subunit; AtfB, the chaperone; and AtfC, the outer membrane molecular usher. No fimbria-related genes are evident either 5 or 3 to the three contiguous genes. AtfA demonstrates significant amino acid sequence identity with type 1 major fimbrial subunits of several enteric species. The 359-bp PCR product hybridized strongly with all Proteus isolates (n ؍ 9) and 25% of 355 Escherichia coli isolates but failed to hybridize with any of 26 isolates among nine other uropathogenic species. Ambient-temperature fimbriae of P. mirabilis may represent a novel type of fimbriae of enteric species.
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