The type III Mxi-Spa secretion machinery of Shigella flexneri is responsible for secretion of Ipa proteins, which are involved in the entry of bacteria into epithelial cells. Ipa proteins accumulate within bacteria growing in laboratory media, and their secretion is activated upon contact of bacteria with eukaryotic cells. In this study, we have identified a group of chemical compounds, including Congo red, Evans blue, and direct orange, which are able to induce secretion of Ipa proteins by bacteria suspended in phosphate-buffered saline. Parameters of kinetics of activation of Ipa secretion by Congo red were determined by measuring by enzyme-linked immunosorbent assay the amount of IpaC secreted and by investigating the increase in susceptibility of Ipa proteins to proteinase K degradation. Ipa secretion occurred at 37 degrees C, was obtained with 5 to 10 microM Congo red, and was complete within 30 min. In addition, activation of Ipa secretion by Congo red was observed with bacteria harvested throughout the exponential phase of growth but not with bacteria in the stationary phase. The interactions of Congo red and Congo red-related compounds with the Mxi-Spa secretion apparatus might be specific hydrophobic interactions similar to those involved in binding of Congo red to amyloid proteins.

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°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 duster 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 -25% amino acid identity with at least one other enteric fimbrial gene product encoded by the pap, fim, smf, fan, or mrk gene clusters.Proteus mirabilis, commonly associated with nosocomially acquired urinary tract infection (12,24,29,37,45), expresses a number of proteins that may contribute to virulence, including urease (15), hemolysin (28,34,41), flagella (7,27,33), and fimbriae (1,7,32,38,39,45,46). Four fimbria-like structures have been purified from P. mirabilis strains for which the major structural subunits have been identified on sodium dodecyl sulfate (SDS)-polyacrylamide gels and subjected to N-terminal analysis (6-8, 20, 43, 46). The gene clusters for two of the fimbriae, MR/P (mannose-resistant/Proteus-like) fimbriae and PMF (P. mirabilis fimbriae), have been cloned, and the nucleotide sequences of the major subunit genes, mrpA (9) and pmfA (6), have been reported.Among the fimbriae of P. mirabilis, MR/P fimbriae are perhaps best understood. The presence of 7-to 8-nm-diameter, channelled fimbriae were first observed on bacterial cells capable of hemagglutinating erythrocytes in a mannose-resistant fashion (31). The MR/P hemagglutination pattern is expressed by virtually all strains (27,31) and elicits a serum immunoglobulin response following experimental ascending urinary tract infection in mice (7, 20). Here we report the complete nucleotide sequence for the mrp operon, the distribution of the genes among Proteus and other genera, and the conditions under which MrpA is expressed in the wild-type isolate.We have previously demonstrated that mice, experimentally infected with P. mirabilis, developed a serum immunoglobulin G response to MrpA, the major subunit of MR/P fimbriae, indicating that the fimbriae are expressed in the urinary tract (7). We sought to mimic these parameters by determining the optimal growth conditions for the in vitro expression of MR/P fimbriae. Expression of MrpA by P. mirabilis H14320, isolated from the urine of a woman with catheter-associated bacteriuria (16, 45), was assessed by Western blotting (immunoblotting) using rabbit polyclonal antiserum raised against purified MR/P fimbriae (Fig. 1A) Under these optimal conditions (Luria broth, 37°C, 48 h, static), expression of MrpA by...

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.

Urinary tract infection with Proteus mirabilis may lead to serious complications, including cystitis, acute pyelonephritis, fever, bacteremia, and death. In addition to the production of hemolysin and the enzyme urease, fimbriae and flagellum-mediated motility have been postulated as virulence factors for this species. We purified mannose-resistant/proteuslike (MR/P) fimbriae and flagella from strains CFT322 and HU2450, respectively. Electron microscopy revealed highly concentrated preparations of fimbriae and flagella. Fimbrial and flagellar structural subunits were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 18.5 and 41 kDa, respectively. N-terminal sequencing revealed that 10 of the first 20 amino acids of the major MR/P subunit matched the sequence of the P. mirabilis uroepithelial cell adhesin N terminus and 11 of 20 amino acids matched the predicted amino acid sequence of the Escherichia coli P fimbriae structural subunit, PapA. In addition, 90 and 80% homologies were found between the first 20 amino acids of P. mirabilis flagellin and those of Salmonella typhimurium phase-i flagellin and the E. coli hag gene product, respectively. An enzyme-linked immunosorbent assay using purified antigens showed a strong reaction between the MR/P fimbriae or flagella and sera of CBA mice challenged transurethrally with P. mirabilis. A possible role for MR/P fimbriae in the pathogenesis of urinary tract infection is supported by (i) a strong immune response to the antigen in experimentally infected animals, (ii) amino acid sequence similarity to other enteric surface structure, and (iii) our previously reported observation that MR/P fimbriae are expressed preferentially as the sole fimbrial type in human pyelonephritis isolates.