The Experts below are selected from a list of 309 Experts worldwide ranked by ideXlab platform
Harry L T Mobley - One of the best experts on this subject based on the ideXlab platform.
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pathogenesis of proteus mirabilis infection
EcoSal Plus, 2018Co-Authors: Harry L T Mobley, Chelsie E Armbruster, Melanie M PearsonAbstract:Proteus mirabilis, a Gram-negative rod-shaped bacterium most noted for its swarming motility and urease activity, frequently causes catheter-associated urinary tract infections (CAUTIs) that are often polymicrobial. These infections may be accompanied by urolithiasis, the development of bladder or kidney stones due to alkalinization of urine from urease-catalyzed urea hydrolysis. Adherence of the bacterium to epithelial and catheter surfaces is mediated by 17 different Fimbriae, most notably MR/P Fimbriae. Repressors of motility are often encoded by these Fimbrial operons. Motility is mediated by flagella encoded on a single contiguous 54-kb chromosomal sequence. On agar plates, P. mirabilis undergoes a morphological conversion to a filamentous swarmer cell expressing hundreds of flagella. When swarms from different strains meet, a line of demarcation, a “Dienes line,” develops due to the killing action of each strain’s type VI secretion system. During infection, histological damage is caused by cytotoxins including hemolysin and a variety of proteases, some autotransported. The pathogenesis of infection, including assessment of individual genes or global screens for virulence or fitness factors has been assessed in murine models of ascending urinary tract infections or CAUTIs using both single-species and polymicrobial models. Global gene expression studies performed in culture and in the murine model have revealed the unique metabolism of this bacterium. Vaccines, using MR/P Fimbria and its adhesin, MrpH, have been shown to be efficacious in the murine model. A comprehensive review of factors associated with urinary tract infection is presented, encompassing both historical perspectives and current advances.
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role of p Fimbrial mediated adherence in pyelonephritis and persistence of uropathogenic escherichia coli upec in the mammalian kidney
Kidney International, 2007Co-Authors: M C Lane, Harry L T MobleyAbstract:P Fimbria, a mannose-resistant adhesin of uropathogenic Escherichia coli (UPEC), has been shown to be associated with acute pyelonephritis. The pap gene cluster encodes the proteins required for P-Fimbrial biogenesis, including papG, which encodes the tip adhesin. The three most studied PapG molecular variants, which are shown to bind distinct isoreceptors, are PapGI, -II, and -III. PapGII preferentially binds globoside, or GbO4, a glycolipid isoreceptor of the human kidney. Studies using different animal models of ascending urinary tract infection (UTI) have demonstrated a variable role for P Fimbriae, and specifically PapGII-mediated adherence, in renal colonization. The disparities in the results obtained from those studies are likely to be attributed to the differences in animal models and UPEC strains utilized. One explanation that is discussed in detail is the contribution of multiple Fimbriae of UPEC that potentially mediate adherence to the mammalian kidney. Overall, P Fimbriae appear to play some role in mediating adherence to uroepithelial cells in vivo and establishing an inflammatory response during renal colonization, thus contributing to kidney damage during acute pyelonephritis. To verify that P Fimbriae contribute to the pathogenesis of UPEC during ascending UTI (and in particular acute pyelonephritis), future studies should be conducted to satisfy fully all three tenets of the molecular Koch's postulates, including complementation of a mutated allele.
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identification of mrpi as the sole recombinase that regulates the phase variation of mr p Fimbria a bladder colonization factor of uropathogenic proteus mirabilis
Molecular Microbiology, 2002Co-Authors: Xin Li, David E Johnson, Virginia C Lockatell, Harry L T MobleyAbstract:Summary 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.
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requirement of mrph for mannose resistant proteus like Fimbria mediated hemagglutination by proteus mirabilis
Infection and Immunity, 1999Co-Authors: Xin Li, David E Johnson, Harry L T MobleyAbstract: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 Galα(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 DH5α, 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 ΔmrpH mutant expressed in E. coli DH5α 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.
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MrpB Functions as the Terminator for Assembly of Proteus mirabilis Mannose-Resistant Proteus-Like Fimbriae
Infection and Immunity, 1998Co-Authors: Xin Li, Harry L T MobleyAbstract:Insertional mutagenesis studies of mrpB, a putative pilin-encoding open reading frame of the mrp gene cluster, which encodes mannose-resistant Proteus-like (MR/P) Fimbriae of Proteus mirabilis, indicate that MrpB functions as the terminator for Fimbrial assembly.
P A Ragsdale - One of the best experts on this subject based on the ideXlab platform.
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identification of a gene involved in assembly of actinomyces naeslundii t14v type 2 Fimbriae
Infection and Immunity, 1998Co-Authors: Maria K Yeung, John O. Cisar, Jacob A Donkersloot, P A RagsdaleAbstract:The nucleotide sequence of the Actinomyces naeslundii T14V type 2 Fimbrial structural subunit gene, fimA, and the 3' flanking DNA region was determined. The fimA gene encoded a 535-amino-acid precursor subunit protein (FimA) which included both N-terminal leader and C-terminal cell wall sorting sequences. A second gene, designated orf365, that encoded a 365-amino-acid protein which contained a putative transmembrane segment was identified immediately 3' to fimA. Mutants in which either fimA or orf365 was replaced with a kanamycin resistance gene did not participate in type 2 Fimbriae-mediated coaggregation with Streptococcus oralis 34. Type 2 Fimbrial antigen was not detected in cell extracts of the fimA mutant by Western blotting with anti-A. naeslundii type 2 Fimbrial antibody, but the subunit protein was detected in extracts of the orf365 mutant. The subunit protein detected in this mutant also was immunostained by an antibody raised against a synthetic peptide representing the C-terminal 20 amino acid residues of the predicted FimA. The antipeptide antibody reacted with FimA isolated from the recombinant Escherichia coli clone containing fimA but did not react with purified type 2 Fimbriae in extracts of the wild-type strain. These results indicate that synthesis of type 2 Fimbriae in A. naeslundii T14V may involve posttranslational cleavage of both the N-terminal and C-terminal peptides of the precursor subunit and also the expression of orf365.
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synthesis and function of actinomyces naeslundii t14v type 1 Fimbriae require the expression of additional Fimbria associated genes
Infection and Immunity, 1997Co-Authors: M K Yeung, P A RagsdaleAbstract:The nucleotide sequence of the chromosomal DNA flanking the Actinomyces naeslundii (formerly A. viscosus) T14V type 1 Fimbrial structural subunit gene (fimP) was determined. Six open reading frames (ORFs), in the order 59 ORF3, ORF2, ORF1,fimP, ORF4, ORF5, ORF6 39, were identified. ORF1 encoded a protein of 408 amino acid residues (Mr = 39,270) and had significant sequence homology with the A. naeslundii T14V type 1 and A. naeslundii WVU45 type 2 Fimbrial structural subunits. An in-frame fusion of ORF1 to the malE gene of the expression vector, pMAL-c2, yielded a protein that was immunostained with antibodies raised against the maltose binding protein and A. naeslundii T14V whole bacteria. Digestion of the fusion protein with factor Xa released a protein (apparent molecular mass of 34 kDa) that was immunostained only with the antibody directed against A. naeslundii T14V whole bacterial cells. Integration plasmids carrying a kanamycin resistance gene (kan) that was used to substitute for ORF1 or for DNA fragments internal to the coding region of the other five ORFs were used to transform A. naeslundii T14V. Neither type 1 Fimbriae nor the 65-kDa Fimbrial structural subunit was detected in mutants obtained by allelic replacement of ORF1 or ORF2. Mutants obtained by allelic replacement of ORF3 or ORF4 expressed only the 65-kDa Fimbrial structural subunit. These mutants did not bind, in vitro, to proline-rich proteins that serve as the receptors for Actinomyces type 1 Fimbriae. In contrast, a mutant in which the integration plasmid DNA had been inserted at a site close to the carboxyl terminus of ORF6 expressed type 1 Fimbriae and had adherence properties similar to those observed in the wild-type strain. These results demonstrate the existence of additional genes near fimP that are likely to be involved in the synthesis and function of cell surface Fimbriae of A. naeslundii T14V.
James P Nataro - One of the best experts on this subject based on the ideXlab platform.
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identification of cell surface exposed proteins involved in the Fimbria mediated adherence of enteroaggregative escherichia coli to intestinal cells
Infection and Immunity, 2014Co-Authors: Mariana Izquierdo, James P Nataro, Fernando Navarrogarcia, Raul Navaacosta, Fernando Ruizperez, Mauricio J FarfanAbstract:Fimbria-mediated adherence to the intestinal epithelia is a key step in enteroaggregative Escherichia coli (EAEC) pathogenesis. To date, four Fimbriae have been described for EAEC; aggregative adherence Fimbria II (AAF/II) is the most important adherence factor for EAEC prototype strain 042. Previously, we described results showing that extracellular matrix (ECM) components might be involved in the recognition of AAF/II Fimbriae by intestinal cells. In this study, we sought to identify novel potential receptors on intestinal epithelial cells recognized by the AAF/II Fimbriae. Purified AafA-dsc protein, the major subunit of AAF/II Fimbriae, was incubated with a monolayer of T84 cells, cross-linked to the surface-exposed T84 cell proteins, and immunoprecipitated by using anti-AafA antibodies. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of cellular proteins bound to AafA-dsc protein identified laminin (previously recognized as a potential receptor for AAF/II) and cytokeratin 8 (CK8). Involvement of the major subunit of AAF/II Fimbriae (AafA protein) in the binding to recombinant CK8 was confirmed by adherence assays with purified AAF/II Fimbriae, AafA-dsc protein, and strain 042. Moreover, HEp-2 cells transfected with CK8 small interfering RNA (siRNA) showed reduced 042 adherence compared with cells transfected with scrambled siRNA as a control. Adherence of 042 to HEp-2 cells preincubated with antibodies against ECM proteins or CK8 was substantially reduced. Altogether, our results supported the idea of a role of CK8 as a potential receptor for EAEC.
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organization of biogenesis genes for aggregative adherence Fimbria ii defines a virulence gene cluster in enteroaggregative escherichia coli
Journal of Bacteriology, 1999Co-Authors: John R Czeczulin, Waldir P Elias, Ian R Henderson, Luiz R Trabulsi, James P NataroAbstract:Several virulence-related genes have been described for prototype enteroaggregative Escherichia coli (EAEC) strain 042, which has been shown to cause diarrhea in human volunteers. Among these factors are the enterotoxins Pet and EAST and the Fimbrial antigen aggregative adherence Fimbria II (AAF/II), all of which are encoded on the 65-MDa virulence plasmid pAA2. Using nucleotide sequence analysis and insertional mutagenesis, we have found that the genes required for the expression of each of these factors, as well as the transcriptional activator of Fimbrial expression AggR, map to a distinct cluster on the pAA2 plasmid map. The cluster is 23 kb in length and includes two regions required for expression of the AAF/II Fimbria. These Fimbrial biogenesis genes feature a unique organization in which the chaperone, subunit, and transcriptional activator lie in one cluster, whereas the second, unlinked cluster comprises a silent chaperone gene, usher, and invasin reminiscent of Dr family Fimbrial clusters. This plasmid-borne virulence locus may represent an important set of virulence determinants in EAEC strains.
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aggregative adherence Fimbria ii a second Fimbrial antigen mediating aggregative adherence in enteroaggregative escherichia coli
Infection and Immunity, 1997Co-Authors: John R Czeczulin, Fernando Navarrogarcia, S Balepur, Susan Hicks, A Phillips, R H Hall, M H Kothary, James P NataroAbstract:Enteroaggregative Escherichia coli (EAEC) has been implicated as an agent of pediatric diarrhea in the developing world. We have shown previously that EAEC adheres to HEp-2 cells by virtue of a plasmid-encoded Fimbrial adhesin designated aggregative adherence Fimbria I (AAF/I), the genes for which have been cloned and sequenced. However, not all EAEC strains express AAF/I. Using TnphoA mutagenesis, we have characterized a novel Fimbria (designated AAF/II) which mediates HEp-2 adherence of the human-pathogenic strain 042. AAF/II is 5 nm in diameter and does not bind AAF/I antiserum, as determined by immunogold transmission electron microscopy. TnphoA identified a gene (designated aafA) which bears significant homology to aggA, the Fimbrial subunit of AAF/I (25% identity and 47% similarity at the amino acid level). When hyperexpressed and purified by polyhistidine tagging, the AafA protein assembled into 5-nm-diameter filaments which bound anti-AAF/II antiserum. The cloned aafA gene complemented a mutation in the aggA gene to confer Fimbrial expression from the AAF/I gene cluster, manifesting phenotypes characteristic of AAF/II but not AAF/I. The aafA mutant did not adhere to human intestinal tissue in culture, suggesting a role for AAF/II in intestinal colonization. By using DNA probes for AAF/I and AAF/II derived from Fimbrial biosynthesis genes, we show that AAF/I and AAF/II are each found in only a minority of EAEC strains, suggesting that still more EAEC adhesins exist. Our data suggest that AAF adhesins represent a new family of Fimbrial adhesins which mediate aggregative adherence in EAEC.
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identification and characterization of a gene cluster mediating enteroaggregative escherichia coli aggregative adherence Fimbria i biogenesis
Journal of Bacteriology, 1994Co-Authors: Stephen J Savarino, Deng Yikang, James P NataroAbstract:Abstract The aggregative pattern of adherence (AA) exhibited by enteroaggregative Escherichia coli upon HEp-2 cells is a plasmid-associated property which correlates with aggregative adherence Fimbria I (AAF/I) expression and human erythrocyte hemagglutination. By using cloning and mutagenesis strategies, two noncontiguous plasmid segments (designated regions 1 and 2) required for AA expression have previously been identified in enteroaggregative E. coli 17-2. TnphoA mutagenesis was performed on clones containing region 1, and 16 TnphoA mutants which were negative for the AA phenotype were analyzed. The TnphoA insertion site for each mutant was determined by junctional DNA sequencing. All 16 mutations occurred within a 4.6-kb span in region 1. Nucleotide sequence analysis of the region revealed four contiguous open reading frames, designated aggDCBA, in the same span. AA-negative TnphoA insertions into all open reading frames except aggB were obtained. On the basis of mutational analysis and protein homology data, it is inferred that aggA, aggC, and aggD are involved in biogenesis of AAF/I, encoding a major Fimbrial subunit, outer membrane usher, and periplasmic Fimbrial chaperone, respectively. By immunogold electron microscopy, polyclonal antiserum raised against the aggA gene product decorated AAF/I Fimbriae, affirming that AggA encodes an AAF/I subunit.
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aggregative adherence Fimbria i expression in enteroaggregative escherichia coli requires two unlinked plasmid regions
Infection and Immunity, 1993Co-Authors: James P Nataro, Stephen J Savarino, Deng Yikang, M H Kothary, J A Giron, R H HallAbstract:Adherence to HEp-2 cells by many enteroaggregative Escherichia coli (EAggEC) strains is associated with the expression of flexible, bundle-forming Fimbriae 2 to 3 nm in diameter, designated aggregative adherence Fimbriae I (AAF/I). We have previously reported the molecular cloning and TnphoA mutagenesis of AAF/I genes from the large plasmid of prototype EAggEC strain 17-2 (J. P. Nataro, Y. Deng, D. R. Maneval, A. L. German, W. C. Martin, and M. M. Levine, Infect. Immun. 60:2297-2304, 1992). Here, we report that further mapping and subcloning of AAF/I regions suggest that expression of the Fimbriae requires two separate plasmid regions (designated regions 1 and 2). Approximately 9 kb of DNA unnecessary for Fimbrial expression separates the two regions; this intervening segment encodes the EAggEC heat-stable enterotoxin (EAST1). Neither region was capable of conferring aggregative HEp-2 adherence (AA) when cloned individually; when the two regions were cloned as a single fragment or when each was cloned into a different vector and introduced into the same E. coli HB101 cell, AA was restored. AA-positive constructs also expressed human erythrocyte hemagglutination, autoagglutination in broth cultures, and the production of AAF/I as detected by immunogold electron microscopy.
John O. Cisar - One of the best experts on this subject based on the ideXlab platform.
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dual function of a tip fimbrillin of actinomyces in Fimbrial assembly and receptor binding
Journal of Bacteriology, 2011Co-Authors: Chenggang Wu, John O. Cisar, Arunima Mishra, Jinghua Yang, Hung TonthatAbstract:Interaction of Actinomyces oris with salivary proline-rich proteins (PRPs), which serve as Fimbrial receptors, involves type 1 Fimbriae. Encoded by the gene locus fimQ-fimP-srtC1, the type 1 Fimbria is comprised of the Fimbrial shaft FimP and the tip fimbrillin FimQ. Fimbrial polymerization requires the Fimbria-specific sortase SrtC1, which catalyzes covalent linkage of Fimbrial subunits. Using genetics, biochemical methods, and electron microscopy, we provide evidence that the tip fimbrillin, FimQ, is involved in Fimbrial assembly and interaction with PRPs. Specifically, while deletion of fimP completely abolished the type 1 Fimbrial structures, surface display of monomeric FimQ was not affected by this mutation. Surprisingly, deletion of fimQ significantly reduced surface assembly of the type 1 Fimbriae. This defect was rescued by recombinant FimQ ectopically expressed from a plasmid. In agreement with the role of type 1 Fimbriae in binding to PRPs, aggregation of A. oris with PRP-coated beads was abrogated in cells lacking srtC1 or fimP. This aggregation defect of the ΔfimP mutant was mainly due to significant reduction of FimQ on the bacterial surface, as the aggregation was not observed in a strain lacking fimQ. Increasing expression of FimQ in the ΔfimP mutant enhanced aggregation, while overexpression of FimP in the ΔfimQ mutant did not. Furthermore, recombinant FimQ, not FimP, bound surface-associated PRPs in a dose-dependent manner. Thus, not only does FimQ function as the major adhesin of the type 1 Fimbriae, it also plays an important role in Fimbrial assembly.
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the actinomyces oris type 2 Fimbrial shaft fima mediates co aggregation with oral streptococci adherence to red blood cells and biofilm development
Molecular Microbiology, 2010Co-Authors: Arunima Mishra, John O. Cisar, Chenggang Wu, Jinghua Yang, Hung TonthatAbstract:Summary Interbacterial interactions between oral streptococci and actinomyces and their adherence to tooth surface and the associated host cells are key early events that promote development of the complex oral biofilm referred to as dental plaque. These interactions depend largely on a lectin-like activity associated with the Actinomyces oris type 2 Fimbria, a surface structure assembled by sortase (SrtC2)-dependent polymerization of the shaft and tip fimbrillins, FimA and FimB respectively. To dissect the function of specific fimbrillins in various adherence processes, we have developed a convenient new technology for generating unmarked deletion mutants of A. oris. Here, we show that the fimB mutant, which produced type 2 Fimbriae composed only of FimA, like the wild type co-aggregated strongly with receptor-bearing streptococci, agglutinated with sialidase-treated red blood cells, and formed monospecies biofilm. In contrast, the fimA and srtC2 mutants lacked type 2 Fimbriae and were non-adherent in each of these assays. Plasmid-based expression of the deleted gene in respective mutants restored adherence to wild-type levels. These findings uncover the importance of the lectin-like activity of the polymeric FimA shaft rather than the tip. The multivalent adhesive function of FimA makes it an ideal molecule for exploring novel intervention strategies to control plaque biofilm formation.
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sortase catalyzed assembly of distinct heteromeric Fimbriae in actinomyces naeslundii
Journal of Bacteriology, 2007Co-Authors: Arunima Mishra, John O. Cisar, Hung TonthatAbstract:Two types of adhesive Fimbriae are expressed by Actinomyces; however, the architecture and the mechanism of assembly of these structures remain poorly understood. In this study we characterized two Fimbrial gene clusters present in the genome of Actinomyces naeslundii strain MG-1. By using immunoelectron microscopy and biochemical analysis, we showed that the fimQ-fimP-srtC1-fimR gene cluster encodes a Fimbrial structure (designated type 1) that contains a major subunit, FimP, forming the shaft and a minor subunit, FimQ, located primarily at the tip. Similarly, the fimB-fimA-srtC2 gene cluster encodes a distinct Fimbrial structure (designated type 2) composed of a shaft protein, FimA, and a tip protein, FimB. By using allelic exchange, we constructed an in-frame deletion mutant that lacks the SrtC2 sortase. This mutant produces abundant type 1 Fimbriae and expresses the monomeric FimA and FimB proteins, but it does not assemble type 2 Fimbriae. Thus, SrtC2 is a Fimbria-specific sortase that is essential for assembly of the type 2 Fimbriae. Together, our experiments pave the way for several lines of molecular investigation that are necessary to elucidate the Fimbrial assembly pathways in Actinomyces and their function in the pathogenesis of different biofilm-related oral diseases.
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identification of a gene involved in assembly of actinomyces naeslundii t14v type 2 Fimbriae
Infection and Immunity, 1998Co-Authors: Maria K Yeung, John O. Cisar, Jacob A Donkersloot, P A RagsdaleAbstract:The nucleotide sequence of the Actinomyces naeslundii T14V type 2 Fimbrial structural subunit gene, fimA, and the 3' flanking DNA region was determined. The fimA gene encoded a 535-amino-acid precursor subunit protein (FimA) which included both N-terminal leader and C-terminal cell wall sorting sequences. A second gene, designated orf365, that encoded a 365-amino-acid protein which contained a putative transmembrane segment was identified immediately 3' to fimA. Mutants in which either fimA or orf365 was replaced with a kanamycin resistance gene did not participate in type 2 Fimbriae-mediated coaggregation with Streptococcus oralis 34. Type 2 Fimbrial antigen was not detected in cell extracts of the fimA mutant by Western blotting with anti-A. naeslundii type 2 Fimbrial antibody, but the subunit protein was detected in extracts of the orf365 mutant. The subunit protein detected in this mutant also was immunostained by an antibody raised against a synthetic peptide representing the C-terminal 20 amino acid residues of the predicted FimA. The antipeptide antibody reacted with FimA isolated from the recombinant Escherichia coli clone containing fimA but did not react with purified type 2 Fimbriae in extracts of the wild-type strain. These results indicate that synthesis of type 2 Fimbriae in A. naeslundii T14V may involve posttranslational cleavage of both the N-terminal and C-terminal peptides of the precursor subunit and also the expression of orf365.
Hung Tonthat - One of the best experts on this subject based on the ideXlab platform.
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dual function of a tip fimbrillin of actinomyces in Fimbrial assembly and receptor binding
Journal of Bacteriology, 2011Co-Authors: Chenggang Wu, John O. Cisar, Arunima Mishra, Jinghua Yang, Hung TonthatAbstract:Interaction of Actinomyces oris with salivary proline-rich proteins (PRPs), which serve as Fimbrial receptors, involves type 1 Fimbriae. Encoded by the gene locus fimQ-fimP-srtC1, the type 1 Fimbria is comprised of the Fimbrial shaft FimP and the tip fimbrillin FimQ. Fimbrial polymerization requires the Fimbria-specific sortase SrtC1, which catalyzes covalent linkage of Fimbrial subunits. Using genetics, biochemical methods, and electron microscopy, we provide evidence that the tip fimbrillin, FimQ, is involved in Fimbrial assembly and interaction with PRPs. Specifically, while deletion of fimP completely abolished the type 1 Fimbrial structures, surface display of monomeric FimQ was not affected by this mutation. Surprisingly, deletion of fimQ significantly reduced surface assembly of the type 1 Fimbriae. This defect was rescued by recombinant FimQ ectopically expressed from a plasmid. In agreement with the role of type 1 Fimbriae in binding to PRPs, aggregation of A. oris with PRP-coated beads was abrogated in cells lacking srtC1 or fimP. This aggregation defect of the ΔfimP mutant was mainly due to significant reduction of FimQ on the bacterial surface, as the aggregation was not observed in a strain lacking fimQ. Increasing expression of FimQ in the ΔfimP mutant enhanced aggregation, while overexpression of FimP in the ΔfimQ mutant did not. Furthermore, recombinant FimQ, not FimP, bound surface-associated PRPs in a dose-dependent manner. Thus, not only does FimQ function as the major adhesin of the type 1 Fimbriae, it also plays an important role in Fimbrial assembly.
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the actinomyces oris type 2 Fimbrial shaft fima mediates co aggregation with oral streptococci adherence to red blood cells and biofilm development
Molecular Microbiology, 2010Co-Authors: Arunima Mishra, John O. Cisar, Chenggang Wu, Jinghua Yang, Hung TonthatAbstract:Summary Interbacterial interactions between oral streptococci and actinomyces and their adherence to tooth surface and the associated host cells are key early events that promote development of the complex oral biofilm referred to as dental plaque. These interactions depend largely on a lectin-like activity associated with the Actinomyces oris type 2 Fimbria, a surface structure assembled by sortase (SrtC2)-dependent polymerization of the shaft and tip fimbrillins, FimA and FimB respectively. To dissect the function of specific fimbrillins in various adherence processes, we have developed a convenient new technology for generating unmarked deletion mutants of A. oris. Here, we show that the fimB mutant, which produced type 2 Fimbriae composed only of FimA, like the wild type co-aggregated strongly with receptor-bearing streptococci, agglutinated with sialidase-treated red blood cells, and formed monospecies biofilm. In contrast, the fimA and srtC2 mutants lacked type 2 Fimbriae and were non-adherent in each of these assays. Plasmid-based expression of the deleted gene in respective mutants restored adherence to wild-type levels. These findings uncover the importance of the lectin-like activity of the polymeric FimA shaft rather than the tip. The multivalent adhesive function of FimA makes it an ideal molecule for exploring novel intervention strategies to control plaque biofilm formation.
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sortase catalyzed assembly of distinct heteromeric Fimbriae in actinomyces naeslundii
Journal of Bacteriology, 2007Co-Authors: Arunima Mishra, John O. Cisar, Hung TonthatAbstract:Two types of adhesive Fimbriae are expressed by Actinomyces; however, the architecture and the mechanism of assembly of these structures remain poorly understood. In this study we characterized two Fimbrial gene clusters present in the genome of Actinomyces naeslundii strain MG-1. By using immunoelectron microscopy and biochemical analysis, we showed that the fimQ-fimP-srtC1-fimR gene cluster encodes a Fimbrial structure (designated type 1) that contains a major subunit, FimP, forming the shaft and a minor subunit, FimQ, located primarily at the tip. Similarly, the fimB-fimA-srtC2 gene cluster encodes a distinct Fimbrial structure (designated type 2) composed of a shaft protein, FimA, and a tip protein, FimB. By using allelic exchange, we constructed an in-frame deletion mutant that lacks the SrtC2 sortase. This mutant produces abundant type 1 Fimbriae and expresses the monomeric FimA and FimB proteins, but it does not assemble type 2 Fimbriae. Thus, SrtC2 is a Fimbria-specific sortase that is essential for assembly of the type 2 Fimbriae. Together, our experiments pave the way for several lines of molecular investigation that are necessary to elucidate the Fimbrial assembly pathways in Actinomyces and their function in the pathogenesis of different biofilm-related oral diseases.