Pic, an Autotransporter Protein Secreted by Different Pathogens in the Enterobacteriaceae Family, Is a Potent Mucus Secretagogue

ABSTRACT A hallmark of enteroaggregative Escherichia coli (EAEC) infection is a formation of biofilm, which comprises a mucus layer with immersed bacteria in the intestines of patients. While studying the mucinolytic activity of Pic in an in vivo system, rat ileal loops, we surprisingly found that EAEC induced hypersecretion of mucus, which was accompanied by an increase in the number of mucus-containing goblet cells. Interestingly, an isogenic pic mutant (EAEC Δpic) was unable to cause this mucus hypersecretion. Furthermore, purified Pic was also able to induce intestinal mucus hypersecretion, and this effect was abolished when Pic was heat denatured. Site-directed mutagenesis of the serine protease catalytic residue of Pic showed that, unlike the mucinolytic activity, secretagogue activity did not depend on this catalytic serine protease motif. Other pathogens harboring the pic gene, such as Shigella flexneri and uropathogenic E. coli (UPEC), also showed results similar to those for EAEC, and construction of isogenic pic mutants of S. flexneri and UPEC confirmed this secretagogue activity. Thus, Pic mucinase is responsible for one of the pathophysiologic features of the diarrhea mediated by EAEC and the mucoid diarrhea induced by S. flexneri.

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A SimpleIn VitroGut Model for Studying the Interaction betweenEscherichia coliand the Intestinal Commensal Microbiota in Cecal Mucus

Applied and Environmental Microbiology ◽

10.1128/aem.02166-18 ◽

2018 ◽

Vol 84 (24) ◽

Cited By ~ 2

Author(s):

Matthew E. Mokszycki ◽

Mary Leatham-Jensen ◽

Jon L. Steffensen ◽

Ying Zhang ◽

Karen A. Krogfelt ◽

...

Keyword(s):

Escherichia Coli ◽

Gene Profiling ◽

Rrna Gene ◽

Mucus Layer ◽

Content Type ◽

E Coli ◽

Commensal Microbiota ◽

Intestinal Mucus

ABSTRACTA novelin vitrogut model was developed to better understand the interactions betweenEscherichia coliand the mouse cecal mucus commensal microbiota. The gut model is simple and inexpensive while providing an environment that largely replicates the nonadherent mucus layer of the mouse cecum. 16S rRNA gene profiling of the cecal microbial communities of streptomycin-treated mice colonized withE. coliMG1655 orE. coliNissle 1917 and the gut model confirmed that the gut model properly reflected the community structure of the mouse intestine. Furthermore, the results from thein vitrogut model mimic the results of publishedin vivocompetitive colonization experiments. The gut model is initiated by the colonization of streptomycin-treated mice, and then the community is serially transferred in microcentrifuge tubes in an anaerobic environment generated in anaerobe jars. The nutritional makeup of the cecum is simulated in the gut model by using a medium consisting of porcine mucin, mouse cecal mucus, HEPES-Hanks buffer (pH 7.2), Cleland’s reagent, and agarose. Agarose was found to be essential for maintaining the stability of the microbial community in the gut model. The outcome of competitions betweenE. colistrains in thein vitrogut model is readily explained by the “restaurant hypothesis” of intestinal colonization. This simple model system potentially can be used to more fully understand how different members of the microbiota interact physically and metabolically during the colonization of the intestinal mucus layer.IMPORTANCEBoth commensal and pathogenic strains ofEscherichia coliappear to colonize the mammalian intestine by interacting physically and metabolically with other members of the microbiota in the mucus layer that overlays the cecal and colonic epithelium. However, the use of animal models and the complexity of the mammalian gut make it difficult to isolate experimental variables that might dictate the interactions betweenE. coliand other members of the microbiota, such as those that are critical for successful colonization. Here, we describe a simple and relatively inexpensivein vitrogut model that largely mimicsin vivoconditions and therefore can facilitate the manipulation of experimental variables for studying the interactions ofE. coliwith the intestinal microbiota.

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Pairing properties of bromouracil and repair of bromouracil-containing DNA. Possible utilization of bromodeoxyuridine triphosphate for site-directed mutagenesis

Biochemical Journal ◽

10.1042/bj2530637 ◽

1988 ◽

Vol 253 (3) ◽

pp. 637-643 ◽

Cited By ~ 5

Author(s):

M Muller ◽

J Martial ◽

W G Verly

Keyword(s):

Dna Polymerase ◽

Site Directed Mutagenesis ◽

Klenow Fragment ◽

Single Experiment ◽

E Coli ◽

Partial Repair ◽

Before And After ◽

Polymerase I

5-Bromo-2′-deoxyuridine triphosphate (Br-dUTP) and dTTP are used interchangeably for DNA synthesis in vitro by the Klenow fragment of Escherichia coli DNA polymerase I. When DNA containing Br-dUMP instead of dTMP at a few preselected sites is transfected into competent bacteria, no mutation occurs, indicating that in vivo E. coli DNA polymerase always places a dAMP residue in front of any unrepaired Br-dUMP residue. On the other hand, in vitro Br-dUTP can also replace dCTP, but only with difficulty: when dCTP is absent, Br-dUMP can be forced in front of a dGMP residue, but the Klenow polymerase pauses before and after addition of Br-dUMP. Transfection into E. coli of the substituted DNA leads to the expected G→A transitions. These mutations can easily be targeted by using a suitable primer and the correctly chosen mix of deoxynucleoside triphosphates containing Br-dUTP. When Br-dUMP has been placed in front of a dGMP residue, the mutation yield is not 100%, showing a partial repair of the transfected DNA before it is replicated. Advantage can be taken of this partial repair to prepare a set of different mutations within a target region in a single experiment.

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Bifidobacterium dentiumFortifies the Intestinal Mucus Layer via Autophagy and Calcium Signaling Pathways

mBio ◽

10.1128/mbio.01087-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 15

Author(s):

Melinda A. Engevik ◽

Berkley Luk ◽

Alexandra L. Chang-Graham ◽

Anne Hall ◽

Beatrice Herrmann ◽

...

Keyword(s):

Calcium Signaling ◽

Signaling Pathways ◽

Goblet Cell ◽

Cell Function ◽

Goblet Cells ◽

Mucus Layer ◽

Content Type ◽

Intestinal Mucus ◽

Intestinal Mucus Layer

ABSTRACTMuch remains unknown about how the intestinal microbiome interfaces with the protective intestinal mucus layer.Bifidobacteriumspecies colonize the intestinal mucus layer and can modulate mucus production by goblet cells. However, selectBifidobacteriumstrains can also degrade protective glycans on mucin proteins. We hypothesized that the human-derived speciesBifidobacterium dentiumwould increase intestinal mucus synthesis and expulsion, without extensive degradation of mucin glycans.In silicodata revealed thatB. dentiumlacked the enzymes necessary to extensively degrade mucin glycans. This finding was confirmed by demonstrating thatB. dentiumcould not use naive mucin glycans as primary carbon sourcesin vitro. To examineB. dentiummucus modulationin vivo, Swiss Webster germfree mice were monoassociated with live or heat-killedB. dentium. LiveB. dentium-monoassociated mice exhibited increased colonic expression of goblet cell markersKrüppel-like factor 4(Klf4),Trefoil factor 3(Tff3),Relm-β,Muc2, and several glycosyltransferases compared to both heat-killedB. dentiumand germfree counterparts. Likewise, liveB. dentium-monoassociated colon had increased acidic mucin-filled goblet cells, as denoted by Periodic Acid-Schiff-Alcian Blue (PAS-AB) staining and MUC2 immunostaining.In vitro,B. dentium-secreted products, including acetate, were able to increase MUC2 levels in T84 cells. We also identified thatB. dentium-secreted products, such as γ-aminobutyric acid (GABA), stimulated autophagy-mediated calcium signaling and MUC2 release. This work illustrates thatB. dentiumis capable of enhancing the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways, with a net increase in mucin production.IMPORTANCEMicrobe-host interactions in the intestine occur along the mucus-covered epithelium. In the gastrointestinal tract, mucus is composed of glycan-covered proteins, or mucins, which are secreted by goblet cells to form a protective gel-like structure above the epithelium. Low levels of mucin or alterations in mucin glycans are associated with inflammation and colitis in mice and humans. Although current literature links microbes to the modulation of goblet cells and mucins, the molecular pathways involved are not yet fully understood. Using a combination of gnotobiotic mice and mucus-secreting cell lines, we have identified a human-derived microbe,Bifidobacterium dentium, which adheres to intestinal mucus and secretes metabolites that upregulate the major mucin MUC2 and modulate goblet cell function. Unlike otherBifidobacteriumspecies,B. dentiumdoes not extensively degrade mucin glycans and cannot grow on mucin alone. This work points to the potential of usingB. dentiumand similar mucin-friendly microbes as therapeutic agents for intestinal disorders with disruptions in the mucus barrier.

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In vivoR–plasmid transfer in a patient with a mixed infection of shigella dysentery

Epidemiology and Infection ◽

10.1017/s0950268800057654 ◽

1994 ◽

Vol 112 (2) ◽

pp. 247-252 ◽

Cited By ~ 9

Author(s):

M. P. Bratoeva ◽

J. F. John.

Keyword(s):

Antimicrobial Agents ◽

Shigella Flexneri ◽

Plasmid Transfer ◽

Shigella Dysenteriae ◽

Type I ◽

E Coli ◽

R Plasmids ◽

Short Time

SUMMARYTransfer of shigella R–plasmidsin vivohas seldom been demonstrated. Strains ofShigella dysenteriaetype 1 andShigella flexneritype 5b were isolated from a Bulgarian traveller who visited Vietnam and developed dysentery, which was treated with trimethoprim/sulfamethoxazole (TMP/SMZ) for a short time. Both species of shigellae are unusual in Bulgaria where strains ofS. sonneipredominate. Both shigella strains were multiresistant to the same antimicrobial agents. Each strain contained a 48–kilobase plasmid that conferred the entire resistance phenotype to a susceptibleEscherichia coli. Restriction endonuclease patterns of plasmid DNA from the respective strains were identical. Transmissible plasmids of the same resistance phenotypes and restriction patterns were isolated from the patient's colonicE. coli. Transconjugants hybridized to a dihydrofolate reductase type I–DNA probe. These studies support the hypothesis that R–plasmid transfer may occur between non-pathogenic, faecal strains and pathogenic shigellae, a process that may have been facilitated by inadequate treatment with TMP/SMZ at the onset of the illness.

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Lysyl-tRNA Synthetase-generated Lysyl-Adenylate Is a Substrate for Histidine Triad Nucleotide Binding Proteins

Journal of Biological Chemistry ◽

10.1074/jbc.m610530200 ◽

2006 ◽

Vol 282 (7) ◽

pp. 4719-4727 ◽

Cited By ~ 44

Author(s):

Tsui-Fen Chou ◽

Carston R. Wagner

Keyword(s):

Binding Proteins ◽

Nucleotide Binding ◽

Trna Synthetase ◽

Site Directed Mutagenesis ◽

Trna Synthetases ◽

E Coli ◽

Protein Protein Interaction ◽

Nucleotide Binding Proteins ◽

Micromolar Range

Histidine triad nucleotide binding proteins (Hints) are the most ancient members of the histidine triad protein superfamily of nucleotidyltransferases and hydrolyases. Protein-protein interaction studies have found that complexes of the transcription factors MITF or USF2 and lysyl-tRNA synthetase (LysRS) are associated with human Hint1. Therefore, we hypothesized that lysyl-AMP or the LysRS·lysyl-AMP may be a native substrate for Hints. To explore the biochemical relationship between Hint1 and LysRS, a series of catalytic radiolabeling, mutagenesis, and kinetic experiments was conducted with purified LysRSs and Hints from human and Escherichia coli. After incubation of the E. coli or human LysRS with Hints and [α-32P]ATP, but not [α-32P]GTP, 32P-labeled Hints were observed. By varying time and the concentrations of lysine, Mg2+, or LysRS, the adenylation of Hint was found to be dependent on the formation of lysyl-AMP. Site-directed mutagenesis studies of the active site histidine triad revealed that Hint labeling could be abolished by substitution of either His-101 of E. coli hinT or His-112 of human Hint1 by either alanine or glycine. Ap4A, believed to be synthesized by LysRS in vivo, and Zn2+ were shown to inhibit the formation of Hint-AMP with an IC50 value in the low micromolar range. Consistent with pyrophosphate being an inhibitor for aminoacyl-tRNA synthetase, incubations in the presence of pyrophosphatase resulted in enhanced formation of Hint-AMP. These results demonstrate that the lysyl-AMP intermediate formed by LysRS is a natural substrate for Hints and suggests a potential highly conserved regulatory role for Hints on LysRS and possibly other aminoacyl-tRNA synthetases.

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On the catalytic mechanism of prokaryotic leader peptidase 1

Biochemical Journal ◽

10.1042/bj2820539 ◽

1992 ◽

Vol 282 (2) ◽

pp. 539-543 ◽

Cited By ~ 51

Author(s):

M T Black ◽

J G R Munn ◽

A E Allsop

Keyword(s):

Catalytic Mechanism ◽

Serine Proteinase ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Calculated Molecular Mass ◽

E Coli ◽

Leader Peptidase ◽

Mutant Forms ◽

Thiol Proteinases

The catalytic mechanism of leader peptidase 1 (LP1) of the bacterium Escherichia coli has been investigated by a combination of site-directed mutagenesis, assays of enzyme activity in vivo utilizing a strain of E. coli which has a conditional defect in LP1 activity, and gene cloning. The biological activity of mutant forms of E. coli LP1 demonstrates that this enzyme belongs to a novel class of proteinases. The possibility that LP1 may be an aspartyl proteinase has been excluded on the basis of primary sequence comparison and mutagenesis. Assignment of LP1 to one of the other three recognized classes of proteinases (metalloproteinases, thiol proteinases and the classical serine proteinases) can also be excluded, as it is clearly demonstrated that none of the histidine or cysteine residues within LP1 are required for catalytic activity. The Pseudomonas fluorescens lep gene has been cloned and sequenced and the corresponding amino acid sequence compared with that of E. coli LP1. The E. coli LP1 and P. fluorescens LP1 primary sequences are 50% identical after insertion of gaps. The P. fluorescens LP1 has 39 fewer amino acids, a calculated molecular mass of 31903 Da and functions effectively in vivo in E. coli. None of the cysteine residues and only one of the histidine residues which are present in E. coli LP1 are conserved in sequence position in the P. fluorescens LP1 enzyme. The possibility that LP1 is a novel type of serine proteinase is discussed.

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DNA Gap Repair-Mediated Site-Directed Mutagenesis is Different from Mandecki and Recombineering Approaches

10.1101/313155 ◽

2018 ◽

Author(s):

George T. Lyozin ◽

Luca Brunelli

Keyword(s):

Dna Sequence ◽

Dna Sequences ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Gene Products ◽

E Coli ◽

Health And Disease ◽

First Time ◽

Disease Understanding

AbstractSite-directed mutagenesis allows the generation of mutant DNA sequences for downstream functional analysis of genetic variants involved in human health and disease. Understanding the mechanisms of different mutagenesis methods can help select the best approach for specific needs. We compared three different approaches for in vivo site-directed DNA mutagenesis that utilize a mutant single-stranded DNA oligonucleotide (ssODN) to target a wild type DNA sequence in the host Escherichia coli (E. coli). The first method, Mandecki, uses restriction nucleases to introduce a double stranded break (DSB) into a DNA sequence which needs to be denatured prior to co-transformation. The second method, recombineering (recombination-mediated genetic engineering), requires lambda red gene products and a mutant ssODN with homology arms of at least 20 nucleotides. In a third method described here for the first time, DNA gap repair, a mutant ssODN targets a DNA sequence containing a gap introduced by PCR. Unlike recombineering, both DNA gap repair and Mandecki can utilize homology arms as short as 10 nucleotides. DNA gap repair requires neither red gene products as recombineering nor DNA denaturation or nucleases as Mandecki, and unlike other methods is background-free. We conclude that Mandecki, recombineering, and DNA gap repair have at least partly different mechanisms, and that DNA gap repair provides a new, straightforward approach for effective site-directed mutagenesis.

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An In Vivo Biocatalytic Cascade Featuring an Artificial Enzyme Catalyzed New-to-Nature Reaction

10.26434/chemrxiv-2021-nppzq ◽

2021 ◽

Author(s):

Linda Ofori Atta ◽

Zhi Zhou ◽

Gerard Roelfes

Keyword(s):

Carbonyl Compounds ◽

Artificial Enzymes ◽

Catalytic Residue ◽

Artificial Enzyme ◽

E Coli ◽

Iminium Ion ◽

Catalyzed Reactions ◽

Enzyme Catalyzed Reactions ◽

Benzaldehyde Derivatives

Artificial enzymes utilizing the genetically encoded non-proteinogenic amino acid p-aminophenylalanine (pAF) as catalytic residue are able to react with carbonyl compounds through an iminium ion mechanism, making reactions possible that have no equivalent in nature. Here, we report an in vivo biocatalytic cascade that is augmented with such an artificial enzyme catalyzed new-to-nature reaction. The artificial enzyme in this study is a pAF containing evolved variant of the Lactococcal multidrug resistance Regulator, designated LmrR_V15pAF_RMH, which efficiently converts in vivo produced benzaldehyde derivatives into the corresponding hydrazone products inside E. coli cells. These in vivo biocatalytic cascades comprising an artificial enzyme catalyzed reactions are an important step towards achieving a hybrid metabolism.

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P071 MODULATION OF THE MUCUS LAYER BY BIFIDOBACTERIUM DENTIUM PROVIDES PROTECTION IN A MODEL OF COLITIS

Inflammatory Bowel Diseases ◽

10.1093/ibd/zaa010.096 ◽

2020 ◽

Vol 26 (Supplement_1) ◽

pp. S38-S38

Author(s):

Melinda Engevik ◽

Wenly Ruan ◽

Faith Ihekweazu ◽

James Versalovic

Keyword(s):

Goblet Cell ◽

Molecular Mechanisms ◽

Goblet Cells ◽

Mucus Layer ◽

Mucin Production ◽

Germ Free ◽

Positive Effects ◽

Intestinal Mucus ◽

Intestinal Mucus Layer

Abstract Background The intestinal mucus layer serves as a critical interface between the environment and the host. Patients with inflammatory bowel disease (IBD), particularly ulcerative colitis, exhibit reduced synthesis and secretion of the mucus protein MUC2 and decreased mucus thickness. This in turn promotes immune activation and inflammation. The clinical relevance of the mucus layer emphasizes the need to address strategies to modulate this barrier. Although bifidobacteria represent only 3–6% of the healthy adult fecal microbiota, their presence has been associated with numerous health benefits, including bolstering mucus production. However, the molecular mechanisms that underlie these positive effects appear to be strain-specific and are not well defined. We hypothesized that the human-derived Bifidobacterium dentium would increase intestinal mucus synthesis and expulsion via specific metabolites. We also speculated that modulation of goblet cells would be beneficial during colitis. Methods & Results In silico genome analysis revealed that B. dentium lacked the enzymatic repertoire required for degradation of mucin glycans. Consistent with these findings, we found that B. dentium could not use mucin glycans as a primary carbon source in vitro. To examine mucus modulation in vivo, germ-free mice were mono-associated with live or heat-killed B. dentium. Live B. dentium mono-associated mice exhibited increased colonic expression of goblet cell markers Krüppel-Like Factor 4 (Klf4), Relmβ, trefoil factor 3 (Tff3), Muc2, and several mucin glycosyltransferases compared to both heat-killed B. dentium and germ-free counterparts. Likewise, live B. dentium mono-associated colon had increased acidic mucin-filled goblet cells as denoted by MUC2 and PAS-AB staining. In vitro, B. dentium secreted products, including acetate, were able to increase MUC2 levels in T84 cells, mouse colonoids and human colonoids. We also identified that B. dentium secreted products, such as GABA, stimulated autophagy-mediated calcium signaling and MUC2 release. To identify whether B. dentium could enhance MUC2 production in mice harboring a complete microbiota, specific pathogen free mice were treated with live B. dentium by oral gavage. Administration of B. dentium increased the inner mucus layer compared to controls. Moreover, in a TNBS model of colitis, B. dentium treated mice had increased goblet cell numbers and MUC2 mRNA. Mirroring these findings, B. dentium treated mice lost less weight, had improved histology and had decreased levels of TNF, KC (IL-8), and IL-6. Conclusions This work illustrates that B. dentium enhances the intestinal mucus layer and goblet cell function via upregulation of gene expression and autophagy signaling pathways with a net increase in mucin production. Ultimately, these pathways may be targeted for the development of novel therapeutics.

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Characterisation of blood coagulation factor XIT475I

Thrombosis and Haemostasis ◽

10.1160/th05-01-0051 ◽

2005 ◽

Vol 93 (06) ◽

pp. 1082-1088 ◽

Cited By ~ 8

Author(s):

Kalpana Lal ◽

Yasufumi Imanaka ◽

Geoffrey Kemball-Cook ◽

Paula Bolton-Maggs ◽

Edward Tuddenham ◽

...

Keyword(s):

Serine Protease ◽

Chinese Hamster Ovary Cells ◽

Consensus Sequence ◽

Chinese Hamster ◽

Coagulation Factor ◽

Glycosylation Site ◽

Site Directed Mutagenesis ◽

Ovary Cells ◽

Serine Protease Domain

SummaryPCR-SSCP and DNA sequence analysis of a factor XI (FXI) deficient patient (FXI:C 39 U/dL; FXI:Ag 27 U/dL) identified a C to T transition in exon 12 of the FXI gene (F11 c.1521C>T) that predicts the substitution of Thr475 by Ile (FXIT475I) within the serine protease domain of FXI. This mutation destroys a consensus sequence for N-linked glycosylation, N473-Y-T475, known to be utilized in vivo. The FXIT475I variant was generated by site-directed mutagenesis, together with other variants that could help explain the phenotype, and recombinant FXI variants were expressed in Chinese hamster ovary cells. FXI:Ag expression was analysed by Western blot analysis, ELISA and immunocyto-chemical staining. Wild-type FXI:Ag was secreted at high levels, however the mutant (FXIT475I) was secreted very poorly. Substitution of Thr475 by Ala, Pro, Lys or Arg (all of which abolish the glycosylation consensus sequence) also severely reduced the level of secreted FXI:Ag suggesting that glycosylation at Asn473 is required for folding or secretion. Concordant with this hypothesis the conservative substitution of Thr475 by Ser (which preserves the glycosylation consensus sequence) had no effect on FXI secretion. Thr/Ser475 is highly conserved in serine protease domains but the glycosylation site (Asn473) is not. Surprisingly, substitution of Asn473 by Ala (which removes the N-linked glycosylation site) had no effect on the levels of FXI:Ag secreted. In conclusion, although the FXI-T475I mutation destroys an N-linked glycosylation consensus sequence, the cause of failure to secrete FXI is not the loss of a glycosylation site but rather a direct effect of the substitution of this highly conserved residue.

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