Characterization of a novel fructosyltransferase InuCA from Lactobacillus crispatus that attaches to the cell surface by electrostatic interaction

Fructosyltransferases (FTases), a group of carbohydrate-active enzymes, synthesize fructooligosaccharides (FOS) and fructans, which are promising prebiotics for human health. Here we originally identified a novel FTase InuCA from L. crispatus , a dominant species in the vaginal microbiotas of human. InuCA was characterized by a shortest C-terminus and the highest isoelectric point among the reported Lactobacillus FTases. InuCA was an inulosucrase and produced a serial of FOS using sucrose as substrate at a moderate temperature. Surprisingly, the C-terminal deletion mutant synthesized oligosaccharides with fructosyl chain longer than that of the wild type, suggesting that the C-terminal part blocked the binding of long-chain receptor. Moreover, InuCA bound to the cell surface by electrostatic interaction, which was dependent on the environmental pH and represented a distinctive binding mode in FTases. The catalytic and structural properties of InuCA will be contributed to the FTases engineering and the knowledge of the adaptation of L. crispatus in the vaginal environment. Importance L. crispatus is one of the most important species in human vaginal microbiotas and its persistence is strongly negatively correlated with the vaginal diseases. Our research reveals that a novel inulosucrase InuCA is present in L. cirspatus . InuCA keeps the ability to synthesize prebiotic fructo-oligosaccharides, although it lacks a large part of the C-terminal region compared to other FTases. Remarkably, the short C-terminus of InuCA blocks the transfructosylation activity for producing oligosaccharides with longer chain, which is meaningful to the directional modification of FTases and the oligosaccharide products. Besides the catalytic activity, InuCA is anchored on the cell surface dependent on the environmental pH and may be also involved in the adhesion of L. crispatus to the vaginal epithelial cells. Since L. crispatus plays an essential role in the normal vaginal micro-ecosystem, the described work will be helpful to elucidate the functional genes and colonization mechanism of the dominant species.

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Loss of the C Terminus of Melanocortin Receptor 2 (MC2R) Results in Impaired Cell Surface Expression and ACTH Insensitivity

Endocrinology ◽

10.1210/endo.151.12.9989 ◽

2010 ◽

Vol 151 (12) ◽

pp. 5971-5971

Author(s):

Andrea Hirsch ◽

Eirini Meimaridou ◽

Monica Fernandez-Cancio ◽

Amit V. Pandey ◽

María Clemente ◽

...

Keyword(s):

Cell Surface ◽

Severe Hypoglycemia ◽

Surface Expression ◽

Melanocortin Receptor ◽

Cell Surface Expression ◽

Wild Type ◽

C Terminus ◽

Exact Function ◽

Acth Insensitivity ◽

Glucocorticoid Deficiency

Objective: Mutations in melanocortin receptor 2 (MC2R) and its related melanocortin receptor accessory protein (MRAP) cause familial glucocorticoid deficiency. We identified a novel MC2R mutation, K289fs. This unique mutation in the C terminus of MC2R is located in the intracellular part of the protein for which the exact function is unknown. Setting: A 6-wk-old boy presented with severe hypoglycemia, unmeasurable cortisol, and grossly elevated ACTH but normal electrolytes. Genetic analysis revealed homozygote K289fs mutation in MC2R. His parents and siblings were heterozygous but phenotypically normal. Intervention and Results: The role of the C terminus of MC2R was studied in two cell systems. Because the K289fs mutant changes the last eight amino acids of the protein and leads to protein elongation, wild-type MC2R and C-terminally mutated constructs were tested for activity to respond to ACTH in an OS3 cell-based reporter assay. Wild-type and alanine-substituted constructs responded normally to ACTH. By contrast K289fs and M290X had a total loss of activity. Cell surface assays and confocal localization studies revealed that K289fs and M290X receptors were not found at the cell surface, indicating that their transport from the endoplasmic reticulum to the cell membrane is disrupted. Interestingly, coimmunoprecipitation experiments showed no alteration in the interaction of mutant MC2R with MRAP, suggesting that interaction between these two proteins does not guarantee normal localization. Conclusions: Loss of the C terminus of MC2R impairs cell surface expression and ACTH sensitivity but does not disrupt interaction of MC2R with MRAP. These findings highlight the extreme sensitivity of MC2R to structural disruption.

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Putative Adhesion Factors in Vaginal Lactobacillus gasseri DSM 14869: Functional Characterization

Applied and Environmental Microbiology ◽

10.1128/aem.00800-19 ◽

2019 ◽

Vol 85 (19) ◽

Cited By ~ 6

Author(s):

Zhu Zeng ◽

Fanglei Zuo ◽

Harold Marcotte

Keyword(s):

Epithelial Cells ◽

Cell Surface ◽

Bacterial Vaginosis ◽

Hela Cells ◽

Molecular Mechanisms ◽

Wild Type ◽

Content Type ◽

Lactobacillus Gasseri ◽

Vaginal Epithelial Cells ◽

Knockout Mutants

ABSTRACT Lactobacilli play an important role in the maintenance of a healthy vaginal microbiota, and some select species are widely used as probiotics. Vaginal isolates of Lactobacillus gasseri DSM 14869 and Lactobacillus rhamnosus DSM 14870 were previously selected to develop the probiotic EcoVag capsules and showed therapeutic effects in women with bacterial vaginosis (BV). However, the molecular mechanisms involved in their probiotic activity are largely unknown. In this study, we identified three cell surface molecules in L. gasseri DSM 14869 that promote adhesion to vaginal epithelial cells (VEC) by constructing dedicated knockout mutants, including exopolysaccharides (EPSs), a protein containing MucBP-like domains (N506_1778), and a putative novel adhesin (N506_1709) with rib/alpha-like domain repeats. EPS knockout mutants revealed 20-fold and 14-fold increases in adhesion to Caco-2 and HeLa cells, respectively, compared with wild type, while the adhesion to VEC was reduced 30% by the mutation, suggesting that EPSs might mediate tissue tropism for vaginal cells. A significant decrease in adhesion to Caco-2 cells, HeLa cells, and VEC was observed in the N506_1778 knockout mutant. The N506_1709 mutant showed no significant difference for the adhesion to Caco-2 and HeLa cells compared with wild type (WT); in contrast, the adhesion to VEC revealed a significant decrease (42%), suggesting that N506_1709 might mediate specific binding to stratified squamous epithelial cells, and this putative novel adhesin was annotated as Lactobacillus vaginal epithelium adhesin (LVEA). Thus, we have discovered an important role for EPSs and a novel adhesin, LVEA, in the adhesive capacity of a vaginal probiotic Lactobacillus strain. IMPORTANCE Lactobacilli are known to contribute to the maintenance of a healthy vaginal microbiota and some are selected as probiotics for the prevention or treatment of urogenital diseases, such as bacterial vaginosis. However, the molecular mechanisms for these health-promoting effects are not fully understood. Here, we functionally identified three cell surface factors of a Lactobacillus gasseri strain potentially involved in its adhesion to vaginal epithelial cells, including exopolysaccharides (EPSs) and two sortase-dependent proteins (N506_1778 and N506_1709). We could demonstrate the tissue-specific adhesion of EPSs to vaginal cells and that N506_1709 might be a novel adhesin specifically mediating bacterial binding to stratified squamous epithelial cells. The results provide important new information on the molecular mechanisms of vaginal Lactobacillus spp. adhesion.

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Functional characterization of FlgM in the regulation of flagellar synthesis and motility in Yersinia pseudotuberculosis

Microbiology ◽

10.1099/mic.0.026294-0 ◽

2009 ◽

Vol 155 (6) ◽

pp. 1890-1900 ◽

Cited By ~ 18

Author(s):

Lisha Ding ◽

Yao Wang ◽

Yangbo Hu ◽

Steve Atkinson ◽

Paul Williams ◽

...

Keyword(s):

Yersinia Pseudotuberculosis ◽

Functional Characterization ◽

Direct Interaction ◽

Site Directed Mutagenesis ◽

Wild Type ◽

C Terminus ◽

In Trans ◽

Differential Gene ◽

Flagellar Biogenesis

We describe here the functional characterization of the flgM gene in Yersinia pseudotuberculosis. Direct interaction of FlgM with the alternative sigma factor σ 28 (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the σ 28 binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with σ 28 were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of flgM and subsequent complementation in trans. Transcriptional fusion analyses showed differential gene expression of flhDC, fliA, flgM and fliC in the fliA and flgM mutants compared with the wild-type. flhDC expression was not influenced by σ 28 or FlgM while fliA expression was abolished in the fliA mutant and considerably reduced in the flgM mutant when compared to the wild-type, indicating that both FliA and FlgM can activate fliA transcription. Conversely, flgM transcription was higher in the fliA mutant when compared to the wild-type, suggesting that flgM transcription was repressed by σ 28. Interestingly, fliC expression was markedly increased in the flgM mutant, suggesting a negative regulatory role for FlgM in fliC expression. The transcription of other σ-dependent genes (cheW, flgD, flaA, csrA and fliZ) was also examined in fliA and flgM mutant backgrounds and this revealed that other σ-factors apart from σ 28 may be involved in flagellar biogenesis in Y. pseudotuberculosis. Taking together the motility phenotypes and effects of flgM mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in Y. pseudotuberculosis may differ from those described for other bacteria.

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Characterization of a Coxiella burnetii ftsZ Mutant Generated by Himar1 Transposon Mutagenesis

Journal of Bacteriology ◽

10.1128/jb.01580-08 ◽

2008 ◽

Vol 191 (5) ◽

pp. 1369-1381 ◽

Cited By ~ 77

Author(s):

Paul A. Beare ◽

Dale Howe ◽

Diane C. Cockrell ◽

Anders Omsland ◽

Bryan Hansen ◽

...

Keyword(s):

Cell Division ◽

Coxiella Burnetii ◽

Fluorescent Protein ◽

Q Fever ◽

Vero Cells ◽

Wild Type ◽

Lower Growth ◽

C Terminus ◽

Lower Growth Rate

ABSTRACT Coxiella burnetii is a gram-negative obligate intracellular bacterium and the causative agent of human Q fever. The lack of methods to genetically manipulate C. burnetii significantly impedes the study of this organism. We describe here the cloning and characterization of a C. burnetii ftsZ mutant generated by mariner-based Himar1 transposon (Tn) mutagenesis. C. burnetii was coelectroporated with a plasmid encoding the Himar1 C9 transposase variant and a plasmid containing a Himar1 transposon encoding chloramphenicol acetyltransferase, mCherry fluorescent protein, and a ColE1 origin of replication. Vero cells were infected with electroporated C. burnetii and transformants scored as organisms replicating in the presence of chloramphenicol and expressing mCherry. Southern blot analysis revealed multiple transpositions in the C. burnetii genome and rescue cloning identified 30 and 5 insertions in coding and noncoding regions, respectively. Using micromanipulation, a C. burnetii clone was isolated containing a Tn insertion within the C terminus of the cell division gene ftsZ. The ftsZ mutant had a significantly lower growth rate than wild-type bacteria and frequently appeared as filamentous forms displaying incomplete cell division septa. The latter phenotype correlated with a deficiency in generating infectious foci on a per-genome basis compared to wild-type organisms. The mutant FtsZ protein was also unable to bind the essential cell division protein FtsA. This is the first description of C. burnetii harboring a defined gene mutation generated by genetic transformation.

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Hedgehog Signaling in the Drosophila Eye and Head: An Analysis of the Effects of Differentpatched Trans-heterozygotes

Genetics ◽

10.1093/genetics/165.4.1915 ◽

2003 ◽

Vol 165 (4) ◽

pp. 1915-1928

Author(s):

Chloe Thomas ◽

Philip W Ingham

Keyword(s):

Cell Fate ◽

Hedgehog Signaling ◽

Missense Mutations ◽

Wild Type ◽

Eye Size ◽

C Terminus ◽

Opposing Effects ◽

Antennal Disc

AbstractCharacterization of different alleles of the Hedgehog receptor patched (ptc) indicates that they can be grouped into several classes. Most mutations result in complete loss of Ptc function. However, missense mutations located within the putative sterol-sensing domain (SSD) or C terminus of ptc encode antimorphic proteins that are unable to repress Smo activity and inhibit wild-type Ptc from doing so, but retain the ability to bind and sequester Hh. Analysis of the eye and head phenotypes of Drosophila melanogaster in various ptc/ptctuf1 heteroallelic combinations shows that these two classes of ptc allele can be easily distinguished by their eye phenotype, but not by their head phenotype. Adult eye size is inversely correlated with head vertex size, suggesting an alteration of cell fate within the eye-antennal disc. A balance between excess cell division and cell death in the mutant eye discs may also contribute to final eye size. In addition, contrary to results reported recently, the role of Hh signaling in the Drosophila head vertex appears to be primarily in patterning rather than in proliferation, with Ptc and Smo having opposing effects on formation of medial structures.

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Mutational analysis of the C-terminal anchoring domains ofStreptococcus mutansP1 antigen: Role of the LPXTGX motif in P1 association with the cell wall

Canadian Journal of Microbiology ◽

10.1139/w00-023 ◽

2000 ◽

Vol 46 (6) ◽

pp. 584-592 ◽

Cited By ~ 4

Author(s):

Song F Lee ◽

Lingqiu Gao

Keyword(s):

Cell Wall ◽

Cell Surface ◽

Streptococcus Mutans ◽

Mutational Analysis ◽

Decisive Role ◽

Site Directed Mutagenesis ◽

Wild Type ◽

Hydrophobic Domain ◽

C Terminus

The salivary agglutinin-interacting adhesin P1 of Streptococcus mutans is anchored to the cell wall via the carboxy (C) terminus, which contains a wall-associated domain, a conserved LPXTGX motif, a hydrophobic domain, and a charged tail. To further investigate the role of the C-terminal anchoring regions in cell wall sorting and anchoring, mutational analysis was performed on P1 in this study. Three truncated P1 mutants and seven site-directed mutants were generated by a polymerase chain reaction-based technique. The mutated P1 genes were returned to the P1-negative S. mutans SM3352 for expression and localization studies by ELISA and Western immunoblotting. The results showed that P1 mutants with deletion of the hydrophobic domain and charged tail, or deletion of the charged tail alone resulted in the secretion of P1 to the culture medium. Results from cellular fractionation experiments with the truncated mutants showed that P1 was not trapped in the membrane or cytoplasm. The site-directed mutants showed normal distribution of P1 to the cell surface as compared to the wild-type. However, when cell walls prepared from the site-directed mutants were boiled with SDS, P1 could be removed readily from the mutants with Thr residue in the LPNTGV motif, altered to either Ser (T1531S) or Phe (T1531F); the mutant with Thr and Gly residues altered to two Phe residues (TG1531-1532FF), and the LPNTGV-deleted mutant (LPNTGV-). In contrast, the wild-type P1 and the other three site-directed P1 mutants (P1529V, N1530I, and G1532F) could not be removed by boiling SDS. When the cell wall P1s from the wild-type, mutants P1529V, N1530I, and G1532F were reacted with an antibody directed against the hydrophobic domain and charged tail, no reaction was detected. However, P1s from mutants T1531S, T1531F, TG1531-1532FF, and LPNTGV-were recognized by the antibody, indicating that the inability of these mutated P1s to firmly link to the cell wall was the result of failure in proteolytic cleavage of the hydrophobic domain and charged tail. In summary, the results suggest that the charged tail plays a decisive role in sorting P1 to the cell surface, while the LPXTGX motif determines the nature of P1-cell wall association. The Thr residue of the LPXTGX motif is required for enzymatic processing to link P1 to the cell wall, presumably via a covalent bond.Key words: antigen P1, cell wall proteins, Streptococcus mutans, protein anchoring, site-directed mutagenesis.

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Loss of the C Terminus of Melanocortin Receptor 2 (MC2R) Results in Impaired Cell Surface Expression and ACTH Insensitivity

Endocrine Reviews ◽

10.1210/edrv.31.6.9987 ◽

2010 ◽

Vol 31 (6) ◽

pp. 943-943

Author(s):

Andrea Hirsch ◽

Eirini Meimaridou ◽

Monica Fernandez-Cancio ◽

Amit V. Pandey ◽

María Clemente ◽

...

Keyword(s):

Cell Surface ◽

Severe Hypoglycemia ◽

Surface Expression ◽

Melanocortin Receptor ◽

Cell Surface Expression ◽

Wild Type ◽

C Terminus ◽

Exact Function ◽

Acth Insensitivity ◽

Glucocorticoid Deficiency

Objective Mutations in melanocortin receptor 2 (MC2R) and its related melanocortin receptor accessory protein (MRAP) cause familial glucocorticoid deficiency. We identified a novel MC2R mutation, K289fs. This unique mutation in the C terminus of MC2R is located in the intracellular part of the protein for which the exact function is unknown. Setting A 6-wk-old boy presented with severe hypoglycemia, unmeasurable cortisol, and grossly elevated ACTH but normal electrolytes. Genetic analysis revealed homozygote K289fs mutation in MC2R. His parents and siblings were heterozygous but phenotypically normal. Intervention and Results The role of the C terminus of MC2R was studied in two cell systems. Because the K289fs mutant changes the last eight amino acids of the protein and leads to protein elongation, wild-type MC2R and C-terminally mutated constructs were tested for activity to respond to ACTH in an OS3 cell-based reporter assay. Wild-type and alanine-substituted constructs responded normally to ACTH. By contrast K289fs and M290X had a total loss of activity. Cell surface assays and confocal localization studies revealed that K289fs and M290X receptors were not found at the cell surface, indicating that their transport from the endoplasmic reticulum to the cell membrane is disrupted. Interestingly, coimmunoprecipitation experiments showed no alteration in the interaction of mutant MC2R with MRAP, suggesting that interaction between these two proteins does not guarantee normal localization. Conclusions Loss of the C terminus of MC2R impairs cell surface expression and ACTH sensitivity but does not disrupt interaction of MC2R with MRAP. These findings highlight the extreme sensitivity of MC2R to structural disruption.

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The beta 4 subunit cytoplasmic domain mediates the interaction of alpha 6 beta 4 integrin with the cytoskeleton of hemidesmosomes.

Molecular Biology of the Cell ◽

10.1091/mbc.4.9.871 ◽

1993 ◽

Vol 4 (9) ◽

pp. 871-884 ◽

Cited By ~ 89

Author(s):

L Spinardi ◽

Y L Ren ◽

R Sanders ◽

F G Giancotti

Keyword(s):

Cell Surface ◽

Cytoplasmic Domain ◽

Wild Type ◽

Type Iii ◽

Amino Acid Region ◽

C Terminus ◽

Mutant Forms ◽

Terminal Pair ◽

Acid Region

The alpha 6 beta 4 integrin is structurally distinct from all the other known integrins because the cytoplasmic domain of beta 4 is unusually large and contains four type III fibronectin-like modules toward its C-terminus. To examine the function of the beta 4 cytoplasmic tail, we have expressed full-length and truncated human beta 4 cDNAs in rat bladder epithelial 804G cells, which form hemidesmosome-like adhesions in vitro. The cDNA encoded wild-type beta 4 subunit associated with endogenous alpha 6 and was recruited at the cell surface within hemidesmosome-like adhesions. A recombinant form of beta 4, lacking almost the entire cytoplasmic domain associated with alpha 6, reached the cell surface but remained diffusely distributed. A beta 4 molecule lacking almost the entire extracellular portion did not associate with alpha 6 but was correctly targeted to the hemidesmosome-like adhesions. Thus, the cytoplasmic portion of beta 4 contains sequences that are required and may be sufficient for the assembly of the alpha 6 beta 4 integrin into hemidesmosomes. To localize these sequences we examined the properties of additional mutant forms of beta 4. A truncated beta 4 subunit, lacking the most C-terminal pair of type III fibronectin homology domains, was incorporated into hemidesmosome-like adhesions, but another recombinant beta 4 molecule, lacking both pairs of type III fibronectin repeats, was not. Finally a recombinant beta 4 molecule, which was created by adjoining the region of the cytoplasmic domain including all type III repeats to the transmembrane segment, was efficiently recruited in hemidesmosome-like adhesions. Taken together these results suggest that the assembly of the alpha 6 beta 4 integrin into hemidesmosomes is mediated by a 303-amino acid region of beta 4 tail that comprises the first pair of type III fibronectin repeats and the segment between the second and third repeats. These data imply a function of a specific segment of the beta 4 cytoplasmic domain in interaction with cytoskeletal components of hemidesmosomes.

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