scholarly journals Mutagenesis and Chemical Cross-Linking Suggest that Wzz Dimer Stability and Oligomerization Affect Lipopolysaccharide O-Antigen Modal Chain Length Control

2010 ◽  
Vol 192 (13) ◽  
pp. 3385-3393 ◽  
Author(s):  
Magdalene Papadopoulos ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Three Dimensional ◽  
Class Ii ◽  
Cross Linking ◽  
Wild Type ◽  
Class V ◽  
O Antigen ◽  
Class Iv

ABSTRACT In Shigella flexneri, the polysaccharide copolymerase (PCP) protein WzzSF confers a modal length of 10 to 17 repeat units (RUs) to the O-antigen (Oag) component of lipopolysaccharide (LPS). PCPs form oligomeric structures believed to be related to their function. To identify functionally important regions within WzzSF, random in-frame linker mutagenesis was used to create mutants with 5-amino-acid insertions (termed Wzzi proteins), and DNA sequencing was used to locate the insertions. Analysis of the resulting LPS conferred by Wzzi proteins identified five mutant classes. The class I mutants were inactive, resulting in nonregulated LPS Oag chains, while classes II and III conferred shorter LPS Oag chains of 2 to 10 and 8 to 14 RUs, respectively. Class IV mutants retained near-wild-type function, and class V mutants increased the LPS Oag chain length to 16 to 25 RUs. In vivo formaldehyde cross-linking indicated class V mutants readily formed high-molecular-mass oligomers; however, class II and III Wzzi mutants were not effectively cross-linked. Wzz dimer stability was also investigated by heating cross-linked oligomers at 100°C in the presence of SDS. Unlike the WzzSF wild type and class IV and V Wzzi mutants, the class II and III mutant dimers were not detectable. The location of each insertion was mapped onto available PCP three-dimensional (3D) structures, revealing that class V mutations were most likely located within the inner cavity of the PCP oligomer. These data suggest that the ability to produce stable dimers may be important in determining Oag modal chain length.

scholarly journals Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues

2003 ◽  
Vol 374 (2) ◽  
pp. 413-421 ◽  
Author(s):  
Amro A. AMARA ◽  
Bernd H. A. REHM
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Chain Length ◽  
Class Ii ◽  
Base Catalyst ◽  
Wild Type ◽  
General Base ◽  
Medium Chain Length

The class II PHA (polyhydroxyalkanoate) synthases [PHAMCL synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHAMCLs using CoA thioesters of medium-chain-length 3-hydroxyfatty acids (C6–C14) as a substrate. Only recently PHAMCL synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHAMCL synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the α/β-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the α/β-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved γ-turns of the α/β-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved residue Trp-398 was replaced by Phe and Ala, respectively, which caused inactivation of the enzyme indicating an essential role of this residue. In the threading model this residue was found to be surface-exposed. No evidence for post-translational modification by 4-phosphopantetheine was obtained. Overall, these data suggested that in class II PHA synthases the conserved histidine which was found as general base catalyst in the catalytic triad of enzymes related to the α/β-hydrolase superfamily, was functionally replaced by His-453 which is conserved among all PHA synthases.

scholarly journals The cellular level of O-antigen polymerase Wzy determines chain length regulation by WzzB and WzzpHS-2 in Shigella flexneri 2a

Microbiology ◽  
2009 ◽  
Vol 155 (10) ◽  
pp. 3260-3269 ◽  
Author(s):  
Javier A. Carter ◽  
Juan C. Jiménez ◽  
Mercedes Zaldívar ◽  
Sergio A. Álvarez ◽  
Cristina L. Marolda ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Chain Length ◽  
Shigella Flexneri ◽  
Length Distribution ◽  
Direct Interaction ◽  
Cellular Level ◽  
Chain Length Distribution ◽  
Inducible Promoters ◽  
O Antigen

The lipopolysaccharide O antigen of Shigella flexneri 2a has two preferred chain lengths, a short (S-OAg) composed of an average of 17 repeated units and a very long (VL-OAg) of about 90 repeated units. These chain length distributions are controlled by the chromosomally encoded WzzB and the plasmid-encoded WzzpHS-2 proteins, respectively. In this study, genes wzzB, wzz pHS-2 and wzy (encoding the O-antigen polymerase) were cloned under the control of arabinose- and rhamnose-inducible promoters to investigate the effect of varying their relative expression levels on O antigen polysaccharide chain length distribution. Controlled expression of the chain length regulators wzzB and wzz pHS-2 revealed a dose-dependent production of each modal length. Increase in one mode resulted in a parallel decrease in the other, indicating that chain length regulators compete to control the degree of O antigen polymerization. Also, when expression of the wzy gene is low, S-OAg but not VL-OAg is produced. Production of VL-OAg requires high induction levels of wzy. Thus, the level of expression of wzy is critical in determining O antigen modal distribution. Western blot analyses of membrane proteins showed comparable high levels of the WzzB and WzzpHS-2 proteins, but very low levels of Wzy. In vivo cross-linking experiments and immunoprecipitation of membrane proteins did not detect any direct interaction between Wzy and WzzB, suggesting the possibility that these two proteins may not interact physically but rather by other means such as via translocated O antigen precursors.

scholarly journals Early Endosomes Are Required for Major Histocompatiblity Complex Class II Transport to Peptide-loading Compartments

1999 ◽  
Vol 10 (9) ◽  
pp. 2891-2904 ◽  
Author(s):  
Valérie Brachet ◽  
Gérard Péhau-Arnaudet ◽  
Catherine Desaymard ◽  
Graça Raposo ◽  
Sebastian Amigorena
Keyword(s):  
Mhc Class Ii ◽  
Class Ii ◽  
Endocytic Pathway ◽  
Cross Linking ◽  
Histocompatibility Complex ◽  
Early Endosomes ◽  
Peptide Loading ◽  
Golgi Network ◽  
Mhc Class Ii Molecules

Antigen presentation to CD4+ T lymphocytes requires transport of newly synthesized major histocompatibility complex (MHC) class II molecules to the endocytic pathway, where peptide loading occurs. This step is mediated by a signal located in the cytoplasmic tail of the MHC class II-associated Ii chain, which directs the MHC class II-Ii complexes from the trans-Golgi network (TGN) to endosomes. The subcellular machinery responsible for the specific targeting of MHC class II molecules to the endocytic pathway, as well as the first compartments these molecules enter after exit from the TGN, remain unclear. We have designed an original experimental approach to selectively analyze this step of MHC class II transport. Newly synthesized MHC class II molecules were caused to accumulate in the Golgi apparatus and TGN by incubating the cells at 19°C, and early endosomes were functionally inactivated by in vivo cross-linking of transferrin (Tf) receptor–containing endosomes using Tf-HRP complexes and the HRP-insoluble substrate diaminobenzidine. Inactivation of Tf-containing endosomes caused a marked delay in Ii chain degradation, peptide loading, and MHC class II transport to the cell surface. Thus, early endosomes appear to be required for delivery of MHC class II molecules to the endocytic pathway. Under cross-linking conditions, most αβIi complexes accumulated in tubules and vesicles devoid of γ-adaptin and/or mannose-6-phosphate receptor, suggesting an AP1-independent pathway for the delivery of newly synthesized MHC class II molecules from the TGN to endosomes.

scholarly journals A Role for Site-Specific Phosphorylation of Mouse Progesterone Receptor at Serine 191 in Vivo

2014 ◽  
Vol 28 (12) ◽  
pp. 2025-2037 ◽  
Author(s):  
Sandra L. Grimm ◽  
Robert D. Ward ◽  
Alison E. Obr ◽  
Heather L. Franco ◽  
Rodrigo Fernandez-Valdivia ◽  
...  
Keyword(s):  
Mammary Epithelial Cells ◽  
Phosphorylation Site ◽  
Three Dimensional ◽  
Progesterone Receptors ◽  
Nuclear Factor Κb ◽  
Mammary Epithelial ◽  
Nuclear Factor ◽  
Wild Type ◽  
Receptor Activator

Progesterone receptors (PRs) are phosphorylated on multiple sites, and a variety of roles for phosphorylation have been suggested by cell-based studies. Previous studies using PR-null mice have shown that PR plays an important role in female fertility, regulation of uterine growth, the uterine decidualization response, and proliferation as well as ductal side-branching and alveologenesis in the mammary gland. To study the role of PR phosphorylation in vivo, a mouse was engineered with homozygous replacement of PR with a PR serine-to-alanine mutation at amino acid 191. No overt phenotypes were observed in the mammary glands or uteri of PR S191A treated with progesterone (P4). In contrast, although PR S191A mice were fertile, litters were 19% smaller than wild type and the estrous cycle was lengthened slightly. Moreover, P4-dependent gene regulation in primary mammary epithelial cells (MECs) was altered in a gene-selective manner. MECs derived from wild type and PR S191A mice were grown in a three-dimensional culture. Both formed acinar structures that were morphologically similar, and proliferation was stimulated equally by P4. However, P4 induction of receptor activator of nuclear factor-κB ligand and calcitonin was selectively reduced in S191A cultures. These differences were confirmed in freshly isolated MECs. Chromatin immunoprecipitation analysis showed that the binding of S191A PR to some of the receptor activator of nuclear factor-κB ligand enhancers and a calcitonin enhancer was substantially reduced. Thus, the elimination of a single phosphorylation site is sufficient to modulate PR activity in vivo. PR contains many phosphorylation sites, and the coordinate regulation of multiple sites is a potential mechanism for selective modulation of PR function.

scholarly journals Characterization of Site-Specific Mutations in a Short-Chain-Length/Medium-Chain-Length Polyhydroxyalkanoate Synthase:In VivoandIn VitroStudies of Enzymatic Activity and Substrate Specificity

2013 ◽  
Vol 79 (12) ◽  
pp. 3813-3821 ◽  
Author(s):  
Jo-Ann Chuah ◽  
Satoshi Tomizawa ◽  
Miwa Yamada ◽  
Takeharu Tsuge ◽  
Yoshiharu Doi ◽  
...  
Keyword(s):  
Chain Length ◽  
Fold Increase ◽  
Short Chain ◽  
Pha Synthase ◽  
Wild Type ◽  
Short Chain Length ◽  
Medium Chain ◽  
Medium Chain Length

ABSTRACTSaturation point mutagenesis was carried out at position 479 in the polyhydroxyalkanoate (PHA) synthase fromChromobacteriumsp. strain USM2 (PhaCCs) with specificities for short-chain-length (SCL) [(R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV)] and medium-chain-length (MCL) [(R)-3-hydroxyhexanoate (3HHx)] monomers in an effort to enhance the specificity of the enzyme for 3HHx. A maximum 4-fold increase in 3HHx incorporation and a 1.6-fold increase in PHA biosynthesis, more than the wild-type synthase, was achieved using selected mutant synthases. These increases were subsequently correlated with improved synthase activity and increased preference of PhaCCsfor 3HHx monomers. We found that substitutions with uncharged residues were beneficial, as they resulted in enhanced PHA production and/or 3HHx incorporation. Further analysis led to postulations that the size and geometry of the substrate-binding pocket are determinants of PHA accumulation, 3HHx fraction, and chain length specificity.In vitroactivities for polymerization of 3HV and 3HHx monomers were consistent within vivosubstrate specificities. Ultimately, the preference shown by wild-type and mutant synthases for either SCL (C4and C5) or MCL (C6) substrates substantiates the fundamental classification of PHA synthases.

scholarly journals The normal chain length distribution of the O antigen is required for the interaction of Shigella flexneri 2a with polarized Caco-2 cells

2012 ◽  
Vol 45 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Anilei Hoare ◽  
Denisse Bravo ◽  
Mara Martinic ◽  
Miguel A Valvano ◽  
Inés Contreras ◽  
...  
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Length Distribution ◽  
Chain Length Distribution ◽  
O Antigen ◽  
Shigella Flexneri 2A

scholarly journals Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2

Microbiology ◽  
2008 ◽  
Vol 154 (4) ◽  
pp. 1104-1116 ◽  
Author(s):  
Leanne Purins ◽  
Luisa Van Den Bosch ◽  
Vanessa Richardson ◽  
Renato Morona
Keyword(s):  
Chain Length ◽  
Shigella Flexneri ◽  
Coiled Coil ◽  
O Antigen

scholarly journals Isolation and characterization of a novel temperate Escherichia coli bacteriophage, Kapi1, which modifies the O-antigen and contributes to the competitiveness of its host during colonization of the murine gastrointestinal tract.

2021 ◽  
Author(s):  
Kat Pick ◽  
Tingting Ju ◽  
Benjamin P. Willing ◽  
Tracy Lyn Raivio
Keyword(s):  
Escherichia Coli ◽  
Gastrointestinal Tract ◽  
Molecular Mechanisms ◽  
Shigella Flexneri ◽  
Simulated Intestinal Fluid ◽  
O Antigen ◽  
Isolation And Characterization

In this study, we describe the isolation and characterization of novel bacteriophage vB_EcoP_Kapi1 (Kapi1) isolated from a strain of commensal Escherichia coli inhabiting the gastrointestinal tract of healthy mice. We show that Kapi1 is a temperate phage integrated into tRNA argW of strain MP1 and describe its genome annotation and structure. Kapi1 shows limited homology to other characterized prophages but is most similar to the seroconverting phages of Shigella flexneri, and clusters taxonomically with P22-like phages. The receptor for Kapi1 is the lipopolysaccharide O-antigen, and we further show that Kapi1 alters the structure of its hosts O-antigen in multiple ways.  Kapi1 displays unstable lysogeny, and we find that lysogeny is favored during growth in simulated intestinal fluid. Furthermore, Kapi1 lysogens have a competitive advantage over their non-lysogenic counterparts both in vitro and in vivo, suggesting a role for Kapi1 during colonization. We thus report the use of MP1 and Kapi1 as a model system to explore the molecular mechanisms of mammalian colonization by E. coli to ask what the role(s) of prophages in this context might be.

Morphology classification of circulating tumor cells could be a predictor of recurrent disease in patients with non-small cell lung cancer after surgery.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e15530-e15530
Author(s):  
Yun Wu ◽  
Yuxu Niu ◽  
Fanzhen Lv ◽  
Wen Gao ◽  
Xiaoyong Shen
Keyword(s):  
Lung Cancer ◽  
Recurrent Disease ◽  
Class Ii ◽  
Class I ◽  
Class Iii ◽  
Class V ◽  
Lung Cancer Patients ◽  
Increased Risk ◽  
Class Iv

e15530 Background: CTCs have been widely used in monitoring the efficacy and prognosis of lung cancer. However, CTCs number count alone cannot accurately predict the recurrent disease in patients. In this study, we investigate whether the morphology classification of CTCs could be as a prognostic marker for increased risk of recurrence after surgery. Methods: In this study, 105 lung cancer patients (median age 68y) who underwent surgery were prospectively enrolled in this study. Samples were obtained before, after, and serially up to 24 months after surgery. CTCs were collected and morphology classified by utilizing a CTC test workflow which uses negative enrichment and immunofluorescence methods to capture and identify CTCs from blood sample. Captured CTCs (epithelial type) were screened with a customized imaging analysis pipeline, a cytological profile of each CTC was created, including cell size, shape, fluorescent intensity and texture etc. Results: The CTC detection rate was 78.1% (78 of 105) prior to surgery, and a total of 726 CTCs were enumerated. Median CTC count number was 3. 5 classes of CTCs with distinct morphological features were observed in lung cancer patients’ CTC tests, briefly, CTC class I and class II possessed large nuclei but relatively lower epithelial expression level, CTC class III, IV, V possessed small nuclei but relatively higher epithelial expression level, CTC class III possessed irregular shaped nuclei, CTC class V possessed relatively lower nuclei/cytoplasm ratio. Class III accounted for the highest proportion of captured CTCs III, about 35.5% with Class I 14.8%, Class II 15.3%,Class IV 17.8% and Class 5 16.6%. Postoperative recurrence and metastasis were observed in 16 patients. CTCs positive were found in 14 patients (87.5%). 145 CTCs were collected, Median CTC count number was 3,Cluster III accounted for 47.3%, with Class I 11.8%,Class II 13.3%,Class IV 14.5% and Class V 11.8%; Patients with Cluster 3 dominant were associated with increased risk of local recurrence ( p < 0.05) and distant metastasis ( p < 0.05). Conclusions: Small and irregular nuclei CTC is significant associated with increased risk of recurrence disease. Morphology Classification of circulating tumor cells is feasible in monitoring the recurrence of disease and may potentially identify the patients who may benefit from further therapy.

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