scholarly journals Characterization and protective activity of monoclonal antibodies directed against Fe (3+) ABC transporter substrate-binding protein of Glaesserella parasuis

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Kexin Zhu ◽  
Dong Yu ◽  
Jiahui An ◽  
Yufeng Li
Keyword(s):  
Monoclonal Antibodies ◽  
Abc Transporter ◽  
Subunit Vaccine ◽  
Binding Protein ◽  
Substrate Binding ◽  
Passive Immunization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
And Control ◽  
Substrate Binding Protein

AbstractGlässer’s disease is caused by the agent Glaesserella parasuis and is difficult to prevent and control. Candidate screening for subunit vaccines contributes to the prevention of this disease. Therefore, in this study, the inactivated G. parasuis reference serovar 5 strain (G. parasuis-5) was used to generate specific monoclonal antibodies (mAbs) to screen subunit vaccine candidates. Six mAbs (1A12, 3E3, 4C6, 2D1, 3E6, and 4B2) were screened, and they all reacted with the G. parasuis serovar 5 strain according to laser confocal microscopy and flow cytometry (FCM). Indirect enzyme-linked immunosorbent assay (ELISA) showed that one mAb 2D1, can react with all 15 reference serovars of G. parasuis. Protein mass spectrometry and Western blot analysis demonstrated that mAb 2D1 specifically reacts with Fe (3+) ABC transporter substrate-binding protein. A complement killing assay found that the colony numbers of bacteria were significantly reduced in the G. parasuis-5 group incubated with mAb 2D1 (p < 0.01) in comparison with the control group. Opsonophagocytic assays demonstrated that mAb 2D1 significantly enhanced the phagocytosis of 3D4/21 cells by G. parasuis (p < 0.05). RAW264.7 cells with stronger phagocytic ability were also used for the opsonophagocytic assay, and the difference was highly significant (p < 0.01). Passive immunization of mice revealed that mAb 2D1 can eliminate the bacteria in the blood and provide protection against G. parasuis-5. Our study found one mAb that can be used to prevent and control G. parasuis infection in vivo and in vitro, which may suggest that Fe (3+) ABC transporter substrate-binding protein is an immunodominant antigen and a promising candidate for subunit vaccine development.

scholarly journals Substrate Binding Protein SBP2 of a Putative ABC Transporter as a Novel Vaccine Antigen of Moraxella catarrhalis

2014 ◽  
Vol 82 (8) ◽  
pp. 3503-3512 ◽  
Author(s):  
Taketo Otsuka ◽  
Charmaine Kirkham ◽  
Antoinette Johnson ◽  
Megan M. Jones ◽  
Timothy F. Murphy
Keyword(s):  
Otitis Media ◽  
Moraxella Catarrhalis ◽  
Binding Protein ◽  
Substrate Binding ◽  
Vaccine Antigen ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Chronic Obstructive ◽  
Content Type ◽  
Substrate Binding Protein

ABSTRACTMoraxella catarrhalisis a common respiratory tract pathogen that causes otitis media in children and infections in adults with chronic obstructive pulmonary disease. Since the introduction of the pneumococcal conjugate vaccines with/without protein D of nontypeableHaemophilus influenzae,M. catarrhalishas become a high-priority pathogen in otitis media. For the development of antibacterial vaccines and therapies, substrate binding proteins of ATP-binding cassette transporters are important targets. In this study, we identified and characterized a substrate binding protein, SBP2, ofM. catarrhalis. Among 30 clinical isolates tested, thesbp2gene sequence was highly conserved. In 2 different analyses (whole-cell enzyme-linked immunosorbent assay and flow cytometry), polyclonal antibodies raised to recombinant SBP2 demonstrated that SBP2 expresses epitopes on the bacterial surface of the wild type but not thesbp2mutant. Mice immunized with recombinant SBP2 showed significantly enhanced clearance ofM. catarrhalisfrom the lung compared to that in the control group at both 25-μg and 50-μg doses (P< 0.001). We conclude that SBP2 is a novel, attractive candidate as a vaccine antigen againstM. catarrhalis.

scholarly journals Substrate recognition and ATPase activity of the E. coli cysteine/cystine ABC transporter YecSC-FliY

2020 ◽  
Vol 295 (16) ◽  
pp. 5245-5256 ◽  
Author(s):  
Siwar Sabrialabed ◽  
Janet G. Yang ◽  
Elon Yariv ◽  
Nir Ben-Tal ◽  
Oded Lewinson
Keyword(s):  
Atpase Activity ◽  
Abc Transporter ◽  
Conformational Changes ◽  
Binding Protein ◽  
Substrate Binding ◽  
Transporter Family ◽  
Wide Range ◽  
Substrate Binding Protein ◽  
Sulfur Containing ◽  
Sulfur Containing Compounds

Sulfur is essential for biological processes such as amino acid biogenesis, iron–sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer–substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine–bound FliY, and maximally by l-cysteine– or l-cystine–bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

scholarly journals Molecular Basis of Unexpected Specificity of ABC Transporter-Associated Substrate-Binding Protein DppA from Helicobacter pylori

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Mohammad M. Rahman ◽  
Mayra A. Machuca ◽  
Mohammad F. Khan ◽  
Christopher K. Barlow ◽  
Ralf B. Schittenhelm ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Binding Pocket ◽  
Amino Acid Sequences ◽  
Content Type ◽  
H Pylori ◽  
Substrate Binding Protein ◽  
Broad Specificity

ABSTRACT The gastric pathogen Helicobacter pylori has limited ability to use carbohydrates as a carbon source, relying instead on exogenous amino acids and peptides. Uptake of certain peptides by H. pylori requires an ATP binding cassette (ABC) transporter annotated dipeptide permease (Dpp). The transporter specificity is determined by its cognate substrate-binding protein DppA, which captures ligands in the periplasm and delivers them to the permease. Here, we show that, unlike previously characterized DppA proteins, H. pylori DppA binds, with micromolar affinity, peptides of diverse amino acid sequences ranging between two and eight residues in length. We present analysis of the 1.45-Å-resolution crystal structure of its complex with the tetrapeptide STSA, which provides a structural rationale for the observed broad specificity. Analysis of the molecular surface revealed a ligand-binding pocket that is large enough to accommodate peptides of up to nine residues in length. The structure suggests that H. pylori DppA is able to recognize a wide range of peptide sequences by forming interactions primarily with the peptide main chain atoms. The loop that terminates the peptide-binding pocket in DppAs from other bacteria is significantly shorter in the H. pylori protein, providing an explanation for its ability to bind longer peptides. The subsites accommodating the two N-terminal residues of the peptide ligand make the greatest contribution to the protein-ligand binding energy, in agreement with the observation that dipeptides bind with affinity close to that of longer peptides. IMPORTANCE The World Health Organization listed Helicobacter pylori as a high-priority pathogen for antibiotic development. The potential of using peptide transporters in drug design is well recognized. We discovered that the substrate-binding protein of the ABC transporter for peptides, termed dipeptide permease, is an unusual member of its family in that it directly binds peptides of diverse amino acid sequences, ranging between two and eight residues in length. We also provided a structural rationale for the observed broad specificity. Since the ability to import peptides as a source of carbon is critical for H. pylori, our findings will inform drug design strategies based on inhibition or fusion of membrane-impermeant antimicrobials with peptides.

Heme-Binding Properties of HupD Functioning as a Substrate-Binding Protein in a Heme-Uptake ABC-Transporter System in Listeria monocytogenes

2014 ◽  
Vol 87 (10) ◽  
pp. 1140-1146 ◽  
Author(s):  
Yasunori Okamoto ◽  
Hitomi Sawai ◽  
Mariko Ogura ◽  
Takeshi Uchida ◽  
Koichiro Ishimori ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Heme Binding ◽  
Heme Uptake ◽  
Binding Properties ◽  
Substrate Binding Protein

Structures of the substrate-binding protein provide insights into the multiple compatible solute binding specificities of the Bacillus subtilis ABC transporter OpuC

2011 ◽  
Vol 436 (2) ◽  
pp. 283-289 ◽  
Author(s):  
Yang Du ◽  
Wei-Wei Shi ◽  
Yong-Xing He ◽  
Yi-Hu Yang ◽  
Cong-Zhao Zhou ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Compatible Solutes ◽  
Binding Pocket ◽  
Compatible Solute ◽  
Structural Basis ◽  
Binding Property ◽  
Substrate Binding Protein

The compatible solute ABC (ATP-binding cassette) transporters are indispensable for acquiring a variety of compatible solutes under osmotic stress in Bacillus subtilis. The substrate-binding protein OpuCC (Opu is osmoprotectant uptake) of the ABC transporter OpuC can recognize a broad spectrum of compatible solutes, compared with its 70% sequence-identical paralogue OpuBC that can solely bind choline. To explore the structural basis of this difference of substrate specificity, we determined crystal structures of OpuCC in the apo-form and in complex with carnitine, glycine betaine, choline and ectoine respectively. OpuCC is composed of two α/β/α globular sandwich domains linked by two hinge regions, with a substrate-binding pocket located at the interdomain cleft. Upon substrate binding, the two domains shift towards each other to trap the substrate. Comparative structural analysis revealed a plastic pocket that fits various compatible solutes, which attributes the multiple-substrate binding property to OpuCC. This plasticity is a gain-of-function via a single-residue mutation of Thr94 in OpuCC compared with Asp96 in OpuBC.

scholarly journals The Escherichia coli metD Locus Encodes an ABC Transporter Which Includes Abc (MetN), YaeE (MetI), and YaeC (MetQ)

2002 ◽  
Vol 184 (19) ◽  
pp. 5513-5517 ◽  
Author(s):  
Christophe Merlin ◽  
Gregory Gardiner ◽  
Sylvain Durand ◽  
Millicent Masters
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Abc Transporter ◽  
Binding Protein ◽  
Substrate Binding ◽  
Uptake System ◽  
Methionine Uptake ◽  
Substrate Binding Protein

ABSTRACT We report that the genes abc, yaeC, and yaeE comprise metD, an Escherichia coli locus encoding a dl-methionine uptake system. MetD is an ABC transporter with Abc the ATPase, YaeE the permease, and YaeC the likely substrate binding protein. Expression of these genes is regulated by l-methionine and MetJ, a common repressor of the methionine regulon. We propose to rename abc, yaeE, and yaeC as metN, metI, and metQ, respectively.

scholarly journals Identification of the Immunogenic Outer Membrane Protein A Antigen of Haemophilus parasuis by a Proteomics Approach and Passive Immunization with Monoclonal Antibodies in Mice

2011 ◽  
Vol 18 (10) ◽  
pp. 1695-1701 ◽  
Author(s):  
Huabin Tian ◽  
Fang Fu ◽  
Xuesong Li ◽  
Xin Chen ◽  
Wei Wang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Field Isolate ◽  
Protein A ◽  
Passive Immunization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Bacterial Cells ◽  
Haemophilus Parasuis ◽  
Two Dimensional Gel Electrophoresis ◽  
Content Type

ABSTRACTMonoclonal antibodies (MAbs) againstHaemophilus parasuiswere generated by fusing spleen cells from BALB/c mice immunized with whole bacterial cells with SP2/0 murine myeloma cells. Desirable hybridomas were screened by enzyme-linked immunosorbent assay (ELISA). Neutralizing MAb 1D8 was selected in protection assays. ELISA results demonstrated that 1D8 can react with all 15 serotypes ofH. parasuisand field isolateH. parasuisHLJ-018. Passive immunization studies showed that mice inoculated intraperitoneally with 1D8 had significantly reduced prevalence ofH. parasuiscolonization in the blood, lung, spleen, and liver and had prolonged survival time compared to that of the control group. Furthermore, the passive transfer experiment indicated that MAb 1D8 can protect mice from both homologous and heterologous challenges withH. parasuis. Using two-dimensional gel electrophoresis (2-DE), the immunoreactive protein target for MAb 1D8 was identified. The data presented confirm the protective role of MAb 1D8 and identify OmpA as the target of the protective monoclonal antibody. The data suggest that OmpA is a promising candidate for a subunit vaccine againstH. parasuis.

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