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Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases.

ABSTRACT: Polysialic acids are bioactive carbohydrates found in eukaryotes and some bacterial pathogens. The bacterial polysialyltransferases (PSTs), which catalyze the synthesis of polysialic acid capsules, have previously been identified in select strains of Escherichia coli and Neisseria meningitidis and are classified in the Carbohydrate-Active enZYmes Database as glycosyltransferase family GT-38. In this study using DNA sequence analysis and functional characterization we have identified a novel polysialyltransferase from the bovine/ovine pathogen Mannheimia haemolytica A2 (PSTMh). The enzyme was expressed in recombinant form as a soluble maltose-binding-protein fusion in parallel with the related PSTs from E. coli K1 and N. meningitidis group B in order to perform a side-by-side comparison. Biochemical properties including solubility, acceptor preference, reaction pH optima, thermostability, kinetics, and product chain length for the enzymes were compared using a synthetic fluorescent acceptor molecule. PSTMh exhibited biochemical properties that make it an attractive candidate for chemi-enzymatic synthesis applications of polysialic acid. The activity of PSTMh was examined on a model glycoprotein and the surface of a neuroprogenitor cell line where the results supported its development for use in applications to therapeutic protein modification and cell surface glycan remodelling to enable cell migration at implantation sites to promote wound healing. The three PSTs examined here demonstrated different properties that would each be useful to therapeutic applications.

SUBMITTER: Lindhout T

PROVIDER: S-EPMC3724679 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases.

Lindhout Theresa T Bainbridge Cynthia R CR Costain Will J WJ Gilbert Michel M Wakarchuk Warren W WW

PloS one 20130726 7

Polysialic acids are bioactive carbohydrates found in eukaryotes and some bacterial pathogens. The bacterial polysialyltransferases (PSTs), which catalyze the synthesis of polysialic acid capsules, have previously been identified in select strains of Escherichia coli and Neisseria meningitidis and are classified in the Carbohydrate-Active enZYmes Database as glycosyltransferase family GT-38. In this study using DNA sequence analysis and functional characterization we have identified a novel poly ...[more]

PMID: 23922842

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Project description:Six Lactobacillus strains originating from the nasopharyngeal microbiota of cattle were previously characterized in vitro and identified as candidate bacterial therapeutics (BTs) for mitigating the bovine respiratory pathogen Mannheimia haemolytica In the present study, these BT strains were evaluated for their potential to (i) reduce nasal colonization by M. haemolytica, (ii) modulate the nasal microbiota, and (iii) stimulate an immune response in calves experimentally challenged with M. haemolytica. Twenty-four Holstein bull calves (1 to 3 weeks old) received either an intranasal BT cocktail containing 6 Lactobacillus strains (3?×?109 CFU per strain; BT + Mh group) 24?h prior to intranasal M. haemolytica challenge (3?×?108 CFU) or no BTs prior to challenge (Mh, control group). Nasal swab, blood, and transtracheal aspiration samples were collected over the course of 16?days after BT inoculation. Counts of M. haemolytica were determined by culturing, and the nasal and tracheal microbiotas were evaluated using 16S rRNA gene sequencing. Serum cytokines (interleukin-6 [IL-6], IL-8, and IL-10) were quantified by enzyme-linked immunosorbent assay (ELISA). Administration of BT reduced nasal colonization by M. haemolytica (P?=?0.02), modified the composition and diversity of the nasal microbiota, and altered interbacterial relationships among the 10 most relatively abundant genera. The BT + Mh calves also had a lower relative abundance of Mannheimia in the trachea (P?<?0.01) but similar cytokine levels as Mh calves. This study demonstrated that intranasal BTs developed from the bovine nasopharyngeal Lactobacillus spp. were effective in reducing nasal colonization by M. haemolytica in dairy calves.IMPORTANCE Bovine respiratory disease (BRD) is one of the significant challenges for the modern dairy industry in North America, accounting for 23 to 47% of the total mortality among pre- and postweaned dairy heifers. Mass medication with antibiotics is a common practice to control BRD in dairy cattle. However, the emergence of multidrug-resistant BRD pathogens highlights the importance of developing alternatives to antibiotics for BRD mitigation. Using a targeted approach, we recently identified 6 Lactobacillus strains originating from the bovine respiratory microbiota as candidates to be used as bacterial therapeutics (BTs) for the mitigation of the BRD pathogen Mannheimia haemolytica Here, we demonstrated that intranasal inoculation of the BT strains reduced nasal colonization by M. haemolytica in dairy calves experimentally challenged with this pathogen. This study, for the first time, shows the potential use of intranasal BTs as an alternative to mitigate BRD pathogens in cattle.

Project description:AimThis study was aimed to detect Mannheimia haemolytica in lung tissues of sheep and from a bacterial culture.IntroductionM. haemolytica is one of the most important and well-established etiological agents of pneumonia in sheep and other ruminants throughout the world. Accurate diagnosis of M. haemolytica primarily relies on bacteriological examination, biochemical characteristics and, biotyping and serotyping of the isolates. In an effort to facilitate rapid M. haemolytica detection, polymerase chain reaction assay targeting Pasteurella haemolytica serotype-1 specific antigens (PHSSA), Rpt2 and 12S ribosomal RNA (rRNA) genes were used to detect M. haemolytica directly from lung tissues and from bacterial culture.Materials and methodsA total of 12 archived lung tissues from sheep that died of pneumonia on an organized farm were used. A multiplex polymerase chain reaction (mPCR) based on two-amplicons targeted PHSSA and Rpt2 genes of M. haemolytica were used for identification of M. haemolytica isolates in culture from the lung samples. All the 12 lung tissue samples were tested for the presence M. haemolytica by PHSSA and Rpt2 genes based PCR and its confirmation by sequencing of the amplicons.ResultsAll the 12 lung tissue samples tested for the presence of PHSSA and Rpt2 genes of M. haemolytica by mPCR were found to be positive. Amplification of 12S rRNA gene fragment as internal amplification control was obtained with each mPCR reaction performed from DNA extracted directly from lung tissue samples. All the M. haemolytica were also positive for mPCR. No amplified DNA bands were observed for negative control reactions. All the three nucleotide sequences were deposited in NCBI GenBank (Accession No. KJ534629, KJ534630 and KJ534631). Sequencing of the amplified products revealed the identity of 99-100%, with published sequence of PHSSA and Rpt2 genes of M. haemolytica available in the NCBI database. Sheep specific mitochondrial 12S rRNA gene sequence also revealed the identity of 98% with published sequences in the NCBI database.ConclusionThe present study emphasized the PCR as a valuable tool for rapid detection of M. haemolytica in clinical samples from animals. In addition, it offers the opportunity to perform large-scale epidemiological studies regarding the role of M. haemolytica in clinical cases of pneumonia and other disease manifestations in sheep and other ruminants, thereby providing the basis for effective preventive strategies.

Dataset Information

Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases.

Publications

Biochemical characterization of a polysialyltransferase from Mannheimia haemolytica A2 and comparison to other bacterial polysialyltransferases.

Similar Datasets

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