scholarly journals Enzymes Involved in Anaerobic Respiration Appear To Play a Role in Actinobacillus pleuropneumoniae Virulence

2005 ◽  
Vol 73 (1) ◽  
pp. 226-234 ◽  
Author(s):  
Ilse Jacobsen ◽  
Isabel Hennig-Pauka ◽  
Nina Baltes ◽  
Matthias Trost ◽  
Gerald-F. Gerlach
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Anaerobic Respiration ◽  
Clinical Score ◽  
Lavage Fluid ◽  
Actinobacillus Pleuropneumoniae ◽  
Lung Lesion ◽  
Infection Model ◽  
Binding Motif ◽  
Anaerobic Conditions

ABSTRACT Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to survive on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated sequesters. It was previously demonstrated that a deletion of the anaerobic dimethyl sulfoxide reductase gene (dmsA) results in attenuation in acute disease (N. Baltes, S. Kyaw, I. Hennig-Pauka, and G. F. Gerlach, Infect. Immun. 71:6784-6792, 2003). In the present study, using two-dimensional polyacrylamide gel electrophoresis and quadrupole time-of-flight mass spectrometry, we identified an aspartate ammonia-lyase (AspA) which is upregulated upon induction with bronchoalveolar lavage fluid (BALF). This enzyme is involved in the production of fumarate, an alternative electron acceptor under anaerobic conditions. The coding gene (aspA) was cloned and shown to be present in all A. pleuropneumoniae serotype reference strains. The transcriptional start point was identified downstream of a putative FNR binding motif, and BALF-dependent activation of aspA was confirmed by construction of an isogenic A. pleuropneumoniae mutant carrying a chromosomal aspA::luxAB transcriptional fusion. Two aspA deletion mutants, A. pleuropneumoniae ΔaspA and A. pleuropneumoniae ΔaspAΔdmsA, were constructed, both showing reduced growth under anaerobic conditions in vitro. Pigs challenged with either of the two mutants in an aerosol infection model showed a lower lung lesion score than that of the A. pleuropneumoniae wild-type (wt) controls. Pigs challenged with A. pleuropneumoniae ΔaspAΔdmsA had a significantly lower clinical score, and this mutant was rarely reisolated from unaltered lung tissue; in contrast, A. pleuropneumoniae ΔaspA and the A. pleuropneumoniae wt were consistently reisolated in high numbers. These results suggest that enzymes involved in anaerobic respiration are necessary for the pathogen's ability to persist on respiratory tract epithelium and play an important role in A. pleuropneumoniae pathogenesis.

scholarly journals Peptides containing the PCNA interacting motif APIM bind to the β-clamp and inhibit bacterial growth and mutagenesis

2020 ◽  
Vol 48 (10) ◽  
pp. 5540-5554 ◽  
Author(s):  
Aina Nedal ◽  
Synnøve B Ræder ◽  
Bjørn Dalhus ◽  
Emily Helgesen ◽  
Rune J Forstrøm ◽  
...  
Keyword(s):  
Dna Replication ◽  
Bacterial Growth ◽  
Cell Penetrating Peptides ◽  
Infection Model ◽  
Binding Motif ◽  
Hydrophobic Pocket ◽  
Surface Plasmon Resonance Analysis ◽  
Bacterial Dna

Abstract In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice. Surface plasmon resonance analysis and computer modeling suggest that APIM bind to the hydrophobic pocket on the β-clamp, and accordingly, we find that APIM-peptides inhibit bacterial DNA replication. Interestingly, at sub-lethal concentrations, APIM-peptides have anti-mutagenic activities, and this activity is increased after SOS induction. Our results show that although the sequence homology between the β-clamp and PCNA are modest, the presence of similar polymerase binding pockets in the DNA clamps allows for binding of the eukaryotic binding motif APIM to the bacterial β-clamp. Importantly, because APIM-peptides display both anti-mutagenic and growth inhibitory properties, they may have clinical potential both in combination with other antibiotics and as single agents.

scholarly journals Deletion of the Ferric Uptake Regulator Fur Impairs the In Vitro Growth and Virulence of Actinobacillus pleuropneumoniae

2005 ◽  
Vol 73 (6) ◽  
pp. 3740-3744 ◽  
Author(s):  
Ilse Jacobsen ◽  
Jörg Gerstenberger ◽  
Achim D. Gruber ◽  
Janine T. Bossé ◽  
Paul R. Langford ◽  
...  
Keyword(s):  
Deletion Mutant ◽  
Constitutive Expression ◽  
Actinobacillus Pleuropneumoniae ◽  
Infection Model ◽  
Ferric Uptake Regulator ◽  
In Vitro Growth ◽  
Transferrin Binding Proteins ◽  
Aerosol Infection

ABSTRACT In order to investigate the role of the ferric uptake regulator Fur in the porcine lung pathogen Actinobacillus pleuropneumoniae, we constructed an isogenic in-frame deletion mutant, A. pleuropneumoniae Δfur. This mutant showed constitutive expression of transferrin-binding proteins, growth deficiencies in vitro, and reduced virulence in an aerosol infection model.

scholarly journals Identification of Dimethyl Sulfoxide Reductase in Actinobacillus pleuropneumoniae and Its Role in Infection

2003 ◽  
Vol 71 (12) ◽  
pp. 6784-6792 ◽  
Author(s):  
Nina Baltes ◽  
Isabel Hennig-Pauka ◽  
Ilse Jacobsen ◽  
Achim D. Gruber ◽  
Gerald F. Gerlach
Keyword(s):  
Dimethyl Sulfoxide ◽  
Actinobacillus Pleuropneumoniae ◽  
Growth Conditions ◽  
Open Reading Frames ◽  
Anaerobic Growth ◽  
Infection Model ◽  
Anaerobic Conditions ◽  
Common Strategy ◽  
Dimethyl Sulfoxide Reductase

ABSTRACT Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is capable of persisting in oxygen-deprived surroundings, namely, tonsils and sequestered necrotic lung tissue. Utilization of alternative terminal electron acceptors in the absence of oxygen is a common strategy in bacteria under anaerobic growth conditions. In an experiment aimed at identification of genes expressed in vivo, the putative catalytic subunit DmsA of anaerobic dimethyl sulfoxide reductase was identified in an A. pleuropneumoniae serotype 7 strain. The 90-kDa protein exhibits 85% identity to the putative DmsA protein of Haemophilus influenzae, and its expression was found to be upregulated under anaerobic conditions. Analysis of the unfinished A. pleuropneumoniae genome sequence revealed putative open reading frames (ORFs) encoding DmsB and DmsC proteins situated downstream of the dmsA ORF. In order to investigate the role of the A. pleuropneumoniae DmsA protein in virulence, an isogenic deletion mutant, A. pleuropneumoniae ΔdmsA, was constructed and examined in an aerosol infection model. A. pleuropneumoniae ΔdmsA was attenuated in acute disease, which suggests that genes involved in oxidative metabolism under anaerobic conditions might contribute significantly to A. pleuropneumoniae virulence.

scholarly journals Two TonB Systems in Actinobacillus pleuropneumoniae: Their Roles in Iron Acquisition and Virulence

2004 ◽  
Vol 72 (2) ◽  
pp. 701-708 ◽  
Author(s):  
Amanda J. Beddek ◽  
Brian J. Sheehan ◽  
Janine T. Bossé ◽  
Andrew N. Rycroft ◽  
J. Simon Kroll ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Iron Acquisition ◽  
Actinobacillus Pleuropneumoniae ◽  
Sole Source ◽  
Infection Model ◽  
Iron Restriction ◽  
Tonb System ◽  
Porcine Hemoglobin

ABSTRACT Iron acquisition in vivo by Actinobacillus pleuropneumoniae depends upon a functional TonB system. Tonpitak et al. (W. Tonpitak, S. Thiede, W. Oswald, N. Baltes, and G.-F. Gerlach, Infect. Immun. 68:1164-1170, 2000) have described one such system, associated with tbpBA encoding the transferrin receptor, and here we report a second, termed tonB2. This gene cluster (exbB2-exbD2-tonB2) is highly homologous to those in other Pasteurellaceae, unlike the earlier system described (now termed tonB1), suggesting that it is the indigenous system for this organism. Both tonB2 and tonB1 are upregulated upon iron restriction. TonB2, but not TonB1, was found to be essential for growth in vitro when the sole source of iron was hemin, porcine hemoglobin, or ferrichrome. In the case of iron provided as iron-loaded porcine transferrin, neither tonB mutant was viable. The tonB1 phenotype could be explained by a polar effect of the mutation on transcription of downstream tbp genes. We propose that TonB2 is crucial for the acquisition of iron provided in this form, interacting with accessory proteins of the TonB1 system that have been demonstrated to be necessary by Tonpitak et al. TonB2 appears to play a much more important role in A. pleuropneumoniae virulence than TonB1. In an acute porcine infection model, the tonB2 mutant was found to be highly attenuated, while the tonB1 mutant was not. We hypothesize that acquisition of the tonB1-tbp gene cluster confers a biological advantage through its capacity to utilize transferrin-iron but that TonB1 itself plays little or no part in this process.

scholarly journals Immunity to Avirulent Enterovirus 71 and Coxsackie A16 Virus Protects against Enterovirus 71 Infection in Mice

2007 ◽  
Vol 81 (19) ◽  
pp. 10310-10315 ◽  
Author(s):  
Te-Chia Wu ◽  
Ya-Fang Wang ◽  
Yi-Ping Lee ◽  
Jen-Ren Wang ◽  
Ching-Chuan Liu ◽  
...  
Keyword(s):  
Passive Immunization ◽  
Enterovirus 71 ◽  
Clinical Score ◽  
Cross Reactivity ◽  
Immune Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infection Model ◽  
Ev71 Strain

ABSTRACT In this study, we sought to determine whether intratypic and intertypic cross-reactivity protected against enterovirus 71 (EV71) infection in a murine infection model. We demonstrate that active immunization of 1-day-old mice with avirulent EV71 strain or coxsackie A16 virus (CA16) by the oral route developed anti-EV71 antibodies with neutralizing activity (1:16 and 1:2, respectively). Splenocytes from both EV71- and CA16-immunized mice proliferated upon EV71 or CA16, but not coxsackie B3 virus (CB3), antigen stimulation. Immunized mice became more resistant to virulent EV71 strain challenge than nonimmunized mice. There was an increase in the percentage of activated splenic T cells and B cells in the immunized mice 2 days after EV71 challenge. The CA16 immune serum reacted with EV71 antigens in an enzyme-linked immunosorbent assay and neutralized EV71 but not CB3 or poliovirus at a titer of 1:4. Passive immunization with the CA16 immune serum reduced the clinical score, diminished the organ viral load, and increased the survival rate of mice upon EV71 challenge. CB3 neither shared in vitro cross-reactivity with EV71 nor provided in vivo protection after both active and passive immunization. These results illustrated that live vaccine is feasible for EV71 and that intertypic cross-reactivity of enteroviruses may provide a way to determine the prevalence of EV71.

scholarly journals Branched-Chain Amino Acids Are Required for the Survival and Virulence of Actinobacillus pleuropneumoniae in Swine

2009 ◽  
Vol 77 (11) ◽  
pp. 4925-4933 ◽  
Author(s):  
Sargurunathan Subashchandrabose ◽  
Rhiannon M. LeVeque ◽  
Trevor K. Wagner ◽  
Roy N. Kirkwood ◽  
Matti Kiupel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Antimicrobial Agents ◽  
Actinobacillus Pleuropneumoniae ◽  
Defined Medium ◽  
Acetohydroxyacid Synthase ◽  
Infection Model ◽  
Wild Type ◽  
Branched Chain

ABSTRACT In Actinobacillus pleuropneumoniae, which causes porcine pleuropneumonia, ilvI was identified as an in vivo-induced (ivi) gene and encodes the enzyme acetohydroxyacid synthase (AHAS) required for branched-chain amino acid (BCAA) biosynthesis. ilvI and 7 of 32 additional ivi promoters were upregulated in vitro when grown in chemically defined medium (CDM) lacking BCAA. Based on these observations, we hypothesized that BCAA would be found at limiting concentrations in pulmonary secretions and that A. pleuropneumoniae mutants unable to synthesize BCAA would be attenuated in a porcine infection model. Quantitation of free amino acids in porcine pulmonary epithelial lining fluid showed concentrations of BCAA ranging from 8 to 30 μmol/liter, which is 10 to 17% of the concentration in plasma. The expression of both ilvI and lrp, a global regulator that is required for ilvI expression, was strongly upregulated in CDM containing concentrations of BCAA similar to those found in pulmonary secretions. Deletion-disruption mutants of ilvI and lrp were both auxotrophic for BCAA in CDM and attenuated compared to wild-type A. pleuropneumoniae in competitive index experiments in a pig infection model. Wild-type A. pleuropneumoniae grew in CDM+BCAA but not in CDM−BCAA in the presence of sulfonylurea AHAS inhibitors. These results clearly demonstrate that BCAA availability is limited in the lungs and support the hypothesis that A. pleuropneumoniae, and potentially other pulmonary pathogens, uses limitation of BCAA as a cue to regulate the expression of genes required for survival and virulence. These results further suggest a potential role for AHAS inhibitors as antimicrobial agents against pulmonary pathogens.

scholarly journals Antipseudomonal Bacteriophage Reduces Infective Burden and Inflammatory Response in Murine Lung

2015 ◽  
Vol 60 (2) ◽  
pp. 744-751 ◽  
Author(s):  
Rishi Pabary ◽  
Charanjit Singh ◽  
Sandra Morales ◽  
Andrew Bush ◽  
Khalid Alshafi ◽  
...  
Keyword(s):  
Bronchoalveolar Lavage Fluid ◽  
Bacterial Load ◽  
Inflammatory Cells ◽  
Lavage Fluid ◽  
Clinical Strain ◽  
Infection Model ◽  
Attractive Alternative ◽  
Content Type ◽  
Cytokine Levels

ABSTRACTAs antibiotic resistance increases, there is a need for new therapies to treat infection, particularly in cystic fibrosis (CF), wherePseudomonas aeruginosais a ubiquitous pathogen associated with increased morbidity and mortality. Bacteriophages are an attractive alternative treatment, as they are specific to the target bacteria and have no documented side effects. The efficacy of phage cocktails was establishedin vitro. TwoP. aeruginosastrains were taken forward into an acute murine infection model with bacteriophage administered either prophylactically, simultaneously, or postinfection. The infective burden and inflammation in bronchoalveolar lavage fluid (BALF) were assessed at various times. With low infective doses, both control mice and those undergoing simultaneous phage treatment clearedP. aeruginosainfection at 48 h, but there were fewer neutrophils in BALF of phage-treated mice (median, 73.2 × 104/ml [range, 35.2 to 102.1 × 104/ml] versus 174 × 104/ml [112.1 to 266.8 × 104/ml],P< 0.01 for the clinical strain; median, 122.1 × 104/ml [105.4 to 187.4 × 104/ml] versus 206 × 104/ml [160.1 to 331.6 × 104/ml],P< 0.01 for PAO1). With higher infective doses of PAO1, all phage-treated mice clearedP. aeruginosainfection at 24 h, whereas infection persisted in all control mice (median, 1,305 CFU/ml [range, 190 to 4,700 CFU/ml],P< 0.01). Bacteriophage also reduced CFU/ml in BALF when administered postinfection (24 h) and both CFU/ml and inflammatory cells in BALF when administered prophylactically. A reduction in soluble inflammatory cytokine levels in BALF was also demonstrated under different conditions. Bacteriophages are efficacious in reducing both the bacterial load and inflammation in a murine model ofP. aeruginosalung infection. This study provides proof of concept for future clinical trials in patients with CF.

In Vitro Stability of a Tissue-Type Plasminogen Activator Mutant, BM 06.022, in Human Plasma

1994 ◽  
Vol 72 (06) ◽  
pp. 906-911 ◽  
Author(s):  
D C Rijken ◽  
E Groeneveld ◽  
M M Barrett-Bergshoeff
Keyword(s):  
Plasminogen Activator ◽  
Half Life ◽  
Polyacrylamide Gel Electrophoresis ◽  
Tissue Type ◽  
Single Chain ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Sds Page ◽  
Chain Form

SummaryBM 06.022 is a non-glycosylated mutant of human tissue-type plasminogen activator (t-PA) comprising only the kringle-2 and proteinase domains. The in vivo half-life of BM 06.022 antigen is 4- to 5-fold longer than that of t-PA antigen. The in vitro half-life of the activity of BM 06.022 at therapeutic concentrations in plasma is shorter than that of t-PA. In this study the inactivation of BM 06.022 in plasma was further investigated.Varying concentrations of BM 06.022 were incubated in plasma for 0-150 min. Activity assays on serial samples showed a dose-dependent decline of BM 06.022 activity with a half-life from 72 min at 0.3 μg/ml to 38 min at 10 μg/ml. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by fibrin autography showed the generation of several BM 06.022-complexes. These complexes could be completely precipitated with antibodies against Cl-inactivator, α2-antiplasmin and α1-antitrypsin.During the incubation of BM 06.022 in plasma, plasmin was generated dose-dependently as revealed by varying degrees of a2-anti-plasmin consumption and fibrinogen degradation. SDS-PAGE and immunoblotting showed that single-chain BM 06.022 was rapidly (i. e. within 45 min) converted into its two-chain form at concentrations of 5 μg/ml BM 06.022 and higher.In conclusion, BM 06.022 at therapeutic concentrations in plasma was inactivated by Cl-inactivator, a2-antiplasmin and a j-antitrypsin. The half-life of the activity decreased at increasing BM 06.022 concentrations, probably as a result of the generation of two-chain BM 06.022 which may be inactivated faster than the single-chain form.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

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