Fhua and HgbA, outer membrane proteins ofActinobacilluspleuropneumoniae: their role as virulence determinants

2006 ◽  
Vol 52 (4) ◽  
pp. 391-396 ◽  
Author(s):  
Lara Shakarji ◽  
Leonie G Mikael ◽  
Ramakrishnan Srikumar ◽  
Marylène Kobisch ◽  
James W Coulton ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Growth Promotion ◽  
Outer Membrane Proteins ◽  
Actinobacillus Pleuropneumoniae ◽  
Membrane Receptors ◽  
Virulence Determinants ◽  
Serotype 1 ◽  
Specific Pathogen ◽  
Polymerase Chain ◽  
Different Sources

For the recently described serotype 15 of biotype I and serotypes 13 and 14 of biotype II of Actinobacillus pleuropneumoniae, fhuA and hgbA were detected by polymerase chain reaction and DNA sequencing. To determine the substrate specificity of the iron receptors FhuA and HgbA and to study their role in the virulence of A. pleuropneumoniae, we used two isogenic A. pleuropneumoniae serotype 1 deletion mutants of fhuA and hgbA. Different sources of iron and siderophores were tested in growth promotion assays. FhuA and HgbA are specific for their ligands ferrichrome and hemoglobin, respectively. The virulence of the two deletion mutant strains was evaluated in experimental infections using specific pathogen-free piglets. While the fhuA mutant (DG02) was as highly virulent as the parental strain S4074, the virulence of the hgbA mutant (ΔhgbA) was reduced. Our data indicate that both FhuA and HgbA are conserved among all serotypes and biotypes of A. pleuropneumoniae and that HgbA, the receptor for porcine hemoglobin, may play a role in virulence.Key words: Actinobacillus pleuropneumoniae, iron uptake, outer membrane receptors, virulence.

scholarly journals Species selectivity of new siderophore-drug conjugates that use specific iron uptake for entry into bacteria.

1996 ◽  
Vol 40 (11) ◽  
pp. 2610-2617 ◽  
Author(s):  
M S Diarra ◽  
M C Lavoie ◽  
M Jacques ◽  
I Darwish ◽  
E K Dolence ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Nalidixic Acid ◽  
Ferric Iron ◽  
Growth Promotion ◽  
Bacterial Species ◽  
Outer Membrane Proteins ◽  
Membrane Receptors ◽  
E Coli ◽  
Drug Conjugates ◽  
Inhibitory Activities

Siderophores selectively bind ferric iron and are involved in receptor-specific iron transport into bacteria. Several types of siderophores were synthesized, and growth-promoting or inhibitory activities when they were conjugated to carbacephalosporin, erythromycylamine, or nalidixic acid were investigated. Overall, 11 types of siderophores and 21 drug conjugates were tested against seven different bacterial species: Escherichia coli, Bordetella bronchiseptica, Pasteurella multocida, Pasteurella haemolytica, Streptococcus suis, Staphylococcus aureus, and Staphylococcus epidermidis. In some species, the inhibitory activities of the drug conjugates were associated with the ability of the bacteria to use the siderophore portion of the molecules for growth promotion in disc diffusion tests (0.04 mumol of conjugate or siderophore per disc). E. coli used catechol-based siderophore portions as well as hydroxamate-based tri-delta-OH-N-OH-delta-N-acetyl-L-ornithine ferric iron ligands for growth under iron-restricted conditions achieved by supplemental ethylenediamine di (O-hydroxyphenylacetic acid) (100 micrograms/ml) and was sensitive to carbacephalosporin conjugated to these siderophore types (up to a 34-mm-diameter inhibition zone). B. bronchiseptica used desferrioxamine B and an isocyanurate-based or trihydroxamate in addition to catechol-based siderophore portions for promotion but was not inhibited by beta-lactam conjugates partly because of the presence of beta-lactamase. P. multocida and P. haemolytica did not use any of the synthetic siderophores for growth promotion, and the inhibitory activities of some conjugates seemed partly linked to their ability to withhold iron from these bacteria, since individual siderophore portions showed some antibacterial effects. Individual siderophores did not promote S. suis growth in restrictive conditions, but the type of ferric iron ligands attached to beta-lactams affected inhibitory activities. The antibacterial activities of the intracellular-acting agents erythromycylamine and nalidixic acid were reduced or lost, even against S. aureus and S. epidermidis, when the agents were conjugated to siderophores. Conjugate-resistant E. coli mutants showed the absence of some iron-regulated outer membrane proteins in gel electrophoresis profiles and in specific phage or colicin sensitivity tests, implying that the drugs used outer membrane receptors of ferric complexes to get into cells.

Molecular cloning of haemoglobin-binding protein HgbA in the outer membrane of Actinobacillus pleuropneumoniae

Microbiology ◽  
2004 ◽  
Vol 150 (6) ◽  
pp. 1723-1734 ◽  
Author(s):  
Ramakrishnan Srikumar ◽  
Leonie G. Mikael ◽  
Peter D. Pawelek ◽  
Ali Khamessan ◽  
Bernard F. Gibbs ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Pasteurella Multocida ◽  
Affinity Purification ◽  
Pcr Amplification ◽  
Actinobacillus Pleuropneumoniae ◽  
Sole Source ◽  
Genomic Libraries ◽  
Iron Deficient ◽  
Serotype 1 ◽  
Internal Peptide

From the porcine pathogen Actinobacillus pleuropneumoniae cultivated in iron-deficient or haem-deficient media, haemoglobin (Hb)-agarose affinity purification was exploited to isolate an outer-membrane protein of ∼105 kDa, designated HgbA. Internal peptide sequences of purified HgbA were used to design oligonucleotide primers for PCR amplification, yielding amplicons that showed partial sequences with homology to hgbA of Pasteurella multocida. Upon screening two genomic libraries of A. pleuropneumoniae serotype 1 strain 4074, positive clones were assembled into an ORF of 2838 bp. HgbA (946 aa) includes a signal peptide of 23 aa and the deduced HgbA sequence (104 890 Da) also demonstrated a possible Ton box. The promoter region of hgbA from A. pleuropneumoniae serotype 1 showed consensus for −35 and −10 sequences and a putative Fur-binding site. RT-PCR confirmed that hgbA of A. pleuropneumoniae is upregulated in response to diminished levels of iron in the culture medium. While an internally deleted hgbA mutant was unable to use pig Hb as sole source of iron for growth, flow cytometry confirmed its Hb binding; the internally deleted sequences may not be required for Hb binding, but appear necessary for the iron supply from Hb. HgbA is required for growth of A. pleuropneumoniae in the presence of Hb as sole iron source.

scholarly journals Genetic and Immunologic Analyses of PlpE, a Lipoprotein Important in Complement-Mediated Killing of Pasteurella haemolytica Serotype 1

1998 ◽  
Vol 66 (12) ◽  
pp. 5613-5619 ◽  
Author(s):  
Karamjeet Pandher ◽  
Anthony W. Confer ◽  
George L. Murphy
Keyword(s):  
Host Defense ◽  
Defense Mechanisms ◽  
Outer Membrane Proteins ◽  
Actinobacillus Pleuropneumoniae ◽  
Immune Serum ◽  
Pasteurella Haemolytica ◽  
Gene Encoding ◽  
E Coli ◽  
Serotype 1 ◽  
Shipping Fever

ABSTRACT Pasteurella haemolytica serotype 1 is the bacterium most commonly associated with bovine shipping fever. The presence of antibodies against P. haemolytica outer membrane proteins (OMPs) correlates statistically with resistance to experimentalP. haemolytica challenge in cattle. Until now, specificP. haemolytica OMPs which elicit antibodies that function in host defense mechanisms have not been identified. In this study, we have cloned and sequenced the gene encoding one such protein, PlpE. Analysis of the deduced amino acid sequence revealed that PlpE is a lipoprotein and that it is similar to an Actinobacillus pleuropneumoniae lipoprotein, OmlA. Affinity-purified, anti-PlpE antibodies recognize a protein in all serotypes of P. haemolytica except serotype 11. We found that intact P. haemolytica and recombinant E. coli expressing PlpE are capable of absorbing anti-PlpE antibodies from bovine immune serum, indicating that PlpE is surface exposed in P. haemolyticaand assumes a similar surface-exposed conformation in E. coli. In complement-mediated killing assays, we observed a significant reduction in killing of P. haemolytica when bovine immune serum that was depleted of anti-PlpE antibodies was used as the source of antibody. Our data suggest that PlpE is surface exposed and immunogenic in cattle and that antibodies against PlpE contribute to host defense against P. haemolytica.

scholarly journals Electrophoretic characterisation of the outer membrane proteins ofYersinia pestisisolated in north-east Brazil

1989 ◽  
Vol 103 (3) ◽  
pp. 595-602 ◽  
Author(s):  
F. G. C. Abath ◽  
A. M. P. Almeida ◽  
L. C. S. Ferreira
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Geographic Origin ◽  
Epidemiological Studies ◽  
Virulence Determinants ◽  
North East ◽  
Sds Page ◽  
Quantitative Alteration

SUMMARYThe outer membrane proteins of 38Yersinia pestisisolates from all known plague foci of north-east Brazil were analysed by SDS-PAGE. Approximately 20 bands were consistently found in all strains analysed and 11 were selected for comparative studies. Although qualitative differences among the electrophoretic profiles of outer membrane proteins of wildY. pestisisolates were not observed, quantitative alterations were clearly noted for most of these proteins. No particular quantitative alteration of the electrophoretic profile of outer membrane proteins could be associated with the period of isolation and geographic origin of the isolates. The 64 kDa outer membrane protein was significantly expressed in higher amounts amongY. pestisstrains isolated from a recent plague outbreak. The possible use of electrophoretic profiles of outer membrane proteins of wildY. pestisisolates as a tool for epidemiological studies and for the analysis of virulence determinants is discussed.

scholarly journals Role of Receptor Proteins for Enterobactin and 2,3-Dihydroxybenzoylserine in Virulence of Salmonella enterica

2003 ◽  
Vol 71 (12) ◽  
pp. 6953-6961 ◽  
Author(s):  
W. Rabsch ◽  
U. Methner ◽  
W. Voigt ◽  
H. Tschäpe ◽  
R. Reissbrodt ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Parental Strain ◽  
Salmonella Enterica ◽  
Growth Promotion ◽  
Outer Membrane Proteins ◽  
Breakdown Product ◽  
Triple Mutant ◽  
Siderophore Receptors ◽  
Systemic Spread ◽  
Serovar Typhimurium

ABSTRACT Single, double, and triple mutants of an enterobactin-deficient mutant strain of Salmonella enterica serovar Typhimurium were constructed that were defective in the expression of the iron-regulated outer membrane proteins (IROMPs) FepA, IroN, and Cir, which are proposed to function as catecholate receptors. Uptake of naturally occurring and chemically synthesized catecholate molecules by these mutants was assessed in standard growth promotion assays. Unique patterns of uptake were identified for each IROMP; specifically, FepA and IroN were confirmed to be required for transport of enterobactin, and all three proteins were shown to function as receptors for the enterobactin breakdown product 2,3-dihydroxybenzoylserine. The fepA, iroN, and cir alleles were transduced to enterobactin-proficient strains of S. enterica serovar Typhimurium and S. enterica serovar Enteritidis, and the resulting phenotypes were confirmed by analysis of outer membrane protein profiles, by sensitivity to KP-736, a catecholate-cephalosporin conjugate, and by growth promotion tests on egg white agar. Intragastric infections of mice with the S. enterica serovar Typhimurium strains indicated that the parental strain and the fepA iroN double mutant were similarly virulent but that the fepA iroN cir triple mutant was significantly attenuated. Moreover, in mixed infections, the fepA iroN mutant showed similar cecal colonization and invasion of the liver to the parental strain, while the triple mutant showed significantly reduced cecal colonization and no measurable spread to the liver. Infections of 4-day-old chicks with S. enterica serovar Enteritidis strains also indicated that mutation of the fepA iroN genes did not significantly reduce cecal colonization and systemic spread compared with those of the parental strain. The results indicate that, while enterobactin uptake is not essential for the virulence of S. enterica serovars in mouse and chicken infection models, the ability to take up 2,3-dihydroxybenzoylserine via any of the three catecholate siderophore receptors appears to play an important role, since the S. enterica serovar Typhimurium triple mutant was significantly attenuated in the mouse model. Salmochelins appear not to be involved in the virulence of S. enterica.

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