Outer membrane proteome ofActinobacillus pleuropneumoniae: LC‐MS/MS analyses validatein silicopredictions

Chung, Jacqueline W.; Ng-Thow-Hing, Christopher; Budman, Lorne; Gibbs, Bernard F.; Nash, John H. E.; Jacques, Mario; Coulton, James W.

doi:10.1002/pmic.200600979

Cited by 50 publications

(58 citation statements)

References 52 publications

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“…Eighty-eight (57%) were in the comprehensive list of in silico predicted OMPs of Bp (Table S1, Supporting Information) and ~40% were PsortB-predicted OMPs. This proportion is similar to that reported for the OM proteomes of Actinobacillus pleuropnemoniae 2 and Erwinia chrysanthemi . 1 We obtained similar results using OMs from B. mallei (see below).…”

Section: Resultssupporting

confidence: 88%

Outer Membrane Proteome of Burkholderia pseudomallei and Burkholderia mallei From Diverse Growth Conditions

2011

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Burkholderia mallei and Burkholderia pseudomallei are closely related, aerosol-infective human pathogens that cause life-threatening diseases. Biochemical analyses requiring large-scale growth and manipulation at biosafety level 3 under select agent regulations are cumbersome and hazardous. We developed a simple, safe, and rapid method to prepare highly purified outer membrane (OM) fragments from these pathogens. Shotgun proteomic analyses of OMs by trypsin shaving and mass spectrometry identified >155 proteins, the majority of which are clearly outer membrane proteins (OMPs). These included: 13 porins, 4 secretins for virulence factor export, 11 efflux pumps, multiple components of a Type VI secreton, metal transport receptors, polysaccharide exporters, and hypothetical OMPs of unknown function. We also identified 20 OMPs in each pathogen that are abundant under a wide variety of conditions, including in serum and with macrophages, suggesting these are fundamental for growth and survival and may represent prime drug or vaccine targets. Comparison of the OM proteomes of B. mallei and B. pseudomallei showed many similarities but also revealed a few differences, perhaps reflecting evolution of B. mallei away from environmental survival toward host-adaptation.

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Section: Resultssupporting

confidence: 88%

Outer Membrane Proteome of Burkholderia pseudomallei and Burkholderia mallei From Diverse Growth Conditions

2011

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“…The cases usually display pleuropneumonia and pulmonary lesions characterized by serious hemorrhage and necrosis. To date, several factors involved in the virulence of A. pleuropneumoniae have been described, including Apx exotoxins, capsular polysaccharides (CPS), lipopolysaccharides (LPS), outer membrane proteins, iron-acquisition proteins and adhesin factors (11,19,24). However, the genetic differences of pathogenesis remain poorly characterized and are worth interpreting from the perspective of comparative genomics for this bacterium.…”

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confidence: 99%

Comparative Genomic Characterization of Actinobacillus pleuropneumoniae

Zhao

Chen

et al. 2010

J Bacteriol

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The Gram-negative bacterium Actinobacillus pleuropneumoniae is the etiologic agent of porcine contagious pleuropneumoniae, a lethal respiratory infectious disease causing great economic losses in the swine industry worldwide. In order to better interpret the genetic background of serotypic diversity, nine genomes of A. pleuropneumoniae reference strains of serovars 1, 2, 4, 6, 9, 10, 11, 12, and 13 were sequenced by using rapid high-throughput approach. Based on 12 genomes of corresponding serovar reference strains including three publicly available complete genomes (serovars 3, 5b, and 7) of this bacterium, we performed a comprehensive analysis of comparative genomics and first reported a global genomic characterization for this pathogen. Clustering of 26,012 predicted protein-coding genes showed that the pan genome of A. pleuropneumoniae consists of 3,303 gene clusters, which contain 1,709 core genome genes, 822 distributed genes, and 772 strain-specific genes. The genome components involved in the biogenesis of capsular polysaccharide and lipopolysaccharide O antigen relative to serovar diversity were compared, and their genetic diversity was depicted. Our findings shed more light on genomic features associated with serovar diversity of A. pleuropneumoniae and provide broader insight into both pathogenesis research and clinical/epidemiological application against the severe disease caused by this swine pathogen.Actinobacillus pleuropneumoniae, a Gram-negative facultative anaerobic encapsulated coccobacillus, belongs to the Actinobacillus genus of the Pasteurellaceae family (19). A. pleuropneumoniae is a primary bacterial etiologic agent of porcine contagious pleuropneumonia, a severe respiratory disease leading to great economic losses to the global swine industry (7). The cases usually display pleuropneumonia and pulmonary lesions characterized by serious hemorrhage and necrosis. To date, several factors involved in the virulence of A. pleuropneumoniae have been described, including Apx exotoxins, capsular polysaccharides (CPS), lipopolysaccharides (LPS), outer membrane proteins, iron-acquisition proteins and adhesin factors (11,19,24). However, the genetic differences of pathogenesis remain poorly characterized and are worth interpreting from the perspective of comparative genomics for this bacterium.Thus far, 15 serovars and two biotypes of A. pleuropneumoniae have been recognized, with great variations in virulence and interlocal distributions (6). The predominant serovar-specific antigens are composed of CPS, which could rigorously define serovars of A. pleuropneumoniae (6, 34). Antigenic differences in the LPS can further determine A. pleuropneumoniae subtypes within a same capsular serovar (13). The metabolic and virulent characteristics of this pathogen have been systematically described based on the prior knowledge and two complete genomes (18, 47), but the molecular basis and evolutionary mechanism of serotypic diversity are still not well explained due to the lack of sequence information. To invest...

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“…Understanding the molecular basis of pathogenicity is important in the design and implementation of vaccine and treatment strategies. Established virulence factors include surface polysaccharides (5,24), Apx toxins (19,29), iron uptake systems (24), components of anaerobic metabolism (3,4,10,11,23), and outer membrane proteins (12). In general, these have been discovered through hypothesis-driven research (9).…”

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New Plasmid Tools for Genetic Analysis of Actinobacillus pleuropneumoniae and Other Pasteurellaceae

Bossé

Durham

Rycroft

et al. 2009

Appl Environ Microbiol

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We have generated a set of plasmids, based on the mobilizable shuttle vector pMIDG100, which can be used as tools for genetic manipulation of Actinobacillus pleuropneumoniae and other members of the Pasteurellaceae. A tandem reporter plasmid, pMC-Tandem, carrying promoterless xylE and gfpmut3 genes downstream of a multiple-cloning site (MCS), can be used for identification of transcriptional regulators and conditions which favor gene expression from different cloned promoters. The ability to detect transcriptional regulators using the tandem reporter system was validated in A. pleuropneumoniae using the cloned rpoE ( E ) promoter (P). The resulting plasmid, pMCrpoEP, was used to identify a mutant defective in production of RseA, the negative regulator of E , among a bank of random transposon mutants, as well as to detect induction of E following exposure of A. pleuropneumoniae to ethanol or heat shock. pMCsodCP, carrying the cloned sodC promoter of A. pleuropneumoniae, was functional in A. pleuropneumoniae, Haemophilus influenzae, Haemophilus parasuis, Mannheimia haemolytica, and Pasteurella multocida. Two general expression vectors, pMK-Express and pMCExpress, which differ in their antibiotic resistance markers (kanamycin and chloramphenicol, respectively), were constructed for the Pasteurellaceae. Both plasmids have the A. pleuropneumoniae sodC promoter upstream of the gfpmut3 gene and an extended MCS. Replacement of gfpmut3 with a gene of interest allows complementation and heterologous gene expression, as evidenced by expression of the Haemophilus ducreyi nadV gene in A. pleuropneumoniae, rendering the latter NAD independent.Actinobacillus pleuropneumoniae is the etiological agent of pleuropneumonia, an economically significant disease responsible for substantial morbidity and mortality in the worldwide pig industry (47). Understanding the molecular basis of pathogenicity is important in the design and implementation of vaccine and treatment strategies. Established virulence factors include surface polysaccharides (5, 24), Apx toxins (19, 29), iron uptake systems (24), components of anaerobic metabolism (3,4,10,11,23), and outer membrane proteins (12). In general, these have been discovered through hypothesis-driven research (9). As with other bacterial pathogens, further advances will be facilitated by the availability of microarrays and genetic tools such as transposons and reporter gene plasmids.Whole-genome sequences are available for A. pleuropneumoniae serovars 3 (accession no. NC_010278), 5b (accession no. NC_009053), and 7 (accession no. NC_010942), with others in progress. Microarrays, developed from the whole-genome sequences, have been used for genotyping (22) as well as to identify genes important for iron uptake (15), anaerobicity (10, 11), interaction with host cells (2), and the role of the global regulators H-NS and RpoE in biofilm formation (J. T. Bossé, S. Sinha, C. A. O'Dwyer, J. H. E. N. Nash, A. N. Rycroft, J. S.Kroll, and P. R. Langford, submitted for publication). It is envisaged that ...

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Outer membrane proteome ofActinobacillus pleuropneumoniae: LC‐MS/MS analyses validatein silicopredictions

Cited by 50 publications

References 52 publications

Outer Membrane Proteome of Burkholderia pseudomallei and Burkholderia mallei From Diverse Growth Conditions

Outer Membrane Proteome of Burkholderia pseudomallei and Burkholderia mallei From Diverse Growth Conditions

Comparative Genomic Characterization of Actinobacillus pleuropneumoniae

New Plasmid Tools for Genetic Analysis of Actinobacillus pleuropneumoniae and Other Pasteurellaceae

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