Genome Analysis and Phylogenetic Relatedness of Gallibacterium anatis Strains from Poultry

Johnson, Timothy J.; Danzeisen, Jessica L.; Trampel, Darrell W.; Nolan, Lisa K.; Seemann, Torsten; Bager, Ragnhild J.; Bojesen, Anders Miki

doi:10.1371/journal.pone.0054844

Cited by 33 publications

(68 citation statements)

References 37 publications

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“…Due to the high degree of predicted hydrophobicity within the Cterminal region of Gab_2312, as well as the size of the whole protein (325.7 kDa), only a small part (338 residues) was included in the final recombinant protein. Homologs of Gab_1309 and Gab_2312 were identified within the genomes of all the 10 strains included in this study, as well as in the recently sequenced G. anatis strain UMN179 (UMN_1211 and UMN_1565) [27], further supporting their potential as broadly protective immunogens. To determine the level of protein sequence conservation of Gab_1309 and Gab_2312, multiple alignments were conducted against the protein homologs from other G. anatis strains.…”

Section: Conservation Of Gab_1309 and Gab_2312supporting

confidence: 64%

“…Of the 42 in silico predicted immunogens (Table 2), 27 proteins were successfully expressed in E. coli (Table 1), and of these, two novel proteins, Gab_1309 and Gab_2312, elicited a significant ELISA response and were also ranked in the Top20 of the G. anatis 12656-12 proteome by VacFinder®. Furthermore, these two proteins were present in all 10 strains included in this study as well as in the recently sequenced genome from G. anatis strain UMN179 [27]. Gab_1309 is a predicted lipoprotein, and this annotation is supported by the DOLOP database [50].…”

Section: Discussionmentioning

confidence: 85%

“…Sequencing and assembly of G. anatis strains 12656-12 and F149 T was performed as described previously [27]. The remaining genomes were sequenced using the Illumina Genome Analyzer IIx (CD genomics, New York, USA).…”

Section: Rv In Silico Analysismentioning

confidence: 99%

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In silico prediction of

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et al. 2014

Vet Res

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The Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in commercial egg-layers, leading to reduced egg production and increased mortality. Unfortunately, widespread multidrug resistance and antigenic diversity makes it difficult to control infections and novel prevention strategies are urgently needed. In this study, a pan-genomic reverse vaccinology (RV) approach was used to identify potential vaccine candidates. Firstly, the genomes of 10 selected Gallibacterium strains were analyzed and proteins selected on the following criteria; predicted surface-exposure or secretion, none or one transmembrane helix (TMH), and presence in six or more of the 10 genomes. In total, 42 proteins were selected. The genes encoding 27 of these proteins were successfully cloned in Escherichia coli and the proteins expressed and purified. To reduce the number of vaccine candidates for in vivo testing, each of the purified recombinant proteins was screened by ELISA for their ability to elicit a significant serological response with serum from chickens that had been infected with G. anatis. Additionally, an in silico prediction of the protective potential was carried out based on a protein property prediction method. Of the 27 proteins, two novel putative immunogens were identified; Gab_1309 and Gab_2312. Moreover, three previously characterized virulence factors; GtxA, FlfA and Gab_2156, were identified. Thus, by combining the pan-genomic RV approach with subsequent in vitro and in silico screening, we have narrowed down the pan-proteome of G. anatis to five vaccine candidates. Importantly, preliminary immunization trials indicated an in vivo protective potential of GtxA-N, FlfA and Gab_1309.

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Section: Conservation Of Gab_1309 and Gab_2312supporting

confidence: 64%

Section: Discussionmentioning

confidence: 85%

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In silico prediction of

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“…Fimbriae are long, hair-like, extracellular appendages on the surfaces of bacterial cells and are important virulence factors in many bacterial species (22). Recently, several F17-like fimbrial biosynthesis gene clusters were identified in the genomes of three different G. anatis strains (23). The most frequently occurring fimbrial gene cluster includes a gene predicted to encode a 20.5-kDa fimbrial subunit designated FlfA (GenBank accession no.…”

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confidence: 99%

The Fimbrial Protein FlfA from Gallibacterium anatis Is a Virulence Factor and Vaccine Candidate

et al. 2013

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cThe Gram-negative bacterium Gallibacterium anatis is a major cause of salpingitis and peritonitis in egg-laying chickens, leading to decreased egg production worldwide. Widespread multidrug resistance largely prevents treatment of this organism using traditional antimicrobial agents, while antigenic diversity hampers disease prevention by classical vaccines. Thus, insight into its pathogenesis and knowledge about important virulence factors is urgently required. A key event during the colonization and invasion of mucosal surfaces is adherence, and recently, at least three F17-like fimbrial gene clusters were identified in the genomes of several G. anatis strains. The objective of this study was to characterize the putative F17-like fimbrial subunit protein FlfA from G. anatis 12656-12 and determine its importance for virulence. In vitro expression and surface exposure of FlfA was demonstrated by flow cytometry and immunofluorescence microscopy. The predicted function of FlfA as a fimbrial subunit protein was confirmed by immunogold electron microscopy. An flfA deletion mutant (⌬flfA) was generated in G. anatis 12656-12, and importantly, this mutant was significantly attenuated in the natural chicken host. Furthermore, protection against G. anatis 12656-12 could be induced by immunizing chickens with recombinant FlfA. Finally, in vitro expression of FlfA homologs was observed in a genetically diverse set of G. anatis strains, suggesting the potential of FlfA as a serotype-independent vaccine candidate This is the first study describing a fimbrial subunit protein of G. anatis with a clear potential as a vaccine antigen.

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“…Virulence factors contributing to the pathogenicity of G. anatis have not yet been well defined, except for an atypical RTX (repeat in toxin) toxin, GtxA, responsible for the hemolytic activity and likely to be a major virulence factor (6,9). Recently, a fimbrial GalF-A was also characterized as a key structure during colonization and invasion events of mucosal surfaces (10).…”

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confidence: 99%