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

Abstract

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.

In silico prediction of

“…The results from these preliminary in vivo immunization trials indicated that at least GtxA-N, FlfA and Gab_1309 are promising vaccine candidates with a good protective potential. These results correspond well with previous results demonstrating the in vivo protective potential of FlfA [24]. Further studies, including larger group sizes, different doses and repeated immunization, are needed to confirm the protective potential of the recombinant proteins.…”

“…By implementing different in silico approaches and in vitro assays, we screened the Gallibacterium panproteome, resulting in a final selection of five proteins with a high predicted potential as vaccine candidates. Importantly, preliminary in vitro immunization results indicate protective potential of at least three of these candidates including FlfA, which has previously been tested and confirmed highly protective against homologous challenge in chickens [24]. Together, these results provide an important step in the development of a new and broadly protective vaccine against G. anatis.…”

“…In support of the ELISA antigen screening approach, three of the 10 proteins identified by the ELISA screening; GtxA, FlfA and Gab_2156, have recently been demonstrated as important, well-conserved G. anatis virulencefactors. GtxA is a large, cytolytic RTX toxin responsible for both the haemolytic and leukotoxic activity of G. anatis [26,35], while FlfA and Gab_2156 are both subunits of F17-like fimbriae [24]. To this, FlfA has been suggested to play a role in the tissue tropism of G. anatis by promoting adhesion to the epithelial lining in the reproductive tract and beyond during the typical course of disease [24].…”

“…GtxA is a large, cytolytic RTX toxin responsible for both the haemolytic and leukotoxic activity of G. anatis [26,35], while FlfA and Gab_2156 are both subunits of F17-like fimbriae [24]. To this, FlfA has been suggested to play a role in the tissue tropism of G. anatis by promoting adhesion to the epithelial lining in the reproductive tract and beyond during the typical course of disease [24]. The identification of GtxA, FlfA and Gab_2156 by ELISA further supports the predictive potential and importance of this screening approach, where particularly the Nterminal part of the GtxA-N elicited an extremely high antibody response in comparison to the remaining 26 proteins investigated (Figure 1 and Table 3), thereby suggesting that this part of the protein is particularly promising as a vaccine candidate.…”

“…A protein was considered present if a significant full length match (E-value < 10 −8 ) was obtained. From a total of 31564 proteins, 162 proteins were predicted as extracellular proteins and 482 proteins as Previously described in [24]. Gab_1164 = FlfA.…”