Expression and secretion of the RTX-toxin GtxA among members of the genus Gallibacterium

Strains from genus Gallibacterium colonize and occasionally cause disease in a range of bird species. The seven species identified vary with respect to haemolytic activity: Gallibacterium genomospecies 1 and 2 are haemolytic, while G. anatis comprise both haemolytic and nonhaemolytic strains. The remaining species are all non-haemolytic. We previously reported that G. anatis strain 12656-12 expresses an atypical RTX-toxin (repeat in toxin), GtxA, responsible for the haemolytic activity and likely to be a major virulence factor. The aim of this study was to investigate the basis of the variation in haemolytic activity observed among Gallibacterium species and strains. Using PCR and dot blotting we found that the gtxA gene was absent from non-haemolytic Gallibacterium species, but present in Gallibacterium genomospecies 1 and 2. Surprisingly, gtxA was present in both haemolytic and non-haemolytic strains of G. anatis. However, in two out of seven of the non-haemolytic G. anatis strains, gtxA was interrupted by an insertion sequence. We identified a new type I secretion system locus (gtxEBD) and showed that this locus is required for export of GtxA. The gtxEBD locus was identified in all strains possessing gtxA, thus, lack of export system genes cannot explain the non-haemolytic phenotype. Instead we examined expression of gtxA at both the transcript- and protein level by Northern- and Western blotting and found that expression of gtxA varied significantly between strains. In conclusion, we have shown that differences in haemolytic properties among strains of Gallibacterium may be explained by both genotypic differences and by differential expression.