Haemophilus ducreyi Requires the flp Gene Cluster for Microcolony Formation In Vitro

Abstract

Haemophilus ducreyi, the etiologic agent of chancroid, has been shown to form microcolonies when cultured in the presence of human foreskin fibroblasts. We identified a 15-gene cluster in H. ducreyi that encoded predicted protein products with significant homology to those encoded by the tad (for tight adhesion) locus in Actinobacillus actinomycetemcomitans that is involved in the production of fimbriae by this periodontal pathogen. The first three open reading frames in this H. ducreyi gene cluster encoded predicted proteins with a high degree of identity to the Flp (fimbria-like protein) encoded by the first open reading frame of the tad locus; this 15-gene cluster in H. ducreyi was designated flp. RT-PCR analysis indicated that the H. ducreyi flp gene cluster was likely to be a polycistronic operon. Mutations within the flp gene cluster resulted in an inability to form microcolonies in the presence of human foreskin fibroblasts. In addition, the same mutants were defective in the ability to attach to both plastic and human foreskin fibroblasts in vitro. An H. ducreyi mutant with an inactivated tadA gene exhibited a small decrease in virulence in the temperature-dependent rabbit model for experimental chancroid, whereas another H. ducreyi mutant with inactivated flp-1 and flp-2 genes was as virulent as the wild-type parent strain. These results indicate that the flp gene cluster is essential for microcolony formation by H. ducreyi, whereas this phenotypic trait is not linked to the virulence potential of the pathogen, at least in this animal model of infection.

Ralstonia solanacearum Needs Flp Pili for Virulence on Potato

“…Nevertheless, the widespread existence of the tad locus (Kachlany et al 2000;Bhattarcharjee et al 2001;Nika et al 2002;de Bentzmann et al 2006). …”

“…actinomycetemcomitans (Schreiner et al 2003), Haemophilus ducreyi (Nika et al 2002;Spinola et al 2003), Yersinia ruckeri (Fernandez et al 2004) and Pasteurella multocida (Fuller et al 2000). In contrast to these human and animal pathogenic bacteria, the functional significance of the tad locus in phytopathogenic bacteria has not yet been reported.…”

“…Flp pili have been shown to mediate biofilm development (Kachlany et al 2000;Nika et al 2002;de Bentzmann et al 2006), we sought to characterize biofilm formation of the wildtype and mutant strains by making use of quantitative PVC microtiter plate assays. After the R. solanacearum strains were incubated for 48 h at 30°C, biofilm bands were observed at the air-liquid interface.…”

Inhibition of Phagocytosis byHaemophilus ducreyiRequires Expression of the LspA1 and LspA2 Proteins

“…Adherence assay. Adherence of H. ducreyi cells to U-937 macrophages was evaluated with a modified version of a previously described attachment assay (38). The differentiation of the U-937 cells was induced as described above at 2 ϫ 10 5 cells/well.…”

Volatile communication between fungi and bacteria

“…A newly described subtype of T2SS, tad locus (tight adherence) (Tomich et al, 2007) was identified in all six genomes. The tad locus encodes the machinery required for the assembly of adhesive Flp (fimbrial low-molecule-weight protein) pili and is necessary for bacterial adhesion to surfaces, biofilm formation, and pathogenesis as shown for Aggregatibacter actinomycetemcomitans (Planet et al, 2003), Haemophilus (Nika et al, 2002), Pseudomonas (Durand et al, 2003), Yersinia etc. (Tomich et al, 2007).…”