Cloning and expression of a Serpula (Treponema) hyodysenteriae hemolysin gene

Abstract

Serpula (Treponema) hyodysenteriae, the etiologic agent of swine dysentery, produces a hemolysin which is thought to be an important factor in the pathogenesis of the disease. We report the cloning, sequencing, and expression of a hemolysin gene (tly) from S. hyodysenteriae B204. A pUC19 gene bank of strain B204 was constructed in the Escherichia coli K-12 strain DH5a, and hemolytic recombinants were identified by plating the library on blood agar plates. From the hemolytic recombinants, a 1.5-kb DNA fragment could be isolated that contained information necessary for the production of a hemolysin/cytotoxin in E. coli. Nucleotide sequence determination of this 1.5-kb fragment showed that it contained an open reading frame capable of encoding a 26.9-kDa protein. The recombinant hemolysin was easily released from E. coli by osmotic shock. As with the native hemolysin, the recombinant hemolysin is EDTA insensitive, thermolabile, and cytotoxic for several eukaryotic cell lines. Southern blot hybridization showed that the cloned S. hyodysenteriae hemolysin gene tly is present in all pathogenic strains of S. hyodysenteriae tested and absent in the nonpathogenic, weakly hemolytic spirochete S. innocens.

Identification of a new hemolysin from diarrheal isolate SSU ofAeromonas hydrophila

“…The Serpula (Treponema) hyodysenteriae hemolysin homolog genes (e.g. tlyA and tlyC) (Muir et al, 1992;Hsu et al, 2001) were recently demonstrated in other pathogenic bacterial species (e.g. Rickettsia, Helicobacter, and Salmonella) (Martino et al, 2001;Whitworth et al, 2005), and TlyC from R. typhi was shown to be functional and to also possess hemolytic activity (Radulovic et al, 1999).…”

Identification of Brachyspira hyodysenteriae-specific DNA fragments using representational difference analysis

“…Treatment is hampered by a limited number of e¡ective antibiotics and an increasing number of resistant strains [3], and vaccination with whole-cell bacterins is not e¡ective [4]. Research concerning potential virulence factors of B. hyodysenteriae has focussed on motility [5] and motility-associated genes [6] as well as on di¡erent hemolysins (tlyA [7], tlyB and tlyC [8], and hlyA [9]). In addition, the NADH oxidase [10] and several surface-associated proteins have been identi¢ed [11], and a physical and genetic map of B. hyodysenteriae B78 T has been constructed [12].…”

Identification ofBrachyspira hyodysenteriae-specific DNA fragments using representational difference analysis

“…Treatment is hampered by a limited number of effective antibiotics and an increasing number of resistant strains[3], and vaccination with whole‐cell bacterins is not effective[4]. Research concerning potential virulence factors of B. hyodysenteriae has focussed on motility[5] and motility‐associated genes[6] as well as on different hemolysins ( tly A[7], tly B and tly C[8], and hly A[9]). In addition, the NADH oxidase[10] and several surface‐associated proteins have been identified[11], and a physical and genetic map of B. hyodysenteriae B78 T has been constructed[12].…”