Molecular cloning, expression, and DNA sequence analysis of the gene that encodes the 16-kilodalton outer membrane lipoprotein of Serpulina hyodysenteriae

Abstract

The gene (smpA) that encodes the 16-kDa outer membrane lipoprotein of Serpulina hyodysenteriae was cloned in Escherichia coli, and its primary structure was determined by nucleotide sequencing. The putative open reading frame encodes a prolipoprotein of 16.8 kDa which in its fully acylated and cleaved form is 15.1 kDa. Analysis of the N-terminal amino acid sequence derived from the DNA sequence revealed the presence of a signal sequence and a putative acylation and signal peptidase II cleavage site (Phe-Ala-Val-Ser-Cys). In E. coli, processing of the prolipoprotein was less efficient than that observed in S. hyodysenteriae, and globomycin, an inhibitor of signal peptidase II, inhibited cleavage of the lipoprotein expressed in E. coli but did not inhibit cleavage in S. hyodysenteriae.When Serpulina hyodysenteriae is inoculated into conventionally reared pigs, clinical signs of dysentery consisting of mucohemorrhagic diarrhea are produced (12, 33). Attempts have been made to develop vaccines against infection by S. hyodysenteriae which have been based on whole-cell bacterins (11,15,21,23) and oral vaccination with attenuated strains of the spirochaete given either alone (16) or together with a parenterally administered bacterin (15). These vaccines have provided some degree of protection from experimental oral challenge with S. hyodysenteriae (36) and in challenge experiments using colonic intestinal loops in pigs (18,19). However, protection was serotype specific and probably relied upon an immune response to endotoxin or lipopolysaccharide components.Antigens other than lipopolysaccharide may reside in the outer membrane or endoflagella of S. hyodysenteriae and may stimulate protective immune responses. Our previous investigations have demonstrated immune responses to the endoflagellum polypeptides of 29 to 44 kDa (20) and also to a 16-kDa membrane-associated polypeptide which was common to strains of S. hyodysenteriae (27). This antigen has been identified as an outer membrane lipoprotein which is exposed on the surface of the spirochete (34, 35). Both polyclonal (27) and monoclonal antibodies (MAbs) (34) to this antigen inhibited the growth of S. hyodysenteriae strains in vitro. Therefore, protective immune responses may be stimulated by this antigen, and this report concerns the molecular cloning, DNA sequence analysis, and expression of the gene that encodes this lipoprotein in Escherichia coli. Cloning strategy. High-molecular-weight genomic DNA was prepared from S. hyodysenteriae P18A (28). The DNA was partially digested with Sau3a and size fractionated on a 10 to 40% (wt/vol) sucrose gradient at 20,000 x g for 18 h. The 2to 6-kbp fraction was partially end filled with nucleotides G and A by using DNA polymerase (Klenow). The XZAP II vector (Stratagene, La Jolla, Calif.) was prepared by digestion with XhoI, and the single-stranded ends were partially end filled with nucleotides T and C. After ligation of * Corresponding author. genomic DNA and the vector, the DNA was packaged in vitro by using packaging ext...

Outer membrane proteins of pathogenic spirochetes

“…For example, the leptospiral outer membrane lipoprotein LipL36, which is exclusively located on the periplasmic face of the outer membrane [187] and down-regulated during infection [188] would be an unlikely protective immunogen. Similarly, the surface-exposed brachyspiral outer membrane lipoprotein SmpA [189] that is also down-regulated during infection [190] is unlikely to elicit immunoprotection.…”

Evaluation of the use of recombinant Bhlp29.7 in immunoblotting with pig serum as a means to identify herds infected with Brachyspira hyodysenteriae

“…Immunoreactive antigens and components involved in host-parasite interaction and pathogenesis may be found in the outer membrane, as well as in the sheath and core of the spirochaetes' endoflagella. Several structural components, such as 16 kDa lipoprotein SmpA or SmpB, 22 kDa protein, 30 kDa lipoprotein BmpB and 39 kDa proteins Vsp, have already been described by several authors (Thomas et al 1992;Thomas and Sellwood 1993;Gabe et al 1995;Lee et al 2000;Ochiai et al 2000;Holden et al 2006). To develop accurate and specific serological tests necessitates the identification of suitable diagnostic antigens which are specific to B. hyodysenteriae.…”

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

“…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].…”