SmpB: a novel outer membrane protein present in some Brachyspira hyodysenteriae strains.

A novel outer membrane protein-encoding gene was identified in Brachyspira hyodysenteriae. The predicted protein, SmpB, was encoded by a gene that contains regions of identity with that encoding the previously identified lipoprotein SmpA. However, the majority of the reading frame encoding SmpA and SmpB share no detectable similarity. Analysis of several strains revealed that B. hyodysenteriae harbours either smpA or the newly identified gene smpB, but not both. smpB encodes for a slightly larger protein than smpA, 17.6 and 16.8 kDa, respectively. The predicted proteins share an identical leader sequence and the first 10 amino acids of the mature protein, however, the remainder of the predicted protein sequence shows no similarity. It is hypothesised that smpA and smpB are present on the same area of the chromosome. The proteins are antigenically unique, as antisera raised against a strain of B. hyodysenteriae that expresses SmpA cannot detect SmpB and vice versa. Although the presence of an identical leader peptide suggests identical localisation of SmpA and SmpB, it is not known if the two predicted proteins share similar function.

The search for Brachyspira outer membrane proteins that interact with the host.

Little is known about the outer membrane structure of Brachyspira hyodysenteriae and Brachyspira pilosicoli or the role of outer membrane proteins (OMPs) in host colonization and the development of disease. The isolation of outer membrane vesicles from B. hyodysenteriae has confirmed that cholesterol is a significant outer membrane constituent and that it may impart unique characteristics to the lipid bilayer structure, including a reduced density. Unique proteins that have been identified in the B. hyodysenteriae outer membrane include the variable surface proteins (Vsp) and lipoproteins such as SmpA and BmpB. While the function of these proteins remains to be determined, there is indirect evidence to suggest that they may be involved in immune evasion. These data may explain the ability of the organism to initiate chronic infection. OMPs may be responsible for the unique attachment of B. pilosicoli to colonic epithelial cells; however, the only B. pilosicoli OMPs that have been identified to date are involved in metabolism. In order to identify further B. pilosicoli OMPs we have isolated membrane vesicle fractions from porcine strain 95-1000 by osmotic lysis and isopycnic centrifugation. The fractions were free of contamination by cytoplasm and flagella and contained outer membrane. Inner membrane contamination was minimal but could not be completely excluded. An abundant 45-kDa, heat-modifiable protein was shown to have significant homology with B. hyodysenteriae Vsp, and monoclonal antibodies were produced that reacted with five B. pilosicoli-specific membrane protein epitopes. The first of these proteins to be characterized is a unique surface-exposed lipoprotein.

Sequence characterization of two new members of a multi-gene family in Serpulina hyodysenteriae (B204) with homology to a 39 kDa surface exposed protein: vspC and D.

Previous cloning and sequencing of clones from a genomic library constructed from Serpulina hyodysenteriae B204 had identified a tandem pair of open reading frames, identified as vspA and vspB (variable surface protein) expected to encode proteins with homology to ( but not identical with) a 39 kDa surface exposed membrane protein from this animal pathogen. Additional screening of the genomic library was performed to retrieve the remainder of the vspB gene using new oligonucleotide probes based upon the cloned gene sequences. Not only was this goal met but we also discovered two more adjacent and related vsp genes (vspC and vspD) and have completely sequenced them. They are all in a parallel orientation and appear to have a set of similar but distinct regulatory elements that may control separate expression of their open reading frames (ORFs). Thus, there are four contiguous vsp genes which are predicted to encode a family of structurally conserved proteins. The four adjacent open reading frames (ORFs) are of similar size (384-389 codons) and share from 83% to 90% identity in their amino acid sequence. Preliminary data suggests there may be yet another homologous gene copy in a distal location of S. hyodysenteriae that faithfully encodes the 39 kDa surface protein. The organization and homologies of these highly conserved multiple gene copies are discussed.

Identification of porcine Serpulina strains in routine diagnostic bacteriology.

Serpulina strains from pigs were identified using simple tests. The large size of S. hyodysenteriae in stainings from colonic contents and faeces was found useful for the presumptive differentiation of this major pathogenic species from the other Serpulinae. However, this morphological characteristic gets lost upon cultivation. The 'ring phenomenon' aided to confirm the strong haemolysis typical for S. hyodysenteriae. The weakly haemolytic species S. innocens, S. pilosicoli, S. intermedia and S. hyodysenteriae could be differentiated with the help of the indole spot test and two or four other simple enzymatic tests. Nearly half of the S. hyodysenteriae strains isolated in Belgium were indole-negative. Such strains have only rarely been reported earlier, and were absent among the strains from other European countries examined.

Characterization of a haemolysin from Mycobacterium tuberculosis with homology to a virulence factor of Serpulina hyodysenteriae.

Scrutiny of sequence data from the Mycobacterium leprae genome sequencing project identified the presence of a gene encoding a 268-amino-acid polypeptide which is highly similar to a pore-forming haemolysin/cytotoxin virulence determinant, TlyA, from the swine pathogen Serpulina hyodysenteriae. Using degenerate oligonucleotide primers based on the TlyA sequences, the Mycobacterium tuberculosis homologue was amplified and this product was used to obtain the clone and sequence a 2.5 kb fragment containing the whole M. tuberculosis tlyA gene. tlyA encodes a 267-amino-acid protein with a predicted molecular mass of 28 kDa. TlyA homologues were identified by PCR in M. leprae, Mycobacterium avium and Mycobacterium bovis BCG, but appeared absent in Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium kansasii, Mycobacterium chelonae and Mycobacterium phlei. The M. tuberculosis gene appeared to be the first gene in an operon containing at least two other genes. Introduction of the M. tuberculosis tlyA gene into M. smegmatis using a mycobacterial shuttle expression plasmid converted non-haemolytic cells into those exhibiting significant haemolytic activity. Similarly, inducible haemolytic activity was observed in sonicated bacteria when tlyA was expressed as a His6-tagged fusion protein in Escherichia coli. tlyA mRNA was detected in both M. tuberculosis and M. bovis BCG using RT-PCR, confirming that this gene is expressed in organisms cultured in vitro.

Pathogenicity of Serpulina hyodysenteriae: in vivo induction of tumor necrosis factor and interleukin-6 by a serpulinal butanol/water extract (endotoxin).

Lipopolysaccharide (LPS) of Gram-negative bacteria is a classic inducer of inflammatory cytokines. In the present experiments, LPS-like (phenol/water extract) or endotoxin-like (butanol/water extract) preparations from Serpulina hyodysenteriae were examined for their ability to induce serum tumor necrosis factor (TNF) or interleukin (IL)-6 bioactivity in mice and swine. Serpulina hyodysenteriae endotoxin (butanol/water extract) elicited increased serum TNF activity in mice, although serpulinal endotoxin was at least 10 times less potent than the LPS preparations of E. coli or S. typhimurium on a weight basis for induction of TNF bioactivity. S. hyodysenteriae LPS induced lower levels of serum TNF in mice than S. hyodysenteriae endotoxin. In contrast, pigs injected with S. hyodysenteriae endotoxin demonstrated no increase in serum TNF activity. However, an induction of IL-6 bioactivity was observed in serum samples from pigs injected with serpulinal endotoxin. In pigs, the serpulinal preparations were five times less potent (on a weight basis) than E. coli or S. typhimurium LPS for induction of IL-6 bioactivity. These data indicate that serpulinal endotoxin, although less bioactive than E. coli or S. typhimurium LPS, is nonetheless capable of inducing the in vivo production of specific pro-inflammatory cytokines.

Outer membrane-associated serine protease of intestinal spirochetes.

Pathogenic intestinal spirochetes cause damage to the intestinal mucosa of humans and animals by an unknown mechanism. The purpose of this study was to assess the pathogenic intestinal spirochetes Serpulina hyodysenteriae, Serpulina pilosicoli, and Brachyspira aalborgi and the non-pathogenic commensal intestinal spirochetes Serpulina innocens and Treponema succinifaciens for protease activity. A partially heat stable, subtilisin-like, serine protease was identified in the outer membrane of all spirochetes and thus may be essential for survival in the intestinal environment. The outer membrane protease may indirectly contribute to intestinal damage caused by pathogenic spirochetes during association with the mucosal surface of the host.

Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.

We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%.

Extracellular DNA from Serpulina hyodysenteriae consists of 6.5 kbp random fragments of chromosomal DNA.

Preparations of chromosomal DNA from a number of Serpulina hyodysenteriae strains have shown, using agarose gel electrophoresis, the presence of an additional band with a mobility similar to that of a 6.5 kbp linear DNA fragment. Analysis showed that this is not a plasmid but rather a form of extracellular DNA like that observed for Gram-negative bacteria. However, unlike the extracellular DNA from Gram-negative bacteria, which showed a similar band profile to that of the DNA from whole cells, that from S. hyodysenteriae consisted primarily of fragments of a fixed 6.5 kbp.

A species-specific periplasmic flagellar protein of Serpulina (Treponema) hyodysenteriae.

We have previously reported that a 46-kDa protein present in an outer membrane protein preparation seemed to be a species-specific antigen of Serpulina hyodysenteriae (Z. S. Li, N. S. Jensen, M. Bélanger, M.-C. L'Espérance, and M. Jacques, J. Clin. Microbiol. 30:2941-2947, 1992). The objective of this study was to further characterize this antigen. A Western blot (immunoblot) analysis and immunogold labeling with a monospecific antiserum against this protein confirmed that the protein was present in all S. hyodysenteriae reference strains but not in the nonpathogenic organism Serpulina innocens. The immunogold labeling results also indicated that the protein was associated with the periplasmic flagella of S. hyodysenteriae. N-terminal amino acid sequencing confirmed that the protein was in fact a periplasmic flagellar sheath protein. The molecular mass of this protein, first estimated to be 46 kDa by Western blotting, was determined to be 44 kDa when the protein was evaluated more precisely by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the protein was glycosylated, as determined by glycoprotein staining and also by N-glycosidase F treatment. Five other periplasmic flagellar proteins of S. hyodysenteriae, which may have been the core proteins and had molecular masses of 39, 35, 32, 30, and 29 kDa, were antigenically related and cross-reacted with the periplasmic flagellar proteins of S. innocens. Finally, serum from a pig experimentally infected with S. hyodysenteriae recognized the 44-kDa periplasmic flagellar sheath protein. Our results suggest that the 44-kDa periplasmic flagellar sheath protein of S. hyodysenteriae is a species-specific glycoprotein antigen.

16 kDa envelope proteins in non-Serpulina hyodysenteriae spirochaetes isolated from pigs.

Spirochaetes isolated from field samples of diarrhoea, 'colitis' and mucoid diarrhoea from pigs were examined by a series of cultural, biochemical and serological tests. In addition sodium dodecyl sulphate polyacrylamide gel electrophoresis was used to determine whether the organisms possessed a 16 kDa protein thought to distinguish Serpulina hyodysenteriae from S innocens. Spirochaetal isolates which differed culturally and biochemically from S hyodysenteriae were found to possess a 16 kDa protein. One of these isolates was examined by electron microscopy and found to have an ultrastructure differing from that of S hyodysenteriae. Antiserum to the 16 kDa antigen of S hyodysenteriae reacted with isolate S80/5, the homologous strain, and with B78, the type species, but not with the 16 kDa antigens of the field isolates considered to be S hyodysenteriae or with the non-S hyodysenteriae spirochaetes. It was concluded that there may be a family of 16 kDa proteins located on the envelope of various spirochaetes responsible for diarrhoea in pigs.

Comparison of outer-membrane fractions of Serpulina (Treponema) hyodysenteriae.

Sarkosyl-insoluble fractions (outer-membrane proteins) and endoflagella (EF) fractions of Serpulina hyodysenteriae serotypes 1-7 were examined for protein differences using SDS-PAGE. Both the outer-membrane proteins (OMP) and endoflagella were also examined for antigenicity using porcine sera from acutely infected and convalescent swine. Seven major staining proteins were resolved in outer-membrane enriched fractions ranging in molecular weight between 42 and 32 kDa. A comparison of purified EF to OMP from serotype 1 and 2 isolates of Serpulina hyodysenteriae demonstrated that six of the seven OMP were actually EF. Sera from swine with acute swine dysentery identified only a portion of the proteins from both preparations. In contrast, immune sera from convalescent swine identified all the proteins in the OMP and EF preparations as well as an additional 16 kDa carbohydrate antigen.

The periplasmic flagella of Serpulina (Treponema) hyodysenteriae are composed of two sheath proteins and three core proteins.

The major components of the periplasmic flagella of the spirochaete Serpulina (Treponema) hyodysenteriae strain C5 were purified and characterized. We demonstrate that the periplasmic flagella are composed of five major proteins (molecular masses 44, 37, 35, 34 and 32 kDa) and present their location, N-terminal amino acid sequence and immunological relationship. The 44 kDa and the 35 kDa protein are on the sheath of the periplasmic flagellum, whereas the 37, 34 and 32 kDa protein reside in the periplasmic flagellar core. The two sheath flagellar proteins are immunologically related but have different N-terminal amino acid sequences. The N-terminus of the 44 kDa protein shows homology with the sheath flagellins of other spirochaetes, but the 35 kDa protein does not. The three core proteins are immunologically cross-reactive and their N-terminal amino acid sequences are almost, but not completely, identical, indicating that the core proteins are encoded by three distinct genes. The core proteins show extensive N-terminal sequence similarities and an immunological relationship with periplasmic flagellar core proteins of other spirochaetes.