Purified outer membranes of Serpulina hyodysenteriae contain cholesterol

Abstract

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 F 1 F 0 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 ( ‫؍‬ 1.10 g/cm 3 ) contained 0. Swine dysentery, caused by Serpulina (formerly Treponema) hyodysenteriae (9, 10), is a highly infectious, acute to chronic disease that can result in a debilitating mucohemorrhagic diarrhea and death. The first stage of the disease is spirochetal colonization and proliferation in the enteric epithelium, resulting in a significant decrease in the absorption of nutrients, ions, and water (12). However, the spirochetes do not invade beyond the lamina propria, and putative virulence factors, such as hemolysins and lipooligosaccharide (LOS), probably cause the cellular damage (16,(21)(22)(23)(24)35). Because a close association of the bacteria with the intestinal lining is critical in the development of disease, surface components of the spirochetes must play a pivotal role in the process.Previously, different groups have attempted to isolate outer membranes (OMs) from S. hyodysenteriae with less than favorable results. This was mainly due to the contamination of OM fractions by periplasmic flagella. For example, Joens et al. (11) found seven major proteins, ranging from 42 to 32 kDa in size, in OMs isolated from S. hyodysenteriae B204 by a Sarkosyl extraction technique. However, six of the seven bands appeared to be derived from the periplasmic flagella. Similarly, Chatfield et al. (5) and Wannemuehler et al. (39) found that the majority of the putative OM proteins isolated with detergent ranged in size from 45 to 29 kDa and strongly resembled the protein profile observed for the flagellar proteins. These data suggested that membranes isolated by these techniques were heavily contaminated by flagella. Therefore, a more effective technique is needed to better define the OM of S. hyodysenteriae.We have developed a membrane separation technique that permits the isolation of S. hyodysenteriae OMs and inner membranes (IMs) with minimal contamination by flagella. Analysis of the OMs indicated that they are quite interesting. First, the OM of S. hyodysenteriae has a lower density than the IM, which is very unusual for bacteria. In addition, the OM contains significant levels of cholesterol. Previously, Mycoplasma species (6), Borrelia hermsii (15), and S. hyodysenteriae (30, 33) had been described as having sterols in their membranes; however, the distributions ...

Borrelia burgdorferi RevA Antigen Is a Surface-Exposed Outer Membrane Protein Whose Expression Is Regulated in Response to Environmental Temperature and pH

“…This is likely due to the lack of classical lipopolysaccharide in the outer membranes of borreliae (58). Additionally, these and other spirochetes contain unusual types of lipids and liposaccharides that might affect membrane buoyancies (7,18,21,41,48,61). The results of our studies also indicate a need for the reexamination of other procedures used for separation of the inner and outer membranes of borreliae (19,27,44,52).…”

“…First, the genome sequence of B. burgdorferi B31 (23) indicates that this bacterium does not encode a homolog of the E. coli F 0 /F 1 ATPase. Second, a similar procedure was used to isolate membrane vesicles from the spirochete Serpulina (now Brachyspira) hyodysenteriae, where it was determined that the relative densities of the inner and outer membranes were opposite to those of other gram-negative bacteria (41). Third, numerous proteins known to be abundant on the B. burgdorferi cell surface (such as OspC and OspA) were seen to localize primarily to what Bledsoe et al called the inner membrane fraction.…”

Characterization of a manganese-dependent regulatory protein, TroR, from Treponema pallidum

“…After transfer, the proteins were visualized with Ponceau red stain so that the protein standards could be marked. The immunoblots were probed with antiTroR serum (diluted 1:1,000) and washed, and immunoreactive protein were visualized as described (10).…”

The search forBrachyspiraouter membrane proteins that interact with the host

“…Stanton (1987) first showed that cholesterol is incorporated into B. hyodysenteriae membranes and that it constitutes approximately 9% of total membrane lipids. Plaza and co-workers (Plaza et al, 1997) extended these observations to show that cholesterol is partitioned exclusively into the outer membrane. Treatment of cells with low concentrations of digitonin, a detergent that interacts with cholesterol, caused the complete disruption of the B. hyodysenteriae outer membrane but had little effect on Borrelia burgdorferi.…”