Pinne and Haake assessed surface exposure of LipL32 by two methods. The first involved adding proteinase K to suspensions of Leptospira to digest proteins exposed on the surface of the spirochete. When they did this, they saw that the known surface-exposed proteins OmpL37 and OmpL47 were degraded. On the other hand, LipL32 didn't break down at all unless the spirochetes were first lysed by boiling them in a detergent (see Western blot below).
|Figure 1B from Pinne and Haake (2013). Increasing concentrations of proteinase K (up to 150 ug/ml) were added to suspensions (first five lanes) or lysates (last five lanes) of L. interrogans. Following incubation, LipL32 was examined in a Western blot. Source.|
They next added LipL32 antibodies to Leptospira to see if they bound to the surface of the spirochete. They did not, providing additional evidence that LipL32 was not exposed on the cell surface. The authors tested LipL32 antibodies from different sources in an attempt to rule out the possibility that failure of antibody binding was due to the surface-exposed portion of LipL32 not being antigenic. LipL32 antiserum raised in rabbits, monoclonal LipL32 antibodies raised in mice, and LipL32 antibodies purified from the sera of leptospirosis patients all failed to bind the surface of Leptospira unless the outer membrane was chemically (with methanol or EDTA) or physically disrupted. Note that antibodies raised against OmpL54, a known suface-exposed protein, reacted strongly with intact Leptospira (last pair of images below).
|Figure 3A from Pinne and Haake (2013). Bound antibody was detected with a secondary fluorescent antibody (green). The spirochetes were also stained with DAPI, a penetrating dye that stains DNA (blue). Left column, intact Leptospira, right column, methanol-treated Leptospira. Source.|
In light of these results, the authors took another look at the 2005 study by Cullen and coauthors, who claimed LipL32 was surface exposed. In contrast to Pinne and Haake, Cullen and colleagues detected binding of LipL32-specific antibodies to intact Leptospira in three different assays. However, Pinne and Haake point out that antibody binding in their assays was extremely weak when the abundance of LipL32 is considered. The most striking example was the immunoelectron microscopy image of Leptospira treated with gold-labeled LipL32 antibody (see next image). Yes, the surface ended up labeled, with a mean of 10.8 gold particles per spirochete cell. However, we now know that there are 38,000 copies of LipL32 in each bacterial cell, making LipL32 the most abundant protein of L. interrogans (see this blog post about the Leptospira protein census). If LipL32 were really surface exposed, the surface of the spirochete should have been packed with gold particles.
|Figure 5 from Cullen et al. (2005).|
Cullen and colleagues also mixed suspensions of Leptospira with a biotin probe that reacts with primary amines (mostly on lysine side chains). The probe should have reacted solely with surface-exposed proteins since it's unable to penetrate the outer lipid bilayer and is assumed to be too large to diffuse through outer membrane porins. Biotinylated proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry (see next figure). Although LipL32 was one of the few proteins labeled with biotin, it's hard to make a firm conclusion about its surface exposure because proteins known to be located underneath the outer membrane, FlaB1 (a flagellar protein) and GroEL (a cytoplasmic heat shock protein), were also labeled with biotin. It's possible that LipL32 was labeled despite being located underneath the surface because the membrane was damaged while the spirochetes were being harvested for the experiment.
The results from Pinne and Haake's study do not rule out the "rainforest canopy" model since they did not test smaller surface probes that could penetrate into the LPS side chain layer. Additional studies are needed to pin down the location of LipL32 relative to the surface of Leptospira.
Pinne, M., & Haake, D.A. (2013). LipL32 is a subsurface lipoprotein of Leptospira interrogans: Presentation of new data and reevaluation of previous studies. PLoS ONE, 8 (1) DOI: 10.1371/journal.pone.0051025
Cullen, P.A., Xu, X., Matsunaga, J., Sanchez, Y., Ko, A.I., Haake, D.A., & Adler, B. (2005). Surfaceome of Leptospira spp. Infection and Immunity, 73 (8), 4853-4863 DOI: 10.1128/IAI.73.8.4853-4863.2005
- Protein census of Leptospira interrogans
- The major outer membrane protein of Leptospira interrogans: Not essential for infection?