Iplasmic) and pellet (total membrane) fractions [28]. The membrane fraction was treated with high pH (0.1 M Na2CO3), high salt (0.6 M NaCl), or urea (1.6 M), to release peripheral membrane proteins not anchored in the lipid bilayer [21,26,29?1]. Immunoblot analysis of the soluble (supernatants) and insoluble (pelleted) membrane fractions revealed that the bulk of LipL32 remained associated with the membrane fraction after all treatments (Fig. 5). Integral outer membrane protein OmpL1, and two OM-lipoproteins; AG-221 site LipL46, and LipL41 were included as positive controls and could not be released from the membrane by any treatment (Fig. 5;[26,30]). As a positive control for release from the membrane, the effect of treatments on the peripheral membrane protein, P31LipL45, also known as Qlp42 [32] was also assessed. Substantial release from the membrane by urea and Na2CO3 was observed (data not shown), as previously described [21,30].LipL32 Is a Subsurface Lipoprotein of LeptospiraFigure 6. Reused from: PLoS One. 2011; 6(7): e21962. Confocal microscopy was performed with live L. interrogans using antisera specific for LIC10258, LIC12880, LIC12238, LipL32 (surface-exposed lipoprotein) and GroEL (protoplasmic cylinder marker). FITC-conjugated secondary antibodies were used to detect the surface-bound antibodies (B). Leptospires were identified by propidium iodide (A) staining of the DNA. Co-localization is shown in the merged images (C). doi:10.1371/journal.pone.0051025.AG-221 gmembrane vesicle fractionation [12], most likely due incomplete separation of outer membrane from inner membrane vesicles rather than inner membrane localization. Our results showing a subsurface location for LipL32 appear to contradict previous studies. This prompted us to reexamine the evidence for LipL32 surface localization presented in previous studies. Immunoelectron microscopy of intact leptospires was presented as evidence for LipL32 surface-exposure [18]. However, given the abundance of LipL32, significantly more immunogold staining should have occurred than what was observed. For example, immunoelectron microscopy of Borrelia burgdorferi using OspC antibodies results in dense staining of the surface of the organism with gold particles [37]. When surface immunofluorescence was performed with rabbit serum recognizing LipL32 [18], much weaker and irregular antibody labeling was obtained in intact cells when compared to permeabilized cells. One possible explanation is that this labeling resulted from damaged organisms presented in that particular microscopic field. When LipL32 was used as a positive control in previously published IFA experiments [19,38], LipL32 surface-exposure was inconclusive as only one of two cells was labeled by antibodies in one study (Fig. 6) [19], while only one cell per microscopic field was shown in the other study [38]. LipL32 monoclonal antibodies [24,25] have also been utilized in IFA, however the interpretation of the data is impossible given the lack of controls 1379592 for the integrity of the outer membrane [24]. In fact, when we assessed LipL32 surface exposure using these same monoclonal antibodies, we found that the antibodies recognized the protein only after the OM have been disrupted (Fig. 3). Out of concern about the ability of antibody reagents to recognize native vs. denatured LipL32 epitopes, we also performed immunofluorescence assays with IgG’s purified from human clinical leptospirosis sera. These results support the conclusion that most.Iplasmic) and pellet (total membrane) fractions [28]. The membrane fraction was treated with high pH (0.1 M Na2CO3), high salt (0.6 M NaCl), or urea (1.6 M), to release peripheral membrane proteins not anchored in the lipid bilayer [21,26,29?1]. Immunoblot analysis of the soluble (supernatants) and insoluble (pelleted) membrane fractions revealed that the bulk of LipL32 remained associated with the membrane fraction after all treatments (Fig. 5). Integral outer membrane protein OmpL1, and two OM-lipoproteins; LipL46, and LipL41 were included as positive controls and could not be released from the membrane by any treatment (Fig. 5;[26,30]). As a positive control for release from the membrane, the effect of treatments on the peripheral membrane protein, P31LipL45, also known as Qlp42 [32] was also assessed. Substantial release from the membrane by urea and Na2CO3 was observed (data not shown), as previously described [21,30].LipL32 Is a Subsurface Lipoprotein of LeptospiraFigure 6. Reused from: PLoS One. 2011; 6(7): e21962. Confocal microscopy was performed with live L. interrogans using antisera specific for LIC10258, LIC12880, LIC12238, LipL32 (surface-exposed lipoprotein) and GroEL (protoplasmic cylinder marker). FITC-conjugated secondary antibodies were used to detect the surface-bound antibodies (B). Leptospires were identified by propidium iodide (A) staining of the DNA. Co-localization is shown in the merged images (C). doi:10.1371/journal.pone.0051025.gmembrane vesicle fractionation [12], most likely due incomplete separation of outer membrane from inner membrane vesicles rather than inner membrane localization. Our results showing a subsurface location for LipL32 appear to contradict previous studies. This prompted us to reexamine the evidence for LipL32 surface localization presented in previous studies. Immunoelectron microscopy of intact leptospires was presented as evidence for LipL32 surface-exposure [18]. However, given the abundance of LipL32, significantly more immunogold staining should have occurred than what was observed. For example, immunoelectron microscopy of Borrelia burgdorferi using OspC antibodies results in dense staining of the surface of the organism with gold particles [37]. When surface immunofluorescence was performed with rabbit serum recognizing LipL32 [18], much weaker and irregular antibody labeling was obtained in intact cells when compared to permeabilized cells. One possible explanation is that this labeling resulted from damaged organisms presented in that particular microscopic field. When LipL32 was used as a positive control in previously published IFA experiments [19,38], LipL32 surface-exposure was inconclusive as only one of two cells was labeled by antibodies in one study (Fig. 6) [19], while only one cell per microscopic field was shown in the other study [38]. LipL32 monoclonal antibodies [24,25] have also been utilized in IFA, however the interpretation of the data is impossible given the lack of controls 1379592 for the integrity of the outer membrane [24]. In fact, when we assessed LipL32 surface exposure using these same monoclonal antibodies, we found that the antibodies recognized the protein only after the OM have been disrupted (Fig. 3). Out of concern about the ability of antibody reagents to recognize native vs. denatured LipL32 epitopes, we also performed immunofluorescence assays with IgG’s purified from human clinical leptospirosis sera. These results support the conclusion that most.
