Reconstitution of surface lipoprotein translocation through the Slam translocon

Article activity feed

1. 

   Version published to 10.7554/elife.72822 on eLife

   Apr 27, 2022
2. 

   eLife

   Mar 14, 2022

   Author Response:

   Evaluation Summary:

   This work provides new insights into how surface-exposed lipoproteins of Gram-negative bacteria reach their destination in the outer membrane. Authors find that the outer membrane protein complex Slam serves as a translocon for the lipoproteins and the periplasmic chaperone Skp mediates their targeting to Slam. This work may contribute to the elucidation of host invasion mechanisms by pathogenic bacteria, in which surface lipoproteins play an important role.

   Reviewer #1 (Public Review):

   Previously, using rigorous genetic, bioinformatic and cell-based biochemical analyses, the same group discovered SLAM1, an outer membrane protein in Neisseria spp., which mediates the membrane translocation of surface lipoproteins (SLPs) (Hooda et al. 2016 Nature Microbiology 1, 16009). Here, authors …

   Author Response:

   Evaluation Summary:

   This work provides new insights into how surface-exposed lipoproteins of Gram-negative bacteria reach their destination in the outer membrane. Authors find that the outer membrane protein complex Slam serves as a translocon for the lipoproteins and the periplasmic chaperone Skp mediates their targeting to Slam. This work may contribute to the elucidation of host invasion mechanisms by pathogenic bacteria, in which surface lipoproteins play an important role.

   Reviewer #1 (Public Review):

   Previously, using rigorous genetic, bioinformatic and cell-based biochemical analyses, the same group discovered SLAM1, an outer membrane protein in Neisseria spp., which mediates the membrane translocation of surface lipoproteins (SLPs) (Hooda et al. 2016 Nature Microbiology 1, 16009). Here, authors reconstituted this system in proteoliposomes using minimal purified components including the translocon Slam1 and the client lipoprotein TbpB. Authors further coupled the system to TbpB-expressing E. coli spheroblasts and LolA, the Slam1-specific periplasmic shuttle system. Using the digestion pattern of TbpB by Proteinase K as a readout, authors confirmed that Slam1 indeed served as a translocon for SLPs. As a step forward, authors found that Skp, a periplasmic chaperone (holdase), was critical to the membrane-assembly and translocation of TbpB. Strengths: Overall, this is a solid biochemical study that demonstrates the role of Slam1 as a translocon for SLPs. The experimental design is neat and straightforward. The specific role of Skp in SLP translocation is interesting. This reconstituted system will serve as a novel platform for further elucidation of the Slam1-mediated SLP translocation mechanisms. The manuscript is overall well written. Weakness: There are several major concerns, however.

   1. It is not fully convincing whether these findings are novel and significantly advance the field. Identification of minimal components in a biological process and their reconstitution are always challenging and thus, this study is an achievement. Nonetheless, I am not sure whether we have learned novel molecular insights besides the confirmation of the group's previous discovery. The specific role of Skp in translocation is interesting but not surprising, considering that periplasmic holdases are already known to be extensively involved in the biogenesis of periplasmic and outer membrane proteins.

   We thank the reviewer for their time and thorough review of the manuscript. In the previous paper (Hooda et al. 2016 Nature Microbiology 1, 16009), we discovered that the outer membrane protein Slam is “important/responsible” for the surface display for SLPs (TbpB, LbpB, fHbp). In this mechanism focused manuscript, we were able to demonstrate Slam’s role as an outer membrane translocon. One of the achievements in this paper is to demonstrate that Slam as an autonomous translocon – importantly this is unlike the two-partner secretion systems, as it does not require the Bam complex for the translocation of TbpB.

   2) Although authors developed nice assays (Figs. 1 and 2), it was not verified whether TbpB protected from Proteinase K digestion had "correct" conformation and membrane-topology. Authors performed a functional assay on TbpB (Fig. 5a), but this result was obtained from a cell-based assay, not from the reconstituted system.

   We have performed pulldown assay for the TbpB that has been translocated into Slam-proteoliposomes using human transferrin conjugated beads to show that this TbpB protein is correctly folded and functional. Blots and explanations are attached in the revised manuscript (see new Figure 2 – figure supplement 2 and line 197-207). (As addressed in major scientific concerns point 2-i).

   Although the data in Figs. 1 and 2 clearly show that the membrane association of TbpB depends on Slam1, it does not mean that the "translocation" has actually occurred in the proteoliposomes. Probably, more rigorous analysis on the Proteinase K-protected portion of TbpB (for example, mass spec) seems necessary (that is, whether the proteolytic product is expected based on the predicted topology).

   The TbpB is flag-tag at its C-terminus and the protected band on our blots (detected by α-flag antibody) corresponds to the expected Mw (~75kDa) for Mcat TbpB flag tagged protein. Therefore, we believed the band at 75kDa is our full length processed TbpB. Moreover, we have confirmed that TbpB can be detected at the top of the sucrose gradient with our Slam-proteoliposomes in this assay. This would only occur if TbpB was actually translocated inside the intact liposomes, otherwise we should not see any TbpB in the top layer of the sucrose gradient (Figure 4d). Furthermore, we have performed a pulldown assay for TbpB in proteoliposomes to check for their functional binding to human transferrin beads after translocation. These results are explained in the updated new Figure 2 – figure supplement 2 and line 197-207.

   3) The manuscript has a couple of missing supporting data. 3a) Lines 87-89: "From our analysis, we found that the Slam1 from Moraxella catarrhalis (or Mcat Slam1) expressed well and the purified protein was more stable than other Slam homologs." I cannot find the expression and stability data of various homologs supporting this sentence.

   In general, what we meant was that we chose Mcat Slam as the target of this study because it is more stable during the purification and resulted in a higher yield of protein. We needed higher yields of Slam to be able to reconstitute the protein into the liposomes for the translocation assay. We have purification data for Mcat Slam1, Nme Slam1 and Ngo Slam2 but we think including them in the supplementary is not necessary. We have changed and rewritten this section dedicated to Mcat Slam1 purification (Figure 1 – figure supplement 1 and 2).

   3b) "Lines 216-219: Furthermore, the processing of TbpB by signal peptidase II and subsequence release from the inner membrane was unaffected suggesting the defect in surface display by Skp occurs after the release of TbpB from the inner membrane (Fig. 4a)." The result supporting this sentence seems missing or this sentence points to a wrong figure.

   Yes, this sentence is misleading. What we meant was that the processed TbpB (TbpB has 2 bands, unprocessed TbpB – upper band and signal peptidase processed, lipidated TbpB - lower band) is similar for all samples indicating that the knockout of Skp did not affect the expression or processing of the signal peptide of TbpB up until it is ready (processed and lipidated in the periplasm) for translocation by Slam to the surface. We have added an explanation in the figure legend of Figure 4a –line 267-269.

   1. Some statistical analysis results are not clear, making some conclusions not convincing. 4a) Figure 4a top "Exposure of TbpB on the surface of K12 E. coli" Apparently, all three data points for (Delta_DegP+Slam1+TbpB) are very closely distributed. Accordingly, (WT+Slam1+TbpB) vs (Delta_DegP+Slam1+TbpB) data look significantly different (difference is ~0.2). But the two data were assigned as "Not Significant". Similarly, in the comparable in vitro data (Figure 4b), the intensity for Slam1 (WT+Proteinase K - Triton) looks larger than that for Slam1 (Delta_DegP + Proteinase K - Triton). So, the DegP contribution should not be ignored.

   For figure 4a, the ONE WAY ANOVA test was performed using Prism with 4 biological replicates (we can include the analysis report in the revised submission if this is requested we have updated the figure to include data points. In general, both our in vitro liposomes translocation assay and in vivo surface exposure assay for TbpB showed that delta-DegP only slightly reduces the translocation of TbpB to the surface but could not detect statistically significant differences.

   4b) Figure 5a top "Exposure of TbpB on the surface of N. meningitidis" What is the p-value for WT vs Delta_Skp data? Are the two data significantly different? The p-value range for (*) is not shown.

   We have included the p-value range for (*) in the revised manuscript, figure 5a.

   Reviewer #2 (Public Review):

   The article addresses the function of SLAM, a protein which the authors have shown previously to be involved in the traffic of lipoproteins to the bacterial surface. The authors have performed a series of experiments to assess the impact of SLAM on the delivery into proteoliposomes of the model lipoprotein TbpB either added exogenously or presented by E coli spheroplasts. They identify a periplasmic chaperone, Skp, which enhances transport of TbpB and other lipoproteins to proteoliposomes, and show the contribution of endogenous Skp to lipoprotein transport in Neisseria meningitidis. The authors set up an in vitro translocation assays using purified components from different bacteria. This is reasonable as the assays can be challenging to establish and require proteins that can be expressed and are stable. It would be helpful however if the sources of the proteins and how they are tagged (for their detection) is clearly documented in the article and the figures. In keeping with this, the figures describing the assays could be improved (ie 1A, 2A, 3A and C). Despite this, the results presented in Fig 1 and 2 clearly demonstrate the role of SLAM as a translocase, and the authors have included appropriate controls for their assays; the translocation of a OmpA to demonstrate that the Bam complex is functional in their hands in an important control and should be included in the main figures. Experiments outlined in Figure 3 and Table 1 demonstrate the interaction specific of TbpB and another lipoprotein HpuA with Skp, a previously characterised periplasmic chaperone. This is performed by pull-downs and MS as well as immunobloting. A critical result is shown in Figure 4 in which SLAM and TbpB are introduced into E coli, and the role of endogenous Skp is assessed. Importantly, the absence of Skp reduces but does not eliminate TbpB surface expression. The authors could speculate on the nature of Skp-indendent surface expression of TbpB, as this result mirrors what they find in a meningococcal strain lacking Skp (Figure 5A). It appears that Skp might be required for the correct insertion/folding of lipoproteins given their result in Figure 5B (currently, this could be changed into 5C) which tests the binding of transferrin to the bacterial surface. Clearly this could be influenced by an effect of Skp on TbpA, which acts as a co-receptor with TbpB. In summary, the authors have used appropriate assays to reach their conclusions about the role of SLAM as a translocase and the contribution of Skp to the localisation of lipoproteins to the surface of bacteria. The findings presented are robust and shed new insights into the sorting of proteins in bacteria, an incompletely understood process which is central to microbial physiology, viurlence and vaccines.

   Reviewer #3 (Public Review):

   Slam was identified as an outer membrane protein involved in the translocation of certain lipoproteins to the cell surface in Neisseria meningitidis. Slam homologs were also identified in other proteobacteria. However, direct evidence that Slam is an outer membrane translocation device is still missing. In this paper, the authors set up an in vitro translocation assay to probe the role of Slam proteins in the translocation of the lipoprotein TbpB. Although they provide strong data supporting the role of Slam in lipoprotein translocation, further molecular dissection is required to unambiguously establish Slam as a lipoprotein translocator. The work is interesting and the paper clearly written. The authors also discovered a functional link between the periplasmic chaperone Skp and Slam-dependent lipoproteins, which is a novel and interesting finding.

   Read the original source
3. 

   eLife

   Mar 14, 2022

   Evaluation Summary:

   This work provides new insights into how surface-exposed lipoproteins of Gram-negative bacteria reach their destination in the outer membrane. Authors find that the outer membrane protein complex Slam serves as a translocon for the lipoproteins and the periplasmic chaperone Skp mediates their targeting to Slam. This work may contribute to the elucidation of host invasion mechanisms by pathogenic bacteria, in which surface lipoproteins play an important role.

   (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

   Read the original source
4. 

   eLife

   Mar 14, 2022

   Reviewer #1 (Public Review):

   Previously, using rigorous genetic, bioinformatic and cell-based biochemical analyses, the same group discovered SLAM1, an outer membrane protein in Neisseria spp., which mediates the membrane translocation of surface lipoproteins (SLPs) (Hooda et al. 2016 Nature Microbiology 1, 16009).

   Here, authors reconstituted this system in proteoliposomes using minimal purified components including the translocon Slam1 and the client lipoprotein TbpB. Authors further coupled the system to TbpB-expressing E. coli spheroblasts and LolA, the Slam1-specific periplasmic shuttle system. Using the digestion pattern of TbpB by Proteinase K as a readout, authors confirmed that Slam1 indeed served as a translocon for SLPs. As a step forward, authors found that Skp, a periplasmic chaperone (holdase), was critical to the …

   Reviewer #1 (Public Review):

   Previously, using rigorous genetic, bioinformatic and cell-based biochemical analyses, the same group discovered SLAM1, an outer membrane protein in Neisseria spp., which mediates the membrane translocation of surface lipoproteins (SLPs) (Hooda et al. 2016 Nature Microbiology 1, 16009).

   Here, authors reconstituted this system in proteoliposomes using minimal purified components including the translocon Slam1 and the client lipoprotein TbpB. Authors further coupled the system to TbpB-expressing E. coli spheroblasts and LolA, the Slam1-specific periplasmic shuttle system. Using the digestion pattern of TbpB by Proteinase K as a readout, authors confirmed that Slam1 indeed served as a translocon for SLPs. As a step forward, authors found that Skp, a periplasmic chaperone (holdase), was critical to the membrane-assembly and translocation of TbpB.

   Strengths:

   Overall, this is a solid biochemical study that demonstrates the role of Slam1 as a translocon for SLPs. The experimental design is neat and straightforward. The specific role of Skp in SLP translocation is interesting. This reconstituted system will serve as a novel platform for further elucidation of the Slam1-mediated SLP translocation mechanisms. The manuscript is overall well written.

   Weakness:

   There are several major concerns, however.

   1. It is not fully convincing whether these findings are novel and significantly advance the field. Identification of minimal components in a biological process and their reconstitution are always challenging and thus, this study is an achievement. Nonetheless, I am not sure whether we have learned novel molecular insights besides the confirmation of the group's previous discovery. The specific role of Skp in translocation is interesting but not surprising, considering that periplasmic holdases are already known to be extensively involved in the biogenesis of periplasmic and outer membrane proteins.

   2. Although authors developed nice assays (Figs. 1 and 2), it was not verified whether TbpB protected from Proteinase K digestion had "correct" conformation and membrane-topology. Authors performed a functional assay on TbpB (Fig. 5a), but this result was obtained from a cell-based assay, not from the reconstituted system.

   Although the data in Figs. 1 and 2 clearly show that the membrane association of TbpB depends on Slam1, it does not mean that the "translocation" has actually occurred in the proteoliposomes. Probably, more rigorous analysis on the Proteinase K-protected portion of TbpB (for example, mass spec) seems necessary (that is, whether the proteolytic product is expected based on the predicted topology).

   1. The manuscript has a couple of missing supporting data.

   3a) Lines 87-89: "From our analysis, we found that the Slam1 from Moraxella catarrhalis (or Mcat Slam1) expressed well and the purified protein was more stable than other Slam homologs."

   >I cannot find the expression and stability data of various homologs supporting this sentence.

   3b) "Lines 216-219: Furthermore, the processing of TbpB by signal peptidase II and subsequence release from the inner membrane was unaffected suggesting the defect in surface display by Skp occurs after the release of TbpB from the inner membrane (Fig. 4a)."

   >The result supporting this sentence seems missing or this sentence points to a wrong figure.

   1. Some statistical analysis results are not clear, making some conclusions not convincing.

   4a) Figure 4a top "Exposure of TbpB on the surface of K12 E. coli"

   > Apparently, all three data points for (Delta_DegP+Slam1+TbpB) are very closely distributed. Accordingly, (WT+Slam1+TbpB) vs (Delta_DegP+Slam1+TbpB) data look significantly different (difference is ~0.2). But the two data were assigned as "Not Significant". Similarly, in the comparable in vitro data (Figure 4b), the intensity for Slam1 (WT + Proteinase K - Triton) looks larger than that for Slam1 (Delta_DegP + Proteinase K - Triton). So, the DegP contribution should not be ignored.

   4b) Figure 5a top "Exposure of TbpB on the surface of N. meningitidis"

   > What is the p-value for WT vs Delta_Skp data? Are the two data significantly different? The p-value range for (*) is not shown.

   Read the original source
5. 

   eLife

   Mar 14, 2022

   Reviewer #2 (Public Review):

   The article addresses the function of SLAM, a protein which the authors have shown previously to be involved in the traffic of lipoproteins to the bacterial surface. The authors have performed a series of experiments to assess the impact of SLAM on the delivery into proteoliposomes of the model lipoprotein TbpB either added exogenously or presented by E coli spheroplasts. They identify a periplasmic chaperone, Skp, which enhances transport of TbpB and other lipoproteins to proteoliposomes, and show the contribution of endogenous Skp to lipoprotein transport in Neisseria meningitidis.

   The authors set up an in vitro translocation assays using purified components from different bacteria. This is reasonable as the assays can be challenging to establish and require proteins that can be expressed and are stable. …

   Reviewer #2 (Public Review):

   The article addresses the function of SLAM, a protein which the authors have shown previously to be involved in the traffic of lipoproteins to the bacterial surface. The authors have performed a series of experiments to assess the impact of SLAM on the delivery into proteoliposomes of the model lipoprotein TbpB either added exogenously or presented by E coli spheroplasts. They identify a periplasmic chaperone, Skp, which enhances transport of TbpB and other lipoproteins to proteoliposomes, and show the contribution of endogenous Skp to lipoprotein transport in Neisseria meningitidis.

   The authors set up an in vitro translocation assays using purified components from different bacteria. This is reasonable as the assays can be challenging to establish and require proteins that can be expressed and are stable. It would be helpful however if the sources of the proteins and how they are tagged (for their detection) is clearly documented in the article and the figures. In keeping with this, the figures describing the assays could be improved (ie 1A, 2A, 3A and C). Despite this, the results presented in Fig 1 and 2 clearly demonstrate the role of SLAM as a translocase, and the authors have included appropriate controls for their assays; the translocation of a OmpA to demonstrate that the Bam complex is functional in their hands in an important control and should be included in the main figures.

   Experiments outlined in Figure 3 and Table 1 demonstrate the interaction specific of TbpB and another lipoprotein HpuA with Skp, a previously characterised periplasmic chaperone. This is performed by pull-downs and MS as well as immunobloting.

   A critical result is shown in Figure 4 in which SLAM and TbpB are introduced into E coli, and the role of endogenous Skp is assessed. Importantly, the absence of Skp reduces but does not eliminate TbpB surface expression. The authors could speculate on the nature of Skp-indendent surface expression of TbpB, as this result mirrors what they find in a meningococcal strain lacking Skp (Figure 5A). It appears that Skp might be required for the correct insertion/folding of lipoproteins given their result in Figure 5B (currently, this could be changed into 5C) which tests the binding of transferrin to the bacterial surface. Clearly this could be influenced by an effect of Skp on TbpA, which acts as a co-receptor with TbpB.

   In summary, the authors have used appropriate assays to reach their conclusions about the role of SLAM as a translocase and the contribution of Skp to the localisation of lipoproteins to the surface of bacteria. The findings presented are robust and shed new insights into the sorting of proteins in bacteria, an incompletely understood process which is central to microbial physiology, viurlence and vaccines.

   Read the original source
6. 

   eLife

   Mar 14, 2022

   Reviewer #3 (Public Review):

   Slam was identified as an outer membrane protein involved in the translocation of certain lipoproteins to the cell surface in Neisseria meningitidis. Slam homologs were also identified in other proteobacteria. However, direct evidence that Slam is an outer membrane translocation device is still missing. In this paper, the authors set up an in vitro translocation assay to probe the role of Slam proteins in the translocation of the lipoprotein TbpB. Although they provide strong data supporting the role of Slam in lipoprotein translocation, further molecular dissection is required to unambiguously establish Slam as a lipoprotein translocator. The work is interesting and the paper clearly written. The authors also discovered a functional link between the periplasmic chaperone Skp and Slam-dependent lipoproteins, …

   Reviewer #3 (Public Review):

   Slam was identified as an outer membrane protein involved in the translocation of certain lipoproteins to the cell surface in Neisseria meningitidis. Slam homologs were also identified in other proteobacteria. However, direct evidence that Slam is an outer membrane translocation device is still missing. In this paper, the authors set up an in vitro translocation assay to probe the role of Slam proteins in the translocation of the lipoprotein TbpB. Although they provide strong data supporting the role of Slam in lipoprotein translocation, further molecular dissection is required to unambiguously establish Slam as a lipoprotein translocator. The work is interesting and the paper clearly written. The authors also discovered a functional link between the periplasmic chaperone Skp and Slam-dependent lipoproteins, which is a novel and interesting finding.

   Read the original source
7. 

   Version published to 10.1101/2021.08.22.457263v1 on bioRxiv

   Aug 23, 2021