Reconstitution of surface lipoprotein translocation reveals Slam as an outer membrane translocon in Gram-negative bacteria

Surface lipoproteins (SLPs) are peripherally attached to the outer leaflet of the outer membrane in many Gram-negative bacteria, playing significant roles in nutrient acquisition and immune evasion in the host. While the factors that are involved in the synthesis and delivery of SLPs in the inner membrane are well characterized, the molecular machineries required for the movement of SLPs to the surface are still not fully elucidated. In this study, we investigated the translocation of a surface lipoprotein TbpB through a Slam1-dependent pathway. Using purified components, we developed an in vitro translocation assay where unfolded TbpB is transported through Slam1 containing proteoliposomes, confirming Slam1 as an outer membrane translocon. While looking to identify factors to increase translocation efficiency, we discovered the periplasmic chaperone Skp interacted with TbpB in the periplasm of Escherichia coli. The presence of Skp was found to increase the translocation efficiency of TbpB in the reconstituted translocation assays. A knockout of Skp in Neisseria meningitidis revealed that Skp is essential for functional translocation of TbpB to the bacterial surface. Taken together, we propose a pathway for surface destined lipoproteins, where Skp acts as a holdase for Slam-mediated TbpB translocation across the outer membrane.

For correspondence, please contact trevor.moraes@utoronto.ca  Introduction: 30 Transport of proteins to their correct spatio-temporal location is imperative for cell 31 survival. This key process often requires the movement of proteins across lipid bilayers 32 through a translocation channel which is also referred to as a translocon (Walter and 33 Lingappa, 1986; Schnell and Hebert, 2003). Translocons are found in all living organisms 34 and include the Sec translocon that is responsible for the bulk of protein transport across ( Fig. 1b, 1c). The addition of the Bam complex contributed little to no effects into the 122 TbpB translocation efficiency, suggesting that Bam complex does not involve in this 123 process. Furthermore, the low efficiency of insertion observed for the defined system 124 together with the observation that translocation across the pore only occurs when the 125 TbpB is denatured by urea lead us to hypothesis that there are likely additional 126 periplasmic factors that keep the SLP unfolded for an efficient translocation.

128
Translocation of TbpB via Slam1 requires periplasmic components but the 129 process is independent from the release of TbpB from the inner membrane. 130 To delve deeper into the mechanism of Slam-mediated SLP translocation and whether  TbpB (spheroplast-independent translocation). As seen previously in the spheroplast-166 dependent translocation assay, we observed TbpB protection from proteinase-K in 167 proteoliposomes containing Slam1 (~40% protection) and Bam+Slam1 (~35%) (Fig. 2b -168 lower panel) but not empty (~7%) nor Bam (~5%) proteoliposomes. Interestingly, we did 169 not observe any loss in translocation efficiency between spheroplast-dependent and 170 spheroplast-independent assay (Fig. 2c), confirming that Slam-mediated SLP 171 translocation is independent of SLP release from the inner membrane. This differs from 172 other secretion systems that require partners in the inner membrane who provide energy 173 through ATP/proton motive force (Sherman et al, 2018;Stubenrauch et al, 2016). This 174 finding suggests Slam-dependent SLP translocation is akin to two-partner secretion Periplasmic chaperone Skp interacts with pre-folded TbpB in the periplasm. 178 As previously mentioned above, the Slam1-dependent translocation requires 179 TbpB to be unfolded and hence, we hypothesized that other factors in the periplasm bind 180 the SLPs and prevent their premature folding prior to Slam mediated translocation. To 181 identify periplasmic factors that might be involved in the translocation, periplasmic TbpB 182 complexes were isolated using an affinity flag-tag on its C-terminus. The pulldown 183 fraction was analyzed using mass spectrometry (MS). In this pulldown assay, AfuAa 184 well-folded periplasmic protein from Actinobacillus pleuropneumoniae was used as a 185 negative control to rule out non-specific periplasmic protein interactions (Sit et al, 2015). 186 Skpa periplasmic chaperone was the only protein that was identified in the pulldown of 187 TbpB but not in the negative control ( Table 1). The mass spectrometry results were 188 validated using α-Skp antibody and confirming Skp interacts with TbpB in the periplasm 189 (Fig. 3a). Skp is a homo-trimeric chaperone that binds to unfolded OMPs in the 190 periplasm and is involved in OMP membrane insertion through the Bam complex (Sklar,191 et al 2007). Our findings suggest that Skp also interacts with TbpB-like SLPs in the 192 periplasm and assists in their translocation across the outer membrane. To further validate the interaction between Skp and SLPs, a reciprocal pulldown assay 195 was performed in which a purified His-tagged chaperone was added into the spheroplast 196 prior to the secretion of SLPs. In this assay, we also examined whether Skp interacts 197 with other SLPs such as hemoglobin-haptoglobin utilization protein (HpuA) -a substrate 198 of Slam2 homolog in N. meningitidis (Hooda et al, 2016) (Fig. 3b). In addition to Skp, two 199 other periplasmic chaperones which are known to be involved in the transport of OMPs,  (Table 1).  To further investigate the role of periplasmic chaperone Skp, we leveraged our in vitro 221 translocation assay using Slam1 proteoliposomes and spheroplast-secreted TbpB .

222
TbpB that was secreted from K12 spheroplast mutants that lacked Skp or DegP, was  with SurA (Fig 4c). Curiously, the addition of Skp to the empty liposomes also increased 240 TbpB's protection by 2 folds. This protection might be from the protease resistance that 241 chaperones provide for their substrates in the periplasm which has previously reported 242 also seen for unfolded OMPs (Yan et al, 2019) (Fig. 4c). Furthermore, this result was 243 consistent with the background protection observed in the spheroplast secretion 244 translocation assays which contained periplasmic components (Fig. 2b). To confirm that 245 the background protection is from the protease resistance of chaperone-substrate 246 complex, the samples were spun down against a sucrose gradient (0-60% w/v) after the 247 proteinase K treatment to isolate the proteoliposomes. The western blots and Coomassie 248 blue stained SDS-PAGE showed a clear separation of the two components (Fig. 4d).    Furthermore, the TbpB on the surface of N. meningitidis Δskp is not functional as these 306 TbpB fail to bind to biotinylated human transferrin. We do not yet know whether these 307 TbpB were misfolded after the translocation or only part of TbpB was exposed on the 308 surface. Further investigation will be needed to understand how TbpB-Skp complex is           The proteins were either stored at -80 o C or sent for antibody production.      collected as elution (E). All samples were treated with 5× SDS loading buffer and pH was 576 adjusted before loading on SDS-PAGE followed by western blotting. TbpB and AfuA (the 577 negative control) was detected using rabbit α-flag antibody, followed by α-rabbit HPR 578 secondary antibody. LolA was detected using mouse α-his antibody and Skp was 579 detected using mouse α-E. coli Skp antibody, followed by α-mouse HPR secondary  In vitro proteoliposomes translocation with addition of periplasmic chaperones: 611 The assay was modified based on previous assay described above for purified TbpB. In The cultures were started similarly to the growth assay. After adjusting the OD600 to 1.0, 666 30μL of cells were used to inoculate 3mL of BHI +/kanamycin (50μg/mL) in a 15mL 667 culture tube. After 4h, 0.1mM deferoxamine was added to induce expression of TbpB.

668
1mM IPTG was also added to Δskp + pGCC4 Nme Skp to induce expression of Skp.

669
The cells were grown for 16 hours at 37 o C, 5% CO2. Cells were adjusted to have OD600     spheroplast-independent assay . Individual data points were included on the graph. Pulldown samples were left on beads, digested with trypsin and analyzed by mass TbpB sample but not AfuA sample, though there were only 9 total spectrum counts.

884
DegP is another periplasmic chaperone that presented in both samples. However, it is 885 known to function as a protease that controls the quantity of over expressed proteins in 886 the periplasm.