SliC is a surface-displayed lipoprotein that is required for the anti-lysozyme strategy during Neisseria gonorrhoeae infection
(A) Diagrams of native SliC, constructs for complementation studies using wild type and mutated versions of sliC placed under the control of the native promoter (P::sliC and P::sliC*, respectively), and recombinant versions of wild type SliC (rSliC), and its mutated variant (rSliC*) bearing alanine substitutions in residues S83 and K103 are shown. Introduced mutations are denoted by arrows. Both recombinant proteins lack a signal peptide and contain a C-terminal Tobacco Etch Virus (TEV) protease cleavage site followed by a C-terminal 6×His-tag (His). The sliC native promoter (Prom) is shown in green; the lipoprotein signal peptide (SP) in aquamarine; the MliC/PliC domain in lavender. Amino acid (aa) domain positions are denoted. (B) Recombinant versions of matured rSliC and rSliC* were purified in a soluble form to homogeneity through affinity and size exclusion chromatography steps and untagged proteins were separated on SDS-PAGE. Proteins were stained with colloidal coomassie. Samples were loaded by protein concentration, as indicated above the gel. Migration of a molecular mass marker (kDa) is indicated on the left. (C) Strains of N. gonorrhoeae FA1090 wild type, isogenic null mutant in ngo1063 (ΔsliC), and complemented strains, ΔsliC/P::sliC and ΔsliC/P::sliC*, harboring either wild type sliC or mutated sliC* bearing S83A K103A substitutions, respectively, were collected from solid media, lysed, separated by SDS-PAGE, and probed with polyclonal rabbit anti-SliC antisera. Whole cell lysates were matched by the same OD600. (D) Cultures of N. gonorrhoeae FA1090 wild type harvested from liquid cultures at mid-logarithmic growth were subjected to subcellular fractionation. Isolated proteomes of cytoplasmic and periplasmic (C+P), cell envelopes (M), naturally released membrane vesicles (MV), and soluble supernatant fractions (S) were matched by the same total protein concentration, separated by SDS-PAGE and probed with polyclonal rabbit antisera against SliC, BamA, BamD, SurA, and Zwf. (E) Wild type gonococci were collected from solid media, sub-cultured in broth for 3 h, diluted to OD600 of 0.1, and cultured until OD600 of ~ 1.0 was reached. Gonococci were washed and after OD600 adjustment to 2.0, the bacterial cells were incubated for 1 h at 37°C with proteinase K at final concentrations as designated above the immunoblots. Protease was deactivated, samples were normalized by the same OD600, subjected to SDS-PAGE, and probed with polyclonal antisera to determine protease susceptibility. (F) N. gonorrhoeae FA1090 strains, as shown above the graphs, were cultured in liquid medium, harvested, and suspended to OD600 of 2.0. Intact and lysed cells were spotted onto nitrocellulose membranes. Individual protein profiles were analyzed by immunoblotting with specific antisera against SliC, BamA, BamD, SurA, and Zwf.