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SliC is a surface-displayed lipoprotein that is required for the anti-lysozyme strategy during Neisseria gonorrhoeae infection

Fig 4

Fitness assessment of N. gonorrhoeae lacking functional SliC and ACP.

(A) N. gonorrhoeae FA1090 wild type, isogenic ΔsliC, ΔsliC/P::sliC, and ΔsliC/P::sliC* were harvested from solid media, suspended to OD600 = 0.1 and incubated under standard aerobic conditions for 3 h. Then, cultures were back-diluted in fresh media and their growth was monitored by measuring OD600 at indicated time points for 6 h. (B) Bacterial strains were collected from solid media, suspended to an OD600 of 0.1, serially diluted and plated for fitness assessments under standard growth conditions on solid media (SGC), during iron limitation (‐Fe), in the presence of 7.5% normal human serum (+NHS), and anaerobically (‐O2). CFU’s were enumerated after 22 h of aerobic and 48 h of anaerobic growth. Experiments were performed in three biological replicates and means with corresponding SEMs are presented. (C) To assess the role of SliC and ACP in in vitro protection of N. gonorrhoeae from the hydrolytic activity of HL, wild type FA1090, ΔsliC, ΔsliC complemented with sliC, Δacp, Δacp/P::acp, ΔacpΔsliC, or ΔacpΔsliC/P::acp P::sliC were suspended in liquid media to an OD600 of 0.1 and cultured at 37°C for 3 h. Bacteria were diluted to 1×104 CFU/mL and 90 μL of each culture was incubated for 3 h at 37°C with the membrane-permeabilizing agent lactoferrin (5 mg/mL) and increasing concentrations of human lysozyme as specified below the graph. Suspensions were plated for CFU scoring (n = 5, mean±SEMs; *p<0.05). (D) Gonococci lacking SliC or ACP were challenged with cell envelope membrane permeabilizing agents (Polymyxin B at 100 U/mL; Tween 20 at 0.001%; bile salts at 0.05%; or all compounds combined together) in the presence of lactoferrin (5 mg/mL) and lysozyme (5 mg/mL) as described above and bacteria were plated for CFU scoring after 4 h of incubation at 37°C (n = 3, mean±SEMs; *p<0.05). (E) Lipooligosaccharide was isolated from indicated N. gonorrhoeae strains collected from solid media. After cell lysis and proteinase K treatment equal amounts were loaded on SDS-PAGE followed by silver staining. (F) Scanning electromicrographs of N. gonorrhoeae wild type FA1090, ΔsliC, ΔsliC complemented with sliC, Δacp, and ΔacpΔsliC. Gonococci were prepared from exponentially-growing cultures, washed in PBS, spotted on copper grids and negatively stained with phosphotungstic acid. Scale bar represents 0.5 μm.

Fig 4