Neisseria gonorrhoeae employs two protein inhibitors to evade killing by human lysozyme
A. Representative images of 1063-FLAG and 1981-FLAG surface expression via imaging flow cytometry. Live Δ1063::1063(WT)-FLAG and Δ1981::1981(WT)-FLAG Gc were labelled with CFSE and subsequently stained with mouse anti-FLAG, followed by goat anti-mouse AlexaFluor647. WT Gc expressing no FLAG-tagged proteins was used as a negative control. n = 4 biological replicates. B. Histogram of anti-FLAG/AlexaFluor647 fluorescence intensity for Δ1063::1063(WT)-FLAG, Δ1981::1981(WT)-FLAG, and WT Gc from Fig. 7A. Shown is 1 representative of 4 biological replicates. C. Geometric mean fluorescence intensity of anti-FLAG-AlexaFluor647 for Δ1063::1063(WT)-FLAG, Δ1981::1981(WT)-FLAG, and WT Gc from Fig. 7A. Values are represented as the mean ± SEM from 4 biological replicates. *p < 0.05; two tailed t-test. D. Immunofluorescence micrographs of 1063-FLAG or 1981-FLAG in Gc with or without cell permeabilization. After paraformaldehyde fixation, Δ1063::1063(WT)-FLAG and Δ1981::1981(WT)-FLAG Gc were permeabilized with methanol and Triton-X-100 (+ perm) or left untreated (- perm). Bacterial DNA was stained with DAPI (blue) and FLAG proteins were detected with an anti-FLAG antibody (red). Scale bar, 5μm. Micrographs are from 1 representative of 3 independent experiments. E. Gc expressing 1063(WT)-FLAG under its native promoter were grown for 3 hr, at which point bacterial whole cell lysates (pellet, P) and conditioned supernatants (SN) were collected for SDS-PAGE and immunoblotting. To detect native Ng_1981 and Ng_1063 protein, blots were probed with anti-r1981 and anti-FLAG, respectively. The cytoplasmic protein Zwf (using anti-Zwf antibody) served as a loading control for proteins present in the whole cell lysate. Shown are the results from 3 biological replicates, labelled #1–3. F. Δ1981Δ1063 mutant Gc, Δ1981Δ1063::1981+ complement Gc, and a 1:1 mix of Δ1981Δ1063 with Δ1981Δ1063::1981+ Gc were grown for 1 hr prior to exposure to 10 μg/mL human lysozyme (HL) for 3 hr. Gc survival was calculated as in Fig 2B. *p < 0.05; two tailed t-test, n = 8 biological replicates.