Neisseria gonorrhoeae MlaA influences gonococcal virulence and membrane vesicle production
Fig 7
Investigations of MlaA effects on cell structure, membrane vesicles, and biofilm formation.
(A) Strains as indicated were cultured in liquid medium without (top row) or with (bottom row) polymyxin B until approximately mid-logarithmic growth. ΔmlaA/Plac::pldA was cultured in the presence or absence of 0.5 mM IPTG, as indicated. Bacteria were subsequently washed twice with PBS, spotted onto 300 mesh copper grids, negatively stained with phosphotungstic acid, and imaged using scanning electron microscopy. (B) Cytoplasmic/periplasmic (C/P), cell envelope (CE), membrane vesicle (MV), and soluble supernatant (SS) subcellular fractions of WT FA1090 collected under standard aerobic conditions were prepared and normalized based on protein concentration, separated by SDS-PAGE, and probed with indicated antisera. (C) MVs collected by ultracentrifugation of culture supernatants derived from strains indicated were quantified by protein concentration (n = 6 for WT and ΔmlaA for − polymyxin B condition; n = 3 for all others; mean ± SEM). (D) WT FA1090 and ΔmlaA bacteria were suspended to an OD550 of 1.5 in GCBL, added to 96 well microtiter plates, and cultured without shaking in 5% CO2 at 37 °C for 24 h. Planktonic bacteria were removed and biofilms were washed with PBS. Biofilms were allowed to dry at room temperature, then stained in 0.1% crystal violet in 2% ethanol. After staining, wells were washed with PBS and dried. Biofilms were dissolved in 30% acetic acid and quantified by A550 measurement. Biofilm experiments were performed 12 times, each with 3 or 4 technical replicates, for a total of 46 datapoints. Mean ± SEM is presented. IPTG, isopropyl β-D-thiogalactopyranoside; C/P, cytoplasmic/periplasmic; C, cytoplasm; CE, cell envelope; OM, outer membrane; MV, membrane vesicle; SS soluble supernatant; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; OD550, optical density at 550 nm; A550, absorbance at 550 nm, GCBL, gonococcal base liquid medium.