This protocol enables researchers to analyze bacteria grown under conditions that more closely mimic those found in a mammalian host, which are metal limited through a process called nutritional immunity. This protocol facilitates generation of consistent reproducible bacterial growth under metal restricted conditions and can be adapted for growth of many different types of bacteria. Two days before beginning the experiment, streak gonococcide from freezer stocks onto GC medium plates for a no more than 24 hour incubation at 37 degrees Celsius.
14 to 16 hours before the growth experiment, steak single colonies onto fresh GC medium plates. On the day of the experiment, add five to 10 milliliters CDM to an acid-washed 125 milliliter baffled side-arm flask and use this medium to blank a Klett colorimeter. Use a sterile cotton tipped swab to inoculate 20 Klett units of CDM from healthy single colonies.
Generation of a healthy Neisseria gonorrhoeae inoculum for subculture into the 96 well plates for liquid growth assays is critical to the success of all downstream experiments. Incubate the cultures at 37 degrees Celsius in 250 revolutions per minute for one to two hours until approximate a one mass doubling before diluting the cultures with a sufficient volume of CDM to reach half of the initial culture density. Then, return the culture to the shaking incubator.
To prepare metal loaded proteins, add phoration solution to a 10 milligram per milliliter human transferrin solution to achieve 30%iron saturation. Add the metal of interest to the protein solution at a 15%molar ratio to obtain a 25%saturation. Use a syringe to add the metal loaded protein to a dialysis cassette and dialyze against two liters of dialysis buffer for four hours at room temperature.
Then, move the cassette to two liters of four degrees Celsius dialysis buffer and dialyze overnight at four degrees Celsius to remove any unbound metals. During the mass doubling, slightly dilute 10X pre-mixes of interest with PBS. Pre-treat the wells of a 96 well microplate with 15 microliters of the diluted pre-mixes and add 10 microliters of 10X pre-mix and 90 microliters of CDM to each of three wells for the blank controls.
Once the cultures in the side-arm flask have doubled, add 100 microliters of each culture to a unused well in the microplate and measure the optical density at 600 nanometers. After calculating the correct amount of dilution required to bring the cultures to an optical density at 600 nanometers of 0.02, dilute the cultures with CDM in small culture tubes and add a sufficient volume to dilute the 10X pre-mixes to 1X in the plate. Then, place the plate in a plate reader for eight to 12 hours with shaking to obtain optical density at 600 nanometers readings at the desired experimental intervals.
After the mass doubling incubation, back dilute the cultures with three volumes of CDM and add the metal treatments of interest. Then, incubate the cultures at 37 degrees Celsius with shaking for four hours. Shortly before the four hour mark, cut three pieces of filter paper and a piece of nitrocellulose to the approximate size needed to fit into a dot-blot apparatus.
Pre-soak the nitrocellulose in deionized water for about 10 minutes before assembling the apparatus with filter paper below the nitrocellulose. At the end of the incubation, record the cell densities and standardize the densities to an appropriate final density. Then, pipette the cell cultures onto the nitrocellulose.
When the filter paper has absorbed all of the liquid disassemble the apparatus and allow the blot to dry. Block the nitrocellulose membrane for one hour with an appropriate blocking solution and reassemble the dot-blot apparatus, replacing the filter paper with paraffin film to create a leak proof seal under the nitrocellulose. Dilute the metal binding ligand of interest to 0.2 micromolar in blocker and probe the cells for one hour with moderate shaking.
At the end of the incubation, siphon off the liquid with a vacuum, wash the blot, and follow standard immunological procedures to develop the signal. Western blot analysis of protein production in variable metal concentrations reveals up regulation of the zinc responsive outer membrane tdfJ in response to zinc chelation by TPEN. TdfJ is essentially undetectable when zinc is added back to the medium.
N.gonorrhoeae grows in the presence of zinc loaded calprotectin, which requires the action of the zinc responsive tdfH. When no usable zinc source is available, growth is restricted. Similar results are observed in the presence of zinc S100 A7, which can serve as a sole zinc source when the outer membrane transporter, tdfJ, is produced.
Gonococcide grown in CDM are able to bind calprotectin when producing tdfH and as a result of zinc scarcity. Increased binding of transferrin is also observed in cultures grown in CDM, indicative of higher levels of protein expression due to the more iron depleted nature of CDM compared to chelated GC broth. This protocol's been optimized for use with the described water sources and additional titration with other chelators or metals will be needed if other types of water are used.
For additional analysis, one could perform quantitative metal uptake assays or quantitative RTPCR for gene expression studies under metal deplete or metal replete conditions.
