The overall goal of this permeable membrane insert-based infection system is to study the effects of secreted bacteria toxins on host cells during infection. This method can help answer key questions in the field of host-pathogen interactions, such as how specific secreted bacterial components contribute to host cell responses during bacterial infection. The main advantage of this technique is that it allows for the study of secreted bacteria components, and more closely models a physiologically relevant host-pathogen environment in the context of human infection.

Demonstrating the procedure will be Rebecca Flaherty, a senior graduate student from my laboratory. To prepare isogenic wild type group A streptococcus, or GAS, and GAS M1T1 5448 mutant strains, start overnight liquid cultures by inoculating five to 10 milliliters of Todd Hewitt broth and incubating at 37 degrees Celsius for 16 hours. Centrifuge the overnight bacterial cultures and use the original volume of fresh bacterial medium to re-suspend the pellet.

Then, normalize the bacterial cultures to equal starting concentrations by diluting the re-suspended cultures in additional medium to arrive at the same OD600. To collect lysates for carrying out signaling analysis and ATP determination assays, plate HaCaT cells in six-well dishes at a seating density of approximately three times 10 to the fifth cells per well, and culture to 90%confluence. To perform ethidium homodimer membrane permeabilization and lactate dehydrogenase release assays, plate HaCaT cells in 24-well dishes at a seating density of approximately five times 10 to the fourth cells per well, and grow to 90%confluence.

Immediately prior to treatment, use 1x PBS to wash the cells. Then apply two milliliters of fresh growth medium with or without pharmacological treatment to the six-well plates, or 0.5 milliliters of medium to the wells of 24-well plates. Next, under a laminar flow hood, use sterile forceps to carefully place a sterile 0.4 micron permeable membrane insert into each well.

Gentle and sterile handling of the permeable inserts during infection preparation is critical to prevent the membrane from becoming compromised or contaminated during this process. Apply fresh cell cultures medium to the upper chamber of each well according to the manufacturer's instructions. Then, apply an appropriate volume of the normalized bacterial cultures to the upper chamber of the permeable membrane insert system, and apply bacterial medium to the control wells.

Careful addition of the bacteria to the upper chamber, as well as gentle transport of the infected cells to the incubator, are critical, because it is essential that bacteria do not contaminate the lower chamber during the experimental setup. To assess for secreted host proteins, use sterile forceps to carefully remove the permeable membrane insert. Then, while not disturbing the monolayer of cells, collect the medium from the lower chamber into 1.5-milliliter tubes.

Centrifuge the samples at 14, 000 RCF and four degrees Celsius for 10 minutes to pellet cellular debris. Then, remove all but 50 microliters of the supernatant, transfer to a fresh 1.5-milliliter tube, and store at minus 20 degrees Celsius, or use immediately. For assessment of host cell lysates, gently aspirate the medium above the monolayer without disturbing the host cells.

Then, use PBS to rinse the cells once. Gently aspirate the PBS, and immediately apply a volume of ice-cold lysis buffer to achieve, for example, a protein concentration of 0.5 to 1.5 milligrams per milliliter. Then, incubate the samples on ice for 15 minutes.

Next, use a cell scraper to detach the cells from the plate surface of each well, and transfer the entire contents of each well to a 1.5-milliliter tube. Then, centrifuge the samples at 14, 000 RCF and four degrees Celsius for 20 minutes. To assess soluble lystate components, transfer the supernatant to a fresh tube, and store at minus 20 degrees Celsius, or use immediately.

To assess nuclear or other insoluable lysate components, reserve the pellet and store at minus 20 degrees Celsius, or use immediately. For assessment by immunofluorescence imaging, aspirate the medium and use 1x cold PBS to wash the cells. Then, with 4%PFA and PBS, fix the cells overnight.

Carry out further analyses according to the text protocol. The western blot data shown here demonstrates significantly enhanced p38 MAP kinase activation and keratinocytes in the presence of streptolysin S or SLS-producing gas strains, indicating that this system may be used to assess changes in the activation of contact-independent host signaling proteins. In this figure, SLS-dependent activation of the key inflammatory mediator nuclear factor-kappa B, which translocates from the cytoplasm to the nucleus upon activation, is shown, indicating that this system can be used to visualize the effects of secreted bacterial factors on host protein responses using immunofluorescence microscopy approaches.

Here, significant increases in both membrane permeabilization and in the release of LDH from host cells are demonstrated following exposure to SLS-producing gas strains, as compared to uninfected cells or cells exposed to an SLS-deficient strain. These results indicate that this system can assess toxin-dependent changes in host cytotoxicity. In this experiment, the levels of ATP in keratinocytes and response to gas infection were determined.

16 hours post-infection, a significant reduction in ATP was observed, and the loss of ATP was more pronounced in the presence of SLS, consistent with the toxin-dependent increases in host stress response signaling and toxicity. Once established, this technique can be utilized to study the specific responses of any secreted microbial factor on a variety of host cell types. While executing this procedure, it's important to practice sterile technique throughout the experimental setup steps, and to maintain careful handling of the permeable membrane inserts, both during the initial setup as well as during sample collection.

Following this procedure, methods such as western blotting, immunofluorescence imaging, and membrane permeabilization assays can be performed, in order to determine how the bacterial factor of interest contributes to changes in signaling cascades and influences overall cytotoxicity during infection. After watching this video, you should have a good understanding of how to prepare host cell samples for the evaluation of a variety of host cell responses following exposure to a specific secreted bacterial factor of interest. Don't forget that handling biohazardous materials, such as bacterial pathogens, requires certain safety considerations.

Appropriate use of safety glasses, gloves, and lab coats, along with the proper use of a biosafety hood, is necessary to perform these experiments safely.