The overall goal of this experiment is to measure the effects cells pre-infected with HSV have on the subsequent attachment of bacterial and fungal opportunistic pathogens, for example, Staphylococcus aureus and Candida albicans. This method can help answer key questions with regards to the role chronic viral infection plays in regulation of the respective members of the host microbiome. The main advantage of this technique is that the interaction of multiple organisms can be examined and quantified at the initiation step for biofilm formation, namely adherence.
The implication of this technique extend towards control of colonizing opportunistic pathogens, because the precondition of HSV infection with asymptomatic shedding is present in the majority of the human population. Though this method can provide insight into HSV control of subsequent microbial attachment, it can also be applied to other systems such as CMV infection and that of adenovirus. To begin, determine HSV1 or HSV2 virus viability and multiplicity of infection, or MOI by x-gal staining for each experimental assay run using a reporter virus entry assay.
Dilute the virus to the desired MOI and place the virus viability controls in a separate microtiter plate in parallel with the poly-microbial assay plates. Then use 100 microliters of paraformaldehyde glutaraldehyde buffer to fix mono layers before staining. To maintain HeLa cells, culture them in DMEM with four point five grams per meter of glucose, ten percent heat inactivated FBS, 50 micrograms per liter of gentamisin and l glutamine.
Use Trypsin solution to passage the cells at 80 percent confluence. To handle C.albicans, culture a frozen stock on sub around dexterous medium at 37 degrees celsius. After 24 hours, subculture the C.albicans on to fungicell medium and incubate for 48 hours before use.
Generate germ tube or GT forms of C.albicans by picking representative fungicell colonies and culturing them in three milliliters of FPS at 37 degrees celsius for three hours. After the incubation, spin down the culture at 4, 000 times g for ten minutes. Then use HBSS to wash the cells two times.
After using a hemocytometer to count the cells, add the washed GT to 37 degrees celsius HBSS to an OD 600 of zero point three two. To make yeast form, or YF stock suspensions, pick representative fungicell colonies and use them to inoculate three milliliters of HBSS. Grow the cultures to an OD 600 of zero point three two using a hemocytometer, count the number of YF forms per milliliter, which should be 99 percent of cells observed under the microscope.
Make a working fungal stock by transferring 250 microliters of GT or YF stock into 25 milliliters of 37 degrees celsius HBSS. This results in ten to the fifth CFUs per milliliter. To prepare SRES ATCC 25923, streak a frozen stock onto five percent sheep blood agar and incubate at 37 degrees celsius for 24 hours.
Within two days, pick representative colonies, transfer to mannitol salts medium, and culture at 37 degrees celsius for 18 hours. To make an SRES stock, suspend representative colonies in three milliliters of 37 degrees celsius HBSS to an OD 600 of zero point one three two or ten to the eighth CFU per milliliter. To prepare a working stock, transfer 100 microliters of the stock suspension to 25 milliliters of HBSS.
This results in ten to the fifth CFUs per milliliter. The initial critical step when applying this method to other organisms or strains of microbes is the determination of the correct number of bacteria and fungi to add so that there are sufficient numbers of colonies to count. To carry out a polymicrobial biofilm assay, seed 96 well plates with 200 microliters of two times ten to the 15th HeLa cells per milliliter.
Rock the plates at 37 degrees celsius for 30 to 45 minutes before incubating in five percent carbon dioxide at 37 degrees celsius for 18 hours. Use One X optimum to wash the mono layers, then seed with the single strain of HSV. Test only one strain at a time.
Incubate the plates at 37 degrees celsius in five percent carbon dioxide for three hours. Using 100 microliters of 37 degrees celsius PPS with magnesium and calcium, wash the infected mono layers. Then use 37 degrees celsius HBSS to replace the PBS.
Leave 25 microliters of the HBSS in each well. Next, add YF, GT, and or SRES working suspension and incubate the plates at 37 degrees celsius in five percent carbon dioxide for 30 minutes. Following incubation, aspirate one column of the plate at a time.
Immediately refill the wells using 300 microliters of PBS with magnesium and calcium. After repeating this wash step twice, add 200 microliters at a one to 50 dilution of filter sterilized RIPA lysis buffer to each well. When working with other strains of bacteria or fungi, make sure that the concentration of RIPA used is sufficient for cell lyses, but still permissive for microbial viability testing.
Rapidly tritorate the HeLa cell lysate and pipette 50 microliters on mannitol salts and or fungicell medium. Then, using a glass rod bent at a 90 degree angle, spread the lysate over the surface of the plate. After incubating the plates for 18 hours, manually count the number of colonies per plate.
Controls consist of SRES an or C.albicans adherents to HSV uninfected HeLa cells. To carry out imaging studies, wash in acetone each round glass cover slip prior to sterilization. Then, using alcohol and flame sterilized forceps, place the dry sterile cover slips into the wells of sterile 24 well plates.
Add HeLa cells to the wells of the plates containing the cover slips. Then, infect the cells with virus. After infection, add bacteria and fungi according to the template and incubate and process the cells as demonstrated earlier for 96 well plate.
After the final wash, fix the cells for microscopy by flooding the cover slips with methanol and then allowing it to evaporate. For bright field microscopy, fill the wells containing the fixed cover slips with deionized water. Immediately aspirate the water and add enough grams crystal violet to cover each cover slip.
After washing the crystal violet stained cover slips with deionized water, dry them in C2 before adhering them with hard set mounting medium to a labelled slide. For fluorescents microscopy, after washing the cover slips free of methanol, dry them in the wells before mounting them on labeled slides with hard set mounting medium. Add a sufficient amount of a one to 20 dilution of FITC conjugated HSV1 plus 2GD antibody to cover the cover slip.
Incubate the slides in a moisture chamber at 37 degrees celsius for 30 minutes. After incubation, use PBS to wash the cover slips four times. Following the final wash, return the slides to the moisture chamber.
Add dab B and incubate for 30 minutes at 37 degrees celsius. After incubation, wash the slides four times with PBS. Allow the mounting medium to cure for 24 hours at room temperature in the dark.
Then, under an oil objective, on either a bright field or fluorescent microscope examine at least 50 fields for colocalization. Representative results of HSV's effect on Staphylococcus and or Candida albicans adherence are shown here. The presence of HSV1 inhibited Staphylococcal adherence to HeLa cells.
The presence of germ tube forms of Candida, but not yeast forms, restored Staphylococcal adherence levels. Similar results were measured for HeLa cells infected with HSV2. As seen in this figure, the level of adherence of Candida germ tube forms is significantly higher for HSV2 infected cells than for HSV1 infected cells.
The reverse effect was measured for HSV1 versus HSV2 on Candida yeast forms. Adherence patterns were determined using crystal violet staining of monolayers to visualize the interaction between bacteria and fungus. Fluorescents microscopy was then used to determine HSV presence in a relation to the other microbes.
In the absence of HSV, Staphylococci and both yeast and germ tube forms of Candida colocalized on HeLa cells. However, only Candida was observed on cells infected with HSV. Once mastered, this technique can be done in eight to nine hours if it is performed properly.
Following this procedure, other methods like differential fluorescent antibody staining can be performed in order to answer additional questions like differential expression of surface receptors that are present after virus cell entry. After it's development, this technique paved the way for researchers in the field of human microbiome to explore post microbe interactions in tissue culture as well as organ culture systems. After watching this video, you should have a good understanding of how to develop a polymicrobial interaction model that spans the taxonomic kingdoms.
Don't forget that working with pathogens can be extremely hazardous, and precautions such as the use of non-spreading virus strains and barrier techniques are required.
