Bacterial surface proteins make direct contact with host cells and are also involved in uptake of nutrients from the environment. Therefore, surface localization of bacterial proteins is a key step towards identification of virulence factors. Here, a method to assess surface exposure of proteins on intact lepto aspires is presented in comparison to previously described immunofluorescence assays.
The details of the method described here are designed to minimize the manipulation of cells in an effort to maintain outer membrane integrity. First lepto spiral suspension is added to glass chamber slides. The slides are incubated to allow the cells to adhere adherent lepto spires are fixed with 2%paraform aldehyde to preserve an intact outer membrane for surface protein assessment, or with 100%methanol to permease the outer membrane to reveal subsurface proteins.
The fixed lepto spires are then labeled with specific antibodies and visualized by fluorescence labeled secondary antibodies. Surface localized proteins can be labeled and observed in intact and permeable cells, while subsurface proteins can only be observed in perme cells. This method can help answer key questions in the microbial pathogenesis field, such as is a protein localized on the bacterial surface where it can play a role in host pathogen interactions.
The method presented here is designed to minimize the manipulation of cells, which should help to maintain alter membrane integrity. This method involves a lower concentration of fixing agent washing with alter membrane, stabilizing buffer, and fewer washing steps than previously described assays. In addition, our surface immunofluorescence method emphasizes the importance of both positive and negative controls that are essential for accurate data interpretation.
Demonstrating the procedure will be Dr.Maria Pinney, a postdoc in my laboratory. Leptospira Interrogans is a BSL two pathogen. Working with live cells requires appropriate handling and precaution.
A lab coat and gloves should be worn at all times. All pipetting and or open container handling of live cells should be performed in a laminar flow. Biosafety hood grow leptospira in EMJH medium supplemented with 1%rabbit serum at 30 degrees Celsius when the bacteria have reached mid to late log phase.
Approximately six days later, harvest the culture by centrifugation at 2000 times G for seven minutes at room temperature following the spin. Remove the supernatant by aspiration and gently resuspend the pellet in PBS with five millimolar magnesium chloride to a final concentration of five times 10 to the eighth cells per milliliter. Next to each well of two well chamber glass slides.
Add one milliliter of the cell suspension here. One test slide and one control slide are prepared for each surface protein assay with the second well used for the controls as shown here, incubate at 30 degrees Celsius to allow the cells to adhere. After 80 minutes, carefully aspirate the liquid to remove unbound cells to the test slides, which will be used to assess surface exposed proteins.
Add one milliliter per well of 2%para formaldehyde in PBS with five millimolar magnesium chloride to fix the remaining intact bacteria to the glass. Incubate for 40 minutes at 30 degrees Celsius to the control slides, which will be used to assess subsurface proteins. Fix the cells and permeate the outer membrane by adding one milliliter of 100%ice cold methanol to each well.
Methanol will permeate the outer membrane, denature the proteins, and fix the cells to the glass. Slides incubate at minus 20 degrees Celsius for 20 minutes. Aspirate the fixing agents after fixation.
The cells are no longer viable and do not need to be handled in a biosafety hood. To label the bacteria with specific antibodies begin by adding one milliliter of blocking buffer to each well on all slides to block non-specific binding. Incubate at 30 degrees Celsius for 90 minutes.
During the incubation, dilute the primary antibody in blocking buffer. The antibodies used here are directed against leptospira interrogans proteins. The MPL 54 antibody recognizes a surface exposed 54 kilodalton outer membrane protein.
The anti flat A one antibody recognizes subunit A of peri plasmic gellan and the anti OL one antibody recognizes lepto spiral poin, which is an outer membrane protein. Also prepare negative controls by diluting pre immune rabbit sera or mouse ascitic fluid containing no antibody in blocking buffer. After incubating for 90 minutes, remove the blocking buffer by aspiration and add one milliliter of diluted primary antibodies or control cera to each well incubate at 30 degrees Celsius for one hour.
Remove the liquid by aspiration and wash the wells three times with PBS, add one milliliter of LOR 4 88 labeled secondary antibodies to each well and the fluorescent nucleic acid stain DPI diluted in blocking buffer. This step ensures detection of antibody binding and the presence of all spiro keets independent of antibody binding respectively. Incubate the slides at 30 degrees Celsius for 45 minutes.
Remove the liquid by aspiration and wash the wells twice with PBS and once with distilled water, remove the chambers and the adhesive strip from the glass slides. Then air dry the slides for about 10 minutes. Add 20 microliters of prolonged gold anti fade mounting medium for each sample on the slide.
Then place a 24 by 50 millimeter cover slip incubate overnight at room temperature in the dark. This step is necessary to allow the mounting medium to cure. The next day.
Seal the cover slip with nail polish. Visualize the staining by fluorescence microscopy using a cyan or blue detection filter for DPI and a green detection filter for LOR 4 88. To ensure that an accurate representation of the samples is made, make sure you evaluate the entire chamber area for each sample.
Record the data by imaging a representative field for each sample. When imaging exor 4 88 fluorescence, use the same exposure time for all samples. Using a consistent exposure time will allow more accurate comparison of results with test antigens versus controls.
Be sure to evaluate the entire chamber area for each sample before making conclusions surface. Immunofluorescence analysis was performed to evaluate the surface exposure of MPL 54 in Leptospira inters. As shown here, a strong fluorescence signal was detected.
This signal was absent in cells incubated with pre immune sera, but remained consistent in perme cells. If protein is on the surface, substantial fluorescence intensity will be detected in intact lept aspires. If protein is subsurface, it will be only detected in permeated cells usually in a positive test.
Approximately the same fluorescence intensity is observed in both intact and perme cells. When nonpermeable cells were incubated against the subsurface protein flat, a one staining was not observed, indicating that the outer membrane remained intact during the experiment. Therefore, it can be concluded that ML 54 is surface exposed when significantly less fluorescence is observed in intact cells than in permeated cells.
Either the protein is in a subsurface location with some non-specific background staining of intact cells or the antibodies are binding preferentially to non-native epitopes that are exposed after methanol permeable. In either case, an ambiguous results such as this requires alternative approaches such as surface biotin or surface proteolysis to determine whether the protein is surface exposed or not. While attempting this procedure, it is important to include sufficient number of controls to ensure that the alter membrane remained intact.
These controls include premium serum methanol, perme cells, and antiserum to subsurface proteins.
