TABLE 2.
Examination of the numbers of Shigella with IpaD or IpaB on the surface in the absence and presence of deoxycholate
|
ipaD mutant expressed in SF622
|
Deoxycholate added
|
Number of bacteria with surface
exposeda
|
|
|---|---|---|---|
| IpaDb | IpaBc | ||
| Wild-type | - | + | - |
| + | ++d | + (100%)e | |
| ipaD null | - | - | - |
| + | - | - | |
| S158A | - | + | - |
| + | + | +/- (70%) | |
| K151E | - | + | - |
| + | +++ | + (100%) | |
| K151E/E229K | - | -/+ | - |
| + | -/+ | -/+ (<20%) | |
| E229K | - | + | - |
| ± | + (80%) | -/+ (20%) | |
The number of bacteria having specific proteins on their surface was determined by immunofluorescence staining of the bacteria as described under “Experimental Procedures.” In each case more than 10 representative fields were examined with at least 20 bacteria per field.
IpaD staining was with monoclonal antibodies (16F8) prepared in the laboratory of Dr. E. V. Oaks with rabbit anti-mouse IgG labeled with Alexa 488 dye as the secondary antibody.
IpaB staining was with rabbit antisera generated against IpaB in the laboratory of Dr. E. V. Oaks with mouse anti-rabbit IgG labeled with Alexa 568 dye as the secondary antibody.
Changes in the intensity of perceived labeling (independent of the percentage of cells stained) are estimated relative to SF622 expressing wild-type ipaD grown in the absence of deoxycholate. Increased apparent labeling is shown with ++ or +++.
In samples where only a limited number of bacteria are stained for a given protein, the approximate percentage of cells showing fluorescence staining as indicated by the values given in parentheses.