TABLE 3.
In vitro and in vivo activities of PaeM mutant proteins
| Protein | Plasmid | Enzymatic activitya | Cytotoxicity against P. aeruginosab |
|---|---|---|---|
| nmol·min−1·mg−1 | μg | ||
| Wild type | pMLD245 | 13 (100%) | 0.04 |
| A238S | pTTB54 | 4.5 (34.5%) | 0.1 |
| D241A | pTTB240 | 0.1 (0.6%) | ND |
| D244A | pTTB243 | 0.6 (4.9%) | ND |
| D241A/D244A | pTTB38 | NDc | ND |
| Y243A | pTTB26 | 0.2 (1.5%) | ND |
| N249A | pTTB60 | 17 (130%) | 0.04 |
| H250A | pTTB30 | 11 (83.2%) | 0.1 |
| R251A | pTTB36 | 0.1 (1.1%) | ND |
| W54K | pTTB67 | 203 | –d |
| E55A | pTTB72 | 206 | – |
| W54K/E55A | pTTB78 | 217 | – |
a The enzymatic activity was measured in vitro in the presence of 2.3 μm of [14C]lipid II and the appropriate amounts of enzyme. Values represent the mean of triplicate determinations, the S.D. being within 15% of the presented values.
b The cytotoxicity of PaeM proteins was measured by spotting various amounts of pure protein samples on a lawn of the susceptible P. aeruginosa DET08 strain. The cytotoxicity was expressed as the minimal amount of protein required to obtain a clear halo indicative of growth inhibition.
c ND, no detectable activity (for in vitro assays, up to 15 μg of protein tested, except for Y243A, 1.5 μg tested; for cytotoxicity assays, up to 3.5 μg of protein tested).
d The cytotoxicity of these variants was not tested.