5 The Experiment
Having selected the etpD gene as a likely contributor to adherence, and so pathogenicity, an experiment was devised that could establish whether this gene makes a contribution to pathogen adherence. The experiment aims to satisfy Falkow’s molecular variant of Koch’s Postulates:
- The wild-type/control pathogen containing etpD must be able to adhere to human tissue/catheter material
- The mutant organism lacking only etpD must not adhere to human tissue/catheter material
- A complemented mutant, with etpD restored, must be able to adhere to human tissue/catheter material
5.1 Testing the postulates
The first of these postulates can be tested using the wild-type/control strain of UPEC.
It is not straightforward to measure adherence directly, so an indirect assay is performed.
Rather than measuring adherence directly, the amount of bacteria recovered from material exposed to the bacteria is measured (as log(CFU) - log of the colony forming unit/mL count)
The assumption is made that if the bacteria do not adhere well to a substrate, like catheter material, then after exposure fewer bacteria can be recovered from that material. If the bacteria do adhere well, then we should be able to recover more bacteria. In this way, logCFU is expected to be a proxy for bacterial adherence.
For this experiment either (i) strips of catheter tubing or (ii) human urinary tract tissue samples were washed and incubated in bacterial suspension (about \(10^7\) CFU in phosphate-buffered saline (PBS), OD=0.02) for two hours at 18degC. On removal from suspension the material was vigorously washed three times with sterile PBS on a vortexer, weighed and homogenised. The samples were then serially diluted and plated on MacConkey’s agar for bacterial counts.
5.1.1 Making a knockout
The etpD sequence was obtained from the sequencing reads in the earlier experiment, and a defined deletion was constructed using allelic exchange. The deletion was confirmed to be successful using PCR (polymerase chain reaction) and Sanger sequencing. The resulting strain is \(\Delta\)etpD, and can be used to test the second part of the postulates above.
5.1.2 Making a complemented strain
To test the third and final postulate, the \(\Delta\)etpD strain must be complemented with an additional plasmid that reintroduces the etpD gene.
Merely having the gene is not sufficient, though - the gene must also be expressed and produce the corresponding gene product (i.e. the protein EtpD). To ensure this happens, the etpD gene is introduced on a plasmid that contains an upstream promoter. It is convenient to make this promoter respond to a chemical signal that the experimenters provide, turning on expression of the gene and production of the EtpD protein.
In this experiment, the etpD gene was cloned into the IPTG (Isopropyl β-d-1-thiogalactopyranoside, a lactose mimic)-inducible plasmid pSE380, and this was transformed into the mutant \(\Delta\)etpD strain.
5.1.3 One more control
To establish whether or not the plasmid vector pSE380 by itself makes any contribution to adherence, the \(\Delta\)etpD strain was also transformed with an “empty” pSE380 plasmid vector containing no etpD gene.
This was not necessary to test the postulates above, but it is good practice to eliminate an unexpected effect due only to the plasmid.
Recovery of bacteria, measured as logCFU, was performed identically for wild-type/control, knockout, empty vector, and complemented strains.
5.2 What next?
Having collected the bacterial counts for all of these experiments, it now falls to you to carry out the analysis and determine whether the postulates are satisfied.