Oct
30

One Step at a Time

It’s been a while since I’ve posted, but I have been hard at work! There has also been some slow-coming, but exciting progress thus far.

As a recap, here are my research questions once more and progress for each research question:

1) In hopZ gene expression regulated by slip-strand mispairing of the poly-A tract?

  • We are working on getting a hopZ phase-on gene (in basic terms, phase-on means that transcription/translation can be completed efficiently and effectively into a functional protein product). Phase is regulated via a poly-CT tract. Currently, our strain J99 is hopZ phase-off with 6-CTs. We are working on mutating this to 7-CTs which puts the gene phase on. We have successfully made a plasmid construct with 7-CTs; however, there were other downstream spontaneous mutations from this CT-tract that made the gene phase off again. I am currently performing more mutagenesis reactions to correct this. However, in previous trials, it has been unsuccessful. Multiple repeats of this are starting to lead us to believe that putting the hopZ gene phase on in E. coli may be toxic to that bacteria. Thus, this may lead to a selection against E. coli  with the hopZ gene phase on. Hopefully this isn’t the case so we can get a good, hopZ phase on plasmid.

2) Does sabB play a role as an adhesin?

  • I have a plasmid construct with the sabB promoter within it. I am currently mutating this plasmid with a BamHI restriction site so I can insert a rdxA and CAT gene cassette into that region. This cassette will allow me to create antibiotic marker-less mutations in these plasmids for the future when I mutate the sabB poly-T tract.

3) Does increased expression of adhesin proteins (via slip-strand mispairing of poly-nucleotide tracts) lead to hyper-adherent strains of H. pylori?

  • I am conducting adhesion studies with my sabA T18 and T23 J99 strains of H. pylori. Interestingly, I have been noticing a pattern of decreased H. pylori adherence of the T23 J99 strain in comparison to the T18 J99 strain. This is contrary to our hypothesis. One of Professor Forsyth’s former students who graduated in 2002 and is currently working at UNC suggested that we do gene expression studies on the nonadherent H. pylori strains to see if there mere exposure to AGS cells influences the expression of other adhesin genes, thus explaining the decreased adhesion of these T23 J99 strains.
  • I am also creating a J99 strain that is phase-on for sabA and either T18 or T23. I successfully constructed plasmids for these and transformed H. pylori. I am gearing up these new mutants to do more adhesion assays as well as prepare to look at CagA translocation and AGS cell IL-8 production.

4) Does this affect CagA translocation?

5) Does this affect IL-8 production by the host cells?

Overall, I have made new mutants and still working on generating the other mutants. This is the hardest and longest phase of my project, but I feel like I am on track for completing these mutants. Hopefully everything goes well and my experiments are successful!