Campylobacter jejuni Survival Strategies and Counter-Attack: An investigation of Campylobacter phosphate mediated biofilms and the design of a high-throughput small-molecule screen for TAT inhibition.

摘要

In these investigations we studied 1) the ability of Campylobacter to modulate its behavior in response to phosphate actuated signals, 2) the modulation of biofilm in response to phosphate related stressors, and 3) we designed and carried out a high-throughput small-molecule screen that targets protein transport via the Twin Arginine Translocation (TAT) system. We identified that the phoX, ppk1 and ppk2 genes were key components of the phosphate response that manifested increased biofilm phenotypes, and were modulated in the presence of inorganic phosphate. We used several molecular and microbiological techniques to investigate the effect of polyP, phosphate uptake inactivation, and inorganic phosphate availability on Campylobacter's response to phosphate stress. Additionally, we counted and measured attached biofilms, as well as measured pellicle size, biofilm shedding over the course of three days, and changes in the expression of genes known to be involved in biofilm formation phenotypes. By resolving biofilm components such as pellicles, attached cells, and shed cells we found that not only did ppk1, phoX, and ppk2 deletion affect the ability of Campylobacter to form biofilms, but biofilm components were not congruently and equally affected in each mutant. Additionally, the presence of phosphate modulated those effects both independently of and additively to gene knockouts.;Furthermore, we observed that biofilm components were additionally affected by biofilm age: where some components had their most robust growth on day 2, biofilm shedding and pellicle growth increased the most on day 3. This growth was not uniform for all mutants, as ppk1 biofilms generally matured more quickly than wild-type cells, but the ppk2 mutant in the presence of phosphate matured more slowly.;In our high-throughput small-molecule screen we designed and carried out a primary screen of small molecules to identify compounds that had anti- Campylobacter activity in the presence of 1mM CuSO4. To screen a greater number of compounds, this study was streamlined from a dual-plate study where each chemical was tested both in the presence and absence of copper sulfate. Our screen resulted in the identification of 680 small-molecule primary hits from the NSRB small-molecule library. These hits were identified from 11 different small-molecule libraries containing more than 50,000 compounds. Using database bioactivity results from past trials, the primary small-molecule positive hits were reduced to 476 targeted hits through in silica primary screens.;We used Golden Triangle in silica medicinal chemistry methods to identify molecules that were likely to be less suitable due to low molecular weight, interactions with solutes, and compound stability. From there, common chemical motifs were identified among the remaining 350 molecules. From these 'chemical families' a representative sample of each group was chosen as likely having similar chemical activity. We chose 54 chemicals as representative of 4 chemical motifs: thiourea, benzimidazoles, oxadiazoles, and acylhydrazones. The rest of the molecules were selected for greatest diversity. Using these techniques, 149 compounds have been chosen that will be used as 'cherry pick' hits for secondary screens in the near future.