Cell based High Throughput Screening Assays of Bacteria.

Learning Objectives:

  1. Identify key steps in a cell-based HT screening approach
  2. Discuss the importance of controls when screening inhibitory molecules
  3. Evaluate the necessity of primary and secondary screens in cell-based HT assays

Bacteria are prokaryotic organisms that affect humans, plants, and animals [1]. These organisms  might be of interest because they might cause a disruption in physiological activities of the  organisms, or they might simply be beneficial for the organism in certain occasions [2]. 

For this matter, screening chemical libraries and monitoring bacterial growth responses to find  potential growth inhibitors is of interest in many pharmaceutical and biotechnological companies [3]. This effort requires large scale experiments, to identify small molecules that might disrupt  either bacterial growth or communication in their communities, a process known as quorum  sensing [4]. 

When designing experiments for small molecule screening, appropriate controls are required. If  screening is performed for the inhibitory effect, then positive controls would consist of a  molecule that is lethal to the bacterium of interest. For instance, a bacterium such as E. coli that  is resistant to ampicillin and kanamycin, if challenged with an antibiotic that the bacterium is  sensitive, then we would not anticipate an increase in the population growth. On the other hand, a negative control would not interfere with the bacterial population and population growth  would be expected [5].  

Another important aspect of cell-based HT assays is the response of the organism of interest through the primary screen. Primary screens are usually testing 1000 to 10000 chemical  compounds and they are performed in duplicates or triplicates with an objective to obtain IC50 or  EC50 values, and then a dose-response curve can be constructed [5]. The dose-response curve  may inform us about the efficacy of a library compound or an unknown small molecule. IC50 (or  EC50) is defined as the half maximal inhibitory (or effective) concentration of a substance (e.g., 

small molecule or drug) which causes a 50% reduction in a biological process [6]. 

Secondary screens in cell based high-throughput screening (HTS), are utilized to characterize the  mode of action of the narrowed-down pool of small molecules that were derived from the  primary screen. Thus, downstream experiments might be performed such as radiolabeling to  track down the small molecule of interest, transcriptomic profiling or metabolomic analysis to  infer the gene expression levels or determine the secondary metabolites respectively [5]. 

Finally, cell-based HT assays may inform us about the response of the whole organism and at the  same time, may enable us narrow down our approach in subsequent experiments to dissect the  witnessed response [5]. It should be highlighted that the physiological stage in a cell-based assay  is of crucial importance, because the biological samples should be comparable.

Figure 1. Workflow of a cell-based HT screening approach in bacteria. A to C. Illustrates a colony  of E. amylovora strain 273 in an agar plate. 106c.f.u per mL were transferred in a 96-well  microtiter plate and the primary screening was performed in the epMOTION 5075. A dose response curve was constructed and the 4 lowest EC50 values were considered for downstream  experiments. D. The secondary screening of the 4 lowest EC50 of the 4 unknown chemical  compounds was performed in a transcriptional profiling experiment. Specifically, D shows a hierarchical clustering of the expression of untreated controls against treated with the 4  unknown compounds. The genes are shown in rows whilst the samples in triplicate in columns. The clustering of the samples and the genes can be seen in the upper and vertical axis  respectively. The colors -red, white, and blue in the heatmap represent the relative expression  levels as high, medium, and low respectively. The heatmap was generated by the CLC Genomic  Workbench software 22.0. Figure was created in PowerPoint.

Questions and Answers:

1. What is a cell-based HT screening approach? 

A cell-based HT screening approach is considered the assay that is performed at a large scale of  compound testing (1000-10000) against a cell or a population of cells. 

2. What controls are required when testing inhibitory chemical molecules? Why do we need  a positive control? Which control do we utilize to perform the comparisons with potential  molecules that we screened out? 

When testing for inhibitory chemical molecules, a negative and a positive control are required. A  positive control in this type of assay ensures that the population will not grow. A negative control  provides the plateau of the population growth under the specified experimental conditions.  Comparisons will be performed with the negative control to specify the effectiveness of the  unknown small molecules. 

3. Define EC50. Consider that we found the following EC50 values 1 ppm, 10 ppm and 25  ppm for the inhibitory molecules A, B and C that were tested in triplicate for the fungus  Colletotrichum acutatum . Which of the above molecules is considered the most  effective? 

EC50 is defined as the half maximal effective concentration of a substance which causes a 50 %  reduction in a biological process. The inhibitory molecule A is the most effective because it can  achieve a 50% reduction of the fungal growth at a much lower concentration. 

4. Why are secondary screens important? Provide two examples. 

Secondary screens are useful because they narrow down our experiments and enable us to  characterize the mode of action small molecule. For instance, the effect of a small molecule on a  gene cluster may be found as well as the cellular compartment where the molecule is revealing  its activity. 

References 

  • Kim, J. S., Yoon, S. J., Park, Y. J., Kim, S. Y., & Ryu, C. M. (2020). Crossing the kingdom border:  Human diseases caused by plant pathogens. Environmental Microbiology, 22(7), 2485–2495.  https://doi.org/10.1111/1462-2920.15028 
  • Linares DM, Ross P, Stanton C. Beneficial Microbes: The pharmacy in the gut. Bioengineered.  2016;7(1):11-20. doi:10.1080/21655979.2015.1126015
  • Silver LL. Challenges of antibacterial discovery. Clin Microbiol Rev. 2011;24(1):71-109.  doi:10.1128/CMR.00030-10 
  • Worthington RJ, Richards JJ, Melander C. Small molecule control of bacterial biofilms. Org Biomol  Chem. 2012;10(37):7457-7474. doi:10.1039/c2ob25835h 
  • Clemons, P. A., Tolliday, N. J., & Wagner, B. K. (2009). Cell-based assays for high-throughput  screening: Methods and protocols. Springer. 
  • Aykul S, Martinez-Hackert E. Determination of half-maximal inhibitory concentration using  biosensor-based protein interaction analysis. Anal Biochem. 2016;508:97-103.  doi:10.1016/j.ab.2016.06.025