Host and Bacterial Cellular Responses After Introduction of Bdellovibrio bacteriovorus into Human Cells Using an Artificial Endosymbiotic Model
Abstract
Bdellovibrio bacteriovorus HD100 is a Gram-negative bacterial predator that attacks other Gram-negative bacteria for its growth and survival in nature. Its prey spectrum includes several pathogens that invade human cells, such as Yersinia and Salmonella spp. Preliminary data found B. bacteriovorus can use these pathogens as vehicles to enter human cells, but is trapped within the endosome. Using a recombinant Escherichia coli strain that expresses both the invasin (INV) and listeriolysin O (LLO) proteins from Y. pseudotuberculosis and Listeria monocytogenes, respectively, we found B. bacteriovorus exits the endosome and enters the cytoplasm of the human cell, where it is still capable of subsequent predation events. The scientific objectives of this study are to expand on these studies to evaluate how the predator responds to being intracellular. This entails looking at the transcriptome of the predator as well as monitoring for extracellular proteases that are produced. We will also perform experiments with a variant of B. bacteriovorus (HI100) that is capable of growing axenically, studying its growth patterns and stability within human cells over an extended period of approximately one week. To accomplish this, we will optimize the invasion process with UV-killed E. coli INV/LLO prey to achieve approximately five predatory bacteria within each human cell. The human cells selected for this study are MCF10a breast epithelial cells since they can be treated with aphidicolin, a chemical which leads to S-phase arrest of growth and division, without loss of viability over a prolonged treatment (doi:10.1038/cddis.2014.315 (2014)). This will allow us to monitor the cellular responses without concern of overgrowth. Since B. bacteriovorus HD100 requires prey for growth and division, these predators will not increase in number over the extent of the experiments. At set times (6, 12, 24 and 48 h), samples of the media will be taken for ELISA to measure the IL-6, IL-8 and/or TNF? cytokine levels using ELISA. We will also determine if the presence of intracellular B. bacteriovorus reduces the viability of the MCF10a cells using both Live/Dead staining and the MTT assay. Previous studies from our group found predatory bacteria respond to extracellular nutrients, particularly amino acids (doi:10.1038/srep03811 (2014); doi:10.1007/s00248-017-1003-1 (2017); doi:10.1038/s41396-018-0154-5 (2018)). These responses include an increased expression and secretion of several proteases. Consequently, we will evaluate the transcriptional and secretome responses of intracellular B. bacteriovorus. The transcriptional responses will be measured using either RNA sequencing or via real time-quantitative PCR (RT-qPCR). Several of these proteases will be expressed within B. bacteriovorus with a his-tag epitope so that we can monitor for their production and presence within the human cells. All of these results (RNA and protein) will be compared with our previously published results. Finally, host-independent variants of B. bacteriovorus will be used to study the growth and stability of this predator when present in human cells. In contrast with B. bacteriovorus HD100, which cannot grow axenically, the host-independent variant (B. bacteriovorus HI100) can but does so at a very slow rate. Using aphidicolin-treated MCF10a cells, we will monitor the growth of HI100 within the human cells over a week period. Likewise, actively growing MCF10a cultures will also be used to see if the HI100 cells are maintained during host cell division. This proposal presents a new paradigm in intracellular bacteria-host interactions, one in which the bacterium offers a clear benefit to the host (attacks and kills other invasive bacteria) but gains only a minimum pay-off (amino acids for HD100 and nutrients for growth of HI100). This study will advance this by determining how both the host and bacterial cells respond to this forced, but potentially natural, interaction.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Feb 14, 2019
- Source ID
- W911NF1910076
Entities
People
- Robert J Mitchell
Organizations
- Army Contracting Command
- Ulsan National Institute of Science and Technology
- United States Army