During the reporting period (2 years) the work carried out have been essential to optimize the conditions to perform unbiased silencing analyses in primary mouse macrophages. Our methodology focused on the use of small hairpin RNAs (shRNAs), molecules that allow to shut down the activity of target genes in host cells. Thus, the optimization of the shRNA technology in bone marrow-derived macrophages (BMDMs) was crucial for the success of the project. We have identified the best conditions to deliver shRNA molecules to BMDMs by using lentiviral particles as a vehicle (transduction). We have constructed a fluorescent S. Typhi strain that carries a chromosomal copy of the mCherry gene (red fluorescence). This strain is highly fluorescent and therefore suitable for further microscopy and flow cytometry analyses. Another critical step of the approach was the sensitivity of the flow cytometer. Depending on whether macrophages can control S. Typhi infection, we expect to obtain different populations of cells containing different amounts of intracellular bacteria. This will result in a different fluorescence of the different population of cells allowing us to identify them and physically separate them for further analyses. To optimize these parameters, we used a shRNA construct targeting HPS-1, a gene known to be essential to restrict S. Typhi infection in mice. In these conditions, we expect to obtain cells unable to kill S. Typhi and therefore containing higher loads of intracellular bacteria as the infection progresses. As a consequence of having higher number of bacteria, their fluorescence will be higher than in cells capable of killing S. Typhi. We have optimized the conditions to perform flow-cytometry experiments where as low as 1 or 2 bacteria per cell are distinguishable from non-infected cells (no bacteria). Also, we found that 24 hours after infection we can detect a population of high fluorescent cells that is not present at early time points nor in control cells. S. Typhi survival assays confirmed that at 24 hours post-infection the bacterium survives within these cells. We also found that this population of cells contains higher number of intracellular bacteria (as expected), confirming the feasibility of the approach. Once we have identified the cells that are unable to kill S. Typhi the next step is to isolate the genomic DNA of those cells to identify the targeted genes. Thus, the last challenge to overcome was the isolation of genomic DNA from fixed cells. Here, we optimized a protocol where DNA isolated from as low as 2,000 fixed cells can be used as template for a further PCR reaction obtaining specific amplification products. Once all the conditions were optimized, we performed a small scale screen (1041 shRNA sequences) where two families of genes (Rab GTPases and deubiquitinases) were targeted. From this screen, cells containing different amount of bacteria at two different times post-infection (1.5 and 24 hours) were sorted, the genomic DNA isolated and the shRNA-encoding regions amplified. The PCR products were sent to the Centre for Genome-Enabled Biology and Medicine (University of Aberdeen) to be sequenced.
In parallel, we evaluated the effect of two known macrophage antimicrobial mechanisms in killing S. Typhi in mouse cells. Specifically, we focused our attention on copper transport and the Cathelicidin related antimicrobial peptide (CRAMP). Copper is known to be delivered to pathogen-containing compartments by a transporter named ATP7A. Thus, we used the shRNA technology developed at the beginning of the project to specifically silence the ATP7A gene. To study the role of CRAMP, BMDMs are isolated from mice defective for CRAMP (CRAMP-/-). ATP7A-depleted BMDM or CRAMP-defective BMDM were infected with S. Typhi to analyse their ability to kill this pathogen. The results obtained showed that, under the experimental conditions tested, absence of CRAMP or depletion of ATP7A have no impact on S. Typhi surviv
