Animal factors could help to understand how to prevent typhoid in humans
Salmonella Typhi (S. Typhi) causes typhoid fever, but only in humans, a phenomenon known as host restriction. Living in macrophages, cells of the immune system devoted to killing pathogens, the socioeconomic cost of this infection is high, claiming the lives of over 200 000 patients every year. Recent research has uncovered a mouse antimicrobial pathway that is required to restrict the growth of S. Typhi in macrophages and contributes to S. Typhi host restriction. Despite this major breakthrough, the molecular mechanisms used by macrophages to kill S. Typhi as well as their role in the adaptation to the human host remain unknown. The factors needed to kill S.Typhi The EU-funded KILLINGTYPHI project has optimised the conditions for a large genome scale analysis on a smaller scale. The target – gene families known to be exploited by Salmonella and other pathogens to cause disease, the RabGTPases and deubiquitinases. “In addition, we wanted to evaluate factors known to have an impact in the survival of bacterial pathogens in macrophages and study their role in the outcome of S. Typhi infection,” says project coordinator, Prof. Stefania Spano. The ingenious use of two state-of-the-art technologies sets this piece of research apart from the others. CRISPR/Cas9 is a very fast, cheap and accurate way of editing genes under surveillance. Small hairpin RNAs (shRNAs) were used to shut down the activity of target genes in host cells. As the fellow who led the research, Dr Virtu Solano-Collado emphasises, “The shRNA technology has rarely been performed with pathogens and never performed with Salmonella. Moreover, CRISPR/Cas9 has not been used before to decipher questions directly related with host-pathogen interactions.” Unbiased silencing analysis in mouse macrophages achieved Using mouse bone-marrow derived macrophages (BMDMs) and shRNA molecules to shut down the expression of host genes, the researchers studied infection rates with S. Typhi. The best way to deliver the shRNAs was via lentivirus particles with the lentiviral infection optimised. To gauge level of infection, KILLINGTYPHI built a fluorescent S. Typhi strain that carries a copy of the mCherry gene – it glows a nice red colour. “The new strain was developed in collaboration with the expert Prof. Leigh Knodler of the Paul G. Allen School for Global Animal Health, Washington after several unsuccessful attempts,” explains Dr Solano-Collado. Further microscopy and flow cytometry analyses helped measure levels of infection. Several milestones were passed by the project researchers using mCherry. “We are able to distinguish cells containing as low as 1 or 2 bacteria per cell from non-infected cells (no bacteria),” Dr Solano-Collado points out. Another small but significant tweak in the method that vastly improves results is to reverse the DNA crosslink caused by paraformaldehyde that is used to fix the cells. The researchers can now use as few as 2 000 cells to obtain DNA products that can be sequenced. Zone in on the genes themselves In parallel to the shRNA screen, the project researchers have evaluated the effect of two known macrophage antimicrobial mechanisms in killing S. Typhi in mouse cells – copper transport and the Cathelicidin related antimicrobial peptide (CRAMP). Copper is known to be delivered to pathogen-containing compartments by a transporter named ATP7A. So far, the results obtained showed that absence of CRAMP or depletion of ATP7A have no impact on S. Typhi survival in mouse macrophages. The results of this screen are currently being further analysed. Next steps to fight a fatal disease “As soon as the sequencing data from the small screen is finished, we will be ready to start the genome-wide screen. The results of this will form the basis of future grants to continue our work,” Dr Solano-Collado explains. Prof. Spano wraps up with the significant impact KILLINGTYPHI promises to have on society in the long term. “We expect that our findings will help to find alternative drugs or develop more efficient vaccines against S. Typhi to prevent infection. Given the speed at which infectious diseases can spread around the world, this is of primary importance.”
Keywords
KILLINGTYPHI, S. Typhi, macrophage, typhoid fever, screen, shRNA, CRISPR/Cas9
