Periodic Reporting for period 1 - GDA in staphylococci (Gen Duplication and Amplification in Staphylococcal populations) Reporting period: 2016-01-01 to 2017-12-31 Summary of the context and overall objectives of the project Prokaryotic organisms represent by far the most abundant living organisms populating our planet. They play important function for the ecology of our ecosystems and environments and without prokaryotes living on earth would not be possible. However, certain bacterial species are associated with severer kinds of human diseases such as tuberculosis or cholera. In addition, invasive infections such as soft tissue infections or bloodstream infection represent a constant threat for human health. Bacterial infections are commonly treated by antibiotics. However, antibiotic therapy becomes more and more challenging as resistant bacterial strains arise. The adaption of bacteria towards antibiotic pressure is a good example of the remarkable ability of prokaryotes to adapt to changing environmental conditions and to secure the thriving of the population. However, environmental pressure is manifold and ranges from the availability of different nutrient sources in the environment to the destructive ability of the human immune system when a pathogen enters the human soft tissue. In all these cases a bacteria community needs to adapt to the new environment which can happen by multiple routs. A prominent way is the so-called horizontal gene transfer were additional genetic material is acquired, transferring novel capabilities. However, it relays on the availability of exogenous genetic material (DNA) in the environment.A mechanism that is independent of exogenous DNA is “gene duplication and amplification” (GDA). GDA uses a genetic mechanism dependent on the SOS recombinase RecA. RecA can promote chromosomal recombination during cell division leading to the duplication of genes within a single chromosome (two copies of an identical gene in a row). Following this duplication, the process can be repeated to extend or to contract the gene array in an accordion-like manner. Long gene arrays can thereby be created and removed rapidly. The amplification of a gene results in strong overexpression of the target protein. GDAs are described to be able transfer antibiotic resistance (overexpression of drug efflux pumps) or to allow better usage of unusual carbon sources. However, it is unclear how frequent GDAs are in natural populations of pathogens and whether GDAs might be important for the adaption of pathogens to certain environmental niches or during the transition from colonization to infectious disease. This lack of knowledge has to be attributed to the difficulty of GDA detection. Even in times of Next Generation Sequencing (NGS) when entire bacterial genomes are sequenced within days, GDAs are only rarely reported. We hypothesized that this is due to the difficulties of their detection. The individual gene copies of GDAs are frequently identical on the DNA level and the short reads (100-200bp) created by NGS technology do therefore fail to indicate that several copies of the same gene are present in a genome. However indications about GDAs can be gained from NGS datasets if analysed accordingly. If more copies of the same gene are present, more individual NGS reads covering this gene will be created (the scaffolding will increase). However, this needs special attention during analysis. In this project we sought to investigate whether NGS technology can be used to conveniently detect GDAs to show how our community can gain additional knowledge from NGS. Furthermore, we wanted to identify the frequency of GDAs in clinical populations and the effects of GDAs on phenotypic characteristics to demonstrate effects of theses mechanism on pathogen evolution. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far We wanted to investigate how frequent GDAs are in clinical populations of the opportunistic human pathogen Staphylococcus aureus. We investigated the scaffolding of ~300 S. aureus genome sequences and identified a number for genomic areas with putative GDAs in individual strains. Most prominently, a high percentage of strains showed putative GDAs in a gene array encoding the Csa1-lipoproteins. These lipoproteins (proteins attached to the bacterial membrane) have been described before to be a target of vaccination strategies, but the biological function of Csa1 remains elusive. We successfully developed experimental techniques to verify the Csa1-copy number variation and showed that indeed NGS-scaffolding allows the prediction of GDAs when analysed accordingly. These results will encourage the scientific community to modify the evaluation of NGS experiments in the future. For the progress of our project, the results promoted us to investigate how the amplification of csa1 locus behaves under laboratory condition and what the biological relevance of this phenomenon might be. We integrated an antibiotic resistance gene into the csa1 locus to gain a selective phenotype. Amplifications of csa1 will thereby accidently also amplify the antibiotic resistance gene leading to changing levels of resistance. This allowed us to trace amplification events in our laboratory. Interestingly we found that amplification events happened constantly during bacterial growth and even within a single overnight culture (10-12 generations of growth), individual lineages harbouring up to 120 copies of the csa1 genes could be identified. Many antibiotics used in clinical practice induce damage to the bacterial DNA thereby inducing bacterial RecA expression. Since GDAs are driven by RecA, we figured that antibiotic pressure might induce the frequency of amplification and indeed we found that presence of ciprofloxacin increased the frequency of csa1 amplification tenfold. Amplification of the csa1 genes had strong effects on the cellular physiology. The isolated copy number variants expressed significantly different amounts of Csa1 proteins. Especially the bacterial membranes of the high copy number variants were overloaded with Csa1 proteins suggesting distinct effects on the properties of the cells. Bacterial lipoproteins are known to be ligands for the human Toll-like receptor 4. This receptor is an important signalling molecule of the innate immune system and allows immune cells to recognize bacterial invaders. Stimulation of immune cells with lipoproteins leads to the secretion of cytokines causing inflammation and appropriate immune responses. Due to this properties of lipoproteins we figured that amplification of csa1 might influence the immunostimmulatory capacity of the bacterial cells. Indeed, we found that high copy number variants caused massive cytokine responses in our experimental systems while low copy number variants showed clearly reduced responses. Currently we investigate the effects the effects of csa1 amplification on the virulence and survival of the bacterial lineages during invasive disease in models of systemic infection of mice. This will clarify whether GDAs can impact the virulence potential of individual lineages. A manuscript to publish and disseminate the gained results is under preparation Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) This project set important foundations for future projects by showing that GDAs are frequent in clinical populations, a phenomenon that is neglected in research up to date. Furthermore, GDAs can be associated with clinically relevant phonotypes. Thereby this project shows that NGS technology might directly be used to predict the pathogenic potential of individual isolates in clinical practice. A novel potential application of NGS whose use needs to be closer investigated in the years to come. Schematic diagram of the project results
