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Identification of genetic determinants involved in Streptococcus pneumoniae pathogenesis

Final Report Summary - TN-SEQ (Identification of genetic determinants involved in Streptococcus pneumoniae pathogenesis)

Respiratory tract infections are a leading cause of global mortality and morbidity. It has been estimated by the WHO that annually 4-5 million people die of pneumonia. Streptococcus pneumoniae, aptly named because it is the most important bacterial cause of pneumonia, is likely primarily responsible for these deaths.

S. pneumoniae is commonly found in the nasopharynx of healthy individuals, ranging from 5 % in adults to up to 40 % in children. Under certain conditions the prevalence of S. pneumoniae colonization is even higher especially in close contact environments such as in day care centres. Infection by S. pneumoniae is generally preceded by colonization. Traditionally, treatment of S. pneumoniae infection involves an antibiotic course, however nowadays up to 20 % of S. pneumoniae strains carry resistance against multiple antibiotics in northern European countries; this number is even higher in Southern European countries.

Preventive measures in the form of vaccinations are frequently employed to protect elderly, adults and children older than 2 years with serious long-term health problems. Vaccination currently protects against up to 80% of all pneumococcal infections. In a recent study however, it was found that the prevalence of non-vaccine strains is on the rise, additionally these strains frequently carry multiple drug resistance.

The high prevalence of S. pneumoniae colonization and the associated rise of non-vaccine drug resistant S. pneumoniae (DRSP) have lead to the undesirable situation that S. pneumoniae is one of the newest “Superbugs”, resistant to multiple antibiotics, making it more difficult to eradicate. Therefore it is of paramount importance that new drug targets are discovered to can lead to new medicines or broad-range vaccines that result in lower infection rates and increased survival, especially in younger infants.

Traditionally, drug target discovery involves extensive testing of a single drug targeting an essential step for (bacterial) pathogenesis or survival with methods that are far from high-throughput. New methods that are capable of high throughput discovery are starting to become more mature, but frequently suffer from a common problem with high throughput techniques in that too much data, with relatively high levels of noise, is generated to process in a feasible manner. The overall goal of this project is to identify S. pneumoniae genes required for growth and pathogenesis.

As both growth of and infection by S. pneumoniae is a complex process dependent on a number of essential metabolic pathways, members of these pathways could form ideal candidates for drug design. To identify microbial genes required for growth and pathogenesis, we have used an insertion knockout strategy that allows rapid identification of disrupted genes and the effect of the disruption on the viability of the bacterium during relevant conditions.

We have successfully performed micro array based approaches and next generation sequencing methodologies to determine which genes are essential under a number of relevant in vitro and in vivo conditions. To identify shared essential pathways we have used statistical analysis, pathway analysis, network reconstruction and functional category enrichment methods to determine the most ideal candidates for drug design.
Novel drug targets and potential vaccine leads generated from this approach will provide alternatives to continue treatment and prevention of life-threatening chronic and acute bacterial infectious diseases.

We have developed and published software to automate and compare gene essentiality screens and determined gene essentiality in various species causing respiratory infections using Tn-seq experiments. Using orthology predictions, pathway predictions and subcellular localization predictions we have propose novel drug targets and used off the shelf inhibitors to test these novel targets and to validate our selection method.

Furthermore, we have determined factors affecting adherence of S. pneumoniae to eukaryotic cells and survival in the presence of these cells. In addition we have performed gene expression studies under these conditions as well to focus on genes responding to exposure or attachment to epithelial cells. Additionally studies have been performed that identify the molecular mechanisms that facilitate pneumococcal growth under CO2-poor and CO-rich conditions, which are encountered in the niche of the bacterium.

Validation of the genes found, is actively being pursued and we have generated a number of knockouts and these have been tested in validation studies, furthermore we have determined new antibiotic drug targets, specifically in metabolic pathways specific for bacteria and have performed proof of concept studies with compounds that inhibit key functions in these pathways.