Fuhrmann Fonden - Pan-genome wide association study in Streptococcus Pneumoniae

Pneumococcus is a gram-positive bacterium that naturally resides on the mucosal surface of the human nasal tract and upper respiratory tract (URT). However, this commensal relationship becomes deadly when Pneumococcus crosses the mucosal barrier. If it enters the blood stream circulation, the pathogen might cause bacteremia or in rare cases spread further to the brain causing meningitis. The greatest cause of morbidity however is when Pneumococcus colonizes the lungs via the bronchi, causing pneumonia [1]. It is estimated that pneumococcal disease is the cause of more than 800.000 deaths amongst children under five years, worldwide each year. This makes Pneumococcus the cause of death in one out of ten times amongst children under the age of five [2].

Currently, infection by Pneumococcus is prevented through antibiotic treatment and the pneumococcal conjugate vaccines (PCVs) targeting the Pneumococcus capsule. Unfortunately, widespread antibiotic resistant strains and non-vaccine serotypes are emerging as a result of the selective pressure put on Pneumococcus [3]–[5]. This rapid adaptability is promoted by the natural competence of Pneumococcus, an ability that enables the pathogen to remodel its genome by uptake and incorporation of exogeneous DNA. This rapid evolution in part, lead WHO to include Pneumococcus to the list of 12 priority pathogens in 2017 [5], [6].

However, very little is known about this pathogen and its molecular biology, making the development of new more efficient treatment options difficult. The aim of this project is therefore to get a better understanding of its interaction with its only natural environment, the human host, specifically of its two component systems (TCS). The TCS serve as the sensory system of Pneumococcus, with an extracellular histidine kinase (HK) sensing a specific environmental que and an internal response regulator (RR) mediating an appropriate genetic response to this signal, this HK and RR make up the two components of each TCS. Currently, 13 different TCS have been identified within the genome of Pneumococcus, but only two of these have been studied in-depth [7], [8].

Instead of going through the meticulous effort of identifying the regulon of each TCS through conventional wet lab techniques, this study aims to streamline this process using bioinformatics. Within recent years pan-genome wide association studies (panGWAS) have become possible in prokaryotes, due to algorithmic advances. This method utilizes the genomic variability in between prokaryotic strains to predict which genes are co-inherited with each other or a trait by aligning pangenomes rather than single reference genomes [9], [10]. These pan-genomes consist of the core genome, which are the genes shared by all strains and the accessory genome which consists of the additional genes This alignment method turns the genomic noise within bacteria into a strength rather than a weakness as relevant genetic traits will stand out amongst the noise [9]–[11].
This makes Pneumococcus the prime candidate for a panGWAS as the natural competence of Pneumococcus and its high rates of homologues recombination has led to a high genomic variability in-between isolates, with strains on average only sharing 74% identity on the nucleotide level [12].

The project aims to determine the distribution of the TCS across all full assemblies of Streptococcus species by screening for each of the 13 HK and RR gene pairs. This absence presence matrix will then be compared to a pangenome created of these strains in a panGWAS. This prediction of which genes are coinherited with each TCS will represent a possible regulon for each TCS and help guide later wet-lab experiments.
This panGWAS study will be complemented with RNA sequencing of Pneumococcus D39 mutants, each overexpressing one RR respectively. In theory this will make the mutants overexpress the regulon amongst other genes and combined with the panGWAS prediction give a strong indicator of the function and regulon of each TCS.

Knowing the environmental ques and responses of the TCS in Pneumococcus will not only give a better understanding of the pathogen and its interactions with the human host but might also aid in future treatment options. The HK of the TCS might prove to be good options for vaccine targets, as they are both extracellular and seemingly indispensable as opposed to the capsule. TCS are also abundant in bacteria but completely absent amongst vertebrates [7].