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MP2 - Theoretical Sciences

A Bioinformatic Analysis of the Bacterial Pathogen Genus Photorhabdus

Project Background

Some of the most devastating pathogens known to man, such as Yersinia pestis - responsible for the black plague - and Plasmodium falciparum, the causal agent of malaria, are obligately associated with invertebrate vectors (the flea and the mosquito respectively in this case). For a great deal of pathogen evolution, invertebrates were the only animal hosts available and this lifestyle will have significantly shaped/driven evolution of their respective pathogens. Upon the emergence of higher organisms, in particular warm-blooded animals, there would have been selection pressure to fill this new niche, but the exact extent and details of the types of adaptations that were required are still unclear.

Organisms capable of jumping the species barrier are of particular clinical importance, and understanding the mechanisms by which they do so has been an intense focus of at least the last 20-30 years - this is epitomised by the vast media attention which the likes of avian influenza and the Human Immunodeficiency Virus (HIV) etc. have garnered. However, these organisms often only transfer from comparatively closely related species, when compared to the jump from cold blooded insects to warm blooded animals. For example, the human immunodeficiency retrovirus is believed to have transferred to humans (as HIV) from monkeys (Simian Immunodeficiency Virus), 2 species lineages that may only differ by as little as 5% of the genome. Even the transfer from birds or pigs, as in the case of avian and swine flu, to humans is comparatively a 'minor' jump. Nevertheless, the changes that must occur at the molecular level to permit survival in the new hosts are often extremely hard to peice together, and this is likely to be even more the case for dramatic shifts such as insect-human transition. Invertebrate hosts provide a well suited pool for horizontal gene transfer to occur, and have also been shown to provide a kind of "virulence bootcamp" for enhancement of pathogenic traits due to conserved features of innate immune systems of invertebrates as well as higher organisms.

Understanding the total genetic content of the genus and having an adequate and well characterised set of genomes for these species is absolutely essential to begin to elucidate the genetic behaviour responsible for differential pathogenicity.

Project Aims

P. asymbiotica is a dual insect and emerging human pathogen that belongs to a genus with two other species, P. luminescens and P. temperata, which are both restricted to insect hosts and cannot survive above 34°C. Conversely, P. asymbiotica can grow at 37°C - the key to its pathogenicity in humans. All Photorhabdus genomes encode large numbers of virulence factors for combating innate immune responses, and it seems likely that in this case, the main barrier to the evolution of human pathogenicity has been an inability of most strains to survive 37°C , thus it is of particular interest to assess what exactly is occurring on a biomolecular level to permit this survival. P. asymbiotca contains a unique plasmid not possessed by the luminescens or temperatas; recent European isolates of P. asymbiotica named HIT and JUN, possess this unique plasmid but aren't capable of human infection. The operating hypothesis entering in to this investigation is that the plasmid is incorporated into the chromosome or a different larger plasmid. Understanding the nature of this plasmid is likely to provide some solid clues as to why the genus exhibits differential host pathogenicity.

This project will analyse a bank of existing Illumina data to assemble high quality genomes and try to define the core genome of the Photorhabdus genus (both chromosomally and plasmidborne). During my first miniproject some further sequencing of HIT and JUN was performed and these sequence data will assembled alongside all the existing sequences. Information regarding miniproject 1 can be found here.

The core genomes of the plasmid and chromosome will be analysed to elucidate orthologous and paralogous sequences, and to compute the average nucleotide identity to establish genome conservation. Alignments and phylogenetic trees will be created to specifically answer the question: have the plasmids co-speciated with genomes or have they been behaving as mobile elements - jumping between enterobacteriaceae populations for example.

Project Outcomes and Conclusions

This was a successful project where it was revealed that plasmids were co-speciating with the genomes of the strains within the Photorhabdus genus. 19 genes were shown to be core on the plasmid, and 2464 chromosomal genes were conserved across the clade. Nucleotide identity ranged from 59% to 99% chromosomally, and from 87%-98% on the plasmid. Further characterisation of gene function is necessary for informative relationships with regards to plasmid core genes however.

Additionally, 11 high quailty genome assemblies were produced for use in the future. Coverage ranged from roughly 14X to 140X. The genome sequences were produced as full genomes, chromosomes only and with plasmid sequences pulled out themselves too.