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MP1 - Experimental Biology

Emerging human pathogens: Photorhabdus asymbiotica as a model organism

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.

Project Aims

To determine how the gene content, gene regulation and cellular biochemistry of P. asymbiotica has changed between species and strains. This is done with a view to unravelling the reasons that have permitted the transition from ubiquitous invertebrate hosts to mammalian hosts and the consequent observed survival at human body temperatures, and thus the emergence of pathogenic properties.

Comparitive genomics of insect restricted strains passaged at a range of temperatures that have had target genes cloned in, will be used to assess the emergent differences and provide information about the potential mechanisms by which P. asymbiotica becomes tolerant to mammalian body temperatures.

Hopefully MiSeq data and transposon libraries will be able to be generated with the time and resources available, and this data will then potentially be analysable in miniproject 2, a bioinformatic analysis - more details of which can be found here.

Results and Conclusions from the Project

Transformants of target operons were generated and exhibited differential phenotypes. Notably, an n-acetyltransferase appears to be acting as a global regulator based on colony morphology and qRT-PCR analyses. The suggested mechanism is an epigentic-like system of acetylation of prokaryotic DNA-binding proteins, not unlike histone modification in higher eukaryotes. Such globally acting shifts in regulation are highly likely to be implicated in multi-faceted emergent properties such as virulence and thermotolerance.

A large, mostly unannotated operon comprising 6 genes, one of which was a topoisomerase-II associated protein homolog, was successfully transformed, but surprisingly resulted in no distinguishable phenotype. While the actions of these genes remains unknown, having no resultant phenotypic changes from the transformation of 6 genes (including promoter regions) with one gene having a putative DNA-binding role, was unexpected. Moreover, in a tightly clustered operon of its size, it seemed reasonable to think that at least one or more additional genes in the operon would likely have DNA-binding modes of action.

Induction experiments have continued outside of this project and hope to shed some further light on the basis of the operons activity.

An additional unexpected finding that came out of qRT-PCR analysis provides evidence about changes in crucial plasmid behaviour in a related, but non-pathogenic strain of P. asymbiotica. Comparisons between ATCC43949, the 'reference' US clinical isolate, and a more recent European isolate, 'JUN', revealed that a phage tail gene, initially picked for its expected role as a plasmid-borne 'housekeeping' gene (intended for use as a 'benchmark') actually showed noticable expression changes between strains. Earlier hypotheses based on some preliminary MiSeq data were that the plasmid in JUN was either incorporated in to the genome or to a larger plasmid, resulting in reduced copy number. Further bioinformatics analyses coming out of miniproject 2 are beginning to confirm this. The plasmid is present in both strains, and so the loss of human pathogenicity could conceivably by tied to reduced genetic copy numbers, and the associated shift in expression.