Peptidoglycan in Orientia tsutsugamushi and other Rickettsiales species

Orientia tsutsugamushi is an obligate intracellular bacterium which causes the neglected human disease scrub typhus. The cell biology of Orientia is relatively poorly studied compared to other human pathogens of equivalent severity and prevalence. Due to the lack of tools for genetic manipulation, this project initially focused on developing a toolkit of probes for labelling bacteria for fluorescence microscopy. By successfully labelling Orientia with antibodies, dyes and chemical probes, key biological questions can be investigated. One of these key questions is whether Orientia synthesises peptidoglycan. Bacterial cell walls typically contain a thick polymer structure of sugars cross-linked by amino acid bridges called peptidoglycan, which is the main determinant of cell shape and which is essential in protecting bacteria from osmotic stress. The fact that it is both essential and unique to bacteria means that eukaryotic cells have evolved to recognize it as a highly immunostimulatory PAMP, and it is also an important target for antibiotic therapy. Obligate intracellular bacteria have evolved to replicate exclusively inside host cells, and the resulting proximity to peptidoglycan receptors such as NOD1/NOD2 may cause selective pressure to reduce the amount of immunostimulatory peptidoglycan in their cell wall. Of the two major groups of obligate intracellular bacteria, the Chlamydiales and Rickettsiales, the peptidoglycan structure in the Chlamydiales has been more extensively studied. It was previously reported that Orientia does not contain a peptidoglycan structure in its cell wall, even though it encodes most of the genes required for production. The research described in this thesis demonstrates that Orientia does possess a peptidoglycan-like structure in its cell wall. Orientia also has an outer membrane comprising of a network of disulphide crosslinked proteins, and this works together with the peptidoglycan-like structure to confer structural rigidity and osmotic protection. The minimal peptidoglycan-like structure of Orientia presents striking similarities with that of the unrelated Chlamydiales group, suggesting convergent evolution to selective pressures of the obligate intracellular lifestyle. Based on the similarities between Orientia and Chlamydia, a bioinformatics analysis of the peptidoglycan biosynthesis pathway was performed on all major obligate intracellular bacteria, including several Rickettsiales species. This led to the prediction that some – but not all – Rickettsiales would possess minimal peptidoglycan-like structures with similarities to those seen in Orientia and Chlamydia. This motivated the third part of this thesis, in which peptidoglycan was studied in five diverse species from the order Rickettsiales (Rickettsia canadensis, Wolbachia, Anaplasma marginale, Anaplasma phagocytophilum, Ehrlichia chaffeensis). This comparative study revealed differences in peptidoglycan-like structures in these organisms that were supported by bioinformatic predictions, leading to hypotheses about how differences in their life cycles and cell tropism led to various selective pressures on their cell wall composition.