Our Research
Developing tools to study obligate intracellular bacteria.
One of the reasons why our understanding of obligate intracellular bacteria lags other pathogens is that they are technically difficult to work with. They are challenging to culture and many, including Orientia tsutsugamushi, remain genetically intractable. Furthermore, Orientia tsutsugamushi is classified as a risk group 3 organism, requiring containment level 3 laboratories.
To address some of these technical challenges, and stimulate research in this field, we developed experimentally validated approaches to improve laboratory propagation of Orientia tsutsugamushi1. We also developed techniques for labelling Orientia tsutsugamushi using chemical probes to carry out live cell imaging2 and a biorthogonal click chemistry-based approach to specifically label metabolically active bacteria3, as well as carrying out the first dual RNAseq study for any Rickettsiales4.
1. Giengkam, S. et al. Improved Quantification, Propagation, Purification and Storage of the Obligate Intracellular Human Pathogen Orientia tsutsugamushi. PLoS Negl Trop Dis 9, e0004009, doi:10.1371/journal.pntd.0004009 (2015).
2. Atwal, S., Giengkam, S., VanNieuwenhze, M. & Salje, J. Live imaging of the genetically intractable obligate intracellular bacteria Orientia tsutsugamushi using a panel of fluorescent dyes. J Microbiol Methods 130, 169-176, doi:10.1016/j.mimet.2016.08.022 (2016).
3. Atwal, S. et al. Clickable methionine as a universal probe for labelling intracellular bacteria. J Microbiol Methods 169, 105812, doi:10.1016/j.mimet.2019.105812 (2020).
4. Mika-Gospodorz, B. et al. Dual RNA-seq of Orientia tsutsugamushi informs on host-pathogen interactions for this neglected intracellular human pathogen. Nat Commun 11, 3363, doi:10.1038/s41467-020-17094-8 (2020).
Genome analysis of Orientia tsutsugamushi
Gene loss in obligate intracellular bacteria has led to heavily reduced genomes. At 2.5 Mbp, the genome of Orientia tsutsugamushi is unusually large for an obligate intracellular bacterium, due to rampant proliferation of a mobile genetic element that comprises around 50% of the Orientia tsutsugamushi genome. Sequencing this highly repetitive genome using short read techniques is difficult5,6.
We used PacBio long read sequencing to generate six complete genomes, including the major reference strains7. We recently identified that two of the strains carry intact mobile genetic elements capable of carrying out lateral gene transfer, previously assumed to be no longer active in this species8. This work has increased the availability of high-quality genomes from two to eight and raised new questions about the dynamics of this genome.
5. Nakayama, K. et al. The Whole-genome sequencing of the obligate intracellular bacterium Orientia tsutsugamushi revealed massive gene amplification during reductive genome evolution. DNA Res 15, 185-199, doi:10.1093/dnares/dsn011 (2008).
6. Cho, N., Kim, H., Lee, J. & Kim, I. The Orientia tsutsugamushi genome reveals massive proliferation of conjugative type IV secretion system and host– cell interaction genes. PNAS 104, 7981-7986 (2007).
7. Batty EM, C. S., Blacksell SB, Richards A, Paris D, Bowden R, Chan C, Lachumanan R, Day N, Donnelly P, Chen SL, Salje J. Long-read whole genome sequencing and comparative analysis of six strains of the human pathogen Orientia tsutsugamushi. Plos Negl Trop Dis (2018).
8. Giengkam, G. et al. Orientia tsutsugamushi: analysis of the mobilome of a highly fragmented and repetitive genome reveals ongoing lateral gene transfer in an obligate intracellular bacterium. mSphere (2023).
Analysis of the cell wall of Orientia tsutsugamushi and other Rickettsiales
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Peptidoglycan is a large macromolecule that encases almost all known bacterial species, conferring rigidity and protection. It is also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria have evolved under pressure to reduce immune activation, whilst maintaining growth and division.
We found that, contrary to previous reports, Orientia tsutsugamushi builds a minimal peptidoglycan wall9. Orientia tsutsugamushi lacks a key enzyme normally used in peptidoglycan synthesis and we showed that this enzyme is also missing in other, unrelated obligate intracellular bacteria10,11. This has led us to a new model for the evolution of peptidoglycan in obligate intracellular bacteria, in which a minimal peptidoglycan-like structure provides a scaffold for growth and division without unduly stimulating the immune response.
9. Atwal, S. et al. Evidence for a peptidoglycan-like structure in Orientia tsutsugamushi. Mol Microbiol 105, 440-452, doi:10.1111/mmi.13709 (2017).
10. Otten, C., Brilli, M., Vollmer, W., Viollier, P. & Salje, J. Peptidoglycan in obligate intracellular bacteria. Mol Microbiol 107, 142-163, doi:10.1111/mmi.13880 (2018).
11. Atwal, S. et al. Discovery of a Diverse Set of Bacteria That Build Their Cell Walls without the Canonical Peptidoglycan Polymerase aPBP. mBio 12, e0134221, doi:10.1128/mBio.01342-21 (2021).
Characterising the developmental cycle in Orientia tsutsugamushi
Several obligate intracellular bacteria are known to undergo biphasic developmental cycles12. However, all these known organisms reside within membrane bound vacuoles, and the same has not been shown for any cytoplasmic bacterium. We found that Orientia tsutsugamushi differentiates into two distinct forms that differ in their protein profiles, cell wall, metabolic activity, stability, and mechanisms of entry into host cells13. This finding has provided us with a framework for the study of the infection cycle of Orientia tsutsugamushi , and we are now working to uncover the mechanisms that regulate this cycle.
12. Salje, J. Cells within cells: Rickettsiales and the obligate intracellular bacterial lifestyle. Nat Rev Microbiol, doi:10.1038/s41579-020-00507-2 (2021).
13. Atwal, S. et al. The obligate intracellular bacterium Orientia tsutsugamushi differentiates into a developmentally distinct extracellular state. Nat Commun 13, 3603, doi:10.1038/s41467-022-31176-9 (2022).
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Developing a new diagnostic assay for scrub typhus using human monoclonal antibodies.
One of the major unmet health challenges in rickettsial diseases is the lack of adequate diagnostic tools14. We are exploring the idea of using human derived monoclonal antibodies to develop a diagnostic test that would directly recognise antigens in human blood. We have isolated a collection of >100 antibodies that interact with Orientia tsutsugamushi and have demonstrated their ability to detect Orientia tsutsugamushi in the blood of infected mice. This antibody-based assay is currently being tested for its ability to detect Orientia tsutsugamushi in blood from scrub typhus patients in Thailand.
