Cell-selective proteomics reveal novel effectors secreted by an obligate intracellular bacterial pathogen
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Abstract
Pathogenic bacteria secrete protein effectors to hijack host machinery and remodel their infectious niche. Rickettsia spp. are obligate intracellular bacteria that can cause life- threatening disease, but their absolute dependence on the host cell environment has impeded discovery of rickettsial effectors and their host targets. We implemented bioorthogonal non-canonical amino acid tagging (BONCAT) during R. parkeri infection to selectively label, isolate, and identify secreted effectors. As the first use of BONCAT in an obligate intracellular bacterium, our screen more than doubles the number of experimentally validated effectors for R. parkeri . The novel secreted rickettsial factors (Srfs) we identified include Rickettsia -specific proteins of unknown function that localize to the host cytoplasm, mitochondria, and ER. We further show that one such effector, SrfD, interacts with the host Sec61 translocon. Altogether, our work uncovers a diverse set of previously uncharacterized rickettsial effectors and lays the foundation for a deeper exploration of the host-pathogen interface.
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Thank you for the kind words and comment! We can only speculate at this point, but those are all interesting models for what SrfA is doing. Since we’re unaware of a mechanism to directly translocate SrfA into a target bacterium (e.g., T6SS), SrfA may instead be able to act on exposed peptidoglycan from other bacteria (or damaged rickettsiae). How often rickettsiae come into contact with other bacteria (e.g., in the tick microbiome) for this to be relevant remains to be determined. Rickettsia spp. appear to encode some components of the peptidoglycan recycling pathway (see the preprint by Oyler et al. https://doi.org/10.1101/2022.03.10.483817), but the extent to which these bacteria release peptidoglycan during infection (either during normal growth/division or from lysis) is unknown. As you suggested, however, we might imagine that SrfA …
Thank you for the kind words and comment! We can only speculate at this point, but those are all interesting models for what SrfA is doing. Since we’re unaware of a mechanism to directly translocate SrfA into a target bacterium (e.g., T6SS), SrfA may instead be able to act on exposed peptidoglycan from other bacteria (or damaged rickettsiae). How often rickettsiae come into contact with other bacteria (e.g., in the tick microbiome) for this to be relevant remains to be determined. Rickettsia spp. appear to encode some components of the peptidoglycan recycling pathway (see the preprint by Oyler et al. https://doi.org/10.1101/2022.03.10.483817), but the extent to which these bacteria release peptidoglycan during infection (either during normal growth/division or from lysis) is unknown. As you suggested, however, we might imagine that SrfA could help limit activation of cytoplasmic peptidoglycan sensors (e.g., Nod1) in the event that peptidoglycan is released. We would love to pursue these kinds of effector mechanism questions in the future!
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Thank you for this thorough and cool story! I love bacterial effectors so this really resonated with me. And I commend you on working in a challenging system and developing new empirical techniques! This is a significant advance in our understanding of Rickettsia biology! Thank you! My hope is that many people will follow suit and apply techniques such as BONCAT to their particular areas of biology. That would be amazing!
I was wondering if I could some questions about SrfA. I find this protein really interesting because its annotation suggests that it acts on bacterial peptidoglycan or peptidoglycan precursors. However it appears to be secreted into the eukaryotic host cell cytoplasm and nucleus. Why do you think it's secreted there if it's acting on a bacteria-specific substrate? I have three ideas so just wanted to list them here (1) …
Thank you for this thorough and cool story! I love bacterial effectors so this really resonated with me. And I commend you on working in a challenging system and developing new empirical techniques! This is a significant advance in our understanding of Rickettsia biology! Thank you! My hope is that many people will follow suit and apply techniques such as BONCAT to their particular areas of biology. That would be amazing!
I was wondering if I could some questions about SrfA. I find this protein really interesting because its annotation suggests that it acts on bacterial peptidoglycan or peptidoglycan precursors. However it appears to be secreted into the eukaryotic host cell cytoplasm and nucleus. Why do you think it's secreted there if it's acting on a bacteria-specific substrate? I have three ideas so just wanted to list them here (1) it's lysing Rickettsia cells, or a subpopulation of Rickettsia cells, (2) it's lysing other non-Rickettsia intracellular bacteria, (3) it's involved in the degradation of released peptidoglycan. Regarding idea (3), is it known if Rickettsia shed (or release) peptidoglycan components as they grow and divide? It may be helpful to have a secreted enzyme that degrades these peptidoglycan components. The peptidoglycan components could act as activators for eukaryotic antibacterial programs which would be undesired from the perspective of a Rickettsia cell. Thanks!
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