Evidence for a peptidoglycan‐like structure in Orientia tsutsugamushi

Abstract

Summary Bacterial cell walls are composed of the large cross-linked macromolecule peptidoglycan, which maintains cell shape and is responsible for resisting osmotic stresses. This is a highly conserved structure and the target of numerous antibiotics. Obligate intracellular bacteria are an unusual group of organisms that have evolved to replicate exclusively within the cytoplasm or vacuole of a eukaryotic cell. They tend to have reduced amounts of peptidoglycan, likely due to the fact that their growth and division takes place within an osmotically protected environment, and also due to a drive to reduce activation of the host immune response. Of the two major groups of obligate intracellular bacteria, the cell wall has been much more extensively studied in the Chlamydiales than the Rickettsiales. Here, we present the first detailed analysis of the cell envelope of an important but neglected member of the Rickettsiales, Orientia tsutsugamushi. This bacterium was previously reported to completely lack peptidoglycan, but here we present evidence supporting the existence of a peptidoglycan-like structure in Orientia, as well as an outer membrane containing a network of cross-linked proteins, which together confer cell envelope stability. We find striking similarities to the unrelated Chlamydiales, suggesting convergent adaptation to an obligate intracellular lifestyle.

The obligate intracellular bacteriumOrientia tsutsugamushidifferentiates into a developmentally distinct extracellular state

“…Highly elongated cells of R. typhi growing in a tick cell line have been reported 53 suggesting the possibility that mechanisms for producing different cell morphologies under different conditions may be widespread across the Rickettsiaceae, The morphology of growing Ot, and possibly other rickettsia, may reflect nutrient availability under different conditions. The observation that elongated Ot cells are rod shaped rather than spherical supports the recent finding that Ot synthesizes a peptidoglycan-like structure, although this is not very abundant 43 .…”

“…The observation that bacteria mostly initiate protein synthesis upon exposure to the cytoplasm raised the question: what triggers the resumption of metabolic activity in intracellular Ot? We have previously shown that the bacterial outer membrane protein TSA56 forms a disulfide crosslinked network 43 . We hypothesized that this, and potentially other proteins containing exposed Cys residues, might become reduced upon exposure to the reducing environment of the host cell cytoplasm and that this might trigger an internal signal to activate metabolism.…”

“…The host cell cytoplasm is a more reducing environment than the extracellular environment. The major surface protein TSA56 of Ot contains three Cys residues and their cross-linking is required for the bacteria to enter host cells 43 . We found that the reducing environment of the host cell cytoplasm is required for activation of metabolic activity upon entry into host cells, as was previously shown for another intracellular bacterium Listeria monocytogenes 51,52 .…”

Peptidoglycan in obligate intracellular bacteria

“…Recent work has provided evidence that O. tsutsugamushi may possess a minimal peptidoglycan-like structure and is sensitive to a range of non-ß-lactam peptidoglycan-targeting antibiotics such as D-cycloserine and phosphomycin, but remains insensitive to all ß-lactams tested, which might be explained by an intrinsic insensitivity by the class B PBPs of this organism (Atwal et al, 2017) or an unknown beta-lactamase. O. tsutsugamushi was also shown to possess a disulphide cross-linked protein network on the outer membrane, analogous to the Chlamydiae (Atwal et al, 2017). The Anaplasmataceae comprise a group of tickborne human and veterinary pathogens.…”

“…O. tsutsugamushi lacks the enzyme catalysing the final step of meso-DAP biosynthesis, DapF. Whilst no direct alternative to this enzyme has been described, a recent study showed the presence of meso-DAP in purified O. tsutsugamushi by mass spectrometry, suggesting the presence of an unidentified alternative gene or pathway (Atwal et al, 2017). Surprisingly, E. ruminantium possesses a complete set of genes required for meso-DAP biosynthesis, although it lacks MurE that would be required to incorporate it into the peptidoglycan precursor.…”