Evidence for a peptidoglycan-like structure in Orientia tsutsugamushi

Abstract

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.

Figure 1. The peptidoglycan biosynthesis pathway in Orientia tsutsugamushi

Genes shown in green are found in the genome of O. tsutsugamushi (Boryong and Ikeda strains), genes shown in red have no identifiable homolog.

Figure 2. Identification of meso-DAP by mass spectrometry analysis

Total Ion Chromatogram (TIC) and Extracted Ion Chromatograms (EIC) of the DAP derivative (N-heptafluorobutyryl 2,6-diaminopimelic acid isobutylester) from acid hydrolyzed O. tsutsugamushi cells showing a peak with retention time of 22.2 minutes and a set of fragment-ions at 380, 324, 306 and 278 m/z.

Figure 3. Expression of genes in the peptidoglycan biosynthesis pathway

Bacterial genome copy number or relative gene expression of O. tsutsugamushi grown in mouse fibroblast L929 cells over 7 days. Relative expression level was determined by qRT-PCR and normalised to the housekeeping gene mipZ. Graph shows each individual data point, as well as the mean and standard deviation.

Figure 4. O. tsutsugamushi treated with cell wall-targeting drugs

A. Table showing the molecular target of drugs and enzymes used in this study. B-E. Bacterial copy number per well of a 24-well plate after 7 days growth in the presence of different drugs. Three replicate wells were used in each experiment. Graph shows each individual data point, as well as the mean and standard deviation. Statistical significance was determined using an unpaired t test analysis. P values are illustrated as follows: ns (P > 0.05); ^* (P ≤ 0.05); ^** (P ≤ 0.01); ^*** (P ≤ 0.001). F. Immunofluorescence microscopy of intracellular O. tsutsugamushi cells treated with 40 μg/ml phosphomycin or 250 μg/ml D-cycloserine for 24 hours. Bacteria are labelled in green (using anti-TSA56 antibody); host cytoplasm in red (Evans blue) and nuclei in blue (DAPI). Scale bar = 10 μm (large images) or 1 μm (insets).

Figure 5. O. tsutsugamushi labelled with peptidoglycan-reactive probes

A. Fluorescence microscopy of O. tsutsugamushi labelled with EDA-DA probe reacted with an azide modified Alex Fluor 488 via click chemistry (green), antibody against the bacterial surface protein TSA56 (red) and the DNA stain DAPI (blue). Bacteria were grown in the presence or absence of drugs for 24 hours. Drug concentrations were as follows: 40 μg/ml phosphomycin; 250 μg/ml D-cycloserine. B. Fluorescence microscopy of O. tsutsugamushi and E. coli in the presence of the fluorescent D-alanine derivative HADA (blue), the fluorescent L-alanine derivative HALA (blue), antibody against the bacterial surface protein TSA56 (red, O. tsutsugamushi) or the bacterial cytoplasm (Evans blue, red, E. coli). Scale bar = 10 μm.

Figure 6. Structural rigidity of O. tsutsugamushi cells

A. Immunofluorescence microscopy showing purified O. tsutsugamushi resuspended in sucrose-phosphate glutamate buffer (SPG), PBS or water, with and without 1 mM DTT. Bacteria are labelled using an antibody against the TSA56 protein. Scale bar = 10 μm. B. Quantitative analysis of images of O. tsutsugamushi treated as described in A. Graph shows morphological differences of O.tsutsugamushi in different solutions as described in A, using Random Forest classification using 18 features. Training dataset includes more than 40 cells for each condition. Bar graph shows fractions of bacteria that exhibit ‘Smooth’, ‘Intermediate’ and ‘Rough’ morphology. See also Fig. S8 for example images from each morphological class. Graph shows mean and SD. C. Western blot of O. tsutsugamushi or uninfected L929 host cells in the presence or absence of ß-mercaptoethanol (BME), using a monoclonal antibody against the TSA56 protein. D. Immunofluorescence microscopy images of L929 cells infected with O. tsutsugamushi that had been mock-treated (left) or pre-treated with 1 mM DTT then washed prior to infection (right). Cells were fixed 24 hours after infection. Bacteria are shown in green (anti-TSA56 antibody), host actin in red (phalloidin) and nuclei in blue (DAPI). Scale bar = 10 μm. E. Average number of bacteria inside host cells after mock treatment or pre-treatment with DTT. 100 host cells were randomly selected, imaged, and manually counted. The mean and standard deviation is shown, with statistical significance determined using an unpaired t test analysis. P values are illustrated as follows: ns (P > 0.05); ^* (P ≤ 0.05); ^** (P ≤ 0.01); ^*** (P ≤ 0.001).