Fig 1.
C. burnetii morphological development in Vero cells.
Vero cell monolayers were incubated with purified SCV for 1 h to allow for adherence and internalization. Extracellular organisms were then washed from cell monolayers and fresh media added. Infected cells were fixed and processed for transmission electron microscopy at 1, 3, 5, and 7, 14, and 21 days post-infection (dpi). Prototypic LCV and SCV are designated in selected panels with an arrow and arrowhead, respectively. Bar, 500 nm.
Fig 2.
Transcriptional profiling of C. burnetii LCV to SCV morphological transition during intracellular growth in Vero cells.
(A) Principal-component analysis showing distinct grouping of microarray expression data at each time point (4 biological replicate samples). (B) Venn diagrams showing the number of genes up or downregulated ≥ 2 fold at 5, 7, 14, and 21 days post-infection of Vero cells relative to 3 days post-infection.
Fig 3.
Expression levels of C. burnetii gene subsets that are upregulated during transition of LCV to SCV.
Fold-increase in mRNA levels at 5, 7, 14, and 21 days post-infection relative to 3 days post-infection with a corresponding color code is depicted. Gene subsets shown are those involved in general and oxidative stress, arginine metabolism and transport, and cell wall remodeling.
Fig 4.
YkuD family proteins of C. burnetii.
(A) Schematic showing location of YkuD domains. Location of signal peptides, predicted by SignalP 4.1, and the LysM domain of CBU1577 are also depicted. (B) Amino acid sequence lineup of the predicted catalytic domains of C. burnetii YkuD family proteins with M. tuberculosis LdtB, E. faecium Ltdfm, and B. subtilis YkuD. Invariant histidine and cysteine proposed as catalytic residues are indicated with asterisks [97].
Fig 5.
QuantiGene and mass spectometry verification of gene expression detected by microarray for cell wall remodeling genes.
RNA from Coxiella-infected Vero cells or bacteria grown in ACCM-2 was subjected to QuantiGene analysis to determine expression levels. Fold-increase in mRNA levels at 5, 7, 14, and 21 days post-infection relative to 3 days post-infection with a corresponding color code is depicted. A value of >100 indicates signal was not detected with the day 3 RNA samples. Mass spectrometry of C. burnetii grown in ACCM-2 for 3, 14, and 21 days. Depicted are normalized peptide scores. Relative expression of protein at 14 and 21 days post-infection is shown as log2 ratio compared to 3 days post-infection. A value of 10 indicates no peptide was detected in day 3 bacteria. A value of -10 indicates no peptide was detected in day 14 and day 21 bacteria. A minus sign indicates no peptide was detected at any time point.
Fig 6.
Muropeptide analysis of C. burnetii peptidoglycan.
(A) HPLC elution profiles of muropeptides of LCV and SCV purified from Vero cells at 5 and 14 days post-infection (dpi), respectively (B) Muropeptides identified by MS and corresponding HPLC peaks in panel (A). (C) Molar percentage of muropeptides in monomer or cross-linked forms. The percentage of 3–3 cross-linked forms is also shown. (D) Schematic of peptidoglycan containing classical 4–3 and non-classical 3–3 cross-links that correspond to peaks R and P, respectively in panel (A). Unlinked stem peptides that are anchored to NAM residues have been removed for clarity. Abbreviations: NAG, N-acetylglucosamine; NAM, N-acetylmuramic acid; DAP, diaminopimelate.
Fig 7.
Ultrastucture of unfixed LCV and SCV imaged by cryo-electron microscopy.
Representative images of LCV and SCV after 4 and 28 days of growth, respectively. A thicker dense cell wall/outer membrane complex distinguishes SCV from LCV. Scale bar, 100 nm.