Genetic mechanisms of Coxiella burnetii lipopolysaccharide phase variation
Fig 5
CBU0678 is essential for production of phase I LPS.
LPS was extracted from NMI, NMC, NMI cbu0678tr, or NMI cbu0678trcomp-I following culture in ACCM-2 for 7 days. LPS was separated by SDS-PAGE then analyzed by (A) silver stain or (B) immunoblot probed with LPS-specific antibodies. NMI cbu0678tr produces intermediate and phase II LPS, but not phase I LPS. Expression of wild type cbu0678 in NMI cbu0678tr restores production of phase I LPS, indicating CBU0678 is essential for synthesis of phase I LPS. LPS from NMII, NMII Δcbu1655, and NMII Δcbu1655comp-II was isolated using a modified microextraction protocol following culture in ACCM-2 for 7 days. LPS was separated by SDS-PAGE and visualized by (C) glycoprotein staining or (D) immunoblot probed with anti-phase II LPS antibody. NMII Δcbu1655 produces a deep-rough phase II LPS (< 3 kDa), smaller than that of NMII, which is not recognized by anti-phase II LPS antibody. Expression of wild type cbu1655 in NMII Δcbu1655 restores production of typical phase II LPS and antibody recognition. These data are consistent with a predicted role of CBU1655 in producing the first heptose of the C. burnetii inner core, which is a component of the epitope recognized by anti-phase II LPS antibody. (E) Model of putative LPS structures of NMI cbu0678tr and NMII Δcbu1655 compared to those of NMI, NMC, and NMII. Bold text in the key indicates highly abundant sugars. KDO is defined as 3-deoxy-D-manno-2-octulosonic acid.