Developmentally regulated synthesis of an unusually small, basic peptide by Coxiella burnetii

Abstract

Coxiella burnetii undergoes a poorly defined developmental cycle within phagolysosomes of eukaryotic host cells. Two distinct developmental forms are part of this cycle: a small-cell variant (SCV) and large-cell variant (LCV). Ultrastructurally, the SCV is distinguished from the LCV by its smaller size and condensed chromatin. At a molecular level, little is known about morphogenesis in C. burnetii, and no proteins specific to the SCV have been identified. Preparative isoelectric focusing was conducted to purify basic proteins possibly involved in SCV chromatin structure. A predominant protein of low M(r) was present in the most basic fraction, eluting with a pH of approx. 11. Degenerate deoxyoligonucleotides corresponding to the N-terminal sequence of this protein were used to recover a cosmid clone from a C. burnetii genomic library. Nucleotide sequencing of insert DNA revealed an open reading frame designated scvA (Small-Cell-variant protein A) with coding potential for a 30 amino acid protein (ScvA) with a predicted M(r) of 3610. ScvA is 46% arginine plus 46% glutamine with a predicted pl of 12.6. SDS-PAGE and silver staining of lysates of SCV and LCV purified by caesium chloride-equilibrium density centrifugation revealed a number of proteins unique to each cell type. Immunoblot analysis with ScvA antiserum demonstrated the presence of ScvA only in the SCV. By Immunoelectron microscopy, ScvA antiserum labelled only the SCV, with the label concentrated on the condensed nucleoid. In addition, ScvA bound double-stranded DNA in gel mobility-shift assays. A 66% reduction in the mean number of gold particles per Coxiella call was observed at 12 h post-infection when compared with the starting inoculum. Collectively, these data suggest that synthesis of ScvA is developmentally regulated, and that the protein may serve a structural or functional role as an integral component of the SCV chromatin. Moreover, degradation of this protein may be a necessary prerequisite for morphogenesis from SCV to LCV.

Temporal Analysis of Coxiella burnetii Morphological Differentiation

“…Conventional methods of C. burnetii cultivation and purification yield a mixture of morphological forms (30). SCV and LCV can be purified to near homogeneity from a mixed population by cesium chloride density gradient centrifugation (20,40); however, in our hands these preparations show little to no infectivity of cultured cells (19). We had previously observed by TEM that the percentage of SCV within the host PV increases with extended incubation times (unpublished observations).…”

“…Purified SCV were resuspended in K-36 buffer (0.1 M KCl, 0.015 M NaCl, 0.05 M potassium phosphate, pH 7.0) and stored at Ϫ80°C. The homogeneity of SCV was assessed by transmission electron microscopy (TEM) as previously described (20).…”

“…Two highly basic proteins, ScvA and Hq1, are DNA binding proteins specific to the SCV that likely play roles in chromatin condensation (18,20). Elongation factors EF-Tu and EF-Ts, the stationary-phase sigma factor RpoS, and a protein with porin activity termed P1 are preferentially expressed by LCV (33,35,39).…”

Host cell-free growth of the Q fever bacterium Coxiella burnetii

“…S2). Developmental transitions were confirmed by immunoblotting for ScvA, a DNA-binding protein specific to the SCV (14,15). ScvA was detected only in the SCV inoculum and the LCV/SCV mixture present at early stationary phase (Fig.…”

Is Q Fever an emerging or re-emerging zoonosis?

“…The formation of the different forms is linked to the lifecycle of Coxiella, a strategy developed to survive in and out of the parasitophorous vacuole. These forms express different proteins specific for each form in the developmental life cycle [55][56][57]143] and recognized by antibodies produced during a Coxiella infection. These differentially expressed antigens could allow the bacteria to escape the immune response by a switch of the exposed proteins on the surface of the bacteria and to survive in the acid vacuole.…”