Antigenic differences between Coxiella burnetii cells revealed by postembedding immunoelectron microscopy and immunoblotting

Abstract

The aim of this study was to investigate the antigenic structures of the morphologically distinct cells of the Coxiella burnetii developmental cycle. Postembedding immunoelectron microscopy with polyclonal antibodies produced in rabbits to (i) phase I cells, (ii) a chloroform-methanol residue fraction of cells, (iii) the cell walls (CW) of large and small cells and small dense cells (SDC), and (iv) the peptidoglycan-protein complexes of small cells and SDC labelled the continuum of morphologically distinct cells. But these antibodies did not distinguish between the antigenic structures of the various cells. Monoclonal antibodies to the phase I lipopolysaccharide labelled the CW of a majority of the smaller cells, but there was diminished reactivity to the larger cells. Although monoclonal antibodies to a 29.5-kDa outer membrane protein labelled the CW of the large mother cells, the large cells, and the small cells, a minority of the SDC with compact CW were not labelled. The endogenous spore within the mother cell was not labelled by the polyclonal or monoclonal antibodies to cellular components. A selected population of SDC was prepared by osmotic lysis of large cells, differential centrifugation, Renografin step-gradient fractionation, and breakage of the small cells in a French press at 20,000 lb/in2. The pressure-resistant SDC collected as fraction CL did not contain the 29.5-kDa protein, as evidenced by the lack of (i) Coomassie brilliant blue staining of protein in the 29.5-kDa region of sodium dodecyl sulfate-polyacrylamide gels and (ii) reactivity of the 29.5-kDa protein antigenic epitopes in immunoblotting with monoclonal antibodies to the protein. In contrast, CW of the pressure-sensitive small cells contained the 29.5-kDa protein. Therefore, the observed ultrastructural differences between large and small cells and SDC reflect differences in sensitivity to breakage by pressure treatment and in cell-associated antigens. Although the process of differentiation in C. burnetii remains an enigma, we have taken steps toward identifying cellular antigens as markers of differentiation. The pressure-resistant SDC in fraction CL that are devoid of the 29.5-kDa protein may be useful for answering questions about the physiological events required for triggering outgrowth and sequential regulation of the Coxiella developmental cycle.

A Sporulation Gene in Coxiella burnetii?

“…C. burnetii does not contain dipicolinic acid as one biochemical indicator of spore forming bacteria while muramic acid and diaminopimelic acid has been detected (MCCAUL et al, 1991). However, the synthesis of dipicolinic acid is coded by a branch for the mother cell compartment which is independent from spoIIIE (ERRINGTON et al, 1988).…”

“…Compared t o other members of the family Rickettsiaceae the organism displays an unusually high resistance against environmental conditions. The existence of "endospored' (MCCAUL and WILLIAMS, 1981) o r "spore-like forms" (SCHAAL et al, 1987;JEKOV, 1985), and "small dense cells" (MCCAUL et al, 1991) has been described and such forms characterized by electron microscopy and immunologically (MCCAUL et al, 1991). As a consequence a complex cycle of differentiation has been postulated (MCCAUL and WIL-Searching for conserved target sequences for a diagnostic PCR to improve the diagnosis of Q fever we have cloned and sequenced a NotIIEcoRV fragment from genomic DNA of C. burnetii, isolate "NINE MILE", phase I.…”

Genome Analysis of Coxiella burnetii Species: Insights into Pathogenesis and Evolution and Implications for Biodefense

“…2 This ability to survive a harsh intra-and extracellular environment may rely, in part, on a developmental life cycle with various forms specialized for propagation, surviving intracellular stress and persisting in the extracellular environment (in a non-replicating state) for long periods between hosts. 2,7 In vitro, C. burnetii passively enters and replicates within a variety of epithelial, fibroblast, and macrophage-like cell lines. 8 Internalization into host cells occurs by a microfilament-dependent parasite-directed endocytic process.…”

Antigenic Characteristic of the Lipopolysaccharides of Coxiella burnetii Isolates.

“…It is generally considered that antigenic structures which play a role in the development of immunity to infection by C. burnetii are the LPS and membrane polypeptides (2,15,25). Many studies on C. burnetii LPS have been performed on LPSs derived from two prototype strains of C. burnetii; Nine Mile, obtained from a tick but which determined acute pulmonary infection in a laboratory contamination, and Priscilla, obtained from the placenta of a goat and considered representative of the chronic group of Q fever (3,12,16).…”