Localization of chlamydial group Antigen in McCoy cell monolayers infected with Chlamydia trachomatis or Chlamydia psittaci

Abstract

Chlamydial inclusions were demonstrated by indirect immunofluorescence (IF) with antiserum to the chlamydial group antigen when McCoy cell monolayers infected with either Chlamydia trachomatis or Chlamydia psittaci were fixed in formaldehyde or paraformaldehyde, provided the monolayer was not allowed to dry. If these monolayers were then air dried and restained by IF with the same antiserum but with a different fluorescence conjugate, group antigen associated with inclusion-containing McCoy cells but independent of the inclusions was revealed. This antigen was not restricted to infected cells but appeared to radiate out from them, suggesting that group antigen was released from infected cells. Similar host cell-associated antigen could be shown by IF of glutaraldehyde-fixed, air-dried monolayers, but inclusions could not be stained by IF before these preparations were dried, presumably because antibody could not penetrate glutaraldehyde-fixed cells. Electron microscopic immunoperoxidase studies of paraformaldehyde-fixed, wet monolayers located group antigen within inclusions on the outer membrane of chlamydial organisms and on single-membrane vesicles. However, when dried monolayers were labeled with the same immunoperoxidase technique, no intracellular labeling occurred, but dense staining was seen at the surface of infected cells and on adjacent membranous material. These observations are compatible with the postulate that replicating chlamydiae produce outer membrane blebs containing group antigen, which are excreted by the host cell during the chlamydial developmental cycle. Chlamydiae are obligate intracellular procaryotic parasites. They possess a double-unit membrane cell envelope similar to that of gramnegative bacteria (4), and they multiply within eucaryotic cytoplasm in phagosomes known as inclusions. These inclusions can be demonstrated in infected eucaryotic cells fixed in methanol or acetone by immunofluorescence (IF) with antiserum to the group antigen (10). This is a lipopolysaccharide antigen common to all members of the genus (7, 8) and is probably located in the outer membrane (OM) of both infectious chlamydial particles (elementary bodies) and replicating forms (reticulate bodies) (6). Recent IF studies of glutaraldehyde (GTA)fixed, air-dried McCoy cells revealed group antigen independent of inclusions that is associated with infected host cell cytoplasm and appears to be released from infected cells (9). It has been suggested that OM blebs which contain group antigen are produced during chlamydial repli

Accumulation of chlamydial lipopolysaccharide antigen in the plasma membranes of infected cells

“…Upon screening a panel of monoclonal antibodies by the immunofluorescence staining technique of Richmond and Stirling (26), one antibody was identified, designated 47A2, which stained the surface of chlamydia-infected cells. Determination of the antigenic specificity of 47A2 indicated that it reacted with chlamydial LPS.…”

“…A modification of the method of Richmond and Stirling (26) was used to examine chlamydia-infected cultures for the distribution of chlamydial antigens. At different times postinoculation, chlamydia-infected and uninfected control monolayers on cover slips were rinsed with phosphate-buffered saline (PBS) and fixed in either 4% formaldehyde or 4% glutaraldehyde in PBS for 20 min (26). Following brief air drying (30 min) at room temperature, monolayers were treated with dilutions of monoclonal antibodies in PBS for 1 h at room temperature.…”

The immunobiology and immunopathology of chlamydial infections

“…Such a mechanism might be important clinically, either by restricting chlamydial-infected cells and the spread of infection, or by playing a role in immunopathology. Initial evidence for the presentation of chlamydial antigen at the host cell surface came from the immunoelectron microscopic demonstration of chlamydial genus-specific antigen, presumably lipopolysaccharide (LPS), at the surface of infected cells and exported from them to adjacent cells (140). However, interpretation of this study is difficult because preliminary air drying or the presence of detergent containing fixative was necessary in order to demonstrate surface antigen, suggesting that the antigen was in hydrophobic micelles not freely exposed at the aqueous surface.…”

The intracellular life of Chlamydia psittaci: how do the bacteria interact with the host cell?

“…Chlamydia genus-speci¢c lipopolysaccharide (LPS) antigen has been observed associated with the cytoplasmic membrane of C. psittaci-and C. trachomatisinfected cell lines by using silver-methenamine staining and electron microscopy [21] as well as indirect immuno£uorescence with polyclonal and monoclonal antibodies [79,80,113]. Additionally, EB envelope antigens have also been detected on the surface of the infected host cell using indirect immuno£uores-cence and polyclonal antibodies [24].…”