Characterization of rickettsial attachment to host cells by flow cytometry

The mechanisms of rickettsial attachment have been studied by measuring quantitative changes in rickettsial binding to host cells by flow cytometry after different treatments of the rickettsiae and host cells. Time-dependent binding of Rickettsia conorii to host cells was demonstrated by the increasing intensity of host cell surface fluorescence of rickettsia-host cell combinations when examined with a rickettsia-specific monoclonal antibody. More than 70% of host cells had intensity of fluorescence above the threshold value after 10 min of incubation, owing to rickettsiae bound to the cell surface, and the greatest fluorescence intensity indicative of binding occurred at 20 min. The binding kinetics was rickettsial dose dependent. The binding of rickettsiae to host cells was greatly decreased when host cells or rickettsiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 5 or 15 min, respectively. Rickettsiae that were heated at 56 degrees C for 15 min lost more than 80% of their ability to attach to host cells. R. rickettsii, an organism closely related to R. conorii, competitively inhibited the attachment of R. conorii (51% inhibition when mixed in equal numbers). These results indicate that the rickettsial binding structures are trypsin and heat sensitive and likely to be surface proteins.