Fate of the 63-kDa periplasmic protein of the infectious form of the endonuclear symbiotic bacterium Holospora obtusa during the infection process.

Holospora obtusa is a macronucleus-specific endosymbiotic bacterium of the ciliate Paramecium caudatum. We report the secretion of a 63-kDa periplasmic protein of an infectious form of the bacterium into the macronucleus of its host. Indirect immunofluorescence microscopy with five monoclonal antibodies against the 63-kDa protein demonstrated that, soon after the bacterial invasion into the host macronucleus, the protein was detected in the infected macronucleus and that levels of the protein increased dramatically within one day of infection. The use of inhibitors for host and bacterial protein synthesis illustrated that, in early infection of H. obtusa, not only the pre-existing but also a newly synthesized 63-kDa protein was secreted into the host macronucleus. A partial amino acid sequence of the protein was determined, and a gene encoding the 63-kDa protein was cloned. The deduced amino acid sequence shows that this protein is a novel protein.

Translocation of an 89-kDa periplasmic protein is associated with Holospora infection.

The symbiotic bacterium Holospora obtusa infects the macronucleus of the ciliate Paramecium caudatum. After ingestion by its host, an infectious form of Holospora with an electron-translucent tip passes through the host digestive vacuole and penetrates the macronuclear envelope with this tip. To investigate the underlying molecular mechanism of this process, we raised a monoclonal antibody against the tip-specific 89-kDa protein, sequenced this partially, and identified the corresponding complete gene. The deduced protein sequence carries two actin-binding motifs. Indirect immunofluorescence microscopy shows that during escape from the host digestive vacuole, the 89-kDa proteins translocates from the inside to the outside of the tip. When the bacterium invades the macronucleus, the 89-kDa protein is left behind at the entry point of the nuclear envelope. Transmission electron microscopy shows the formation of fine fibrous structures that co-localize with the antibody-labeled regions of the bacterium. Our findings suggest that the 89-kDa protein plays a role in Holospora's escape from the host digestive vacuole, the migration through the host cytoplasm, and the invasion into the macronucleus.