Growth of typhus group and spotted fever group rickettsiae in insect cells.

To analyze the host dependency of rickettsial growth, NIAS-AeAl-2 insect cells (AeAl2) derived from mosquito were first used in this study. It was demonstrated that typhus group rickettsiae (TGR) grew well in AeAl2 cells, but spotted fever group rickettsiae (SFGR) failed. To elucidate the inhibitory process of the growth of SFGR in AeAl2 cells, the adherence and invasion were first analyzed. SFGR possessed abilities to adhere to and invade AeAl2 cells as well as TGR in contrast to their inability of the growth in the cells. Morphologically, generation of microvilli could not be observed on AeAl2 cells inoculated with either group of rickettsiae. On the contrary, Vero cells inoculated with rickettsiae generated a great number of microvilli that adhered to rickettsiae and engulfed them into the cells. The roles of rickettsial major outer membrane protein A and B (rOmpA and rOmpB) were later investigated using E. coli expressing either rOmpA or rOmpB on their surface. Bacteria expressing either one of the major outer membrane proteins of rickettsiae as well as bacteria not expressing these proteins showed adherence to and invasion of AeAl2 cells. Thus, it is yet to be elucidated whether these major outer membrane proteins have any roles in these steps.

Subcellular localization of rickettsial invasion protein, InvA.

To understand further the molecular basis of rickettsial host cell invasion, Rickettsia prowazekii invasion gene homolog (invA) has been characterized. Our previous experiments have shown that InvA is an Ap5A pyrophosphatase, a member of the Nudix hydrolase family, which is up-regulated during the internalization, early growth phase, and exit steps during rickettsial mammalian cell infection. In addition to the molecular characterization, subcellular localization of InvA was investigated. InvA-specific antibodies were raised in mice and used for immunoelectron microscopy. The generated antibodies were shown to recognize InvA and by immunogold labeling showed InvA in the cytoplasm of rickettsiae. A cytoplasmic location for InvA would allow for a rapid response to any internal substance and efficient functioning in hydrolysis of toxic metabolic by-products that are accumulated in the rickettsial cytoplasm during host cell invasion. Protecting bacteria from a hazardous environment could enhance their viability and allow them to remain metabolically active, which is a necessary step for the rickettsial obligate intracellular lifestyle.

Electron microscopic study on the interaction between normal guinea pig peritoneal macrophages and Coxiella burnetii.

An electron microscopic study was conducted to explore the interaction between normal guinea pig peritoneal macrophages and phase I and II Coxeilla burnetii previously treated with either normal or immune serum. A comparison was made on the efficiency of phagocytosis and subsequent killing of rickettsiae by macrophages. Both phases of rickettsiae previously treated with normal serum multiplied within phagosomes after phagocytosis with resultant destruction of macrophages. In contrast, suspending rickettsiae in immune serum rendered them more susceptible to phagocytosis and potentiated their destruction within macrophages.