Rebecca Wattam 07-08-2004 0.83 The methods of cell invasion and bacterial multiplication within vertebrate cells are described for the family Rickettsiaceae, which includes the three tribes Rickettsieae, Ehrlichieae, and Wolibachieae. Specifically, this interaction deals with Rickettsia rickettsii. wattam@vbi.vt.edu Adhesin, rOmpA Extracellular Ligand binding or carrier With monoclonal antibodies and the technological approach of flow cytometry, rOmpA was demonstrated to play a critical role in the attachment of R. rickettsii to host cells (Li and Walker, 1998). rOmpA and rOmpB are immunodominant, surface-exposed proteins of Rickettsia rickettsii. Prior evidence suggests that adhesion of R. rickettsii to the host cell is mediated by a rickettsial protein. Five monoclonal antibodies to rOmpA, five to rOmpB, and one to the rickettsial lipopolysaccharide (LPS) were tested for inhibition of rickettsial attachment. All the monoclonal antibodies to rOmpA inhibited adhesion of rickettsiae to the L-929 cells with some inhibition rates as high as 90%. In contrast, monoclonal antibodies to rOmpB and LPS did not block attachment. When Fab fragments of monoclonal antibodies against rOmpA and rOmpB were used, similar results were observed as for the intact monoclonals, non-adhesion and adhesion, respectively. Purified rOmpA showed a competitive inhibitive effect on the attachment of R. rickettsii to host cells. Trypsin completely digested rOmpA but not rOmpB from the surface of intact R. rickettsii, resulting in loss of the ability of the rickettsiae to attach to the host cell (Li and Walker, 1998). Endothelial cells of small- and medium-sized vessels are the primary target cells for Rickettsia rickettsii during infection of vertebrate hosts (Eremeeva and Silverman, 1998). Protein-dependent Receptor Cell membrane Ligand binding or carrier Rickettsia attach to a protein dependent receptor on the host cell membrane and induce focal host cell cytoskeletal rearrangements at the site of attachment, resulting in their entry into the host cell even in nonprofessional phagocytes by a mechanism requiring rickettsial metabolic activity (Walker et al., 2003). Important unidentified elements of the rickettsia-host cell interaction include the host cell membrane receptor for the rickettsial adhesin(s) (Walker et al., 2003). Rickettsia in Phagocyte Phagosome Other Internalization of obligate intracellular bacteria belonging to the genus Rickettsia by eukaryotic cells requires participation of both the parasitized host and the microorganism. The term "induced phagocytosis" has been used specifically to describe the entry of Rickettsia prowazekii, although a similar mechanism is likely for R. rickettsii. A role for a phospholipase in the internalization process has been proposed for both of these organisms, with the strongest supporting evidence provided for R. prowazekii. Despite general acceptance of the notion that phospholipase activity is involved in the internalization process of these bacteria, the origin of the enzyme is not known. The results of the study presented here, which used R. rickettsii and Vero cells, suggest that a rickettsial phospholipase, rather than a host cell phospholipase, mediates internalization of the organism. This conclusion is based upon results which show that pretreatment of R. rickettsii, but not of host cells, with a specific chemical inhibitor of phospholipase, and also antiserum to this enzyme, significantly reduces uptake of the organism and its ability to cause plaque formation (Silverman et al., 1992). The entry of rickettsiae into eukaryotic cells is mediated by an induced phagocytosis, but rickettsiae have never been observed in a closed phagocytic vacuole. In this study, Rickettsia conorii entry into Vero cells was observed by transmission electron microscopy during a period of 3 to 20 min after bacterium-cell contact. The entry occurred within 3 min after bacterium-cell contact, and R. conorii was observed in the process of engulfment, within a phagocytic vacuole, or free in the cytosol. Escape from the phagosome is a very rapid step since phagosome lysis was only occasionally observed. By 12 min, 90% of bacteria were internalized and half were free in the cytosol. This report confirms that rickettsiae penetrate nonphagocytic cells by induced phagocytosis and is the first demonstration of rickettsiae within a complete phagocytic vacuole (Teysseire et al., 1995). Rickettsia in Cytoplasm Cytoplasm Other Rickettsia rapidly lyse the phagosomal membrane and escape into the cytosol prior to phagolysomal fusion avoiding exposure to the lysosomal enzymes. In the cytosol they acquire their nutrients (e.g., glutamate), a part of their energy requirements (ADP/ATP transporter), and many components required for growth (e.g., amino acids) (Walker et al., 2003). The ultrastructural changes that occur during parasitism by R. rickettsii include a marked dilation of internal membranes, particularly the rough endoplasmic reticulum. The explicit mechanism of rickettsia-induced cellular injury has not been defined, but hypothetical mechanisms include trypsin-like protease activity, free-radical-induced lipid peroxidation, and phospholipase A (PLA) activity directly or through the generation of diacylglycerol and arachidonic acid metabolites (Hackstadt, 1996). Rickettsia rickettsii infection of endothelial cells is manifested in very distinctive changes in cell morphology, consisting of extensive dilatation of the membranes of the endoplasmic reticulum and outer nuclear envelope and blebbing of the plasma membrane, as seen by transmission electron microscopy. These changes in cellular architecture are thought to be due to oxidant-mediated cell injury, since their occurrence correlates with dramatic alterations in cellular metabolism, particularly with regard to antioxidant systems (Eremeeva and Silverman, 1998). Rickettsia rickettsii, a gram-negative and obligate intracellular bacterium, is the causative agent of Rocky Mountain spotted fever. In human infections, the primary target of R. rickettsii infection is vascular endothelium. Our laboratory has shown that activation of nuclear transcription factor-kappa B (NF-kappaB) during R. rickettsii infection of cultured human endothelial cells protects against apoptosis by preventing the activation of apical caspases-8 and -9, and the effector caspase-3 (Joshi et al., 2004). Rickettsia in Nucleus TEXT Nucleus Other Intranuclear growth of R. rickettsii occurred, but it appeared to be a relatively rare phenomenon in both CE and L cells. Intranuclear growth could be identified with reasonable certainty only after compact masses of organisms appeared within the borders of the nuclear membrane. It was impossible to classify single or a few dispersed organisms within the nuclear membrane border into intranuclear and superimposed intracytoplasmic populations. Hence, it was not possible to determine how early in the growth cycle rickettsiae entered the nucleus or to measure early intranuclear growth kinetics. However, by 45 to 48 h of incubation, compact intranuclear clumps of rickettsiae were rarely but readily identified. In contrast to the dispersed distribution of rickettsial body count (RLB) in the cytoplasm, the intranuclear rickettsiae remained in compact masses and tended to increase in numbers, which at times almost completely filled the nucleus with a dense mass of organisms (Wisseman et al., 1976). The low frequency of intranuclear rickettsiae suggests that R. rickettsii does not enter the nucleus from the cytoplasm as readily as it enters through the plasma membrane into the cytoplasm from the extracellular environment. Moreover, the accumulation of large numbers of intranuclear rickettsiae in those nuclei that are identifiably infected suggests that, if the organisms escape from the nucleus, the rate of passage out of the nucleus is probably much slower than that out of the cytoplasm into the extracellular space, at least relative to the rates of multiplication in the respective cellular compartment (Wisseman et al., 1976). Extracellular Rickettsia TEXT Extracellular Other Insight into the virulence of spotted fever group rickettsiae, such as R. rickettsii, was achieved with the discovery that these organisms utilize an intracellular actin-based motility (ABM) system to promote direct cell-to-cell spread. This mechanism of pathogenesis is also exploited by the facultative intracellular bacteria Listeria monocytogenes and Shigella flexneri. By using the propulsive force supplied by parasite-directed polymerization of host cell actin, motile bacteria move into pseudopodia that can be subsequently engulfed by neighboring cells. Escape from the double membrane vacuole allows infection of the new cytoplasm. The ability of spotted fever group rickettsiae to spread via ABM within the endothelium (the target host tissue of rickettsia) by directly passing from one cell to another allows evasion of the host humoral immune response, minimizes exposure to cell-impermeant antibiotics, and maintains rickettsiae within their required intracellular niche (Heinzen et al., 1999). At 96 to 120 h of incubation, the host cells showed progressive degeneration and destruction. However, light microscopy showed that very large numbers of rickettsiae were extracellular at this time. Many were free, as single organisms or clumps, unassociated with cell fragments, and intact extracellular doughnuts were occasionally seen (Wisseman et al., 1976). TEXT >Rickettsia attach to a protein dependent receptor on the host cell membrane and induce focal host cell cytoskeletal rearrangements at the site of attachment, resulting in their entry into the host cell even in nonprofessional phagocytes by a mechanism requiring rickettsial metabolic activity (Walker et al.,2003). Rickettsial invasion of susceptible host cells is an active process that requires active participation of both the host cell and the rickettsiae. Starved rickettsiae or rickettsiae inhibited by heat, formalin, KCN, 2,4-dinitrophenol, N-ethylmaleimide,or UV-irradiation do not penetrate cells efficiently or even adhere as well. Similarly, inhibition of the host cell by NaF or N-ethylmaleimide treatment or inhibition of actin polymerization by cytochalasin B or D, greatly reduced rickettsial entry, although the rickettsiae were able to adhere to the treated cells. The requirement for active involvement of both the parasite and the host has lead to this process being termed induced phagocytosis, or parasite-directed endocytosis (Hackstadt, 1996). TEXT The precise mechanism of entry has itself been an area of some controversy. Whether rickettsiae enter cells by direct penetration of the plasma membrane or simply escape rapidly from an endocytic vesicle has been a matter of dispute. That rickettsiae gain access to the cytoplasm very rapidly after internalization is not questioned. The difficulty is that rickettsiae have only rarely been observed within vesicles in the early stages of entering cells. Even when such micrographs exist, the question of viability of the particles and whether occupancy of a vacuole reflects a normal phase of rickettsial entry or simply the trafficking of nonviable organism to a lysosomal vesicle cannot be easily answered (Hackstadt, 1996). TEXT The low frequency of intranuclear rickettsiae suggests that R. rickettsii does not enter the nucleus from the cytoplasm as readily as it enters through the plasma membrane into the cytoplasm from the extracellular environment. Moreover, the accumulation of large numbers of intranuclear rickettsiae in those nuclei that are identifiably infected suggests that, if the organisms escape from the nucleus, the rate of passage out of the nucleus is probably much slower than that out of the cytoplasm into the extracellular space, at least relative to the rates of multiplication in the respective cellular compartment (Wisseman et al., 1976). TEXT Passage or R. rickettsii through the plasma membrane in the opposite direction, i.e., from cytoplasm to the extracellular compartment, without detectable gross damage to the host cell, was a most striking and important feature of the infection (Wisseman et al., 1976). Insight into the virulence of spotted fever group rickettsiae, such as R. rickettsii, was achieved with the discovery that these organisms utilize an intracellular actin-based motility (ABM) system to promote direct cell-to-cell spread. This mechanism of pathogenesis is also exploited by the facultative intracellular bacteria Listeria monocytogenes and Shigella flexneri. By using the propulsive force supplied by parasite-directed polymerization of host cell actin, motile bacteria move into pseudopodia that can be subsequently engulfed by neighboring cells. Escape from the double membrane vacuole allows infection of the new cytoplasm. The ability of spotted fever group rickettsiae to spread via ABM within the endothelium (the target host tissue of rickettsia) by directly passing from one cell to another allows evasion of the host humoral immune response, minimizes exposure to cell-impermeant antibiotics, and maintains rickettsiae within their required intracellular niche (Heinzen et al., 1999). TEXT TEXT Rickettsia rickettsii molecular pathway The infection cycle of Rickettsia rickettsii, studied in slide chamber cultures of chicken embryo and L-929 cells, was found to be complex and did not conform to a one-step growth cycle. Initial uptake kinetics resembled those established for Rickettsia prowazekii, but subsequent events showed very marked differences. Intracytoplasmic growth commenced exponentially without measurable lag. However, very soon after infection, intracytoplasmic rickettsiae began to escape from the host cell into the medium in large numbers, resulting in (i) failure of large numbers of rickettsiae to accumulate in the cytoplasm, (ii) sustained rapid division of the organisms in the cytoplasm, (iii) substantial accumulation of extracellular rickettsiae, and (iv) rapidly spreading infection in the culture, with most cells infected in 48 to 72 h. In the occasional cell, rickettsiae were found in the nucleus, where they multiplied to form compact masses. Thus, analysis of the growth characteristics of R. rickettsii must consider the entire culture as a unit in which the rickettsiae are distributed among three compartments in which they behave in different ways: (i) intranuclear, (ii) intracytoplasmic, and (iii) extracellular. The rickettsial traffic is bidirectional across the host cell plasma membrane and dominantly monodirectional across the nuclear membranes (Wisseman et al., 1976). 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