Stabilizing Microtubular Network Facilitates the Intracellular Growth of Orientia tsutsugamushi

Microtubule network provides many intracellular microbes with an efficient way to move within host cells. Orientia tsutsugamushi move from the cell periphery to the microtubule organizing center (MTOC) by dynein-dependent mechanism. In this study, we investigated the role of microtubule on the growth of O. tsutsugamushi. The treatment of infected cells with taxol as well as daunomycin enhanced the bacterial growth in contrast to colchicine. Immunofluorescent (IF) staining of taxol-treated cells exhibited that O. tsutsugamushi clustered tightly near the nucleus with thick bundles of microtubules, whereas dispersed in the cytoplasm in colchicine-treated cells. These results suggest that microtubule network facilitate the growth of O. tsutsugamushi.

Synergistic Effect of Ionizing Radiation and beta-lapachone against RKO Human Colon Adenocarcinoma Cells / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: To reveal the interaction between beta-Lapachone (beta-lap) and ionizing radiation in causing cell death in RKO human colon adenocarcinoma cells, and to elucidate the potential usefulness of combined beta-lap treatment and radiotherapy for cancer treatment. MATERIALS AND METHODS: The cytotoxicities of various treatments were determined in vitro using clonogenic and apoptotic cell death. The changes in cell cycle distribution were studied using flow cytometry and an in vitro kinase assay. The tumor growth was studied using RKO tumors grown s.c. in the hind leg BALB/c- nuslc nude mice. RESULTS: beta-lap caused clonogenic cell death and rapid apoptosis in RKO cells in vitro, in a dose dependent manner. The repair of sublethal radiation damage was almost completely inhibited when cells were maintained in beta-lap during the interval between the two-dose irradiation. Flow cytometry study demonstrated that beta-lap induced apoptosis, independent of the cell cycle phase, and completely prohibited the induction of radiation- induced G2 arrest in irradiated cells. The prohibition of radiation-induced G2 arrest is unclear, but may be related to the profound suppression of the p53, p21 and cyclin B1-Cdc2 kinase activities observed in cells treated with beta-lap. The combination of beta-lap and radiation markedly enhanced the radiation-induced growth suppression of tumors. CONCLUSION: beta-lap is cytotoxic against RKO cells, both in vitro and in vivo, and also sensitized cells to ionizing radiation by inhibiting sublethal radiation damage repair. beta-lap is potentially useful as a potent anti-cancer chemotherapy drug and potent radiosensitizer against caner cells.

The Attachment of Detergent-Extracted Outer Membrane Proteins of Orientia tsutsugamushi to the Host Cell Surface

Orientia tsutsugamushi, a causative agent of scrub typhus, is an obligate intracellular parasite. The mechanisms by which O. tsutsugamushi invade host cells are unknown. Given the importance of surface-exposed proteins in the pathogenesis of microbial pathogens, outer membrane proteins (OMP) of O. tsutsugamushi were extracted with detergents and their cellular binding was studied. Outer membrane fraction of O. tsutsugamushi was enriched by a sodium-lauryl sarcosinate (Sarkosyl) treatment of total membranes. Outer membrane proteins were extracted by the treatment with sodium dodecyl sulfate (SDS) and Sarkosyl. The resulting soluble fractions were examined for their cellular binding by the immunofluorescence microscopy. A fifty six kilodalton protein was found to bind to fixed ECV304 cells only when the outer membrane preparation was not treated by DTT or heat. These results suggest that the conformation the 56-kDa OMP is important for the attachment to the host cell surfaces.

A case of ruptured infected aneurysm of abdominal aorta caused by septic salmonellosis / 대한내과학회지

Salmonella infected aneurysm of aorta is one of the local complications by systemic salmonellosis. We have experienced a case of the rupture of infected abdominal aortic aneurysm in 47-year old man. He was admitted because of 39degrees C fever, abdominal pain, back pain of 7 day's duration. Diabetes mellitus had been diagnosed previously, but not managed. Abdominal CT scans showed aneurysmal dilatation with periaortic air bubbles and severe atherosclerotic change of abdominal aorta from renal hilum to the iliac bifurcation level. Blood cluture yielded Salmonella group B strains. He was treated with intravenous ceftriaxone, metronidazole, ciprofloxacin. On the seventh hospital day, the patient showed sign of shock with blood pressure 80/60 mmHg and comatous conciousness. Abdominal CT scans revealed the rupture of infected aneurysm of abdominal aorta just superior to the iliac artery bifurcation. The patient had not underwent surgery, then he was expired.

A case of ruptured infected aneurysm of abdominal aorta caused by septic salmonellosis / 대한내과학회지

Salmonella infected aneurysm of aorta is one of the local complications by systemic salmonellosis. We have experienced a case of the rupture of infected abdominal aortic aneurysm in 47-year old man. He was admitted because of 39degrees C fever, abdominal pain, back pain of 7 day's duration. Diabetes mellitus had been diagnosed previously, but not managed. Abdominal CT scans showed aneurysmal dilatation with periaortic air bubbles and severe atherosclerotic change of abdominal aorta from renal hilum to the iliac bifurcation level. Blood cluture yielded Salmonella group B strains. He was treated with intravenous ceftriaxone, metronidazole, ciprofloxacin. On the seventh hospital day, the patient showed sign of shock with blood pressure 80/60 mmHg and comatous conciousness. Abdominal CT scans revealed the rupture of infected aneurysm of abdominal aorta just superior to the iliac artery bifurcation. The patient had not underwent surgery, then he was expired.

Proliferation of ECV304 cells Infected Persistently with Orientia tsutsugamushi

Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular parasite. Previously we have shown that it persistently infect the human endothelial cell line ECV304. In this study, we have investigated the mechanism of in vitro persistent infection, which could be maintained for over a year without addition of normal cells. The persistently infected cultures exhibited cyclic changes in the host cell number, which resulted in a net increase of cell number. Floating cells detached from the culture plate bottom were filled with rickettsial cells and lost normal morphology. Only part of attached EVC304 cells was infected with O. tsutsugamushi. Some of ECV304 cells that still attached to the bottom were free of rickettsial cells. Rickettsial cells in the floating cells could not effectively infect new EVC304 cells while those in the attached cell could infect new cells with high efficiency. Host cells that allowed vigorous rickettsial multiplication and resultantly lost viability, and low of infectivity of the bacteria in the dead cells might have allowed in vitro persistent infection of O. tsutsugamushi.

Expression of Eta-1 in Escherichia coli and Production of Monoclonal Antibody / 감염

BACKGROUND: Early T-lymphocyte activation-1 (Eta-1) is a secreted phosphoprotein which regulates a variety of cells involved in the immune and nonimmune systems. It is unique in the sense that it regulates various immune functions, as well as acting as an extracellular matrix protein. The Eta-1 gene has been mapped to the same genetic locus as the Rickettsia resistance gene (Ric), and Eta-1 expression is a part of an early T-dependent response to Orientia tsutsugamushi infection in susceptible hosts. In an initial effort to study Eta-1's mechanism of protection against Orientia tsutsugamushi infection, we attempted to produce Eta-1 in E. coli and to produce monoclonal antibodies against recombinant Eta-1. METHODS: Expression plasmids containing GST-Eta-1 were generated by cloning the polymerase chain reaction-amplified N-and C-terminal Eta-1 fragments into the cloning sites of pGEX-3X. The expressed protein was purified using a GST column and injected into BALB/c mice. Hybridoma clones reactive to Eta-1 were produced and analyzed with ELISA and Western blot. RESULTS: Expression plasmids containing GST-Eta-1 were generated by cloning the polymerase chain reaction-amplified N-and C-terminal Eta-1 fragments into the cloning sites of pGEX-3X. N-and C-terminal fragments of Eta-1 were generated as bacterially expressed GST fusion proteins. However, the expression of full-length Eta-1 was very poor. We immunized BALB/c mice with purified Eta-1 N-terminal fragments. Their spleen cells were used for cell fusion. We obtained two hybridoma cell lines secreting antibodies against Eta-1, but not against GST. Conclusions:We produced Eta-1 protein produced in E. coli. The expression of C-terminal Eta-1 fragments was poor, therefore it appeared that this part of Eta-1 was toxic to E. coli. We obtained monoclonal antibodies which were reactive in ELISA test and Western blot. These monoclonal antibodies could be useful in the analysis of the function of Eta-1 in the pathogenesis of tsutsugamushi disease as well as other diseases.

Apoptosis of Murine Macrophage-like Cells Infected with Orientia tsutsugamushi

A large number of bacterial pathogens have been identified as mediators of apoptosis in vitro and the induction of apoptosis might be an important step in the pathogenesis of these bacteria. In this study, we analyzed the interactions of Orientia tsutsugamuchi with J774 murine macrophage-like cells. The J774 cells were infected with Boryong strain of O. tsutsugamushi and the DNA was analyzed with agarose gel electrophoresis. We observed the typical laddering pattern of DNA fragmentation indicative of apoptosis in infected cells but not cells infected with heat-killed O. tsutsugamushi. We performed terminal deoxynucleotidyl transferase (TdT) assay to label the 3'-hydroxy ends of DNA breaks and observed intense brown staining of nuclei of infected macrophages. With Hoechst 33258 for staining nucleus, strong chromatin condensation was observed only in infected J774 cells. We also examined the cytokine secretion pattern of J774 cells during the rickettsial infection. The large amount of TNF-alpha and IL-10 were secreted after 24 hrs of infection, but the secretion of IL-1beta was increased in small amount. These results showed that O. tsutsugamushi induce apoptosis in murine macrophage-like cells by different mechanism from that of shigella which cause secretion of large amount of IL-1beta.

Production of Monoclonal Antibody Against Human 14 - 3 - 3 Zeta Isoform Expressed in Escherichia coli / 대한면역학회지

14-3-3 proteins are cytoplasmic proteins of about 29 kDa and have a minimum of seven isoforms. This protein is important in signal transduction with the ability of binding with phosphoserine of many signalling proteins. We expressed 14-3-3 protein tagged with 6 histidine residues in E. coli and purified the protein by nickel affinity chromatography. Using this purified protein as an antigen, we made rabbit antisera and mouse monoclonal antibodies to 14-3-3 zeta isoform. We subcloned cDNA of 14-3-3 zeta isoform derived from HeLa cell lamda gt 11 library into an E. coli expression vector which is designed to express heterologous protein with N- terminal 6 hidtidine tag. BALB/c mice were immunized with purified 14-3-3 protein and the hybridoma clones which produce monoclonal antibodies angainst 14-3-3 protein were selected. These monoclonal antibodies reacted with the recombinant protein expressed in E. coli as well as the 29-kDa native protein in various cell lines. However, they did not immunoprecipitate 14-3-3 protein. The monoclonal antibodies produced in this study can be valuable tools for the identification of the 14-3-3 in signal transduction study.

Pathologic study of mice infected with Rickettsia tsutsugamushi R19 strain

Scrub typhus, an acute febrile infectious disease caused by R. tsutsugamushi, has been reported from various parts of the far east and pacific rim of Asia including Korea. It is well known that all human pathogenic rickettsia share an affinity to endothelial cells of the small blood vessels and evoke vascular inflammation variably associated with a rash, microthrombi, and hemorrhage. We infected the ICR mice by inoculating sublethal doses of R. tsutsugamushi R19 strain intraperitoneally and observed the pathologic changes by time sequence. The histopathologic features of experimentally induced scrub typhus in the mice were generally nonspecific interstitial inflammations characterized by interstitial pneumonitis, periportal inflammation, multifocal hepatic necrosis, interstitial nephritis, sinusoidal engorgement, and lymphohistiocytic cell infiltration in lymph nodes and spleen. Contrary to the general features of other rickettsial diseases, the pathologic process of scrub typhus experimentally induced by R. tsutsugamushi R19 strain mainly involved the interstitial connective tissue but not the blood vessels.