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1.
The mechanisms of entry for the obligate intracellular bacterium C. trachomatis were examined by functional disruption of proteins essential for various modes of entry. RNA interference was used to disrupt proteins with established roles in clathrin-mediated endocytosis (clathrin heavy chain, dynamin-2, heat shock 70-kDa protein 8, Arp2, cortactin, and calmodulin), caveola-mediated endocytosis (caveolin-1, dynamin-2, Arp2, NSF, and annexin II), phagocytosis (RhoA, dynamin-2, Rac1, and Arp2), and macropinocytosis (Pak1, Rac1, and Arp2). Comparative quantitative PCR analysis was performed on small interfering RNA-transfected HeLa cells to accurately determine the extent of C. trachomatis entry after these treatments. Key structural and regulatory factors associated with clathrin-mediated endocytosis were found to be involved in Chlamydia entry, whereas those for caveola-mediated endocytosis, phagocytosis, and macropinocytosis were not. Thus, clathrin and its coordinate accessory factors were required for entry of C. trachomatis, although additional, uncharacterized mechanisms are also utilized. 相似文献
2.
Ultrastructural studies of the entry of Chlamydia strain TWAR into HeLa cells showed that the elementary bodies first attach to host cells by the pointed end, secure other binding sites on the host cells by forming cell wall protrusions, enter host cells by invaginating the host cell membrane, and form vacuolated endocytic vesicles. Differences were demonstrated between TWAR and other chlamydiae in the mode of attachment and endocytosis. 相似文献
3.
The infection of HeLa 229 cells in monolayer culture with trachoma (B/TW-5/OT) and lymphogranuloma venereum (LGV) (L2/434/Bu) organism was studied in terms of two parameters: radioactivity counts of cell-associated tritium labeled organisms at the initial stage of inoculation for measurement of attachment, and inclusion counts of infection cells after incubation for measurement of growth. Factors affecting attachment and inclusion formation and correlation of the two are presented. It was shown that attachment is an important initial step in infection by Chlamydia trachomatis. The rate of attachment was temperature dependent. The attachment of LGV organisms was affected more profoundly by temperature than was that of trachoma organisms. Attachment and inclusion formation of trachoma and LGV organisms were inhibited by heparin. Diethylaminoethyl-dextran was again shown to enhance attachment and inclusion formation of trachoma but not LGV organisms. NaF had no effect on attachment, but inhibited inclusion formation of both trachoma and LGV organisms. Both attachment and inclusion formation of trachoma organisms were strongly enhanced by centrifugation of the inoculum onto the cell monolayer. Although inclusion formation of trachoma organism was much greater in susceptible cells (HeLa 229) than relatively insusceptible cells (fetal tonsil), attachment was only slightly greater. The results based on the test of two cell lines suggested that attachment prpbably is not a critical factor in determing a cell line's susceptibility to infection with trachoma organisms. 相似文献
4.
Chlamydia trachomatis infections are an important problem inhuman reproduction and family planning. In this study, the significanceof chlamydial infection in male infertility and artificial inseminationhas been investigated. Electron microscope observations on maleejaculates have revealed the presence of the elementary andreticulate body forms of C.trachomatis in spermatozoa. Furthermore,the entry of the elementary body into the human spermatozoonhead has been demonstrated. After the passage of the infectiouselementary body into the nucleus, all stages of reticulate bodyformation in the head of the spermatozoon were detected. Accordingto ultrastructural findings, C.trachomatis not only adheredto but also penetrated into the tail structure. Thus two differentfunctional and morphological forms of C.trachomatis can infectand be transmitted by spermatozoa and may cause infertility. 相似文献
5.
Parasite-specified phagocytosis of Chlamydia psittaci and Chlamydia trachomatis by L and HeLa cells. 总被引:2,自引:37,他引:2
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Phagocytosis of the 6BC strain of Chlamydia psittaci and the lymphogranuloma venereum 440L strain of Chlamydia trachomatis by L cells and HeLa 229 cells occurred at rates and to extents that were 10 to 100 times greater than those observed for the phagocytosis of Escherichia coli and polystyrene latex spheres. Both species of Chlamydia were efficiently taken up by host cells of a type they had not previously encountered. Phagocytosis of chlamydiae was brought about by the interaction of parasite surface ligands with elements of the host cell surface. The chlamydial ligands were readily denatured by heat, were masked by antibody, and were resistant to proteases and detergents. The host cell components were reversibly removed by proteases. Chlamydial phagocytosis was inhibited when host cells were incubated for many hours with cycloheximide. It was suggested that the presence on the chlamydial cell surface of ligands with high affinity for normal, ubiquitously occurring structures on the surface of host cells is an evolutionary adaptation to intracellular existence. The term parasite-specified phagocytosis was used to describe the efficient phagocytosis of chlamydiae by nonprofessional phagocytes and to distinguish it from the host-specified immunological and non-immunological phagocytosis carried out by professional phagocytes. 相似文献
6.
Selective translocation of annexins during intracellular redistribution of Chlamydia trachomatis in HeLa and McCoy cells.
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When Chlamydia trachomatis elementary bodies enter epithelial cells, they occupy membrane-bound vesicles that aggregate with each other in a calcium-dependent manner but that do not fuse with lysosomes. As members of the annexin family of calcium- and membrane-binding proteins have been implicated in mediating calcium-regulated membrane traffic during endo- and exocytosis, we examined the intracellular localization of certain annexins following invasion of HeLa and McCoy cells by C. trachomatis serovar L2. Immunofluorescence staining with a panel of polyclonal antibodies against five human annexins revealed that annexins III, IV, and V translocate within the cytoplasm to the proximity of intracellular chlamydiae whereas the distribution of annexins I and VI was unaffected. The distinct distribution of annexins I and III was further analyzed by confocal microscopy, which revealed an intimate association between chlamydial aggregates or inclusions and annexin III. Confocal microscopy also confirmed the nonassociation of annexin I with chlamydial aggregates. Depletion of intracellular Ca2+ did not prevent association of annexin III with individual elementary body-containing endosomes but did prevent formation of chlamydial aggregates and translocation of annexin III. Furthermore, chloramphenicol-treated cells also showed association between chlamydial aggregates and annexin III, indicating that the annexins are of host cell origin. These data suggest that certain cytosolic annexins may be involved in the Ca(2+)-dependent aggregation and fusion of chlamydia-containing vesicles. The fact that these Ca(2+)-binding proteins differ in their ability to associate with chlamydia-containing vesicles and inclusions implies that the factors that regulate the interaction of annexin I and annexin III with membrane are different and suggests a selective regulatory mechanism for endosome aggregation and avoiding lysosome fusion during chlamydia infection. 相似文献
7.
P Weber J Sinaceur C Ribière M P Le Pennec J C Ghnassia R Nordmann 《Pathologie-biologie》1985,33(3):179-182
Cytosolic superoxide dismutase (SOD) activity was found to increase with time during HeLa cell culture, this increase being due exclusively to Mn-SOD. Infection of the cells by Chlamydia trachomatis resulted in a further enhancement of this Mn-SOD activity, whereas cytosolic catalase activity was decreased in these infected cells. Superoxide (O-2.) being able to induce Mn-SOD and to inhibit catalase, these data suggest that Chlamydia trachomatis infection could be responsible for an increase in O-2. production by the infected HeLa cells. 相似文献
8.
Rödel J Grosse C Yu H Wolf K Otto GP Liebler-Tenorio E Forsbach-Birk V Straube E 《Infection and immunity》2012,80(1):195-205
Intracellular persistence of Chlamydia trachomatis has been implicated in the development of chronic infection that can result in pelvic inflammatory disease and tubal sterility. By inhibition of host cell apoptosis, chlamydiae have evolved a strategy to maintain the intracellular environment for replication and persistence. Both antiapoptotic host cell-derived factors and the chlamydial protease-like activity factor (CPAF) are involved in Chlamydia-mediated apoptosis resistance. Here, we show that in HeLa cells infected with gamma interferon (IFN-γ)-induced persistent C. trachomatis serovar D, the expression of CPAF is downregulated, and proapoptotic protease substrates are not cleaved. Persistent infection protected HeLa cells from apoptosis when they were exposed to staurosporine. Small-interfering RNA-mediated inhibition of myeloid cell leukemia 1 (Mcl-1) protein upregulation sensitized persistently infected cells for apoptosis. The inhibitor of apoptosis protein 2 (IAP-2) seems not to be relevant in this context because IAP-2 protein was not induced in response to IFN-γ treatment. Although apoptosis was inhibited, persistent infection caused cell membrane disintegration, as measured by the increased release of cytokeratin 18 from HeLa cells. Moreover, persistently infected cells released significantly increased amounts of high mobility group box 1 (HMGB1) protein which represents a proinflammatory damage-associated pattern molecule. The data of this study suggest that cells infected with persistent C. trachomatis are protected from apoptosis independently of CPAF but may promote chronic inflammation through HMGB1 release. 相似文献
9.
Enhancement of adherence and growth of Chlamydia trachomatis by estrogen treatment of HeLa cells. 总被引:3,自引:2,他引:3
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Treatment of HeLa 229 cultures with 17-beta-estradiol or with diethylstilbestrol, a synthetic estrogen analog, prior to infection with Chlamydia trachomatis UW31 (serovar K) or LGV440 (serovar L1) led to a 50 to 60% enhancement of chlamydial inclusion formation. After infection, the presence of estrogen was required for the enhancement. The optimal concentration of estrogen required was 10(-10) M. At least 18 h of preinfection treatment plus 12 h of postinfection treatment was necessary. The adherence of purified radioactive elementary bodies of C. trachomatis to estrogen-treated HeLa cells was stimulated in an estrogen dose- and exposure-dependent manner. The requirements for both pre- and postinfection exposure to the hormone suggest that alterations in the cell membrane as well as in the metabolic capacity of the host cells is required for intracellular chlamydial development. Cycloheximide did not prevent estrogen enhancement of chlamydial adherence or subsequent intracellular development of inclusions. 相似文献
10.
Mechanism of entry of human rhinovirus 2 into HeLa cells 总被引:19,自引:0,他引:19
Internalized human rhinovirus 2 (HRV2) undergoes a rapid conformational change leading to recognition by the C-determinant-specific monoclonal antibody 2G2. In the presence of the ionophore monensin, the virus accumulates in the cells in its native conformation and infection is strongly inhibited. At 20 degrees but not at 34 degrees the inhibitory effect of monensin can be overcome by a short incubation of the infected cells at low pH as late as 2 hr after inoculation. Incubation of infected cells at 20 degrees prior to addition of monensin permits virus synthesis to occur, depending on the time of preincubation. 相似文献
11.
Korhonen JT Puolakkainen M Haveri A Tammiruusu A Sarvas M Lahesmaa R 《Microbial pathogenesis》2012,52(3):157-164
A gram-negative obligate intracellular bacterium, Chlamydia pneumoniae, is a common respiratory pathogen. Here, we examined the invasion and attachment of C. pneumoniae K6 into nonphagocytic HL epithelial cell line by manipulating host plasma membranes by using cholesterol-depleting methyl-beta-cyclodextrin (MβCD) and cholesterol-loading MβCD complexed cholesterol (chol-MβCD). The invasion was attenuated by MβCD-treatment while chol-MβCD augmented the attachment and invasion. In addition, the invasion was inhibited by cholesterol sequestering reagents, nystatin and filipin. Furthermore, exposure of host cells to sphingomyelinase inhibited the invasion. RNA interference was used to assay the role of clathrin and human scavenger receptor B, type I (SR-BI) in the entry of C. pneumoniae into A549 lung epithelial adenocarcinoma cells. In contrast to Chlamydia trachomatis L2, the entry of C. pneumoniae was found to be independent of clathrin. In addition, the entry was found to be SR-BI-independent, but interestingly, the chlamydial growth was attenuated in the SR-BI-silenced cells. These findings suggest that the attachment and invasion of C. pneumoniae into nonphagocytic epithelial cells is dependent on the formation of cholesterol- and sphingomyelin-rich plasma membrane microdomains, and the entry is a clathrin-independent process. In addition, our data indicate that SR-BI supports the growth of C. pneumoniae in epithelial cells. 相似文献
12.
目的 探讨沙眼衣原体 (Chlamydiatrachomatis ,Ct)K型感染对HeLa细胞MHCⅠ、Ⅱ类分子表达的影响。方法 用免疫荧光法和流式细胞术等方法 ,对Ct感染和未感染的HeLa细胞MHCⅠ类分子表达水平和IFN γ诱导的HeLa细胞MHCⅡ类分子表达水平进行检测 ,同时对IL 10抗体的影响也作了研究。结果 Ct感染细胞MHCⅠ类分子表达水平和IFN γ诱导的感染细胞MHCⅡ类分子表达水平 ,随Ct感染剂量增加和感染时间延长而下降 ,与正常未感染细胞比较上述差异均具有统计学意义 (P <0 .0 1)。IL 10抗体能部分抑制感染细胞MHCⅠ类分子表达下调 ,但对IFN γ诱导的感染细胞MHCⅡ类分子表达下调无显著影响。结论 Ct感染可下调感染细胞MHCⅠ类分子和IFN γ诱导的细胞MHCⅡ类分子表达水平 ,这可能是造成衣原体持续感染的重要原因。感染过程中分泌的IL 10在下调感染细胞MHCⅠ类分子表达过程中起一定作用 ,而对IFN γ诱导的感染细胞MHCⅡ类分子表达水平下调无显著性影响 相似文献
13.
Binding of the glycan of the major outer membrane protein of Chlamydia trachomatis to HeLa cells. 总被引:3,自引:1,他引:3
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Recent studies have shown that the major outer membrane protein (MOMP) of Chlamydia trachomatis is glycosylated. The glycan of the MOMP of C. trachomatis serovar L2 was separated from the glycoprotein with N-glycanase, reduced with tritiated NaBH4, and tested for its ability to interact with HeLa cells. The [3H]glycan was shown to attach readily to HeLa cells at 25 or 37 degrees C. This process was slower at 4 degrees C. Competition for possibly similar receptor sites on HeLa cells between the glycan and a sugar, an aminosaccharide, or elementary bodies (EBs) was then studied. D-Galactose, D-mannose, or N-acetylglucosamine was shown to reduce the attachment of the glycan to HeLa cells at concentrations of 0.1 to 0.5 M. Sedoheptulose, D-fructose, or sialic acid did not inhibit the binding of glycan to HeLa cells. The presence of at least 100 native or UV-inactivated EBs per HeLa cell interfered with the glycan's ability to bind to HeLa cells. Heat-inactivated EBs did not compete with the glycan for binding. In the reverse situation, nonradiolabeled glycan prevented the EBs from infecting and forming inclusions in HeLa cells. Incubation of [3H]glycan with rabbit immune serum prepared against antigens of whole EB and the MOMP inhibited attachment. In contrast, incubation of glycan with mouse monoclonal antibodies against the protein portion of the MOMP or the chlamydial lipopolysaccharide did not inhibit attachment. These results suggest that the glycan portion of the MOMP is involved in the attachment process of C. trachomatis organisms to HeLa cells. 相似文献
14.
Fusion of inclusions following superinfection of HeLa cells by two serovars of Chlamydia trachomatis. 总被引:5,自引:7,他引:5
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We used a double-label immunofluorescence assay to examine the ability of Chlamydia trachomatis serovar F to infect and develop within HeLa 229 cells previously infected with serovar E. No exclusion to superinfection occurred for up to 24 h following infection by serovar E. The percentage of HeLa cells infected in cultures inoculated with both strains was identical to that of cells in cultures inoculated with one strain as a control. Organisms of both serovars were located within the same intracellular inclusion in 88 to 95% of HeLa cells infected with both serovars. The proportion of superinfected HeLa cells containing both strains in separate inclusions increased when there was exposure to inhibitors of cytoskeletal structure and transport. We used this inhibition to demonstrate that fusion of C. trachomatis phagosomes occurs throughout the developmental cycle. 相似文献
15.
Using pharmacologic and biochemical criteria, we evaluated whether uptake of four different Chlamydia trachomatis serovars, D, E, K, and L2, was dependent upon lipid rafts. Our data suggest that lipid raft-mediated entry is not required for C. trachomatis infection of cultured epithelial cells. 相似文献
16.
Entry of genital Chlamydia trachomatis into polarized human epithelial cells. 总被引:3,自引:17,他引:3
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P B Wyrick J Choong C H Davis S T Knight M O Royal A S Maslow C R Bagnell 《Infection and immunity》1989,57(8):2378-2389
To study the initial invasion process(es) of genital chlamydiae, a model system consisting of hormonally maintained primary cultures of human endometrial gland epithelial cells (HEGEC), grown in a polarized orientation on collagen-coated filters, was utilized. After Chlamydia trachomatis inoculation of the apical surface of polarized HEGEC, chlamydiae were readily visualized, by transmission electron microscopy, in coated pits and coated vesicles. This was true for HEGEC maintained in physiologic concentrations of estrogen (proliferative phase) and of estrogen plus progesterone (secretory phase), despite the finding that association of chlamydiae with secretory-phase HEGEC is significantly reduced (P = 0.025; A.S. Maslow, C.H. Davis, J. Choong, and P.B. Wyrick, Am. J. Obstet. Gynecol. 159:1006-1014, 1988). In contrast, chlamydiae were rarely observed in the clathrin-associated structures if the HEGEC were cultured on plastic surfaces. The same pattern of coated pit versus noncoated pit entry was reproducible in HeLa cells. The quantity of coated pits associated with isolated membrane sheets derived from HeLa cells, grown on poly-L-lysine-coated cover slips in medium containing the female hormones, was not significantly different as monitored by radiolabeling studies and by laser scanning microscopy. These data suggest that culture conditions which mimic in vivo cellular organization may enhance entry into coated pits for some obligate intracellular pathogens. 相似文献
17.
Contrast of Glycogenesis and protein synthesis in monkey kidney cells and HeLa cells infected with Chlamydia trachomatis lymphogranuloma venereum 总被引:2,自引:0,他引:2
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Glycogen metabolism of monkey kidney (LLC-MK-2) cells and HeLa 229 cells infected with a Chlamydia trachomatis lymphogranuloma venereum 440 L (LGV) was studied. The growth cycle of LGV in both host cells was similar; however, a greater number of infectious organism developed intracellularly and were released into the medium during LGV infection of HeLa 229 cells than MK-2 cells. A rapid infection accompanied by a high rate of glycogen synthesis and a short period of accumulation was found in GeLa 229 cells infected with LGV. LGV infected MK-2 cells started to accumulate glycogen about the same time as HeLa 229 cells; however, the rate of glycogen synthesis was lower and the period of accumulation was longer. The LGV agent grew in cycloheximide-treated cells in the absence of host cell protein synthesis. Protein synthesis associated with LGV throughout the developmental cycle was similar in both cell types and could be abolished by chloramphenicol. The continued synthesis of glycogen in the presence of cycloheximide suggested that the synthesis of glycogen was directed by the organism in both MK-2 cells and HeLa 229 cells. 相似文献
18.
Amino acid transport into McCoy cells infected with strains representative of the two major biovars of Chlamydia trachomatis has been studied to determine if uptake is increased during infection. Preliminary work suggested that the transport systems L, A/ASC (for neutral amino acid transport), N (for transport of Asn, Gln, and His) and y+ (for cationic amino acids) were present in McCoy cells. With lymphogranuloma venereum biovar strain 434, little difference in the influx of representative amino acids Trp, His, and Lys or the analogue 2-aminoisobutyric acid (AIB) was observed during infection. With trachoma biovar strain DK20, a small increase in the initial entry rate and equilibrium concentration of each amino acid was found. McCoy cells appear to have great capacity for concentrating amino acids, which might obviate the need for transport induction by chlamydiae under conditions favoring the growth of infectious organisms. 相似文献
19.
Human Mannose-Binding Protein Inhibits Infection of HeLa Cells by Chlamydia trachomatis 总被引:2,自引:0,他引:2
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Albertina F. Swanson R. Alan B. Ezekowitz Amy Lee Cho-chou Kuo 《Infection and immunity》1998,66(4):1607-1612
The role that collectin (mannose-binding protein) may play in the host’s defense against chlamydial infection was investigated. Recombinant human mannose-binding protein was used in the inhibition of cell culture infection by Chlamydia trachomatis (C/TW-3/OT, E/UW-5/Cx, and L2/434/Bu), Chlamydia pneumoniae (AR-39), and Chlamydia psittaci (6BC). Mannose-binding protein (MBP) inhibited infection of all chlamydial strains by at least 50% at 0.098 μg/ml for TW-3 and UW-5, and at 6.25 μg/ml for 434, AR-39, and 6BC. The ability of MBP to inhibit infection with strain L2 was not affected by supplementation with complement or addition of an L2-specific neutralizing monoclonal antibody. Enzyme-linked immunosorbent assay and dot blot analyses showed MBP bound to the surface of the organism to exert inhibition, which appeared to block the attachment of radiolabeled organisms to HeLa cells. Immunoblotting and affinity chromatography indicated that MBP binds to the 40-kDa glycoprotein (the major outer membrane protein) on the outer surface of the chlamydial elementary body. Hapten inhibition assays with monosaccharides and defined oligosaccharides showed that the inhibitory effects of MBP were abrogated by mannose or high-mannose type oligomannose-oligosaccharide. The latter carbohydrate is the ligand of the 40-kDa glycoprotein of C. trachomatis L2, which is known to mediate attachment, suggesting that the MBP binds to high mannose moieties on the surface of chlamydial organisms. These results suggest that MBP plays a role in first-line host defense against chlamydial infection in humans. 相似文献
20.
Santos RI Rodrigues AH Silva ML Mortara RA Rossi MA Jamur MC Oliver C Arruda E 《Virus research》2008,138(1-2):139-143
Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1h. 相似文献