Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: mechanisms of endocytosis

The mechanism by which Chlamydia trachomatis is endocytosed by host cells is unclear. Studies of the kinetics of chlamydial attachment and uptake in the susceptible HeLa 229 cell line showed that chlamydial endocytosis was rapid and saturable but limited by the slow rate of chlamydial attachment. To overcome this limitation and to investigate the mechanism of endocytosis, chlamydiae were centrifuged onto the host cell surface in the cold to promote attachment. Endocytosis of the adherent chlamydiae was initiated synchronously by rapid warming to 36 degrees C. Electron micrographs of chlamydial uptake 5 min after onset showed that chlamydial ingestion involves movement of the host cell membrane, leading to interiorization in tight, endocytic vacuoles which were not clathrin coated. Chlamydial ingestion was not inhibited by monodansylcadaverine or amantadine, inhibitors of receptor-mediated endocytosis and chlamydiae failed to displace [3H]sucrose from micropinocytic vesicles. Chlamydial endocytosis was markedly inhibited by cytochalasin D, an inhibitor of host cell microfilament function, and by vincristine or vinblastine, inhibitors of host cell microtubules. Hyperimmune rabbit antibody prevented the ingestion of adherent chlamydiae, suggesting that endocytosis requires the circumferential binding of chlamydial and host cell surface ligands. These findings were incompatible with the suggestion that chlamydiae enter cells by taking advantage of the classic mechanism of receptor-mediated endocytosis into clathrin-coated vesicles, used by the host cell for the internalization of beta-lipoprotein and other macromolecules, but were consistent with the hypothesis that chlamydiae enter cells by a microfilament-dependent zipper mechanism.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: the role of calmodulin

Adhesion of the obligate intracellular bacterium Chlamydia trachomatis to host cells is associated with a flux of Ca2+ across the cell membrane, and infection is enhanced by treatment of host cells with Ca2+ ionophore. The possibility that Ca2+ might interact with host cell Ca2+ regulatory proteins to promote chlamydial infection was investigated. Treatment of HeLa 229 cells with the calmodulin inhibitors pimozide, trifluoperazine, chlorpromazine, promethazine or haloperidol reduced chlamydial infectivity as measured by inclusion counting or the specific incorporation of [3H]threonine. The inhibitory effect was reversible, dose-related and shown to be associated with impairment of chlamydial adhesion and uptake by the host cells. This effect was clearly distinguished from the delayed maturation of chlamydiae due to continuous exposure to calmodulin inhibitors which may result from a decrease in the availability of high energy compounds from the host cells necessary for chlamydial growth. The possible mechanisms for calmodulin-mediated chlamydial endocytosis are discussed.     

Phospholipase A2 modulation by calmodulin, prostaglandins and cyclic nucleotides

Phospholipase A2 in the presence of Ca2+ was stimulated by calmodulin and by prostaglandin F2 alpha. Prostaglandin E2, cyclic-AMP and cyclic-GMP inhibited phospholipase A2 in the presence or absence of calmodulin. Dimethylsuberimidate cross-linking of phospholipase A2 with calmodulin was found to be Ca2+ dependent. These results indicate that phospholipase A2 is directly regulated by a host of key intracellular regulators and is one of the calmodulin-regulated enzymes.     

Trifluoperazine inhibits the infectivity of Chlamydia trachomatis for McCoy cells

Abstract Trifluoperazine (TFP), an inhibitor of the Ca²⁺-binding protein calmodulin, was used to study the infectivity of Chlamydia trachomatis for McCoy cells. TFP inhibited the number of chlamydial inclusions and the chlamydia-dependent amino acid incorporation when added within 9 h after inoculation with chlamydiae. However, TFP did not affect the attachment of chlamydiae to the cells or the protease-removable fraction of cell-bound chlamydiae.
These results suggest that an early step in the intracellular development of chlamydiae, partly coinciding with the elementary body-reticulate body conversion, is sensitive to TFP and that clathrin coats are not crucial in the ingestion of chlamydiae by McCoy cells.     

Characterization of estrogen-responsive epithelial cell lines and their infectivity by genital Chlamydia trachomatis

Chlamydial attachment and infectivity in vitro and ascending disease and sequelae in vivo have been reported to be enhanced/modulated by estrogen. Endometrial carcinoma cell lines Ishikawa and HEC-1B and the breast cancer lines MCF-7 and HCC-1806 were examined for Chlamydia trachomatis E infectivity. Estrogen receptor (ER) presence was confirmed by Western blot and qRT-PCR analyses. FACS analysis was used to determine the percent of plasma membrane-localized ERs (mERs), and their activity was tested by estrogen binding and competitive estrogen antagonists assays. Chlamydiae grew in all cell lines with HEC (90%) > MCF-7 (57%)>Ishikawa (51%) > HCC-1806 (20%). The cell line ER isoform composition was re-defined as: ERalpha + ERbeta + for MCF-7, HCC-1806 and Ishikawa; and ERbeta only for HEC-1B. HeLa cells were also tested and found to express ERbeta, but not ERalpha. A small percentage of both ERs were surface-exposed and functionally active. The endometrium-predominant ERbeta isoform was found in all cell lines, including those most representative of the common sites of C. trachomatis infection. Thus, the role of chlamydial attachment/infectivity will now be analyzed in ERbeta+and-isogenic HEC-1B cells.     

Inhibition of growth of Chlamydia trachomatis by the calcium antagonist verapamil

Treatment of BGM (African Green Monkey kidney) cells with the calcium antagonist Verapamil resulted in a reduced yield of chlamydial infectious particles. The inhibitory effect was concentration-dependent, the maximal effect being achieved at 200 microM-Verapamil, which produced a 99.99% reduction of infectious particle yield. Electron microscopy showed that control Chlamydia trachomatis-infected BGM cells contained typical large inclusions in which most of the particles were elementary bodies, whereas Verapamil-treated infected cells contained small inclusions consisting predominantly of reticulate bodies. The findings indicate a possible therapeutic use of this calcium antagonist as an anti-chlamydial drug.     

Molecular characterization and subcellular localization of macrophage infectivity potentiator, a Chlamydia trachomatis lipoprotein

Macrophage infectivity potentiator (MIP) was originally reported to be a chlamydial lipoprotein from experiments showing incorporation of radiolabeled palmitic acid into native and recombinant MIP; inhibition of posttranslational processing of recombinant MIP by globomycin, known to inhibit signal peptidase II; and solubility of native MIP in Triton X-114. However, the detailed structural characterization of the lipid moiety on MIP has never been fully elucidated. In this study, bioinformatics and mass spectrometry analysis, as well as radiolabeling and immunochemical experiments, were conducted to further characterize MIP structure and subcellular localization. In silico analysis showed that the amino acid sequence of MIP is conserved across chlamydial species. A potential signal sequence with a contained lipobox was identified, and a recombinant C20A variant was prepared by replacing the probable lipobox cysteine with an alanine. Both incorporation of U-(14)C-esterified glycerol and [U-(14)C]palmitic acid and posttranslational processing that was inhibitable by globomycin were observed for recombinant wild-type MIP but not for the recombinant C20A MIP variant. The fatty acid contents of native and recombinant MIP were analyzed by gas chromatography-mass spectrometry, and the presence of amide-linked fatty acids in recombinant MIP was investigated by alkaline methanolysis. These results demonstrated a lipid modification in MIP similar to that of other prokaryotic lipoproteins. In addition, MIP was detected in an outer membrane preparation of Chlamydia trachomatis elementary bodies and was shown to be present at the surfaces of elementary bodies by surface biotinylation and surface immunoprecipitation experiments.     

Significance of intracellular calcium and cyclic adenosine 3',5'-monophosphate in the mechanisms by which prostaglandins influence human sperm function

The selective ability of PGE-1 and PGE-2 to enhance the capacity of human spermatozoa for hamster oocyte penetration was dependent upon high concentrations (1.7 mM) of extracellular Ca2+ and a prolonged (4 h) duration of exposure, and insensitive to the Ca2+ channel antagonist, verapamil. Studies with the intracellular calcium indicator Quin-2 indicated that exposure to PGE-1 and PGE-2, but not PGF-2 alpha, induced a significant rise in the levels of cytoplasmic Ca2+, suggesting that an ionophore-like action might be responsible for the ability of the E-series prostaglandins to influence sperm function. Stimulation of oocyte penetration with PGE-1 and PGE-2 was significantly enhanced when these compounds were presented to the spermatozoa in medium of high osmolarity (410 mosmol). A combination of PGE-1 in hyperosmotic medium did not significantly influence sperm function in cases of oligozoospermia, although it was effective with patients exhibiting idiopathic infertility. Exposure to high doses of PGE-1 and PGE-2, but not 19-hydroxy PGE or PGF-2 alpha, also induced a significant rise in the cyclic AMP content of human spermatozoa. This effect did not appear to be involved in the enhancement of fertilization rates because it did not exhibit the same absolute dependence on high levels of extracellular Ca2+ as did the fertilization responses and the enhancement of oocyte penetration and the elevation of cAMP were independent of each other within the patient population.     

Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation

The slow Ca²⁺ channels (L-type) of the heart are stimulated by cAMP. Elevation of cAMP produces a very rapid increase in number of slow channels available for voltage activation during excitation. The probability of a Ca²⁺ channel opening and the mean open time of the channel are increased. Therefore, any agent that increases the cAMP level of the myocardial cell will tend to potentiate I_Ca, Ca²⁺ influx, and contraction. The action of cAMP is mediated by PK-A and phosphorylation of the slow Ca²⁺ channel protein or an associated regulatory protein (stimulatory type). The myocardial slow Ca²⁺ channels are also rogulated by cGMP, in a manner that is opposite orantagonistic to that of cAMP. We have demonstrated this at both the macroscople level (whole-cell voltage clamp) and the single-channel level. The effect of cGMP is mediated by PK-G and phosphorylation of a protein, as for example, a regulatory protein (inhibitory-type) associated with the Ca²⁺ channel. Introduction of PK-G intracellularly causes a relatively rapid inhibition of I_Ca(L) in both chick and rat heart cells. Such inhibition occurs for both the basal and stimulated I_Ca(L). In addition, the cGMP/PK-G system was reported to stimulate a phosphatase that dephosphorylates the Ca²⁺ channel. In addition to the slower indirect pathway—exerted via cAMP/PK-A—there is a faster more-direct pathway for I_Ca(L) stimulation by the -adrenergic receptor. This latter pathway involves direct modulation of the channel activity by the alpha subunit (_s*) of the G_s-protein. In vascular smooth muscle cells the two pathways (direct and indirect) also appear to be present, although the indirect pathway producesinhibition of I_Ca(L). PK-C and calmodulin-PK also may play roles in regulation of the myocardial slow Ca²⁺ channels. Both of these protein kinases stimulate the activity of these channels. Thus, it appears that the slow Ca²⁺ channel is a complex structure, including perhaps several associated regulatory proteins, which can be regulated by a number of factors intrinsic and extrinsic to the cell, and thereby control can be exercised over the force of contraction of the heart.This review-type article was prepared by modifying an article published in a book by Sperelakiset al., 1994.     

Control of blastema cell proliferation by possible interplay of calcium and cyclic nucleotides during newt limb regeneration

The effects of the divalent cation ionophore A23187, papaverine, and chlorpromazine on the mitotic index and cyclic nucleotide levels in newt limb regeneration blastemata (Notophthalmus viridescens) were assessed. The results of the experiments suggest that an intracellular increase in divalent cation (Ca2+) concentration results in elevated cGMP levels, suppressed cAMP levels, and a corresponding increase in blastema cell proliferation. The results also suggest that the converse conditions, namely, calcium efflux or inhibition of calmodulin activation (i.e., inhibition of Ca2+ binding), yields elevated cAMP levels, suppressed cGMP levels, and a corresponding decrease in blastema cell divisions.     

Simultaneous extraction and assay of cyclic nucleotides, prostaglandins, and DNA from cat alveolar bone

A method for the simultaneous extraction of cAMP, cGMP, PGE₂, PGF_2α, and DNA from a small sample of mineralized bone and the subsequent assay of these substances is described. Various solvents were tested for efficiency of extraction for the fatty acids, and water or 40% ethanol was found to extract more than 90% of labeled prostaglandin. In order to avoid enzymatic degradation, the substances were extracted at ?5°C requiring a solvent which would not freeze during extraction. Frozen alveolar cat bone samples were homogenized in 40% ethanol in the presence of 5 mm EDTA to inhibit phosphodiesterase. Small aliquots of the homogenate were withdrawn for the spectrofluorophotometric assay of DNA. After centrifugation, the supernatant was extracted first with petroleum ether, in order to take out neutral lipids, followed by ethyl acetate partition. The ethyl acetate layer was dired with N₂ gas, reconstituted with assay buffer, and assayed for PGE₂ and PGF_2α. A portion of the aqueous fraction was used for cAMP binding assay, while the rest was column chromatographed to elute the cGMP for radioassay. On the basis of per microgram of DNA, values for each of the following in cat alveolar bone were: 0.346 ± 0.049 pmol for cAMP, 0.026 ± 0.001 pmol for cGMP, 5.52 ± 1.46 pg for PGE₂, and 1.00 ± 0.29 pg for PGF_2α. Values calculated after the dilution of the sample aliquots or addition of standards to cAMP, cGMP, or PGE₂ showed no significant difference (P < 0.05) to their respective values. Within the limits of the sensitivity for each of the assay systems, it is feasible to measure cAMP, cGMP, PGE₂, and PGF_2α in alveolar bone from the same sample.     

Inoculation of Chlamydia pneumoniae or Chlamydia trachomatis with ligands that inhibit attachment to host cells reduces infectivity in the mouse model of lung infection: implication for anti-adhesive therapy

Previous studies have shown that the chlamydial glycan contains a high-mannose oligosaccharide, which mediates attachment and infectivity of the organism. Removal of the glycan decreases infectivity in vitro and in vivo. The present study demonstrates that simultaneous inoculation of chlamydial organisms and a ligand that prevents glycan binding reduces lung burden in infected animals.     

Macrophage plasminogen activator: modulation of enzyme production by anti-inflammatory steroids, mitotic inhibitors, and cyclic nucleotides.

Plasminogen activator production by cultured mouse peritoneal macrophages can be modulated in vitro by low concentrations of various pharmacologically active molecules. Glucocorticoid hormones and their synthetic derivatives, as well as cholera toxin, colchicine, and vinblastine markedly inhibit production of this enzyme without affecting other important macrophage functions. The effect of glucocorticoids is of particular interest, both because their relative in vivo anti-inflammatory potencies correlate exactly with their effect on plasminogen activator production in culture and because this effect occurs at near physiological concentrations. In view of the correlations established in other systems between plasminogen activator production and cell migration, we have also examined the age of the macrophages in thioglycollate-induced exudates. Confirming the results of Van Furth and Cohn (1968), we have found that the majority of these cells are young, having recently replicated and arrived in the peritoneal cavity. Using a fibrinagar overlay technique which allowed us to determine the production of plasminogen activator by individual cells. we have found that the majority of these cells produce the enzyme. The potential roles of plasminogen activator in monocyte migration and the relationship of this enzyme to the anti-inflammatory effect of gluccorticoids are correlated and emphasized.     

Differences in infectivity and induction of infertility: a comparative study of Chlamydia trachomatis strains in the murine model

Chlamydia trachomatis, although commonly asymptomatic in women, can result in chronic sequelae, such as pelvic inflammatory disease, ectopic pregnancy and infertility. However, a clear relationship has not been determined between specific serovars and the ability to lead to upper genital tract infection or infertility. Thus, in order to investigate differences in pathogenicity, C3H/HeN mice were infected in the ovarian bursa with the C. trachomatis strains D (UW-3/Cx), F (N.I.1), F (IC-Cal-3) and E (Bour). Differences both in the amount of vaginal shedding as well as subsequent fertility rates were observed between D (UW-3/Cx) and F (N.I.1) compared to F (IC-Cal-3) and E (Bour). Approximately 50% of the mice infected with the D (UW-3/Cx) and F (N.I.1) strains had vaginal shedding for up to 3–4 weeks after infection and fertility rates of less than 25%. Furthermore, mice inoculated with D (UW-3/Cx) and F (N.I.1) showed infertility even in the absence of medroxy progesterone acetate (MPA) treatment. In contrast, both MPA and non-MPA treated mice infected with F (IC-Cal-3) or E (Bour) did not show vaginal shedding and had fertility rates between 45 and 88%. Mutations in the CT135 open reading frame have been associated with virulence. However, no nucleotide differences were found among the four isolates for CT135. This murine model of infection with C. trachomatis may help with the understanding of disease pathology in humans and ultimately vaccine development.     

Histamine, endogenous prostaglandins and cyclic nucleotides in the regulation of airway muscle responses in the guinea pig

Indomethacin (30 mg/kg, i.p.) reduced pulmonary resistance in guinea pigs but did not affect their sensitivity to histamine. This treatment preferentially reduced the generation of PGE2 by isolated tracheal preparations. The ratios of PGF2 alpha/PGE2 before and after treatment were 1/1 and 6/1, respectively. Chronic indomethacin treatment (30 mg/kg, i.p., twice a day for 4 days) increased histamine sensitivity in vivo 2 fold while a longer treatment (10 days) was without effect. The efficacy of histamine and the potency of isoproterenol in tracheal tissues were unaffected by either treatment. Indomethacin (17 microM for 30 min) relaxed tracheal tissues but not bronchial tissues. Responses of both tissues to contractile agonists were potentiated after indomethacin treatment. The efficacy of histamine was smaller in bronchi than in tracheas. Similarly, PGE2, PGI2 and isoproterenol were less potent in bronchi. Basal amounts of cyclic AMP were higher in bronchi than in tracheas; indomethacin did not affect the basal amounts of cyclic AMP in tracheal tissues but reduced them in bronchial preparations. Histamine elevated cyclic AMP content in both preparations; this elevation was reduced by indomethacin. While prostaglandins play a role in modulating airway responses in vitro, their role in airways in normal animals in vivo is more difficult to demonstrate.     

Requirement for the Rac GTPase in Chlamydia trachomatis invasion of non-phagocytic cells

Chlamydiae are gram-negative obligate intracellular pathogens to which access to an intracellular environment is paramount to their survival and replication. To this end, chlamydiae have evolved extremely efficient means of invading nonphagocytic cells. To elucidate the host cell machinery utilized by Chlamydia trachomatis in invasion, we examined the roles of the Rho GTPase family members in the internalization of chlamydial elementary bodies. Upon binding of elementary bodies on the cell surface, actin is rapidly recruited to the sites of internalization. Members of the Rho GTPase family are frequently involved in localized recruitment of actin. Clostridial Toxin B, which is a known enzymatic inhibitor of Rac, Cdc42 and Rho GTPases, significantly reduced chlamydial invasion of HeLa cells. Expression of dominant negative constructs in HeLa cells revealed that chlamydial uptake was dependent on Rac, but not on Cdc42 or RhoA. Rac but not Cdc42 was found to be activated by chlamydial attachment. The effect of dominant negative Rac expression on chlamydial uptake is manifested through the inhibition of actin recruitment to the sites of chlamydial entry. Studies utilizing Green Fluorescent Protein fusion constructs of Rac, Cdc42 and RhoA, showed Rac to be the sole member of the Rho GTPase family recruited to the site of chlamydial entry.     

Regulation of platelet cytosolic free calcium by cyclic nucleotides and protein kinase C

PAF elicits a rapid, concentration-dependent elevation of platelet cytosolic free calcium ([Caf]), measured by quin2. Elevation of [Caf] is transient, and the rate of reversal increases with agonist concentration. Adenylate cyclase stimulants (PGI2, PGD2) and 8-bromo cAMP; a guanylate cyclase stimulant (sodium nitroprusside) and 8-bromo cGMP; and a protein kinase C stimulant (phorbol myristate acetate) block the elevation of [Caf] induced by PAF, and accelerate its reversal. These results suggest that cAMP, cGMP and 1,2-diacylglycerol (DAG) could act as second messengers to regulate [Caf] in platelets. As PAF is known to stimulate platelet phosphoinositide hydrolysis (ergo DAG formation) but fails to elevate platelet cAMP or cGMP, it is proposed that DAG, via activation of protein kinase C, may act as an endogenous modulator of platelet [Caf]: an action that contributes to the role of DAG as a bi-directional regulator of platelet reactivity.     

Stimulation of the cytosolic receptor for peptidoglycan, Nod1, by infection with Chlamydia trachomatis or Chlamydia muridarum

Infection of epithelial cells by the intracellular pathogen, Chlamydia trachomatis, leads to activation of NF-kappaB and secretion of pro-inflammatory cytokines. We find that overexpression of a dominant-negative Nod1 or depletion of Nod1 by RNA interference inhibits partially the activation of NF-kappaB during chlamydial infection in vitro, suggesting that Nod1 can detect the presence of Chlamydia. In parallel, there is a larger increase in the expression of pro-inflammatory genes following Chlamydia infection when primary fibroblasts are isolated from wild-type mice than from Nod1-deficient mice. The Chlamydia genome encodes all the putative enzymes required for proteoglycan synthesis, but proteoglycan from Chlamydia has never been detected biochemically. Since Nod1 is a ubiquitous cytosolic receptor for peptidoglycan from Gram-negative bacteria, our results suggest that C. trachomatis and C. muridarum do in fact produce at least the rudimentary proteoglycan motif recognized by Nod1. Nonetheless, Nod1 deficiency has no effect on the efficiency of infection, the intensity of cytokine secretion, or pathology in vaginally infected mice, compared with wild-type controls. Similarly, Rip2, a downstream mediator of Nod1, Toll-like receptor (TLR)-2, and TLR4, increases only slightly the intensity of chlamydial infection in vivo and has a very mild effect on the immune response and pathology. Thus, Chlamydia may not produce sufficient peptidoglycan to stimulate Nod1-dependent pathways efficiently in infected animals, or other receptors of the innate immune system may compensate for the absence of Nod1 during Chlamydia infection in vivo.