1 Department of Microbiology, Yamaguchi University School of Medicine, 1-1-1, Minamikogushi, Ube, Yamaguchi 755-8505, Japan; 2 Department of Clinical Research, National Sanyou Hospital, Yamaguchi 755-0241, Japan; 3 Department of Veterinary Microbiology, Gifu University, 1-1 Yanagido, Gifushi, Gifu 501-1193, Japan
Received 4 July 2005; returned 29 July 2005; revised 16 August 2005; accepted 19 August 2005
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Abstract |
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Methods: Rates of chlamydial infection and sizes of the inclusions were evaluated by in vitro infection using three Chlamydiaceae species, Chlamydia trachomatis, Chlamydophila pneumoniae and Chlamydophila felis, which show significant divergence of tryptophan synthesis genes and different susceptibilities to IFN-.
Results: Melatonin and serotonin, which are recognized as neural hormones for maintenance of organism homeostasis, reduced chlamydial infection but not other bacterial growth tested here. Unlike IFN-, melatonin limited infection of all three chlamydiae and the effects were not recovered by tryptophan supplementation. Melatonin treatment only of host cells could diminish infection and the infection reduction was neutralized by a pertussis toxin, an inhibitor of G proteins. Ligands of melatonin and serotonin receptors also hampered infection.
Conclusions: Inhibition mechanisms of chlamydial infection by melatonin and serotonin appear to be different from those of IFN- and involve specific G-protein-coupled receptors. Melatonin is deemed to inhibit early progression of the chlamydial development cycle, such as establishment of intracellular infection and/or conversion from elementary body to reticulate body. Utilization of melatonin, serotonin or their derivatives may be advantageous for harmless prevention of chlamydial infection.
Keywords: Chlamydophila pneumoniae , Chlamydia trachomatis , tryptophan , antimicrobial agents , G-protein coupled receptor
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Introduction |
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One of the most important host defence systems against the chlamydial infection is interferon-gamma (IFN-) synthesis, which induces indoleamine 2,3-dioxygenase (IDO) and thus depletes tryptophan from human cells, since many human pathogenic chlamydiae rely on the tryptophan.811 Besides the function of IFN-
-inducible tryptophan shortage as a host defence, insufficient IFN-
exposure may lead the chlamydial growth to a persistent state, which is characterized by atypical inclusions that are generally smaller in diameter and contain larger aberrant RBs.12 This is why chlamydial IFN-
resistance and host/tissue tropism are thought to depend on chlamydial tryptophan synthesis.13,14 Persistent chlamydial infection is thought to be involved in the pathophysiology of some chronic inflammatory diseases, e.g. asthma and atherosclerosis for C. pneumoniae, and blindness and infertility for C. trachomatis.15
Human tryptophan catabolism mainly consists of two pathways, the kynurenine pathway and the serotonin pathway. In the kynurenine pathway, tryptophan is processed to N-formylkynurenine by two types of rate-limiting enzymes, tryptophan 2,3-dioxygenase (TDO) induced by tryptophan in the liver and the IDO induced by IFN- during the immune response in many tissues. Tryptophan is finally metabolized to an essential cellular cofactor, nicotinamide adenine dinucleotide (NAD+). The other pathway is the serotonin pathway that produces a variety of neural activators such as a neurotransmitter, serotonin and, a pineal hormone, melatonin.10
Serotonin is a well-known ubiquitous monoamine, which is derived from tryptophan and functions as a neurotransmitter, hormone and mitogenic factor that mediates a wide range of physiological activities in different cells through multiple receptor subtypes. Serotonin is produced and stored within several tissues throughout the human body, such as the central neural cells,16 blood platelets,17 gastrointestinal enterochromaffin cells18 and prostatic neuroendocrine cells.19 Furthermore, serotonin plays a fundamental role in regulation of growth, differentiation and gene expression.2022 Melatonin, originally identified as an effector for circadian rhythms,23 is now known to be a hormone involved in a vast range of homeostasis maintenance activities, for example seasonal timing, sexual development, the antioxidant defence system and immune response.2426 Melatonin is synthesized from tryptophan within the serotonin pathway mainly in the pineal gland, and in a number of extrapineal organs such as retina, lens, bone marrow, intestine, skin and so on.27 To date, three mammalian melatonin receptors, G-protein coupled receptors MT1 and MT2, and a quinone reductase family receptor, MT3, have been identified.19,28,29
Recently, several chlamydial genome sequencing projects have been completed and have revealed the divergence of the gene repertoire for tryptophan synthesis.13,14,30 It is not known why chlamydial species contain different tryptophan gene repertoires, but it is possible that some host factors related to tryptophan naturally restrict gene compositions of the chlamydial tryptophan operon in chlamydial evolution. Here we report the finding that the tryptophan derivatives serotonin and melatonin reduce chlamydial infection in HEp-2 cells in a manner different from the IFN--inducible host defence.
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Materials and methods |
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L-Tryptophan, and its derivative compounds, such as 5-hydroxy tryptophan, 5-hydroxy tryptophol, 5-methoxyindole acetic acid, melatonin and serotonin, and ligands of melatonin and serotonin receptors, IIK7, luzindole, buspirone and NAN190, were purchased from SigmaAldrich (Saint Louis, MO). Gentamicin, cycloheximide, DAPI and Dulbecco's modified Eagle medium (DMEM) were purchased from SigmaAldrich. Pertussis toxin was purchased from List Biological Laboratories, Inc. (Campbell, CA), human recombinant IFN- was from Peprotech Ec Ltd (London, UK), and fetal calf serum (FCS) was from Cansera International Inc. (Etobicoke, Canada). The family Chlamydiaceae-specific fluorescein isothiocyanate (FITC)-labelled antibody was from Denkaseiken (Tokyo, Japan).
Cell line and bacterial strains
HEp-2 cells (ATCC CCL-23) were grown in HEp-2 medium (DMEM supplemented with 10% heat-inactivated FCS and 50 mg/L gentamicin) at 37°C with 5% CO2. C. pneumoniae J138 isolated in Japan in 1994,30 C. felis Japanese strain Fe/C-5631 and C. trachomatis serovar D were used in this work. These strains were purified by sucrose-gradient centrifugation and stored at 80°C in the SPG buffer (pH 7.2), which consists of 250 mM sucrose, 10 mM sodium phosphate and 5 mM glutamate. Chlamydial titres were adjusted for C. pneumoniae J138 to 2.0 x 108 inclusion formation units (IFU)/mL, and C. felis and C. trachomatis to 1.0 x 108 IFU/mL. Other bacterial strains used here are Escherichia coli, Staphylococcus aureus, Bacillus subtilis E192, Streptococcus pneumoniae, Pseudomonas aeruginosa PAO1, Helicobacter pylori CPY3401, Mycobacterium tuberculosis H37Rv, and Legionella pneumophila ATCC33153.
Chlamydial infection
Chlamydial infection was performed by methods described elsewhere.32 Briefly, 2.0 x 104 HEp-2 cells in 0.10 mL of the medium were seeded into each well of flat-bottomed 96-well tissue culture plates, and allowed to adhere for 24 h prior to use. Infection was performed by addition of chlamydial EBs to achieve a multiplicity of infection (MOI) of 0.20. In this condition, inclusion formation units, number of host cells and infection rate may turn to 1 x 105 IFU/mL, 5 x 104 cells and 20%, respectively. Relative infection rate was determined as an average of at least three replicate tests and confirmed with two other independent experiments performed under the same conditions. After centrifugation at 700g for 60 min at 22°C and then incubation for 30 min at 36°C with 5% CO2, the inoculum was replaced with the post-infection medium (DMEM with 5% heat-inactivated FCS, 50 mg/L gentamicin and 1 µg/mL cycloheximide). The infected cells were incubated for 48 h at 36°C with 5% CO2, following fixation of the infected cells for 30 min in methanol.
For measurement of IFUs and rates of infected host cells, the infected cells were stained with the family Chlamydiaceae-specific FITC-labelled antibody for 60 min at 37°C, and then stained with 0.1 µg/mL of DAPI for 10 min. Numbers of host cell nuclei and chlamydial inclusions were counted under fluorescence microscopy, BX50 (Olympus, Japan) at a magnification of x200. Under treatments with chemicals and IFN-, typical and atypical (or small) inclusions were observed. Here, inclusions larger than approximately 1 µm in diameter were counted as successful infections.33,34
Chemical treatment
In most experiments, treatments with 50200 µM test compound were started at 24 h prior to infection and ended at 48 h post-infection, otherwise treatment schedules and concentrations of chemicals are described in the figure legends for each experiment.
For other bacteria, E. coli, S. aureus, B. subtilis, P. aeruginosa were cultured in LB broth, whereas S. pneumoniae was cultured in Brain Heart Infusion and H. pylori in Brucella broth supplemented with 5% horse serum and 0.1% ß-cyclodextrin. Inocula of each strain were incubated in their respective media treated with melatonin, gentamicin or mock at 37°C, and then the OD at 600 nm was measured as the indication for bacterial growth up to 8 h except H. pylori to 24 h. The BACTEC MGIT 960 Automated System (Becton Dickinson, Cockeysville, MD) was adapted for susceptibility tests with M. tuberculosis and L. pneumophila. For L. pneumophila growth, Legionella BCYE growth supplement (Oxoid Ltd, Hampshire, UK) was added to the MGIT medium for M. tuberculosis.
Assay of infectious progeny
To analyse the production of infectious progeny under the treatment of melatonin, serotonin and IFN-, chlamydial progeny were collected from the culture medium at 72 h post-infection by centrifugation at 10 000g for 15 min at 4°C. To measure the amount of infectious progeny, the collected chlamydiae were suspended in the DMEM buffer and adapted to the secondary infection using HEp-2 cells growing in 96-well plates. The procedures described above were used to determine the infection rates.
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Results |
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Tryptophan and six derivatives (Figure 1a) were administered at 50 µM onto HEp-2 cells infected with C. pneumoniae J138 at 24 h prior to infection and maintained until the infection was terminated by fixation. The rates of infected host cells were calculated and are shown in Figure 1(b). Among the compounds tested here, serotonin and melatonin reduced the chlamydial infection by 50% compared with controls. 5-Hydroxytryptophan at 200 µM also showed an inhibitory effect on chlamydial infection (data not shown). Optimum concentrations of melatonin and serotonin were analysed in a range of 25400 µM, at which no host toxicity was observed under microscopic studies. Treatments with melatonin and serotonin caused a dose-dependent inhibition of C. pneumoniae infection of HEp-2 cells and at 100 µM both reached the optimum effect (Figure 1c). In most experiments below, melatonin and serotonin were used at 100 µM.
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Elevation of IFN- levels by melatonin has been reported during viral infection in a mouse model.35 To investigate the relationship between melatonin and IFN-
, excess tryptophan was added independently to melatonin and IFN-
in chlamydial infection. The inhibition of chlamydial infection by IFN-
was observed as previously reported33 and was reversed by adding tryptophan, consistent with the well-known schema of tryptophan depletion by IFN-
.9,11 In contrast, tryptophan addition did not reverse melatonin-mediated inhibition (Figure 2a). This suggests that the inhibitory mechanism of melatonin is different from that of IFN-
in this case.
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IFN- exposure and melatonin treatment cause atypical chlamydial inclusions, supposedly showing the typical persistent state of chlamydial infection and producing few progeny.12,33,34 The re-infection ability of progeny produced under melatonin and serotonin treatment was analysed. At 72 h post-infection, infected host cells which were treated with melatonin and serotonin had released approximately half the infectious progeny compared with untreated control (Figure 2d), while IFN-
-treated cells had released one-fourth of the control. Chlamydial progeny in inclusions under melatonin and serotonin treatment are infectious, not in the persistent state, and are released at approximately the same time as in controls. This suggests that melatonin and serotonin may affect the early establishment stage in the chlamydial development cycle, but not intracellular growth or later development.
Specificity of melatonin on microbes
To determine melatonin specificity for chlamydial species, infection experiments were performed with two more chlamydiae with worldwide distribution, C. trachomatis and C. felis, which cause a human sexually transmitted disease and cat conjunctivitis, respectively. Infection rates of the two chlamydiae were reduced depending on melatonin concentrations. C. felis is resistant to IFN- but susceptible to melatonin, and C. trachomatis is relatively resistant to both melatonin and IFN-
, whereas C. pneumoniae is susceptible to both IFN-
and melatonin (Figure 3). This indicates that the mechanism of melatonin to inhibit chlamydial growth is different from that of IFN-
, and the chlamydial gene repertoires, but not tryptophan synthesis, may affect susceptibility to melatonin.
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Target(s) of melatonin and serotonin
It has been reported that melatonin influences the hydrophobic characteristics of Escherichia coli cell surfaces.36 Therefore, chlamydial cells and host cells were pre-treated with melatonin and serotonin before infection. Chlamydial infection was reduced by pre-treatment of HEp-2 cells with melatonin and also serotonin. Pre-treatment of chlamydial cells showed no effect (Figure 4a). In addition, post-infection treatment with melatonin also reduced chlamydial infection (data not shown). This suggests that melatonin and serotonin interact with human cells but not with chlamydial cells, and some melatonin and serotonin receptors are involved in the resistance to chlamydial infection.
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Discussion |
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Pre-infection melatonin treatment of host cells, but not chlamydial cells, inhibits chlamydial infection. Post-infection treatment with melatonin also inhibited chlamydial infection, illustrating that melatonin may not block interaction between cell surfaces of chlamydiae and host cells. Moreover, addition of pertussis toxin with melatonin (but not serotonin) treatment, cancelled the melatonin effect, and ligands of melatonin and serotonin receptors also inhibit chlamydial infection, suggesting that some melatonin and serotonin receptors are involved in the inhibition of chlamydial infection.
Melatonin is synthesized within the serotonin pathway from tryptophan mainly in the pineal gland and also in a number of tissues, such as retina, intestine, and so on.27 Three mammalian melatonin receptors have been identified to date. MT1 and MT2 receptors are G-protein-coupled receptors sensitive to the pertussis toxin28,29 and MT3 receptor is a quinone reductase family receptor.19 It is reported that the receptors are widely exhibited on peripheral tissues, such as brain, liver and kidney, and an epithelial cell line, HEK-293.38,39 Gene expression of MT1 and MT2 receptors was not detected by RTPCR in our experiments using a human epithelial cell line, HEp-2 (data not shown). In addition, distribution analysis of inclusion sizes demonstrates distinctive groups of inclusions. One of these may be a group (50%) in which susceptibility to melatonin is high and prevented from visible inclusion formation. The other two groups may be resistant and partially susceptible to melatonin, resulting in typical and small inclusion formations, respectively. It may implicate the existence of a melatonin-specific receptor(s), whose signal transduction system or gene expression determines the susceptibility to melatonin relying on the host cell cycle.
In contrast to melatonin, chlamydial growth inhibition by serotonin is insensitive to pertussis toxin. However, it is still highly possible that the inhibitory mechanism of serotonin involves specific receptor(s) coupled with G-proteins resistant to pertussis toxin, firstly because ligands of serotonin receptors have inhibited chlamydial infection, and secondly because most physiological serotonin reactions are processed through G protein-coupled serotonin-specific receptors. In the large family of G protein-coupled serotonin receptors, Ga12/13, has already been reported to be a type of G protein insensitive to pertussis toxin.40
As common features, the melatonin and serotonin signal transductions through each specific receptor result in an increase in intracellular Ca2+ concentration and regulation of adenylyl cyclase activity, which produces adenosine 3':5'-cyclic monophosphate (cAMP). Both concentrations of Ca2+ and cAMP produced by adenylyl cyclase subsequently modulate the gene expression status of the host cell.41,42 Interestingly, chlamydial infection is closely related to intracellular levels of cAMP and Ca2+. Chlamydial infection is inhibited by high concentrations of extracellular cAMP or cGMP.43 Ca2+ mobility across the cell membrane is rapidly increased by chlamydial attachment on to host cells.43 Recently, Ca2+-dependent phagolysosomal fusion has been illustrated as a host defence strategy, in which host cells may monitor Ca2+ influx accompanying injuries by invasion of intracellular pathogens and the Ca2+ influx may promote lysosomal exocytosis.44 The type III secretion system is a candidate to injure the host cell membrane and is thought to be an invasion system of chlamydiae as well. It is likely that melatonin modulates intracellular Ca2+ and/or cAMP levels and leads to increased phagolysosomal activity of the host cells against the chlamydial infection.
To detect modification of gene expression in HEp-2 cells by the melatonin and serotonin treatments, microarray analysis was carried out with AceGene oligo-DNA chips (HitachiSoft, Japan) arraying approximately ten thousand known human genes. Positively and negatively regulated genes in the treated cells were nominated when the signals are more than 1.5 times or less than 0.6 times compared with untreated cells, respectively. Melatonin and serotonin treatments resulted in modifying mRNA accumulation moderately, except for some genes, e.g. myosin and kinesin family members and platelet-derived growth factors, implying that the melatonin and serotonin treatments may remodel the intracellular matrix to a state inadequate for chlamydial intracellular growth (data not shown).
Melatonin, originally identified as an effector for circadian rhythms,23 is now known as a hormone involved in a vast range of homeostasis maintenance, i.e. human mood,24 seasonal timing,45 anti-ageing,46 oncostatic,47 antioxidant defence,27,48 and immunomodulation.49 Many of the melatonin effects seem to relate to mitochondrial functions as a direct scavenger for a variety of toxic oxygen and nitrogen-based reactants.50 Enhancements of the non-specific and specific immune responses by exogenous melatonin are reported as stimulation for proliferation/production of NK cells and monocytes,51 and prevention from the reduction in B- and T-cell proliferation and Th1 cytokine secretions caused by retroviral infection.52 Despite the many investigations on melatonin and serotonin that have been carried out, they have not been generally recognized as antimicrobial agents, apart from a few reports for melatonin showing protective effects in mice against viral infections by Venezuelan equine encephalomyelitis virus,53 Semliki Forest virus54 and West Nile virus,55 and against infection by a helminth parasite, Schistosoma mansoni.56 Melatonin is used in Mycobacterium tuberculosis infection to increase the chemotherapeutic activity of isoniazid (pyridine-4-carbohydrazide), which is an inhibitor of tryptophan metabolism to NAD.57 So, the inhibition of infection for the intracellular bacterium chlamydia is a new function of melatonin and serotonin.
Variation of tryptophan biosynthesis genes is the most remarkable feature in the comparison of gene repertoires among chlamydial genomes. Considering that tryptophan depletion by IFN- is a crucial host defence mechanism against chlamydiae, it evokes all evolutional relationships between strategies of host defence and parasite infection, leading to chlamydial host and tissue tropisms. There is an apparent paradox: C. trachomatis and C. pneumoniae, worldwide common parasites to human but not to animals, contain partial and no tryptophan biosynthesis genes in their genomes, respectively, and they are susceptible to both IFN-
and melatonin; on the other hand, C. felis, a worldwide common parasite of cats but rarely humans, seems to possess an almost complete gene set for tryptophan biosynthesis (Y. Azuma, H. Hirakawa, A. Yamashita, Y. Cai, M. A. Rahman, H. Suzuki, S. Mitaku, H. Toh, T. Murakami, K. Sugi, H. Hayashi, H. Fukushi, M. Hattori, S. Kuhara and M. Shirai, unpublished data) and show marked resistance to IFN-
, but not to melatonin. Whereas, tryptophan might play a key role for the molecular basis of hostparasite interaction in terms of IFN-
resistance, the gene repertoires of tryptophan biosynthesis cannot entirely explain host tropisms of individual chlamydial species. The different susceptibilities to melatonin or serotonin, or any tryptophan derivatives may be involved in determination of the host tropisms. Different melatonin susceptibilities among chlamydial species may depend on gene variations of the chlamydial species responsible for host defence systems through melatonin receptors. Genes discriminating C. trachomatis from C. felis and C. pneumoniae consist of 69 genes, whose products are annotated as 60 unknown proteins, a membrane thiol protease, two transporters, two polymorphic outer membrane proteins and four phospholipase D superfamily proteins. Although our studies have provided evidence of possible anti-chlamydial functions of melatonin and serotonin, further investigations are still needed to determine the chlamydial factors that are involved in the various diseases and host/tissue tropism.
Supplementary data
Figure 2(b) is available in colour as online supplementary data at http://jac.oxfordjournals.org/.
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Acknowledgements |
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