| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Microbiology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614-0579, USA
2 ViroMed Laboratories, Minnetonka, MN, USA
3 Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, USA
Correspondence
Robert V. Schoborg
schoborg{at}etsu.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
(IFN-), IFN-
, IFN-β, and tumour necrosis factor- (TNF-) exposure, and iron, amino acid or glucose deprivation. A tissue-culture model of Chlamydia trachomatis/herpes simplex virus type-2 (HSV-2) co-infection indicates that viral co-infection stimulates the formation of persistent chlamydiae. This study was designed to ascertain whether co-infection-induced persistence is mediated by a previously characterized mechanism. Luminex assays indicate that IFN-, IFN-, and TNF- are not released from co-infected cells. Semiquantitative RT-PCR studies demonstrate that IFN-β, IFN-, indoleamine 2,3-dioxygenase, lymphotoxin- and inducible nitric oxide synthase are not expressed during co-infection. These data indicate that viral-induced persistence is not stimulated by any persistence-associated cytokine. Supplementation of co-infected cells with excess amino acids, iron-saturated holotransferrin, glucose or a combination of amino acids and iron does not restore chlamydial infectivity, demonstrating that HSV-2-induced persistence is not mediated by depletion of these nutrients. Finally, inclusions within co-infected cells continue to enlarge and incorporate C6-NBD-ceramide, indicating that HSV-2 co-infection does not inhibit vesicular transport to the developing inclusion. Collectively these data demonstrate that co-infection-induced persistence is not mediated by any currently characterized persistence inducer or anti-chlamydial pathway. Previous studies indicate that HSV-2 attachment and/or entry into the host cell is sufficient for stimulating chlamydial persistence, suggesting that viral attachment and/or entry may trigger a novel host pathway which restricts chlamydial development.
, lymphotoxin-; MOMP, major outer-membrane protein; NO, nitric oxide; RB, reticulate body; TNF-, tumour necrosis factor A supplementary table of primers and three supplementary figures are available with the online version of this paper.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
).
Herpes simplex viruses begin their infection of epithelial cells by binding to the host-cell receptor, heparan sulfate. After this initial binding, viral envelope glycoproteins interact with one of four known co-receptors [herpes viral entry mediator (HVEM), nectin-1 and -2, and 3-O-sulfated heparan sulfate], facilitating viral entry (Spear, 2004). Following fusion of the viral envelope with the host cell, viral tegument proteins are released into the cytoplasm and the capsid is transported to the nucleus, where viral genome replication occurs. The viral genome is transcribed and viral proteins are synthesized using host-cell machinery. New virions are then assembled and exit the host cell (Roizman & Knipe, 2001).
Chlamydia trachomatis is a Gram-negative, obligate intracellular bacterium which is responsible for 4 million sexually transmitted infections each year in the USA (Butler, 1997; Weinstock et al., 2004). Genital chlamydial infections are often chronic and asymptomatic, leading to severe complications including pelvic inflammatory disease, ectopic pregnancy and infertility (Darville, 2000). In vivo and in culture, chlamydiae carry out a characteristic biphasic developmental cycle. Extracellularly, chlamydiae exist as infectious elementary bodies (EBs). EBs attach to and enter a host cell, where they differentiate within an inclusion into non-infectious reticulate bodies (RBs). The infectious cycle spans 30–72 h (depending upon the chlamydial species/serovar). Near the end of the cycle, RBs develop back into EBs and are released from the infected host cell. When developing chlamydiae are exposed to certain adverse environmental factors [such as interferon- (IFN-) or penicillin G] or are deprived of nutrients (such as amino acids, iron or glucose), they deviate from the normal developmental cycle into a state termed persistence (Hogan et al., 2004). Once in the persistent state, RBs continue to grow but do not divide, resulting in the formation of enlarged, abnormally shaped RBs. Although they remain viable and continue genomic DNA replication and rRNA synthesis, persistent chlamydiae are no longer infectious. Interestingly, persistent chlamydiae can re-enter the normal developmental cycle and recover infectivity if the environmental stressor is removed. For example, nutrient-deprived, persistent chlamydiae resume development into infectious EBs if the appropriate nutrients are replenished (Harper et al., 2000; Matsushima et al., 1999; Raulston, 1997).
Several host-cell pathways have evolved to limit the spread of chlamydial infections. In human cells, IFN- exposure increases host-cell indoleamine 2,3-dioxygenase (IDO) expression, leading to depletion of host-cellular tryptophan. Without this essential amino acid, developing chlamydiae enter into persistence and cease productive replication (Beatty et al., 1994). In murine cells, IFN- exposure increases synthesis/activity of the p47 GTPase, Iigp1. Iigp1 inhibits chlamydial development by restricting vesicular trafficking to developing inclusions (Nelson et al., 2005). IFN-, IFN-β and tumour necrosis factor- (TNF-) interfere with chlamydial development by induction of IDO, induction of inducible nitric oxide synthase (iNOS) and/or restriction of intracellular iron stores (Hogan et al., 2004; Matsushima et al., 1999). When iNOS is upregulated, the host cell produces nitric oxide (NO), which kills developing chlamydiae. Although these pathways are intended to eliminate the bacterial infection, chlamydiae are often able to survive the onslaught of the host cell's arsenal by entering into a persistent state until conditions become favourable for their continued development.
Several studies have shown that C. trachomatis and HSV-2 co-infections occur in vivo (Paavonen et al., 1985; Tait et al., 1985). In vitro models of HSV-2/C. trachomatis co-infections have indicated that HSV-2 co-infection alters chlamydial development; however, previous studies did not examine the co-infection process in detail (Chiarini et al., 1996; Pontefract et al., 1989; Superti et al., 2001). Data from a tissue culture model of C. trachomatis and HSV-2 co-infection established in our laboratory indicate that, during C. trachomatis serovar E and HSV-2 co-infection, HSV attachment and/or entry transmits a signal which interrupts the normal chlamydial developmental cycle and induces persistence (Deka et al., 2007). These and other data have led us to hypothesize that HSV attachment to and/or entry activates a novel anti-chlamydial defence pathway in mucosal epithelial cells.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
). HSV-2 strain 333 and HSV-1 strain KOS stocks were obtained from Mary K. Howett (Drexel University) and Udayasankar Kumaraguru (James H. Quillen College of Medicine), respectively. Viral stocks were prepared in monolayers of Vero cells (African green monkey kidney cells ATCC CCL-81) as described by Duff & Rapp (1971).
Co-infection experimental design and nutrient supplementation.
HeLa cells, a human cervical adenocarcinoma epithelial cell line (ATCC CCL2), or HEC-1B cells, a human endometrial epithelial cell line (ATCC HTB-113), were used for all infection experiments. In each experiment, the appropriate host cells were divided into four groups for mock infection, chlamydial infection, HSV infection and C. trachomatis/HSV double infection. Host cells were incubated with a dilution of crude EB stock calculated to infect >80 % of the cells. After 1 h of adsorption, monolayers were refed with Minimal Essential Medium (MEM; Gibco) and incubated at 35 °C for either 4 or 24 h. Cultures were then infected with either HSV-2 or HSV-1 at a m.o.i. of 10 p.f.u. per cell. Mock-infected HeLa or HEC-1B cells were treated similarly except they were exposed to either 2SPG (0.2 M sucrose, 6 mM NaH2PO4, 15 mM Na2HPO4, 5 mM L-glutamine, pH 7.2; mock C. trachomatis infection) or growth medium (mock viral infection). In most experiments, monolayers were refed with MEM following HSV adsorption. For nutrient supplementation studies, cultures were refed with either MEM or MEM supplemented with excess essential and non-essential amino acids (Gibco), iron-saturated holotransferrin (hTF; Sigma), glucose, or a combination of amino acids and hTF as described in the legend to Supplementary Fig. S3, available with the online version of this paper.
Luminex assay.
Aliquots of culture supernatants were examined for IFN-, IFN-, TNF-, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-6 (IL-6) using the BioSource Multiplex Bead Immunoassay (BioSource International) according to the manufacturer's instructions (Deka et al., 2007).
RNA and DNA isolation.
Total RNA and DNA were isolated simultaneously from experimental samples using the RNeasy Mini (Qiagen) and QIAmp DNA Blood Mini (Qiagen) kits as described previously (Deka et al., 2006). Total RNA and DNA preparations were quantified by measuring A260 and A280; all samples had A260/A280 ratios >1.9.
Reverse transcription, PCR and RT-PCR.
Reverse transcription of total RNA, PCR and RT-PCR were performed using identical conditions to those previously described (Deka et al., 2006). PCR was performed using purified total cellular DNA or cDNA as a template. Control and experimental template DNAs were diluted from 1/10 to 1/1000 (in double-distilled H2O) and synthetic control DNA targets diluted from 10 to 0.1 pg ml–1, ensuring that each reaction was quantified in the linear amplification range. Published primer sets included chlamydial 16S rRNA, human glyceraldehyde-3-phosphate dehydrogenase and IDO (Deka et al., 2006; Nelson et al., 2005). We designed specific primer sets and, in some cases, synthetic DNA targets for chlamydial trpA, human 18S rRNA, ifn-β, inf-, lymphotoxin- and inos (see Supplementary Table S1 for sequences). All synthetic DNA targets and primer sets were designed using Ensembl and Vector NTI Advance V10 (Invitrogen). After PCR, all reactions were electrophoresed and quantified as described previously (Deka et al., 2006).
Griess reaction.
Aliquots of select culture supernatants (50 µl) were examined for nitrite using the Griess Reagent System (Promega) according to the manufacturer's instructions.
Fluorescence microscopy and image analysis.
Fluorescence analyses were performed as described previously (Deka et al., 2006) except that FITC-conjugated monoclonal antibodies generated against C. trachomatis major outer-membrane protein (Pathfinder Chlamydia trachomatis monoclonal MOMP antibody 30702, Bio-Rad) were used to stain chlamydial inclusions. Images were captured using a Zeiss Axiovert S100 microscope and Axiocam camera. Measurement of inclusion sizes was performed as described by Deka et al. (2006) except that selected photomicrographs were converted to greyscale using Adobe Photoshop V5.0. The relative area of each inclusion was determined using Dymension II Imaging Software V2.06 (SynGene).
Cer6-NBD-ceramide labelling.
HeLa cells were plated in 24-well plates (1.5x105 cells per well), and mock, singly- or co-infected as described above. At 16.5 h post-HSV-2 infection, cells were stained with 200 µg ml–1 BSA pre-conjugated C6-NBD-ceramide stain (Molecular Probes) for 30 min at 4 °C. The stain was then back-extracted for 3 h at 37 °C until 20 h post-HSV-2 infection (Alzhanov et al., 2007; Hackstadt et al., 1995). Cells were mounted with GelMount (Fisher) and examined by fluorescence microscopy using FITC filters. Images were captured as described above with a 4 s exposure and 320x magnification.
Chlamydial titrations by subpassage.
Chlamydial titrations were performed as previously described (Deka et al., 2006) using Pathfinder anti-chlamydial stain (Bio-Rad) to stain chlamydial inclusions formed from subpassaged EBs. The number of inclusion-forming units (i.f.u.) in the undiluted inoculum was then derived from triplicate counts and expressed as i.f.u. ml–1.
Spectro Ferritin ELISA.
Intracellular ferritin in cell lysates collected from mock-, chlamydiae-singly-infected with or without 50 µM Desferal, and C. trachomatis/HSV-2 co-infected cells was measured using the Spectro Ferritin ELISA kit (Ramco) as described in the legend to Supplementary Fig. S3. Addition of the iron-chelating agent Desferal (Sigma) to chlamydiae-infected cultures was used as a positive control for iron depletion (Raulston, 1997).
Statistical analyses.
Statistical analyses were performed using Microsoft Excel. Comparison of means was done by using a 2-sample t-test for independent samples. P values 
0.05 were considered significant. All plotted values are means±SEM of either eight or nine biological replicates divided between three separate experiments.
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
, is one of the most extensively studied mechanisms of inducing chlamydial persistence. However, other cytokines can also negatively affect C. trachomatis development. TNF-, IL-1 and IL-1β can synergize with IFN-, lowering the concentration at which IFN- exposure affects developing chlamydiae (Carlin & Weller, 1995; Shemer-Avni et al., 1988; Summersgill et al., 1995). Lymphotoxin- (LT-), TNF- and IFN-/β exposure can also upregulate iNOS expression and cellular NO production, inhibiting chlamydial development (Devitt et al., 1996; Matsushima et al., 1999). Cycloheximide exposure experiments (Deka et al., 2007) suggest that HSV-induced persistence occurs in the absence of de novo host protein synthesis. However, the observation that cycloheximide exposure did not completely abrogate host protein synthesis (Deka et al., 2007) made it important to confirm that ‘anti-chlamydial’ cytokines were not released from co-infected cells. HSV-infected human corneal epithelial cells produce IFN-β, as well as the cytokines TNF-, interleukin-8 (IL-8) and IL-6 (Li et al., 2006). However, co-infected epithelial cells may produce cytokines that are not elicited by infection with either pathogen alone (or by other commonly utilized experimental stimuli, such as cytokine exposure). Therefore, production of all cytokines known to elicit an anti-chlamydial response was examined in triplicate mock-, singly- or co-infected cultures as described in Methods. At 20 h post-viral infection, supernatants were collected and assayed for IFN-, IFN-, TNF-, GM-CSF, IL-1/β and IL-6. IFN-, IFN-, TNF- and GM-CSF were not detected in HeLa supernatants, regardless of infection status. IL-1 (11.1–13.4 pg ml–1), IL-1β (34.7–46.9 pg ml–1) and IL-8 (1.7–3.6 pg ml–1) were detected, but at similar levels in C. trachomatis singly-infected and co-infected HeLa cultures, indicating that they were unlikely to be involved in HSV-2-induced persistence. As expected, IL-6 was strongly induced by HSV-2 infection and was increased in HSV singly- and co-infected HeLa cultures compared to that in mock- and C. trachomatis singly-infected cultures (Fig. 1a). However, in duplicate singly- and co-infected HEC-1B cultures, similar quantities of IL-6 were detected in all experimental samples (Fig. 1b). Because HSV-induced persistence also occurs in HEC-1B cells, it is unlikely that IL-6 production stimulates HSV-induced persistence (Deka et al., 2007). Co-infection experiments using HSV-1 yielded similar results (data not shown).
|
, IFN-β and LT- mRNAs during co-infection was measured by semiquantitative RT-PCR. As shown in Fig. 1(c)
, ifn-, ifn-β, and lt- were not expressed in mock-, singly- or co-infected cells. Amplification of diluted synthetic DNA targets specific to the ifn-, ifn-β, and lt- genes demonstrated that PCR reactions were sensitive and specific. Human 18S rRNA targets were also amplified to ensure that the cDNAs were not degraded (see Supplementary Fig. S1b). Amplification of a dilution series of control HeLa cell genomic DNA ensured that all 18S rRNA amplification reactions were quantified within the linear range of the PCR (Deka et al., 2006). All template-negative (Fig. 1c) and reverse-transcriptase-negative (data not shown) samples were negative, as expected. Finally, all amplimers were of the expected size and sequence (data not shown). Taken together, these data indicate that HSV/C. trachomatis co-infection does not stimulate host epithelial cells to produce any known ‘anti-chlamydial’ cytokine.
C. trachomatis/HSV-2 co-infected cells do not produce iNOS
The iNOS/NO pathway is another important component of the anti-chlamydial defence (Devitt et al., 1996; Matsushima et al., 1999). Although iNOS activity is not stimulated by either IFN- or chlamydial infection in cultured human epithelial cells (Roshick et al., 2006), the possibility that the additional stimulus provided by viral co-infection might activate iNOS/NO production in human cells is untested and should be evaluated. Therefore, C. trachomatis/HSV-2 co-infected cells were examined for iNOS mRNA upregulation by semiquantitative RT-PCR and for nitrite accumulation in supernatants using the Griess reaction. Neither iNOS mRNA nor nitrite was detected in any experimental cell cultures (Supplementary Fig. S1a and data not shown), confirming that HSV-2-induced chlamydial persistence is not mediated by iNOS induction or NO production.
Inclusions within co-infected cells continue to enlarge and incorporate C6-NBD-ceramide
In murine cells, IFN- exposure activates the p47 GTPase, Iigp1, which halts C. trachomatis development by interrupting sphingomyelin transport and vesicular trafficking to the inclusion. If C. trachomatis-infected murine cells are exposed to IFN- early in the developmental cycle, inclusions remain small (Nelson et al., 2005). Recent studies indicate that Iigp1 is upregulated in HSV-1-infected murine cells at 3 h post-infection (Pasieka et al., 2006). Although a human Iigp1 homologue has not been identified, it is possible that HSV/host cell interaction activates human p47 GTPases that function similarly. Therefore, C. trachomatis-infected HeLa cells were co-infected with HSV-2 at either 4 or 24 h post-chlamydial infection. Replicate cultures were harvested either immediately after HSV adsorption or 20 h post-HSV-2 infection. Monolayers were fixed, immunostained with anti-MOMP antibodies, photographed, and relative inclusion size was determined (Deka et al., 2006). Mean inclusion size was similarly increased in both singly- and co-infected cells over the course of the experiment when HSV-2 was added 4 h post-chlamydial infection (Fig. 2a). In cells co-infected with HSV-2 at 24 h post-C. trachomatis infection, the starting inclusion size was identical in both cultures. However, inclusions in co-infected cells were significantly larger than those in singly-infected controls after the 20 h HSV-2 infection period, as previously reported (Deka et al., 2006). Additional cultures were infected as described above, labelled with BSA-conjugated C6-NBD-ceramide stain, and visualized by fluorescence microscopy (Alzhanov et al., 2007; Hackstadt et al., 1995). No obvious difference was observed in the intensity or distribution of ceramide staining between chlamydial inclusions in singly- and co-infected cells at either time interval tested (Fig. 2b and data not shown). Although it is possible that vesicular trafficking to the inclusion is subtly altered by HSV infection, these data demonstrate that neither gross vesicular trafficking nor sphingomyelin transport to the inclusion is disrupted by viral co-infection.
|
). In response to tryptophan deprivation, chlamydiae upregulate expression of the tryptophan synthase gene, trpA (Belland et al., 2003). To determine whether our HeLa cell clone can express IDO, mock- and C. trachomatis-infected HeLa monolayers were exposed to IFN- (50 U ml–1) for 48 h followed by RNA isolation. Expression of ido and trpA mRNA in IFN--exposed control cultures was then evaluated by semiquantitative RT-PCR. IDO expression was significantly increased in both mock- (data not shown) and C. trachomatis-infected HeLa cells in response to IFN-, compared to that in unexposed cells (Supplementary Fig. S2a). A significant increase in trpA expression was also observed in C. trachomatis-infected cells following IFN- exposure versus unexposed, chlamydiae-infected cultures (data not shown).
To determine if co-infection similarly elevates ido or trpA levels, total RNA was isolated from triplicate sets of mock-, singly- or co-infected HeLa cells and subjected to reverse transcription and semiquantitative RT-PCR with primers specific for the human ido and chlamydial trpA genes. Again, amplification of a dilution series of cDNA from C. trachomatis-infected, IFN--exposed HeLa cells indicated that the IDO RT-PCR was specific and sensitive. IDO mRNA was not detected in any of the experimental samples tested, regardless of infection status (Supplementary Fig. S2b). Various dilutions of chlamydial genomic DNA were amplified to ensure that all trpA amplification reactions were quantified within the linear range of the PCR (data not shown). Additionally, chlamydial genome copy number in each sample was determined by amplification of the chlamydial 16S rRNA gene (data not shown). All trpA RT-PCR values were normalized to the chlamydial genome copy number (Deka et al., 2006). In contrast to trpA expression during IFN- exposure, no statistical difference in trpA expression was observed between C. trachomatis singly-infected and co-infected samples (Supplementary Fig. S2c). These data confirm that C. trachomatis/HSV-2 co-infected cells are not deprived of tryptophan by the action of IDO.
Global deprivation of amino acids and other nutrients, especially iron and glucose, can also cause developing chlamydiae to enter persistence (Harper et al., 2000; Raulston, 1997). To investigate the possibility that HSV-2 co-infection induces chlamydial persistence by limiting iron availability, we first determined whether co-infected cells were iron-deprived. Intracellular ferritin levels can be used as an indirect measure of host intracellular iron levels (Dill & Raulston, 2007). As a positive control for iron depletion, duplicate C. trachomatis-infected HeLa cultures were exposed to the iron-chelating agent Desferal following chlamydial infection. Intracellular ferritin levels were not significantly reduced in chlamydiae singly-infected or co-infected cells, compared to the level in mock-infected controls (Supplementary Fig. S3a), indicating that co-infected cells were not iron-deprived. In contrast, Desferal exposure significantly reduced intracellular ferritin levels, as expected (Supplementary Fig. S3a).
During HSV-2 replication, cellular amino acids are depleted through the synthesis of viral proteins (Roizman & Knipe, 2001). While it is possible that co-infection induces persistence by depriving the chlamydiae of amino acids, it remains unlikely, given previous data that UV-inactivated, replication-incompetent HSV-2 stimulates chlamydial persistence (Deka et al., 2007). However, several incoming HSV virion proteins, such as the virion host shut-off protein (VHS), produce significant host cellular physiological alterations in the absence of viral replication (Roizman & Knipe, 2001). Thus, the possibility that viral infection induces nutrient deprivation via an indirect pathway cannot be excluded. Notably, supplementation of nutrient-starved chlamydiae with amino acids, iron or glucose reverses the negative effect of the nutrient deficiency (Hogan et al., 2004; Raulston, 1997). Additionally, the anti-chlamydial effect stimulated by IFN- can only be completely reversed by addition of both excess amino acids and iron (Devitt et al., 1996). Based upon this information, we performed co-infection supplementation studies with excess amino acids, iron, glucose, or a combination of amino acids plus iron. Immediately following HSV-2 infection, mock-, singly- or co-infected cells were refed with MEM or MEM supplemented with a 5x concentration of both essential and non-essential amino acids, 6 mg ml–1 iron-saturated holotransferrin (hTF), 450 mg glucose ml–1, or a combination of 5x amino acids plus 6 mg hTF ml–1. This concentration of hTF has been previously shown to rescue chlamydial infectivity in iron-deprived, infected epithelial cells (Raulston, 1997). Twenty hours after HSV-2 infection, the monolayers were collected and processed for chlamydial titration. The titre of infectious EBs was significantly reduced in epithelial cells co-infected with HSV-2 compared to that from cultures singly-infected with C. trachomatis (Supplementary Fig. S3b). Supplementation of co-infected host cells with hTF did not rescue chlamydial infectivity (Supplementary Fig. S3b), indicating that supplementation with excess iron was not able to restore production of infectious chlamydial progeny during co-infection. Similar results were obtained with excess amino acids, glucose (Supplementary Fig. S3c) and a combination of amino acids plus iron supplementation (data not shown). These data demonstrate that co-infection-induced chlamydial persistence is not mediated by limitation of amino acids, iron, glucose, or the combination of amino acids plus iron within the host cell. Because the anti-chlamydial effect of IFN- is mediated by restriction of both tryptophan and iron, these results support previous data indicating that co-infection does not induce persistence through an IFN--dependent mechanism.
Concluding remarks
Collectively, our data demonstrate that co-infection-induced persistence is not mediated by: (i) any known anti-chlamydial cytokine; (ii) activation of iNOS or IDO; (iii) inhibition of vesicular trafficking or sphingomyelin transport to the inclusion or (iv) amino acid, iron or glucose deprivation. The reduction in chlamydial infectivity stimulated by HSV-2 co-infection is of smaller magnitude than that observed in other models of persistence (Beatty et al., 1994; Pantoja et al., 2001). Additionally, the negative effects of HSV-2 co-infection on chlamydial development are observed when the stimulus is added at 24 h post-C. trachomatis infection, whereas IFN- must be added to chlamydia-infected cultures either before infection or very early during the developmental cycle for maximal effect (Nelson et al., 2005). Taken together, these observations support the hypothesis that HSV-2 co-infection induces C. trachomatis persistence through an undescribed, and therefore novel mechanism.
Previously, we reported that both cycloheximide exposure during co-infection with replication-competent HSV-2 and co-infection with UV-inactivated HSV-2 can stimulate chlamydial persistence, indicating that early events during HSV-2 infection are sufficient for this effect (Deka et al., 2007). During the host-cell entry process, HSV binds to one of several co-receptors: HVEM, nectin-1, nectin-2 or 3-O-sulfated heparin sulfate (Spear, 2004). Previous studies have demonstrated that each of these co-receptors can alter cellular signalling pathways when bound to their natural ligands (Granger & Rickert, 2003; Hsu et al., 1997; Mauri et al., 1998; Nakanishi & Takai, 2004). Therefore, HSV-2 attachment to the host cell during co-infection could alter cellular signalling and activate a novel host anti-chlamydial response, leading to persistence. Another possibility is that HSV stimulates Toll-like receptors, initiating such an anti-chlamydial pathway (Aravalli et al., 2005; Compton et al., 2003; Kurt-Jones et al., 2005; Pyles et al., 2002; Wang et al., 2005). Conversely, there is the potential that viral tegument proteins, released into the cell during entry, could stimulate chlamydial persistence by altering the host cytoskeleton, degrading host mRNAs or altering host gene transcription (Elliott & O'Hare, 1998; Kwong & Frenkel, 1989; Roizman & Knipe, 2001). Investigations of anti-chlamydial host cell responses have provided valuable information regarding C. trachomatis biology, pathogenesis and interactions with the host cell. Illumination of the mechanism behind HSV-2-induced persistence will allow us to evaluate the contribution of this novel anti-chlamydial response to C. trachomatis pathogenesis and host defence.
|
ACKNOWLEDGEMENTS |
|---|
Edited by: T. P. Hatch
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTS AND DISCUSSIONREFERENCES |
|---|
Aravalli, R. N., Hu, S., Rowen, T. N., Palmquist, J. M. & Lokensgard, J. R. (2005). TLR2-mediated proinflammatory cytokine and chemokine production by microglial cells in response to herpes simplex virus. J Immunol 175, 4189–4193.
Beatty, W. L., Belanger, T. A., Desai, A. A., Morrison, R. P. & Byrne, G. I. (1994). Tryptophan depletion as a mechanism of gamma interferon-mediated chlamydial persistence. Infect Immun 62, 3705–3711.
Belland, R. J., Nelson, D. E., Virok, D., Crane, D. D., Hogan, D., Sturdevant, D., Beatty, W. L. & Caldwell, H. D. (2003). Transcriptome analysis of chlamydial growth during IFN-gamma-mediated persistence and reactivation. Proc Natl Acad Sci U S A 100, 15971–15976.
Butler, T. R. (1997). The Hidden Epidemic: Confronting Sexually Transmitted Diseases, Table 2-5, p. 59. Washington, DC: National Academy Press.
Carlin, J. M. & Weller, J. B. (1995). Potentiation of interferon-mediated inhibition of Chlamydia infection by interleukin-1 in human macrophage cultures. Infect Immun 63, 1870–1875.[Abstract]
Chiarini, F., Mansi, A., Pisani, S., Seganti, L., Brunori, S., Gentile, V. & Di Silverio, F. (1996). In vitro study of a double infection by herpes simplex virus type 2 and Chlamydia trachomatis. New Microbiol 19, 263–266.[Medline]
Compton, T., Kurt-Jones, E. A., Boehme, K. W., Belko, J., Latz, E., Golenbock, D. T. & Finberg, R. W. (2003). Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2. J Virol 77, 4588–4596.
Darville, T. (2000). Chlamydia spp. In Persistent Bacterial Infections, pp. 229–261. Edited by J. P. Nataro. Washington, DC: American Society for Microbiology Press.
Deka, S., Vanover, J., Dessus-Babus, S., Whittimore, J., Howett, M. K., Wyrick, P. B. & Schoborg, R. V. (2006). Chlamydia trachomatis enters a viable but non-cultivable (persistent) state within herpes simplex virus type 2 (HSV-2) co-infected host cells. Cell Microbiol 8, 149–162.[CrossRef][Medline]
Deka, S., Vanover, J., Sun, J., Kintner, J., Whittimore, J. & Schoborg, R. V. (2007). An early event in the herpes simplex virus type-2 replication cycle is sufficient to induce Chlamydia trachomatis persistence. Cell Microbiol 9, 725–737.[CrossRef][Medline]
Devitt, A., Lund, P. A., Morris, A. G. & Pearce, J. H. (1996). Induction of alpha/beta interferon and dependent nitric oxide synthesis during Chlamydia trachomatis infection of McCoy cells in the absence of exogenous cytokine. Infect Immun 64, 3951–3956.[Abstract]
Dill, B. D. & Raulston, J. E. (2007). Examination of an inducible expression system for limiting iron availability during Chlamydia trachomatis infection. Microbes Infect 9, 947–953.[CrossRef][Medline]
Duff, R. & Rapp, F. (1971). Properties of hamster embryo fibroblasts transformed in vitro after exposure to ultraviolet-irradiated herpes simplex virus type 2. J Virol 8, 469–477.
Elliott, G. & O'Hare, P. (1998). Herpes simplex virus type 1 tegument protein VP22 induces the stabilization and hyperacetylation of microtubules. J Virol 72, 6448–6455.
Granger, S. W. & Rickert, S. (2003). LIGHT-HVEM signaling and the regulation of T cell-mediated immunity. Cytokine Growth Factor Rev 14, 289–296.[CrossRef][Medline]
Hackstadt, T., Scidmore, M. A. & Rockey, D. D. (1995). Lipid metabolism in Chlamydia trachomatis-infected cells: directed trafficking of Golgi-derived sphingolipids to the chlamydial inclusion. Proc Natl Acad Sci U S A 92, 4877–4881.
Harper, A., Pogson, C. I., Jones, M. L. & Pearce, J. H. (2000). Chlamydial development is adversely affected by minor changes in amino acid supply, blood plasma amino acid levels, and glucose deprivation. Infect Immun 68, 1457–1464.
Hogan, R. J., Mathews, S. A., Mukhopadhyay, S., Summersgill, J. T. & Timms, P. (2004). Chlamydial persistence: beyond the biphasic paradigm. Infect Immun 72, 1843–1855.
Hsu, H., Solovyev, I., Colombero, A., Elliott, R., Kelley, M. & Boyle, W. J. (1997). ATAR, a novel tumor necrosis factor receptor family member, signals through TRAF2 and TRAF5. J Biol Chem 272, 13471–13474.
Kurt-Jones, E. A., Belko, J., Yu, C., Newburger, P. E., Wang, J., Chan, M., Knipe, D. M. & Finberg, R. W. (2005). The role of Toll-like receptors in herpes simplex infection in neonates. J Infect Dis 191, 746–748.[CrossRef][Medline]
Kwong, A. D. & Frenkel, N. (1989). The herpes simplex virus virion host shutoff function. J Virol 63, 4834–4839.
Li, H., Zhang, J., Kumar, A., Zheng, M., Atherton, S. S. & Yu, F. X. (2006). Herpes simplex virus 1 infection induces the expression of proinflammatory cytokines, interferons and TLR7 in human corneal epithelial cells. Immunology 117, 167–176.[CrossRef][Medline]
Matsushima, H., Shirai, M., Ouchi, K., Yamashita, K., Kakutani, T., Furukawa, S. & Nakazawa, T. (1999). Lymphotoxin inhibits Chlamydia pneumoniae growth in HEp-2 cells. Infect Immun 67, 3175–3179.
Mauri, D. N., Ebner, R., Montgomery, R. I., Kochel, K. D., Cheung, T. C., Yu, G. L., Ruben, S., Murphy, M., Eisenberg, R. J. & other authors (1998). LIGHT, a new member of the TNF superfamily, and lymphotoxin alpha are ligands for herpesvirus entry mediator. Immunity 8, 21–30.[CrossRef][Medline]
Nakanishi, H. & Takai, Y. (2004). Roles of nectins in cell adhesion, migration and polarization. Biol Chem 385, 885–892.[CrossRef][Medline]
Nelson, D. E., Virok, D. P., Wood, H., Roshick, C., Johnson, R. M., Whitmire, W. M., Crane, D. D., Steele-Mortimer, O., Kar, L. & other authors (2005). Chlamydial IFN-gamma immune evasion is linked to host infection tropism. Proc Natl Acad Sci U S A 102, 10658–10663.
Paavonen, J., Teisala, K., Heinonen, P. K., Aine, R., Miettinen, A., Lehtinen, M. & Gronroos, P. (1985). Endometritis and acute salpingitis associated with Chlamydia trachomatis and herpes simplex virus type two. Obstet Gynecol 65, 288–291.[Medline]
Pantoja, L. G., Miller, R. D., Ramirez, J. A., Molestina, R. E. & Summersgill, J. T. (2001). Characterization of Chlamydia pneumoniae persistence in HEp-2 cells treated with gamma interferon. Infect Immun 69, 7927–7932.
Pasieka, T. J., Baas, T., Carter, V. S., Proll, S. C., Katze, M. G. & Leib, D. A. (2006). Functional genomic analysis of herpes simplex virus type 1 counteraction of the host innate response. J Virol 80, 7600–7612.
Pontefract, R. D., Ng, C. W. & Bergeron, G. (1989). Vero cells co-infected with Chlamydia trachomatis and herpes simplex virus type 2: a scanning and transmission electron microscope study. Sex Transm Dis 16, 74–78.[Medline]
Pyles, R. B., Higgins, D., Chalk, C., Zalar, A., Eiden, J., Bown, C., Van Nest, G. & Stanberry, L. R. (2002). Use of immunostimulatory sequence containing oligonucleotides as topical therapy for herpes simplex virus type 2 infection. J Virol 76, 11387–11396.
Raulston, J. E. (1997). Response of Chlamydia trachomatis serovar E to iron restriction in vitro and evidence for iron-regulated chlamydial proteins. Infect Immun 65, 4539–4547.[Abstract]
Roizman, B. & Knipe, D. M. (2001). Herpes simplex viruses and their replication. In Field's Virology, pp. 2399–2459. Edited by D. M. Knipe & P. M. Howley. Baltimore, MD: Lippincott Williams & Wilkins.
Roshick, C., Wood, H., Caldwell, H. D. & McClarty, G. (2006). Comparison of gamma interferon-mediated antichlamydial defense mechanisms in human and mouse cells. Infect Immun 74, 225–238.
Shemer-Avni, Y., Wallach, D. & Sarov, I. (1988). Inhibition of Chlamydia trachomatis growth by recombinant tumor necrosis factor. Infect Immun 56, 2503–2506.
Spear, P. G. (2004). Herpes simplex virus: receptors and ligands for cell entry. Cell Microbiol 6, 401–410.[CrossRef][Medline]
Summersgill, J. T., Sahney, N. N., Gaydos, C. A., Quinn, T. C. & Ramirez, J. A. (1995). Inhibition of Chlamydia pneumoniae growth in HEp-2 cells pretreated with gamma interferon and tumor necrosis factor alpha. Infect Immun 63, 2801–2803.[Abstract]
Superti, F., Longhi, C., Di Biase, A. M., Tinari, A., Marchetti, M., Pisani, S., Gallinelli, C., Chiarini, F. & Seganti, L. (2001). Herpes simplex virus type 2 modulates the susceptibility of human bladder cells to uropathogenic bacteria. Med Microbiol Immunol 189, 201–208.[CrossRef][Medline]
Tait, J., Peddie, B. A., Bailey, R. R., Arnold, E. P., Russell, G. L., Bishop, V. A. & Burry, A. F. (1985). Urethral syndrome (abacterial cystitis) – search for a pathogen. Br J Urol 57, 552–556.[Medline]
Wang, J. P., Kurt-Jones, E. A., Shin, O. S., Manchak, M. D., Levin, M. J. & Finberg, R. W. (2005). Varicella-zoster virus activates inflammatory cytokines in human monocytes and macrophages via Toll-like receptor 2. J Virol 79, 12658–12666.
Weinstock, H., Berman, S. & Cates, W., Jr (2004). Sexually transmitted diseases among American youth: incidence and prevalence estimates, 2000. Perspect Sex Reprod Health 36, 6–10.[CrossRef][Medline]
Wyrick, P. B., Gerbig, D. G., Jr, Knight, S. T. & Raulston, J. E. (1996). Accelerated development of genital Chlamydia trachomatis serovar E in McCoy cells grown on microcarrier beads. Microb Pathog 20, 31–40.[CrossRef][Medline]
Received 2 August 2007;
revised 15 October 2007;
accepted 27 November 2007.
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
