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Infection and Immunity, July 2007, p. 3478-3489, Vol. 75, No. 7
0019-9567/07/$08.00+0     doi:10.1128/IAI.00023-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Reversal of the Antichlamydial Activity of Putative Type III Secretion Inhibitors by Iron{triangledown}

Anatoly Slepenkin,1 Per-Anders Enquist,2 Ulrik Hägglund,3 Luis M. de la Maza,1 Mikael Elofsson,2 and Ellena M. Peterson1*

Department of Pathology and Laboratory Medicine, University of California, Irvine, California,1 Department of Chemistry, Umea University, SE-90187 Umea, Sweden,2 Innate Pharmaceuticals AB, Umestan Foretagspark, SE-90347 Umea, Sweden3

Received 5 January 2007/ Returned for modification 30 January 2007/ Accepted 12 April 2007


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ABSTRACT
 
INPs, which are chemically synthesized compounds belonging to a class of acylated hydrazones of salicylaldehydes, can inhibit the growth of Chlamydiaceae. Evidence has been presented that in Yersinia and Chlamydia INPs may affect the type III secretion (T3S) system. In the present study 25 INPs were screened for antichlamydial activity at a concentration of 50 µM, and 14 were able to completely inhibit the growth of Chlamydia trachomatis serovar D in McCoy and HeLa 229 cells. The antichlamydial activities of two of these INPs, INPs 0341 and 0400, were further characterized due to their low cytotoxicity. These compounds were found to inhibit C. trachomatis in a dose-dependent manner; were not toxic to elementary bodies; were cidal at a concentration of ≥20 µM; inhibited all Chlamydiaceae tested; and could inhibit the development of C. trachomatis as determined by the yield of progeny when they were added up to 24 h postinfection. INP 0341 was able to affect the expression of several T3S genes. Compared to the expression in control cultures, lcrH-1, copB, and incA, all middle- to late-expressed T3S genes, were not expressed in the INP 0341-treated cultures 24 to 36 h postinfection. Iron, supplied as ferrous sulfate, as ferric chloride, or as holo-transferrin, was able to negate the antichlamydial properties of the INPs. In contrast, apo-transferrin and other divalent metal ions tested were not able to reverse the inhibitory effect of the INPs. In conclusion, the potent antichlamydial activity of INPs is directly or indirectly linked with iron, and this inhibition of Chlamydia has an effect on the T3S system of this intracellular pathogen.


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INTRODUCTION
 
The type III secretion (T3S) system is known to be a potent virulence mechanism shared by several pathogenic bacteria, including the Chlamydiaceae (10). All T3S systems share common structural components, while their effector proteins and methods of gene regulation vary widely. Targeting and inactivating common T3S components has been proposed as a strategy to fight infections caused by pathogens that require a T3S system for virulence (13). In an attempt to identify such compounds, Kauppi et al. (13) used a chemical genetics approach to screen a large number of synthetic compounds for the ability to inhibit Yersinia T3S gene expression. They identified compounds with the general structure of an acylated hydrazone of salicylaldehydes that were able to inhibit the pathogenic Yersinia T3S system, neutralizing the virulence while not affecting the growth of the organism (13, 17).

We have previously reported that INP 0400 was able to inhibit the growth of Chlamydophila pneumoniae (27a). We reported that this compound inhibited C. pneumoniae development in a dose-dependent manner, was not cytotoxic, was not directly toxic to elementary bodies (EBs), and was effective at inhibiting the growth of Chlamydia trachomatis and Chlamydia muridarum. The appearance of inclusions at lower concentrations (<20 µM) of INP 0400 resembled the appearance of inclusions seen in persistent infections resulting from gamma interferon, iron deprivation, or penicillin treatment (1, 5-7, 24). Our work was soon corroborated by Muschiol et al. (16), who also showed that there was dose-dependent inhibition of Chlamydia growth in the presence of INP 0400 and who were able to demonstrate that the putative T3S effector proteins IncG and IncA failed to localize to the inclusion membrane, demonstrating a link to the T3S system of Chlamydia. Wolf et al. (32), using a similar compound, C1 (INP 0007), identified from the work of Kauppi et al. (12, 13), also reported on the anti-Chlamydia effects of this compound. They presented evidence for the accumulation of two T3S effectors, IncA and Tarp, in the inhibited reticulate bodies (RBs), suggesting that there was a defect in deployment of the Chlamydia T3S system.

In this study we expanded our screening for and characterization of this class of compounds that are inhibitory to Chlamydia. In an attempt to determine a mechanism for the inhibition resulting from these compounds, we present data that suggest an effect on the T3S gene expression during middle to late events in the Chlamydia developmental cycle. We also show that the inhibitory effect on Chlamydiaceae is directly or indirectly linked to iron.


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MATERIALS AND METHODS
 
Chlamydiaceae and cell lines. C. trachomatis serovar D strain UW-3/Cx, C. muridarum mouse pneumonitis (MoPn) strain Nigg II, and C. pneumoniae CM-1 were obtained from the American Type Culture Collection (Manassas, VA). Chlamydophila caviae GPIC was a kind gift from Roger Rank (University of Arkansas, Little Rock). Stocks of Chlamydiaceae were propagated in HeLa 229 cells (American Type Culture Collection). Cells were grown in Eagle's minimal essential medium (Gibco, Invitrogen Corporation, Grand Island, NY) supplemented with 5% fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 2 mM L-glutamine (Meditech, Herndon, VA), and 50 µg/ml of gentamicin (Meditech) (MEM-FBS). Cells used for propagation of stocks of C. pneumoniae were first pretreated for 10 min at room temperature with 30 µg/ml DEAE-dextran (Sigma-Aldrich Co., St. Louis, MO). Upon addition of Chlamydia to the monolayers, cells were centrifuged at room temperature for 1 h at 800 x g, followed by addition of medium containing cycloheximide (1 µg/ml). Cultures were incubated for 36 h (C. caviae and C. muridarum), 44 to 48 h (C. trachomatis), or 72 h (C. pneumoniae) at 37°C, and then the medium was removed, the cultures were sonicated in sucrose-phosphate-glutamate (SPG) (pH 7.4) and centrifuged (800 x g, 10 min), and the supernatant was stored at –80°C.

Cells and stocks of the Chlamydiaceae were determined to be free of Mycoplasma contamination by PCR using primers for the Mycoplasma 16S rRNA (5'-GGG AGC AAA CAG GAT TAG ATA CCC T and 5'-TGC ACC ATC TGT CAC TCT GTT ACC CTC) (18).

Chemicals and INPs. INPs were kind gifts from Pia Keyser (Innate Pharmaceuticals, Umea, Sweden). INPs were dissolved in dimethyl sulfoxide (DMSO) (Fisher Scientific, Fair Lawn, NJ) at a concentration of 25 mM and stored at –20°C. Immediately before use aliquots of the compounds were diluted in MEM-FBS to obtain the desired concentration. Other chemicals used, including FeSO4, FeCl3, MgCl2 (Fisher Scientific), ZnCl2, CaCl2 (Sigma-Aldrich), and MnCl2 (Mallinckrodt. Inc., Paris, KY), were diluted in distilled water and filter sterilized (0.22 µm) prior to use. Stock solutions of human holo-transferrin and apo-transferrin (Sigma-Aldrich) were dissolved in sterile water to obtain a concentration of 0.5 µM and stored at –80°C. Immediately prior to use deferoxamine methanesulfonate (Desferal; Sigma-Aldrich) was dissolved and diluted in distilled water.

Cell cultures. HeLa 229 and McCoy cells were seeded into 1-dram glass shell vials in 1 ml of MEM-FBS and incubated overnight at 37°C. Subsequently, confluent overnight monolayers were infected by removing the medium and adding 0.5 ml of MEM-FBS containing Chlamydia and compounds under investigation (e.g., INPs, metal ions, or transferrin). The shell vials were then centrifuged for 1 h at room temperature at 800 x g. After centrifugation 0.5 ml of MEM-FBS was added, and the cultures were incubated at 37°C. Cycloheximide was not used in any of the experiments except the iron deprivation model described by Raulston (24).

When preparations were used for microscopy, the medium was removed, and the cultures were fixed with methanol. After fixation, monolayers were washed twice with phosphate-buffered saline (PBS), and monoclonal antibodies (MAbs) recognizing lipopolysaccharide of the Chlamydiaceae and/or the major outer membrane protein of C. trachomatis (MAbE4) diluted in PBS, were added to the monolayers and incubated for 1 h at 37°C (20, 21). Cultures were then rinsed twice with PBS, overlaid, and incubated for 1 h at 37°C with goat anti-mouse antibody tagged with horseradish peroxidase (MP Biomedicals, Aurora, OH). Color was developed using chloronaphthol and peroxidase as previously described (21).

To assess the progeny of C. trachomatis resulting from an infection, 44 h after infection monolayers were rinsed twice with cold SPG. Shell vials were then sonicated for 20 s using a Braun-Sonic 2000 needle probe. Subsequently, fresh monolayers were infected as previously described, incubated for 44 to 48 h at 37°C, and then fixed and stained.

In all experiments, control cultures were treated like the experimental cultures and received the same volume of DMSO diluent used for the chemical or compound under study.

The iron deprivation model using Desferal described by Raulston (24) was used, with a slight modification. Briefly, subconfluent monolayers of HeLa 229 cells were grown in medium containing 400 µM Desferal. Upon infection with C. trachomatis, medium containing 400 µM Desferal and 1 µg/ml of cycloheximide was added. In the original description of this model (24), cycloheximide was added to cultures to minimize the production of transferrin receptors by the host, which may decrease the effects of Desferal.

Cytotoxicity assays. Cytotoxicity was measured using WST-1 (Roche Diagnostics, Indianapolis, IN) as suggested by the manufacturer. Briefly, medium from overnight monolayers grown in 96-well plates was replaced with fresh medium containing INPs (20 and 50 µM) and incubated for 24 h at 37°C in 5% CO2. Subsequently, 0.01 ml of WST-1 was added to each well and incubated for 1.5 h, and then the optical density at 450 nm was determined using a Multiskan RC plate reader (Thermo Electron, Milford, MA). The absorbance values for control monolayers not containing INP were compared to those for cells exposed to the various concentrations of INPs.

The effects of INPs on the proliferation of HeLa 229 cells with and without added iron were measured by first seeding cells into 1-dram glass vials and allowing them to grow overnight at 37°C, thus forming a subconfluent monolayer. Subsequently, the medium was replaced by fresh MEM-FBS supplemented with 20 µM of either INP 0341, INP 0400, INP 0406, or DMSO (control). Each INP tested was incubated alone and in the presence of 0.5 mM ferrous sulfate. Cell monolayers were then incubated at 37°C in 5% CO2 for 24 and 48 h. At each time point cells were washed with PBS, removed with trypsin, and suspended in 1 ml of PBS containing 0.2% trypan blue (BioWhittaker, Walkersville, MD), and viable cells were counted.

Nucleic acid isolation. HeLa 229 cell monolayers were infected as described above with C. trachomatis serovar D using a multiplicity of infection (MOI) of 3 and were treated with 20 µM INP 0341, INP 0406, or DMSO suspended in MEM-FBS. Infected cells were collected and processed for nucleic acid analysis at 4, 8, 24, and 36 h postinfection. At designated times, cell monolayers were washed twice with ice-cold PBS. TRIzol (Invitrogen Life Technologies, Carlsbad, CA) was used according to the manufacturer's instructions for isolating total DNA and RNA. RNA preparations were treated with RNase-free DNase RQ1 (Promega Corporation, Madison, WI) by following the manufacturer's instructions and were purified with an RNeasyPlus kit (QIAGEN GmbH, Hilden, Germany) used according to the manufacturer's protocol. RNA samples were confirmed to be DNA free by performing a PCR for the Chlamydia rs16 gene. The concentration of isolated DNA and RNA in samples was established by spectrophotometry, and preparations were stored frozen (DNA, –20°C; RNA, –80°C).

Analysis of gene expression. Optimal amplification conditions for each gene examined were established. Semiquantitative PCR assays were performed using Taq polymerase (New England Biolabs, Ipswich, MA). In general, the reaction mixtures included ThermoPol reaction buffer, 2 mM MgCl2, 0.5 µM primers, 0.5 U Taq DNA polymerase, 0.2 mM deoxynucleoside triphosphates, 1% DMSO, serial dilutions of total DNA preparations, and enough diethyl pyrocarbonate (Sigma-Aldrich)-treated water to bring the volume to 25 µl. The hot start procedure was used to attain maximum efficiency of the PCR. The PCR conditions were denaturation at 94°C for 2.0 min, primer annealing at 50°C for 1 min, and primer extension at 72°C for 1.0 min, followed by 34 cycles of 94°C for 40 s, 50°C for 45 s, and 72°C for 45 s and then by a final extension for 10 min at 72°C. PCR products were resolved and visualized on 2% agarose gels with ethidium bromide.

To quantitate C. trachomatis DNA for each time point examined, PCR targeting the Chlamydia rs16 gene was employed. Briefly, using serial dilutions of the total DNA preparation, the minimum amount of DNA that could be amplified by PCR was determined. Dilutions of pure C. trachomatis DNA (50, 25, 5, and 1 copies/reaction mixture) that were used as standards were amplified in parallel with the experimental DNA preparations.

Transcription of RNA was assayed by reverse transcription (RT)-PCR. Specific messages were amplified using an Access RT-PCR kit (Promega) from material corresponding to 5 x 103 Chlamydia genomes. Genes of interest and sequence-specific primers used in this work are shown in Table 1. RT reactions were performed according to the manufacturer's instructions. Briefly, RT extension was carried out for 45 min at 48°C, followed by 2.5 min at 94°C and PCR for 40 cycles consisting of denaturation at 94°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C for 35 s and then by a final extension at 72°C for 10 min. This was followed by denaturation of the mixture of RNA templates and specific primers (70°C, 5 min) before the RT-PCR. PCR products were separated on 2% agarose gels using ethidium bromide staining. Each RT-PCR was accompanied by a reaction without added RT to control for DNA contamination. Amplification of the 323-bp product, using the specific 1.2-kb positive control RNA transcript and gene-specific primers provided in the Access RT-PCR kit, was used as a positive control for RT-PCR.


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  		TABLE 1. Primers used for PCR and RT-PCR assays

To avoid mRNA degradation and DNA contamination, DNase- and RNase-free plastic and glassware and diethyl pyrocarbonate-treated water were used throughout the experiment. The RNasin RNase inhibitor (Promega) was used during the RQ1 DNase treatment (1 U of RNasin/µl of reaction mixture) and in each RT-PCR (0.5 U of RNasin/µl of reaction mixture) to inhibit mRNA degradation.

Iron chelation. The method previously reported by Ponka et al. (22) was used to measure the relative abilities of INP 0406 and INP 0341 to chelate iron. The wavelengths at which the difference in the absorbance spectra between each INP-Fe3+ complex and the corresponding free INP species and Fe3+ solution was maximal were established. The INP mixture contained 0.5 mM INP 0341 or INP 0406 and 10 mM trisodium citrate in 50 mM Tris-HCl buffer (pH 7.4). The INP-Fe3+ solution contained 0.5 mM INP 0341 or INP 0406, 0.25 mM FeCl3, and 10 mM trisodium citrate in 50 mM Tris-HCl buffer (pH 7.4). The Fe3+ solution contained 0.25 mM FeCl3 and 10 mM trisodium citrate in 50 mM Tris-HCl buffer (pH 7.4). The spectra were recorded using distilled water as a blank with a UV-3101PC spectrophotometer (Shimadzu, Tokyo, Japan). When a difference spectrum was calculated based on spectral data from the solutions described above, a new absorbance maximum appeared, indicating that INP 0341-Fe3+ absorbed most efficiently at 450 nm and INP 0406-Fe3+ absorbed most efficiently at 460 nm. The formation of INP-Fe3+ complexes was then monitored by measuring the absorbance at the specified wavelengths for mixtures containing 150 µM INP 0341 or INP 0406, 10 mM trisodium citrate, and 30 to 230 µM FeCl3 in polystyrene flat-bottom 96-well plates (Greiner, Frankfurt, Germany). The mixtures were allowed to equilibrate for at least 3 days before measurement of the absorbance with a SPECTRAmax 340 plate reader (Molecular Devices, Sunnyvale, CA).

Statistical analysis. Statistical significance was determined by Student's t test and the Mann-Whitney U test using SigmaStat 3.5 software (Systat Software, Inc., Point Richmond, CA). A P value of ≤0.05 was considered significant.


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RESULTS
 
Screening INPs for antichlamydial activity. Twenty-five derivatives of acylated hydrazones of salicylicaldehydes referred to as INPs, used at concentrations of 20 and 50 µM, were screened for the ability to affect the developmental cycle of C. trachomatis serovar D. Assays were performed with both McCoy and HeLa 229 cells. There was 100% inhibition of Chlamydia in both cell types with 14 INPs at a concentration of 50 µM compared to the results for controls to which the diluent for the INPs, DMSO, was added alone (Table 2). Five of the INPs, INPs 0007, 0149, 0161, 0341, and 0400, also completely inhibited C. trachomatis in McCoy cells at the lower concentration tested (20 µM). Since these five INPs exhibited the greatest antichlamydial activity at both concentrations in the two cell lines, they were tested further for cytotoxicity using HeLa cells along with INP 0406, that failed to inhibit C. trachomatis (Table 3). To measure this cytotoxicity, mitochondrial dehydrogenase activity was determined after the cells were exposed to an INP for 24 h. Four of the INPs tested, INPs 0161, 0341, 0400, and 0406, did not exhibit appreciable cytotoxicity (<10%), whereas INPs 0007 and 0149 were cytotoxic. Therefore, the inhibition of Chlamydia by INPs 0161, 0341, and 0400 could not be explained by a direct cytotoxic effect on the host cells. Since INP 0406 was neither cytotoxic nor antichamydial and yielded the same results as the DMSO diluent, it was included as a negative INP control for all experiments.


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  		TABLE 2. Inhibition of C. trachomatis by INPs in McCoy and HeLa 229 cells


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  		TABLE 3. Cytotoxicity of INPs for HeLa 229 cellsa

To determine whether the observed inhibition of C. trachomatis was due to direct toxicity of the compounds on the EBs, C. trachomatis was incubated with INPs 0341, 0400, and 0406 and DMSO before it was used to infect HeLa 229 cells. EBs were mixed with 20 and 50 µM of the INPs and incubated for 1 and 24 h at 22°C and for 1 h at 37°C. Subsequently, the EBs were pelleted by centrifugation, washed twice, suspended in SPG, and used to infect monolayers of HeLa 229 cells. Infected cultures were then incubated for 44 h, fixed, stained, and examined for inclusions. Overall, there was no significant difference between the cultures in which the EBs were pretreated with the antichlamydial INPs, INPs 0341 and 0400, and the cultures in which the EBs were pretreated with the control INP, INP 0406 (P ≤ 0.05). The numbers of inclusion-forming units (IFU) in cultures infected with EBs incubated with the compounds at 37°C for 1 h were lower than the numbers of IFU in cultures incubated at 22°C, as would be expected; however, the three INPs resulted in comparable numbers of Chlamydia IFU. Therefore, the observed antichlamydial effect did not appear to be due to direct toxicity of the compounds for the EBs.

Dose-dependent inhibition of Chlamydia. The noncytotoxic, antichlamydial compounds INP 0341 and INP 0400 had also previously been shown to inhibit the Yersinia T3S system (Innate Pharmaceuticals, unpublished data) and therefore were further tested to characterize the antichlamydial effect. A dose response experiment, using INP concentrations ranging from 0.5 to 50 µM, was performed to establish the MIC needed to attenuate the development of C. trachomatis serovar D. With increasing concentrations of the INPs the inclusions became smaller, until at 50 µM they were no longer detected microscopically (Fig. 1). At lower concentrations, there was great variation in size and possible maturation of the chlamydial inclusions. Therefore, with INP 0341 and INP 0400 a dose-dependent antichlamydial effect was observed, and complete inhibition of development was observed at a concentration of 50 µM.


Figure 1
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  		FIG. 1. Dose-dependent inhibitory effect of INP 0341 on C. trachomatis serovar D. HeLa 229 cells were infected with C. trachomatis serovar D using an MOI of 0.2. The final concentrations of INP 0341 present in the culture media ranged from 0.5 to 50 µM, as indicated above the images. Cultures were fixed after 44 h of incubation at 37°C in 5% CO2. Infected monolayers were stained using MAb E4. The arrowheads indicate small, abnormal inclusions. The inclusions seen with 0.5 µM INP 0341 appear to be the same as the inclusions in cultures without INP 0341 present. Similar results were obtained with INP 0400 (not shown).

INP effect on Chlamydiaceae. The ability of INPs 0341 and 0400 to inhibit other Chlamydiaceae was tested. HeLa 229 cells were infected with C. muridarum, C. caviae, and C. pneumoniae in the presence of 20 and 50 µM INP 0341 and INP 0400. INP 0406 was included since it had little activity against C. trachomatis serovar D and thus served as a negative INP control (Table 2). None of the Chlamydiaceae tested was inhibited by INP 0406 or DMSO alone at either concentration used. In contrast, C. pneumoniae and C. caviae were completely inhibited by both 20 and 50 µM of INP 0341 and INP 0400. C. muridarum was completely inhibited by 50 µM of INP 0341, but at the lower concentration, 20 µM, the inhibition was 70%. INP 0400 at a concentration of 20 µM effectively inhibited >95% of C. muridarum. Therefore, INPs 0341 and 0400 were able to significantly inhibit all members of the Chlamydiaceae examined.

Rescue of Chlamydia from INP inhibition. Having established that INP 0341 and INP 0400 were able to completely inhibit the growth of Chlamydia, we next wanted to determine if C. trachomatis serovar D could be rescued from the inhibited cultures. To accomplish this, 44 h after infection in the presence of an INP at a concentration of 20 or 50 µM, infected monolayers were washed, and either fresh medium without the INPs was added and the cultures were reincubated for an additional 48 h or the cell monolayers were disrupted and transferred to fresh monolayers. As expected, control INP 0406-treated cultures showed secondary infection in cells washed and reincubated in fresh medium. In addition, for INP 0406-treated cultures that were transferred, the yield of infectious progeny from the original inclusions ranged from 239 to 252 IFU. In striking contrast, with INP 0341 and INP 0400, there was no evidence that Chlamydia could be rescued from cultures in which 20 µM of the INP had been removed at 44 h postinfection and which were incubated for an additional 44 h or from cultures that were transferred to fresh monolayers. Therefore, it appears that with these two compounds, there was a cidal effect at INP concentrations of ≥20 µM.

Ability of INP to arrest Chlamydia development at various times postinfection. We next wanted to establish whether INP 0341 could affect the development of Chlamydia when it was added at different times postinfection and to determine the number of resulting progeny. Therefore, HeLa 229 cells were infected with C. trachomatis serovar D, INP 0341 and INP 0406 (control) were added from 1 to 24 h postinfection, and cultures were incubated for 44 h. Subsequently, one set of cultures was fixed and stained, and a duplicate set was transferred to assess the resulting progeny. As expected, INP 0406 (control)-treated cultures appeared to develop mature inclusions by 44 h regardless of when INP 0406 was added to the culture (Table 4). The concentration of the resulting infectious progeny, depending on the time of addition of INP 0406, ranged from 254 to 502 IFU/primary culture inclusion. Therefore, even with INP 0406, although the primary inclusions appeared to be mature regardless of when the compound was added after infection, early addition resulted in a decrease in the number of progeny; the earlier the INP was added, the fewer progeny were recovered. Addition of the antichlamydial compound INP 0341 at 2 and 4 h after infection resulted in complete inhibition of the growth of Chlamydia. Addition of INP 0341 at 8 h postinfection significantly attenuated the development of the Chlamydia inclusions; while microscopic inclusions could be seen, they were substantially smaller than those seen in the INP 0406-treated control cultures, but they were counted for presentation of the data in Table 4. Cultures in which the addition of INP 0341 was delayed until 16 and 24 h postinfection yielded the same numbers and equivalent sizes of inclusions as controls. The results obtained for INP 0341 and INP 0400 were similar (data not shown). Therefore, in the primary cultures the main inhibitory event(s) due to the INPs appeared to take place within the first 8 to 16 h of the C. trachomatis developmental cycle since addition of an INP after this time had no effect on the number and size of the resulting inclusions. As expected, there were no or rare progeny resulting from original cultures treated at 2 and 4 h postinfection with INP 0341. In cultures that were treated with INP 0341 at 8 and 16 h postinfection, the numbers of inclusions were equivalent to the numbers in control cultures, but overall the inclusions were smaller; these cultures yielded none and 1.3 progeny IFU/original inclusion, respectively, thus corroborating the hypothesis that there was a correlation between small size and halted or delayed C. trachomatis development when cultures were treated up to 16 h postinfection with INP 0341. The numbers of progeny resulting from cultures treated at 24 h postinfection with INP 0341 were about one-half the numbers obtained with control cultures, which may be a reflection of the asynchronous nature of the development within a chlamydial inclusion.


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  		TABLE 4. Addition of INPs after infection and effect on the yield of Chlamydia in primary cultures

Expression of T3S genes. In order to determine if INP 0341 affected expression of T3S genes, three sets of HeLa 229 cells infected with C. trachomatis serovar D were run in parallel. Each set received one of the following at the initiation of the infection: DMSO, INP 0406, or INP 0341. For each time point examined (4, 8, 24, and 36 h postinfection), RT-PCR was performed with purified nucleic acid containing 5,000 genome equivalents of C. trachomatis. In both sets of controls, DMSO- and INP 0406-treated cultures, identical results were seen for all genes. The results obtained for 4 and 8 h postinfection and for 24 and 36 h postinfection, in general, showed little difference within each time period. Therefore, the overall results for the genes examined for both the cultures incubated with INP 0406 as the control and the cultures incubated with INP 0341 at 4 and 24 h postinfection are illustrated in Fig. 2. The genes used as markers of the early, middle, and late developmental cycle were groEL-1, ompA, and omcB, respectively (26). As expected, groEL-1 was expressed early and throughout the times examined, and ompA was expressed from 8 h on. Expression of omcB in both INP 0406- and INP 0341-treated cultures was detected at 24 and 36 h postinfection, and there was slight down-regulation in INP 0341-treated cultures compared to controls. The rs16 gene was also monitored for all the time points examined, and there was a steady level of gene expression in all cultures for all time points.


Figure 2
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  		FIG. 2. Expression of T3S genes analyzed throughout the developmental cycle of C. trachomatis serovar D using RT-PCR, as well as expression of three chlamydial genes, groEL, ompA, and omcB, that have been shown to correlate with the early, middle, and late developmental cycle, respectively (26). The gene for the 16S ribosomal protein (CT026) was also included as an internal control for quantitation. As indicated at the top, HeLa 229 cells infected with C. trachomatis serovar D were grown in the presence of 20 µM INP 0406, INP 0341, or Desferal (400 µM) and harvested at 4 and 24 h postinfection (hpi). The RNA was isolated, and the equivalent of 5,000 genomic copies of C. trachomatis was used in an RT-PCR with primers for the genes indicated on the left (Table 1). For each time point both the RT-PCR results (+) and the results without added RT (–), to control for DNA contamination, are shown. The DMSO-treated samples yielded the same gene expression pattern as INP 0406 (data not shown). Amplicons were separated on a 2% agarose gel and stained with ethidium bromide. The TriDye 2-Log DNA ladder (0.1 to 10.0 kb) (New England Biolabs) was used as markers (lane M). Lane PCR contained a control for the PCR within the RT-PCRs. ORF, open reading frame.

Of the T3S chaperone genes examined, sycE, lcrH-1, and lcrH-2, in control cultures lcrH-2 appeared early (4 h), while lcrH-1 and sycE appeared late (24 h). In contrast, in INP 0341-treated cultures, while the results for lcrH-2 were the same as the results observed for the controls, sycE also appeared late (24 h) but was slightly down-regulated, and lcrH-1 was not expressed at any time point examined.

Genes that code for T3S proteins that are secreted and make up part of the T3S structure (yscC, copB, and copB2) or serve as the "plug" for the secretory apparatus (lcrE) were also examined. In control cultures, lcrE and copB2 appeared by 8 h (data not shown) and at all subsequent time points, and copB and yscC were expressed late, at 24 and 36 h. In INP 0341-treated cultures, lcrE expression was the same as it was controls; copB2 expression was delayed, with expression appearing by 24 h, but compared to controls the level of expression was slightly decreased; and yscC expression appeared late, by 24 h, as it did in control cultures, but was decreased. In contrast to controls, copB was not expressed at any time point examined in INP 0341-treated cultures.

Among the genes encoding proteins that have been proposed to be secreted effectors of the Chlamydia T3S system, incA, incC, incG, and the Tarp gene were examined. In both control cultures and INP 0341-treated cultures incC and incG were expressed by 4 h and at all other times examined. In striking contrast, while both incA and the Tarp gene were expressed by 24 h in control cultures, there was no expression (incA) or there were very low levels of expression (Tarp gene) of these two effector genes in INP 0341-treated cultures.

Ability of iron to reverse the inhibitory effect of INPs. Hydrazone compounds have been shown to chelate iron (11, 15, 22). Therefore, in order to explore whether the mechanism for the inhibition of Chlamydia by INPs could be iron deprivation, HeLa 229 cells were infected with C. trachomatis serovar D with and without INP 0341 or INP 0406 (control) and 500 µM ferrous sulfate. Compared to controls incubated in medium without supplemental iron, cultures incubated without an INP but with added iron yielded the same number of inclusions and resulting progeny. Therefore, additional iron did not affect the infectivity or development of Chlamydia. In contrast, when infected cultures incubated with INP 0341 alone were compared to cultures to which iron was also added, there were dramatic increases in the number and overall inclusion size, which approached those of control cultures. Concentrations of ferrous sulfate ranging from 0.25 to 250 µM were tested for the ability to negate the inhibitory effect of INP 0341 (Fig. 3 and 4). Concentrations of Fe2+ of ≤1 µM were not able to restore the development of the Chlamydia inclusion, whereas there was approximately 60% recovery of visible inclusions with 5 µM and 80% to 100% recovery with concentrations of ≥10 µM (Fig. 4). Iron supplied as ferrous ammonium sulfate resulted in a dose response identical to that shown in Fig. 3 for ferrous sulfate. Fe3+, supplied as ferric chloride, was also able to negate the inhibitory INP effect at concentrations of ≥20 µM, (Fig. 4). However, compared to the concentration of Fe2+, a fourfold-higher concentration of Fe3+ was required to achieve the same result.


Figure 3
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  		FIG. 3. Iron supplied as Fe2+ was able to abrogate the inhibitory effect of INP 0341 in a dose-dependent manner. HeLa 229 cells were infected with C. trachomatis serovar D using an MOI of 0.2. The medium contained 20 µM INP 0341, and ferrous sulfate was added to the medium to obtain final concentrations that ranged from 0.25 to 250 µM, as indicated above the images. Cultures were fixed after 44 h of incubation at 37°C in 5% CO2. Infected monolayers were stained using MAb E4. The arrowheads indicate small, abnormal inclusions.


Figure 4
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  		FIG. 4. Iron supplied as both Fe2+ and Fe3+ is able to reverse the inhibitory effect of INP 0341. HeLa 229 cells were infected with C. trachomatis serovar D. Upon infection medium containing 20 µM INP 0406 (control) or INP 0341 with and without additional ferrous sulfate (Fe2+) or ferric chloride (Fe3+) was added. After 44 h of incubation at 37°C in 5% CO2, cultures were fixed and stained using MAb E4. The number of IFU was determined, and the results for INP 0341 were compared to and expressed as a percentage of the INP 0406 control results.

To determine if other cations were able to negate the INP effect or if this phenomenon was specific for iron, 50 µM each of Ca2+, Mn2+, Mg2+, and Zn2+ were added at the time of infection along with INP 0341 (Fig. 5). In contrast to Fe2+, none of the other divalent cations tested were able to restore the development of Chlamydia, and the appearance and size of inclusions were similar to the appearance and size of inclusions in the cultures containing INP 0341 alone. Therefore, the ability to restore the development of C. trachomatis, as judged by inclusion appearance, appeared to be specific to iron.


Figure 5
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  		FIG. 5. Comparison of the abilities of different divalent metal ions to rescue Chlamydia from the inhibitory effect of INP 0341. HeLa 229 cells were infected with C. trachomatis serovar D using an MOI of 0.2. The medium contained INP 0341 at a final concentration of 20 µM, and the ions indicated above the images were added to the medium at a concentration of 50 µM. The salts used to supply the ions are listed in Materials and Methods. Cultures were fixed after 44 h of incubation at 37°C in 5% CO2. Infected monolayers were stained using MAb E4.

To establish whether iron could reverse the antichlamydial effect of 20 µM of INPs 0341 and 0400, regardless of the length of exposure to INP, iron was added to cultures 2 to 24 h postinfection (Fig. 6). All cultures were incubated for a total of 44 h, and one set that received iron at 24 h postinfection was incubated for an additional 24 h to account for the possible delay in chlamydial inclusion development. INP-treated cultures that did not receive additional iron failed to develop any inclusions. In contrast, addition of iron 2 h after infection restored the inclusion number and size to >95% of the control cultures. Addition of iron after this time gradually decreased the ability to restore cultures to control values, and by 24 h after infection <50% of the number of inclusions in the controls were recovered. Upon incubation for an additional 24 h this number did not increase. Therefore, while iron can reverse the inhibitory effect of INPs, as the time of addition of iron postinfection is delayed, there is an overall decrease in the recovery of Chlamydia.


Figure 6
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  		FIG. 6. Ability of iron over time to rescue Chlamydia from inhibition by INP 0341 and INP 0400. HeLa 229 cells were infected with C. trachomatis serovar D. Upon infection, medium containing either 20 µM INP 0406 (control), INP 0341, or INP 0400 was added to cell monolayers. At various times after infection iron supplied as ferrous sulfate (250 µM) was added. Cultures were fixed after 44 h of incubation at 37°C in 5% CO2. In addition, a set of cultures to which iron was added 24 h postinfection was incubated for 68 h [24(+)]. Infected monolayers were stained using MAb E4. The resulting numbers of IFU were compared to and expressed as percentages of the INP 0406 control value.

Iron binding of INPs. The abilities of the antichlamydial compound INP 0341 and INP 0406 (control), which did not inhibit chlamydial growth, to bind iron were compared. Using the method of Ponka et al. (22), a set of mixtures with fixed concentrations of INP 0341 or INP 0406 but different concentrations of ferric ions were prepared and scanned in an UV spectrophotometer at the wavelength corresponding to the specific INP-Fe3+ absorption maximum. When the measured absorbance is proportional to the INP-Fe3+ concentration, the slope of the scans should decline to zero when no more INP-Fe3+ complex is formed (i.e., no free INPs are present). This plateau was clearly observed for both INP 0341 and INP 0406 at a ratio of approximately 0.5, indicating that INP 0341 and INP 0406 both are tridentate ligands relative to iron, establishing that these two compounds can chelate iron to the same extent.

T3S gene expression with Desferal treatment. It has been established (1, 24) that Desferal can also inhibit the growth of Chlamydiaceae, presumably through iron deprivation in the host and intracellular Chlamydiaceae. To see whether Desferal treatment resulted in a gene expression pattern similar to that observed with the inhibitory INPs, RT-PCR was performed with cultures of C. trachomatis serovar D grown in the presence of 400 µM Desferal (Fig. 2). At this concentration of Desferal, inclusion development was inhibited, and the microscopic appearance was similar to that of INP-inhibited chlamydial growth (data not shown). Distinct differences in gene expression between the Desferal- and antichlamydial INP-treated cultures were seen. Notably, in contrast to INP 0341-treated cultures, in which there was clearly down-regulation of lcrH-1 and copB, in Desferal-treated cultures the expression levels of these genes were equivalent to those in the controls. Therefore, while chlamydial growth inhibition mediated by INP 0341 appeared to ultimately result in an alteration in T3S gene expression, Desferal treatment did not affect the T3S genes examined.

Effect of INPs on host cell proliferation. Up to this point INP cell toxicity studies, as described above, tested exposure of host cells to the anti-Chlamydia INPs for 24 h, followed by measurement of mitochondrial dehydrogenase activity. In light of the data on the rescue of Chlamydia by iron in INP-treated cultures indicating that INPs may interfere with the availability of iron, we examined cell proliferation as another parameter of possible INP host cell toxicity. Therefore, we next determined the effect of INPs on the proliferation of host cells with and without iron supplementation. A total of 1.28 x 105 HeLa 229 cells were seeded into 1-dram glass vials in order to obtain a subconfluent monolayer after overnight incubation at 37°C. At the time of seeding INP 0406, which served as an INP control, and the antichlamydial compound INP 0341 were each added at a concentration of 20 µM. Monolayers were given the INP with and without 0.5 mM of supplemental ferrous sulfate. Cells from treated monolayers were counted after 24 and 48 h of incubation. By 24 h the antichlamydial compound INP 0341 had a significant effect on the proliferation of HeLa 229 cells, in contrast to the control, INP 0406 (P < 0.05). By 48 h of exposure to INP 0341, the number of HeLa 229 cells had slightly decreased, to 8.7 x 104 ± 0.2 x 104 cells (mean ± standard deviation), compared to the number of cells present at the start of the experiment. This is in contrast to cells treated with INP 0406, which exhibited a nearly twofold increase, to 2.28 x 105 ± 0.1 x 105 cells, at 48 h (P < 0.05). However, when cells were treated with INP 0341 and 0.5 mM of supplemental ferrous sulfate, there was not a significant difference between the number of cells treated with iron and INP 0341 (2.12 x 105 ± 0.5 x 105 cells) and the number of control cells treated with INP 0406 (2.28 x 105 ± 0.1 x 105 cells) (P >0.05). Therefore, in the presence of the inhibitory INP, host cells did not proliferate. Instead, they appeared to remain in a static state since they remained viable as determined by trypan blue staining; however, iron was able to negate this growth inhibition.

Ability of transferrin to reverse inhibition by INP. Since it has previously been shown that Chlamydia is able to obtain iron from the transferrin of host cells (24, 25), to further establish the ability of added iron to negate the effect of INPs, HeLa 229 cells were infected with C. trachomatis serovar D in the presence of INP 0341, INP 0400, and INP 0406 (control) to which either 10 nM human apo-transferrin or holo-transferrin was added. No inclusions were detected in INP 0341- and INP 0400-treated cultures with or without supplementation with apo-transferrin (Fig. 7 and Table 5). This was in contrast to the 100% recovery for cultures to which holo-transferrin was supplied. Furthermore, the yields of infectious progeny were 162 and 163 IFU/original inclusion for the cultures originally propagated with INP 0341 and INP 0400 supplemented with holo-transferrin, respectively. These yields were 55 to 64% of the progeny yields for control cultures supplemented with holo-transferrin. These data corroborated the results obtained using ferrous sulfate, in that iron supplied by holo-transferrin was able to partially negate the INP antichlamydial effect.


Figure 7
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  		FIG. 7. Ability of holo-transferrin to rescue Chlamydia from the inhibitory effect of INP 0341. HeLa 229 cells were infected with C. trachomatis serovar D using an MOI of 0.2. At the time of infection INP 0341 (20 µM) was added to the medium. To some cultures human apo-transferrin (Apo-Tr) or holo-transferrin (Holo-Tr) (10 nM) was also added, as indicated above the images. Cultures were fixed after 44 h of incubation at 37°C in 5% CO2. Infected monolayers were stained using MAb E4. The arrowheads indicate small, abnormal inclusions.


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  		TABLE 5. Effects of INPs and apo- and holo-transferrin on the development of C. trachomatis in primary cultures and the resulting progeny


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DISCUSSION
 
Chlamydiaceae possess components of a T3S system, structural genes, chaperones, and putative effectors; however, the role that the T3S system plays in the virulence or development of these pathogens remains to be established (10, 29). The inability to manipulate a genetic system in these obligate intracellular bacteria has impacted our ability to directly address some of the questions related to the role of the Chlamydia T3S system. Therefore, using complementation and in vitro techniques, investigators have attempted to identify the T3S components and their roles and regulation during the developmental cycle (3, 8, 27, 30, 31). Another approach to address some of these questions is to use agents that interfere with the T3S system of the Chlamydiaceae. This was the rationale for our initial investigation (27a) in which we used INP 0400, a compound belonging to a class of acylated hydrazones of salicylaldehydes which had previously been identified as an inhibitor of the T3S system of Yersinia (Innate Pharmaceuticals, unpublished data). We found that INP 0400 inhibited the growth of all Chlamydiaceae tested, which included C. trachomatis serovar D, C. muridarum, and C. pneumoniae. This inhibition was dose dependent, significantly decreased the yield of infectious progeny, and was not due to direct toxicity for the EBs or short-term (24 h) toxicity for the host cell. Subsequently, our work was corroborated by Muschiol et al. (16), who used INP 0400 with C. trachomatis L2-infected McCoy cells. In addition, Wolf et al. (32), using C1 (INP 0007), described the same dose-dependent inhibition of C. trachomatis L2 that we had previously reported with INP 0400 (27a). Recently, Bailey et al. (2) reported that they were able to inhibit C. pneumoniae but not C. trachomatis L2 using INP 0010 at concentrations up to 30 µM. In our study, however, we found complete inhibition of C. trachomatis serovar D at concentrations of INP 0010 of 50 µM (Table 2). We have titrated INP 0010 against C. pneumoniae and C. trachomatis serovar D and found that while it is true that C. pneumoniae is more susceptible to INP 0010, C. trachomatis serovar D is also inhibited. C. pneumoniae inclusion size is affected by concentrations as low as 2.5 µM, and a dramatic decrease is seen at 20 µM, whereas C. trachomatis serovar D is affected at concentrations of ≥20 µM and there are no recognizable inclusions at 50 µM. Therefore, with both organisms, the INP showed a dose-dependent effect, and C. pneumoniae exhibited this inhibition at a lower concentration. This finding is not too surprising since in our experience C. pneumoniae is, in general, more fastidious in that it requires a higher inoculum for in vivo work, takes longer to grow, and is more difficult to propagate in cell culture (19, 20).

In this report we expanded our screening to identify similar compounds that potentially may help provide insight into the role of the T3S system of the Chlamydiaceae. Of the 25 compounds that we screened for the ability to inhibit C. trachomatis, 14 completely inhibited growth at a concentration of 50 µM in two cell lines. Using these compounds, we showed that INPs do not appear to exert their effect by being directly cytotoxic to the infectious EBs, for INP-pretreated EBs infected cells to the same extent as nontreated control EBs. In addition, INPs had little effect on the entry of EBs into the host cell. In cultures in which an INP was present throughout the early stages of infection and then washed out and replaced with fresh medium not containing the INP, the same number and size of inclusions were restored in the INP-treated cultures as in the control cultures. In addition, if concentrations of the INP of ≤20 µM were used, although the inclusions may have been dramatically smaller than those in the control cultures, the actual numbers of inclusions were similar to the numbers in control cultures, thus supporting the statement that INPs did not affect the entry of the EBs into the host. Muschiol et al. (16) concluded that there was a 40% reduction in the entry of EBs when 40 µM of INP 0400 was used during infection of McCoy cells with C. trachomatis L2. However, we suggest an alternate explanation since at high concentrations of INP 0400 (>20 µM), inclusion size varies within the same culture, making some inclusions difficult to detect microscopically, and therefore there is not a reduction in entry, but rather upon entry some EBs are unable to progress to form a microscopically recognizable inclusion even upon limited exposure to INPs. In addition, our ability to "rescue" inclusions when iron was added to INP 0400 (20 µM)-treated cultures 2 h after infection provides more evidence that entry is not affected, since 100% of the inclusions in INP 0400-treated cultures could be recovered. Therefore, it is our conclusion that if a T3S system plays a role in the infectivity or entry of Chlamydia, the mechanism is not affected by INPs. Another possibility is that the "trafficking" of the EB into the host cell may have been affected by the INP; however, again the rescue experiments with iron and also the ability to wash out and thus negate the INP effect early in the development of the inclusion argue against an effect on trafficking.

Once the bacterium is in the host cell inclusion, another major step in Chlamydia development is the differentiation of the EB to the RB. Wolf et al. (32) present evidence that in C1 (INP 007)-treated cultures, EBs differentiate into RBs. Supporting this conclusion, we have shown (Fig. 1) that the abnormal inclusions resulting from INP treatment appear to contain RB-like structures based on size and appearance. However, once a Chlamydia cell develops and transforms from an RB to an EB, based on our data it does not appear that INPs can significantly affect the intracellular mature EB. This conclusion was drawn from the results that we obtained by adding INPs later in the development of Chlamydia (24 h postinfection), for under these conditions the yield of inclusions containing infectious EBs approached that of control cultures. Although one may argue that the yield was slightly less than that of controls, this can be explained by the asynchronous nature of a 24-h Chlamydia inclusion in that it contains Chlamydiaceae at all stages of development, including noninfectious RBs whose development may be inhibited upon addition of INP, even at 24 h. Supporting this concept is the fact that upon addition of INPs at 8, 16, and 24 h, although the same number of inclusions could be counted by 44 h postinfection, the yield of infectious EBs increased 100-fold with every 8-h delay in adding INP to the cultures. Therefore, our data and those of Wolf et al. (32) suggest that the inhibition of Chlamydia development is in either the replication of the RBs within the inclusion and/or the maturation of the RB to an infectious EB.

Having established that the INPs can inhibit the development of the RB, we next wanted to explore their mode of action. Examining Chlamydia T3S gene expression, we found that compared to controls, select T3S genes were down-regulated in the presence of INP 0341. Interestingly, two genes that are adjacent, lcrH-1 and copB, which code for a class 2 T3S chaperone and a secreted structural protein, respectively, were completely inhibited by INP 0341. These two genes are normally expressed in the middle to late stages of the developmental cycle (28). While not down-regulated to the same extent as these two T3S genes, three other T3S genes normally expressed later in the developmental cycle, sycE, lcrE, and copB2, were also affected. Of the putative Chlamydia T3S effector genes tested, incC and incG, two genes that are normally expressed early, were unaffected by the presence of INP 0341, whereas the expression of incA, a gene expressed in the middle to late stages, was dramatically down-regulated (9). Therefore, it appears that the T3S genes that are normally expressed earlier in the chlamydial developmental cycle were unaffected, whereas those expressed later were either turned off completely or down-regulated.

Wolf et al. (32) measured putative Chlamydia T3S system secreted proteins in cultures of C. trachomatis L2 grown with and without C1 (INP 0007). Using immunoblots, they reported accumulation of the T3S substrates CADD, IncA, and Tarp, while they were unable to detect a difference in the T3S structural proteins, CopN and CopB2, compared to controls. Providing further evidence for an effect on the T3S system, they reported the failure of IncA to colocalize with the inclusion membrane, normally an event in the middle of the developmental cycle, which was seen in untreated controls. In their treated cultures, IncA was seen in association with the Chlamydia structures within the abnormal inclusions, yet not in association with the inclusion membrane, suggesting that the C1 (INP 0007)-treated Chlamydia was not able to transport the IncA through the T3S system to the inclusion membrane. A possible interpretation of their findings with IncA and our gene expression data involves feedback inhibition in that upon accumulation of IncA in the INP-treated RB, the expression of incA may be dramatically down-regulated. Since it was the T3S genes that we know to be expressed at the middle to late stages in the developmental cycle that were down-regulated while the genes normally expressed early seemed unaffected, this may suggest that in Chlamydia the T3S system is used at different stages of development for distinct purposes (28). Wolf et al. (32) found that Tarp, which has been proposed to be a T3S effector deployed in the entry events during Chlamydia infection, was secreted early in infection. This finding suggests that the functioning T3S structures and proteins needed to transport Tarp into the host cell were unaffected by C1 (INP 0007) or possibly already present in a mature EB or that this protein is secreted by an alternate system.

In comparing our gene expression data for lcrH-1 and lcrH-2 in C. trachomatis harvested from INP-treated cultures, we obtained the same results that we previously reported for gamma interferon-treated C. pneumoniae (28). The T3S chaperone gene lcrH-2, normally expressed early, was unaffected by gamma interferon or INP treatment of cultures. In contrast, expression of lcrH-1, normally expressed later in Chlamydia development, was not detected in treated cultures. In addition, the stunted appearance of the inclusions resulting from gamma interferon treatment and the stunted appearance of the inclusions resulting from INP treatment were strikingly similar (4, 7). Abnormal inclusions have also been reported in cultures treated with antibiotics, such as penicillin, or when Chlamydia cultures have been deprived of iron (14, 24). It is intriguing that the class of compounds represented by the INPs resemble those that have the ability to bind iron (11, 15). Therefore, to investigate a possible mechanism for the inhibition of Chlamydia growth seen with the INPs, we explored the role of iron in this antichlamydial effect. As shown here, we were able to abrogate the inhibitory properties of the INPs by providing iron in the form of iron salts or supplied by holo-transferrin to the infected cells. Therefore, this strongly suggests that, either directly or indirectly, INPs work by altering the intracellular iron available to Chlamydia and/or the host cell.

When cell proliferation was measured in the presence of the INPs, we found that the host cells were not able to proliferate, yet they remained viable, when they were incubated with the antichlamydial INPs. When iron was added to the medium along with the INP, then the cells proliferated. One could speculate that the INPs bound iron, thus limiting the host iron supply, and therefore the cells remained suspended in the G0 phase until iron supplies were restored. With this line of reasoning, Chlamydia that is viable but latent in an iron-starved host can resume development once the host reenters the G1 phase. Iron has long been known to be critical to both the host cell and the pathogen, and the multiple schemes that bacteria have devised to acquire and utilize iron to regulate essential functions have been reviewed previously (23) and are beyond the scope of this discussion. Host cells use iron withholding as a strategy to fight off pathogenic bacteria. Both hosts and bacteria have highly specialized proteins to fight this tug-of-war for limited iron supplies. Therefore, exploiting the availability of this essential nutrient and tipping the balance to the host have been considered to be a strategy for development of novel antimicrobial agents. It thus appears that the INP compounds might have the ability to limit the iron supply by successfully outcompeting bacterial proteins designed to sequester iron from the host. Alternatively, it is possible that the T3S system may function to acquire iron from the host and that the INP compounds may actually interfere with the function or regulation of the T3S system, with addition of supplemental iron to the cultures overcoming this T3S block and thus restoring chlamydial growth.

The observation that iron can attenuate or reverse the action of the INPs raises several possibilities. It is plausible that the iron effect seen with the INPs may be due to simple chelation of intracellular iron supplies, similar to the chelation observed by Raulston (24) and Al-Younes et al. (1) using Desferal. Here the iron chelator Desferal was found to inhibit the normal development of Chlamydia. In examining T3S gene expression in C. trachomatis serovar D raised in HeLa 229 cells using the Desferal model, we observed no appreciable difference in gene expression compared to that in control cultures. This is in sharp contrast to INP 0341-treated cultures, where several T3S genes were clearly down-regulated or repressed. Based on this finding, it appears that the underlying mechanism of the INPs differs from that proposed for Desferal. In the Desferal model described by Raulston (24), a low level of cycloheximide was used with the rationale that it would decrease host cell receptors that may be able to compete with Desferal to maintain intracellular iron supplies. Whether this may have ultimately affected the outcome of the T3S gene expression is unknown. To strengthen the theory that the INPs do not work by a nonspecific iron depletion mechanism but rather target the T3S system in a specific manner, iron chelation experiments with the antichlamydial compound INP 0341 and the negative control compound INP 0406 were conducted using UV spectrophotometry. The results of the absorption spectral analysis confirmed that both INP 0341 and INP 0406 bind Fe3+ as tridentate ligands. These data suggest that the inhibitory effect on intracellular growth of Chlamydia is not simply the result of iron depletion caused by the INPs since the negative control INP 0406 chelates Fe3+ as well as the potent inhibitor INP 0341. Another possible explanation for the ability of iron to reverse the INP inhibition of Chlamydia is that, as we observed here, iron addition actually hinders or inactivates the INP antichlamydial properties. However, when we added excess iron to the Yersinia pseudotuberculosis E-lux expression model described by Kauppi et al. (13), there was no appreciable effect on the ability of the INPs tested to inhibit the expression of the Y. pseudotuberculosis T3S genes examined or the secretion of YopH as determined by measuring the phosphatase activity of the secreted YopH (data not shown). These results argue against the hypothesis that iron has a direct anti-INP effect. In addition, Wolf et al. (32) tested the ability of the equivalent of INP 0007 to inhibit another intracellular bacterium, Coxiella burnetti. Although this organism is quite different from Chlamydia, if the INPs simply chelated intracellular iron, then one might expect Coxiella growth to also be inhibited due to reduced iron availability. Therefore, the fact that Wolf and colleagues (32) reported that Coxiella growth was unaffected by INP 0007 also argues against the explanation that INPs inhibit simply through iron chelation. However, even here one must be cautious in interpreting data for different pathogens since inclusion properties and the location within the host cell may influence the responses to inhibitors such as Desferal or INPs.

In conclusion, while the findings presented here, together with those of Wolfe et al. (32), are intriguing and have implicated the Chlamydia T3S system as being affected and possibly critical to the full maturation of RBs, it is premature at this point to suggest that INPs have a direct effect on Chlamydia T3S system function. However, either directly or indirectly, these inhibitory compounds appear to have the ability to limit intracellular iron supplies, thus affecting Chlamydia growth.


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ACKNOWLEDGMENTS
 
We thank Pia Keyser of Innate Pharmaceuticals AB, Umea, Sweden, for providing advice and reagents used in this investigation.

This work was supported by NIH/NIAID grant AI-71104 (to E.M.P.). P.-A.E. was supported by grant 28755-1 from the Swedish Governmental Agency for Innovation Systems (VINNOVA).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathology and Laboratory Medicine, Medical Science Building, Room D-440, University of California, Irvine, Irvine, CA 92697-4800. Phone: (949) 824-4169. Fax: (949) 824-2160. E-mail: epeterso{at}uci.edu Back

{triangledown} Published ahead of print on 30 April 2007. Back

Editor: R. P. Morrison


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Infection and Immunity, July 2007, p. 3478-3489, Vol. 75, No. 7
0019-9567/07/$08.00+0     doi:10.1128/IAI.00023-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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