. 2006 Sep 14;103(39):14566–14571. doi:10.1073/pnas.0606412103

A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle ofChlamydia trachomatis

Sandra Muschiol^*,^†,Leslie Bailey^‡,Åsa Gylfe^‡,Charlotta Sundin^§,Kjell Hultenby^¶,Sven Bergström^‡,Mikael Elofsson^‖,Hans Wolf-Watz^‡,Staffan Normark^*,^†,Birgitta Henriques-Normark^*,^†,^**

*Swedish Institute for Infectious Disease Control, SE-171 82 Solna, Sweden;

^†Department of Microbiology, Tumor Biology, and Cell Biology, Karolinska Institutet, SE-171 77 Stockholm, Sweden;

^‡Department of Molecular Biology, Umeå University, SE-901 87 Umeå, Sweden;

^§Innate Pharmaceuticals, Umestan Företagspark, SE-903 47 Umeå, Sweden;

^¶Department of Laboratory Medicine, Karolinska Institutet, SE-141 86 Huddinge, Sweden; and

^‖Organic Chemistry, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden

^✉

**To whom correspondence should be addressed at:Department of Bacteriology, Swedish Institute for Infectious Disease Control, Nobels väg 18, SE-171 82 Solna, Sweden. E-mail:birgitta.henriques@smi.ki.se

Communicated by Rino Rappuoli, Chiron Corporation, Siena, Italy, July 27, 2006

Author contributions: S.M., S.N., and B.H.-N. designed research; S.M., L.B., Å.G., and K.H. performed research; C.S. and M.E. contributed new reagents/analytic tools; S.M., S.B., H.W.-W., S.N., and B.H.-N. analyzed data; and S.M., S.N., and B.H.-N. wrote the paper.

Received 2006 Jul 5; Issue date 2006 Sep 26.

Freely available online through the PNAS open access option.

PMC Copyright notice

PMCID: PMC1566191 PMID:16973741

Abstract

The intracellular pathogenChlamydia trachomatis possesses a type III secretion (TTS) system believed to deliver a series of effector proteins into the inclusion membrane (Inc-proteins) as well as into the host cytosol with perceived consequences for the pathogenicity of this common venereal pathogen. Recently, small molecules were shown to block the TTS system ofYersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity ofC. trachomatis at micromolar concentrations resulting in small inclusion bodies frequently containing only one or a few reticulate bodies (RBs). INP0400, at high concentration, given at the time of infection, partially blocked entry of elementary bodies into host cells. Early treatment inhibited the localization of the mammalian protein 14-3-3β to the inclusions, indicative of absence of the early induced TTS effector IncG from the inclusion membrane. Treatment with INP0400 during chlamydial mid-cycle prevented secretion of the TTS effector IncA and homotypic vesicular fusions mediated by this protein. INP0400 given during the late phase resulted in the detachment of RBs from the inclusion membrane concomitant with an inhibition of RB to elementary body conversion causing a marked decrease in infectivity.

Keywords: type III secretion system

Screening for novel antimicrobials has traditionally been done by scoring for growth inhibitionin vitro on artificial media. This approach has over the years led to a limited number of antimicrobial classes. To combat increasing antibacterial resistance, development has focused on modifying compounds within the existing classes rather than identifying small molecules with a completely novel mode of action. Targeting growth and virulence underin vivo-like conditions will likely identify completely new sets of molecules, as recently shown forVibrio cholerae where a small-molecule inhibitor of the transcriptional activator ToxT, virstatin, prevented both toxin and pili expression, protecting infant mice from colonization (1). Small molecules belonging to a class of acylated hydrazones of salicyl aldehydes were recently identified (2,3) that inhibited type III secretion (TTS)-dependent delivery ofYersinia pseudotuberculosis Yop effectors into target cells without inducing a measurable toxicity on the host cells. Neither virstatin nor theYersinia TTS inhibitors affected bacterial growthin vitro. It is likely that alsoin vivo, the bacterial multiplication rates are not affected by these novel classes of antivirulence drugs but only their ability to cause pathophysiology and/or to evade the host innate immune response.

Chlamydia trachomatis is the most common sexually transmitted bacterial disease and the leading cause of preventable blindness worldwide (4).Chlamydia are Gram-negative, obligate, intracellular bacteria that share a unique biphasic developmental cycle (5). Infection is initiated by attachment of elementary bodies (EBs) to eukaryotic host cells. A few hours after internalization, infectious but metabolically inactive EBs differentiate into reticulate bodies (RBs), the metabolically active form ofChlamydia. RBs will then replicate within a cytoplasmic vacuole termed inclusion before they redifferentiate to EBs. Upon EB release from the infected host cell, a new round of infection can begin. Throughout their entire time in the host cell,Chlamydia remain within the confinements of the parasitophorous vacuole, which very early during infection exits the endocytic pathway and becomes instead fusiogenic with a subset of exocytic vesicles originating from the ER/Golgi network and late endosomes (6,7).

Like many other Gram-negative pathogenic bacteria,Chlamydia possess a TTS system that enables them to deliver effector proteins into the host cell (8,9).C. trachomatis EBs rapidly induces its own entry into host cells, through an internalization process believed to be promoted by the TTS effector protein TARP (10). During the early phase of infection,C. trachomatis induces and secretes a set of putative type III effectors (Inc-proteins) (11), of which IncG has been shown to specifically interact with the mammalian signal transducer protein 14-3-3β at the inclusion membrane (12). Little is known about the function of these early-phase proteins that are displayed at the interface of intravacuolarChlamydia and host cell. IncA, a protein induced and secreted during chlamydial mid-cycle, has been demonstrated to be involved in homotypic fusion betweenChlamydia-containing vesicles at high multiplicities of infection (13). In addition to the family of Inc-proteins, other chlamydial proteins secreted into the inclusion membrane have been identified (14,15). Moreover, proteins targeted into the host-cell cytosol have been described (16–18).

In the absence of a genetic system to modifyChlamydia, the function of most of the Inc proteins still remains unknown, and it has not been possible to elucidate whether Inc-proteins or other type III effector proteins secreted into the inclusion membrane or the host cytosol are required forChlamydia to undergo a normal infectious cycle.

In this article, we demonstrate that INP0400, a small molecule identified in a TTS inhibitor screen ofY. pseudotuberculosis, without affectingin vitro multiplication, causes a dose- and growth phase-dependant inhibition ofC. trachomatis RB multiplication. Drug treatment at different stages in the chlamydial developmental cycle reveals a partial block of entry, an inhibition of the translocation of the TTS effectors IncG and IncA during the early and middle phase, respectively, and a bacterial detachment from the inclusion membrane during the late stage concomitant with an inhibition of terminal differentiation from RBs to infectious EBs.

Results

The Small-Molecule TTS Inhibitor INP0400 Given at the Time of Infection Inhibits RB Multiplication Resulting in Small Inclusion Bodies (SIB).

C. trachomatis serovar L2 was used to infect McCoy cells at a low multiplicity of infection (MOI 0.5–1.0). At the time of infection, cells were treated with INP0400, a compound isolated to specifically block TTS ofY. pseudotuberculosis. INP0400 (Fig. 1B) was found to inhibit bacterial replication as shown by immunofluorescence microscopy (Fig. 1A). Inclusion size was affected at concentrations as low as 10 μM, without a detectable effect on the number of infected cells. At a concentration of 20 μM, inclusions were barely visible (Fig. 1A). However, the number of infected cells was only 5–10% lower as compared with untreated control (data not shown). Electron microscopy, after 30 h of infection in the presence of INP0400, showed a dose-dependant reduction in the size of the inclusion bodies and a concomitant reduction in the number of intracellular bacteria (Fig. 1C). At 20 μM or higher of INP0400, the SIB contained only one or few RBs (Fig. 1C).

Fig. 1. — The small molecule INP0400 inhibits RB multiplication. (A) Dose-dependent growth inhibition ofC. trachomatis serovar L2 by INP0400. McCoy cells were infected withC. trachomatis serovar L2 (MOI 0.5–1) and cultured in the presence of INP0400 at the concentrations indicated. At 24 h p.i.,*Chlamydia* were labeled with anti-*Chlamydia*-LPS antibody (green) and analyzed by immunofluorescence microscopy. (Scale bar, 25 μm.) Cells were counterstained with Evan’s blue (red). (B) Chemical structure of INP0400. (C) Electron micrographs of chlamydial inclusions treated with INP0400. McCoy cells were infected withC. trachomatis serovar L2 (MOI 0.5) and cultured in the presence of INP0400 at the concentrations indicated. At 30 h p.i., infected cells were analyzed by electron microscopy. Electron micrographs reveal a dose-dependent reduction of intracellular bacteria, resulting in inclusion bodies with a gradual decrease in size. Chlamydial inclusions in INP0400-treated cultures contain primarily RBs, whereas in untreated controls, RBs have already started to redifferentiate to infectious EBs (arrows indicate RBs and EBs of untreated cells).

Reinfection in the absence of drug after 48 h treatment with INP0400 revealed a dose-dependant reduction in the number of inclusions (Fig. 2). A 20-fold reduction was seen with 20 μM and an impressive 3-log reduction was observed when using 25 μM (Fig. 2). Cytotoxicity assays performed on the McCoy cells were negative for INP0400 at concentrations <70 μM (data not shown).

Partial Inhibition of EB Entry.

C. trachomatis EBs have been shown to be equipped with a TTS apparatus able to secrete one protein, TARP, thought to interact with the actin cytoskeleton, thereby activating the host cell to internalize the organism (10). To see whether INP0400 had an effect on uptake, McCoy cells were infected byChlamydia at a MOI of 0.5 by centrifugation of bacteria onto the cells or by a temperature shift. INP0400 at different concentrations was given at the time of infection and removed after 3 h by washing (Fig. 3). Both methods of infection resulted in a dose-dependant decrease in the number of cells containing detectable inclusion bodies after 40 h growth in the absence of the drug. At the highest concentration used (40 μM), inhibition of entry was ≈40%.

Fig. 3. — Effect of INP0400 on chlamydial entry. McCoy cells were infected withC. trachomatis serovar L2 (MOI 0.5) in the presence of INP0400 at the concentrations indicated. Infection was performed by centrifugation of inoculum onto McCoy cell monolayers or by temperature shift from 4°C to 37°C. At 3 h p.i., infected cells were washed and further grown in the absence of INP0400 for 40 h.Chlamydia were then labeled with anti-*Chlamydia*-LPS antibody, and the number of infected cells was determined by immunofluorescence microscopy. Data are represented as means ± SEM of three independent experiments, each with 2,000 single cells analyzed. A dose-dependent reduction of infected cells was observed for both experimental procedures.

Early Drug Treatment Prevents the IncG-Interacting Host Protein 14-3-3β from DecoratingChlamydia-Containing Inclusion Bodies.

The TTS effector protein IncG is synthesized already 2 h postinfection (p.i.) (19). It localizes to the inclusion membrane and there interacts with the mammalian protein 14-3-3β (12). To see whether INP0400 inhibits IncG translocation, we performed immunofluorescence with antibodies to 14-3-3β 24 h after infection withChlamydia and treatment with INP0400 at 50 μM (Fig. 4). Whereas untreated infected cells accumulated most of their cellular content of 14-3-3β to the inclusion membrane, there was no detectable decoration of SIB, leaving 14-3-3β evenly distributed in the cytosol as in noninfected cells (Fig. 4).

Fig. 4. — Mammalian 14-3-3β is not localized to SIB. (A,D, andG) Uninfected control. McCoy cells were infected withC. trachomatis serovar L2 (MOI 1) and grown in the absence (B,E, andH) or presence (C,F, andI) of INP0400. Infected cells were fixed 24 h p.i. and analyzed by indirect immunofluorescence microscopy with anti-14-3-3β polyclonal antiserum (D–F). Host-cell nuclei and bacterial DNA were stained with DAPI (A–C). (G) 14-3-3β is a cytosolic protein that localizes to the chlamydial inclusion membrane by its interaction with IncG (H). Observe the typical rim-like structure. In drug-treated cells (C,F, andI), no specific interaction with the chlamydial inclusion of SIB (arrows) is found. 14-3-3β remains evenly distributed in the host-cell cytosol. (Scale bar, 10 μm.)

INP0400 Treatment During the Chlamydial Mid-Cycle Blocks IncA Translocation and Homotypic Fusion Without Affecting Inclusion Membrane Targeting of the Early-Phase Effector Protein IncG.

A number of putative chlamydial TTS effector proteins have been identified by screenings in heterologous secretion systems like that ofShigella flexneri orSalmonella typhimurium (16,20). However, the function of most of these effector proteins has still to be assigned. ClinicalC. trachomatis isolates, mutated in the gene encoding the putative TTS effector protein IncA, have been shown to give rise to multiple inclusions after infection with a high MOI in contrast to the single inclusions typically seen in IncA-producing wild-type isolates (21,22). However, intracellular replication ofC. trachomatis was not inhibited in the absence of homotypic fusions. IncA transcript can be detected 10–12 h p.i. and continue to be produced during the mid and late phase of the infectious cycle (13). We therefore infected McCoy cells withC. trachomatis at a MOI of 5 and treated the cells with INP0400 at 50 μM after 8 h of infection. As seen inFig. 5, this concentration of drug inhibited IncA-mediated homotypic fusion, resulting in a number of distinctly separated SIB, each decorated by 14-3-3β as evidence for a successful translocation of IncG, an early-phase TTS effector. In accordance with this finding, we were unable to detect IncA in the inclusion membrane of SIB when following the same treatment protocol (Fig. 6). Intermediate concentrations of INP0400 were also tested, showing that concentrations around 50 μM INP0400 are required to observe these phenotypes (data not shown).

Fig. 5. — INP0400 inhibits homotypic vesicle fusion of SIB. Infected McCoy cells (MOI 5) were grown in the absence of drug for 8 h (A,C, andE). At 8 h p.i., 50 μM INP0400 was added, and cells were allowed to grow for an additional 22 h (B,D, andF). Homotypic vesicle fusion was then analyzed by indirect immunofluorescence staining with anti-14-3-3β polyclonal antibodies (C andD). Host-cell nuclei and bacterial DNA were stained with DAPI (A andB). In untreated cultures, 14-3-3β localizes to the inclusion membrane (E). In drug-treated cultures, single SIB were identified, indicative for impaired vesicle fusion due to the absence of IncA (F). (Scale bar, 10 μm.) 14-3-3β decorates the individual SIB, suggesting that IncG is localized to the inclusion membranes of SIB when INP0400 is added at 8 h p.i.

Fig. 6. — IncA is absent on SIB. McCoy cells were infected withC. trachomatis serovar L2 (MOI 5) and grown in the absence of INP0400 for 8 h (A,C, andE). At 8 h p.i., 50 μM INP0400 was added and cells were allowed to grow for an additional 22 h before analysis by indirect immunofluorescence microscopy (B,D, andF) using IncA polyclonal antibodies (C andD). Host-cell nuclei and bacterial DNA were stained with DAPI (A andB). Although IncA is localized to the chlamydial inclusion membrane of untreated cells (E), no specific IncA staining pattern was observed for SIB in cultures treated with 50 μM INP0400 (F). (Scale bar, 10 μm.)

INP0400 Treatment During the Late Phase of the Infectious Cycle Results in Bacterial Detachment from the Inclusion Membrane and an Inhibition of Terminal Differentiation into Infectious EBs.

Electron-microscopic studies have revealed thatC. trachomatis RBs are typically found juxtaposed to the inner surface of the inclusion membrane during mid-cycle development (23). It is not known, however, whether this attachment requires effectors delivered by the chlamydial TTS system. Likewise, it is not known whether RB association with the inclusion membrane is required for bacterial multiplication and/or RB-to-EB conversion. To specifically examine the effect of INP0400 on chlamydial interaction with the inclusion membrane, McCoy cells were infected withC. trachomatis for 24 h (MOI 0.5–1.0) in the absence of drug to obtain inclusions that could be readily visualized by immunofluorescence microscopy (Fig. 7A). INP0400 at different concentrations was then added to the infected cells. After another 24 h, the infected cells were examined. Untreated cells contained a large inclusion with bacteria filling up the entire inclusion body (Fig. 7B). In contrast, bacteria in drug-treated cells (25 μM and higher) failed to associate with the inclusion membrane (Fig. 7C), leaving a free rim between the inclusion membrane and the bacteria. If INP0400 was removed after 44 h and infected cells incubated in the absence of the drug for another 4 h, bacteria reassociated with the inclusion membrane (Fig. 7D).

Fig. 7. — INP0400 treatment during the late cycle leads to bacterial dissociation from the inclusion membrane and a reduction in recovery of infectious bacteria. McCoy cells infected withC. trachomatis serovar L2 (MOI 0.5–1) were grown for 24 h (A) or 48 h (B) in the absence of INP0400 and stained with anti-*Chlamydia*-LPS antibody (green). McCoy cells are counterstained with Evan’s blue (red). At 24 h p.i., 25 μM INP0400 was added to infected cells. (C) Cells were further grown in the presence of INP0400 and stained 48 h p.i. A dissociation ofChlamydia from the inclusion membrane was observed, resulting in a rim (arrows). (D) In an additional experiment, INP0400 was added 24 h p.i. and removed 44 h p.i. Infected cells were then further grown in fresh medium, and cells were stained 48 h p.i. (Scale bar, 10 μm.)*Chlamydia* were found to be reassociated with the inclusion membrane. (E) Addition of INP0400 to established inclusions at 24 h leads to a dose-dependent reduction of infectious progeny after 48 h. Data are means ± SEM of three independent experiments. (F) Loss of infectivity after INP0400 treatment during the late cycle is reversible. McCoy cells infected withC. trachomatis serovar L2 (MOI 0.5–1) were grown for 48 h in presence of 30 μM INP0400 as indicated. A reduction of infectious progeny was observed in treated cultures as assessed at 48 h p.i. If INP0400 was removed after 12 h, more infectious bacteria were recovered compared with infected cells treated for 24 h with INP0400.

After incubation for 48 h in the absence of INP0400, conversion of RBs to EBs has occurred, resulting in a high number of infectious units upon reinfection (Fig. 7E). Addition of the drug after 24 h of infection resulted in a dose-dependant decrease in infectivity after 48 h despite the fact that drug treatment between 24 and 48 h in the infectious cycle had no apparent effect on bacterial growth (Fig. 7C). Hence, INP0400 inhibits terminal RB-to-EB differentiation during the late cycle. This effect of the drug was reversible (Fig. 7F). Thus, compared with cells treated from 24 to 48 h in the infectious cycle, infectivity increased by 1-log when INP0400 added at 24 h was removed after 12 h and infected cells were allowed to incubate for another 12 h in the absence of drug (Fig. 7F).

Discussion

A class of acylated hydrazones was recently identified that inhibited TTS ofY. pseudotuberculosis without affecting thein vitro growth rate (2,3). The same class of compounds has also been shown to inhibit TTS ofPseudomonas aeruginosa as well asS. typhimurium (unpublished data), suggesting that these compounds target the secretory apparatus itself rather than individual effector molecules. The obligate intracellular pathogenC. trachomatis also possesses a TTS system believed to deliver a large number of chlamydial proteins into either the inclusion membrane or the host cytosol. So far, only one protein, Tarp, has been shown to be delivered from extracellular EBs upon contact with target cells, whereas the other putative TTS effectors are produced at specific stages during the chlamydial infectious cycle. The differential expression pattern of TTS effectors suggests that they play an instrumental role at different stages in the chlamydial developmental cycle. However, lack of genetic tools has made it difficult to determine whether TTS effectors are needed forChlamydia to undergo its different developmental phases. There are, however, evidences that TTS effectors promote entry (TARP) and modulate properties of the inclusion membrane to escape from the endosomal pathways, and to promote heterotypic as well as homotypic interactions.

Here, we initiate a chemical genetic approach to dissect a role forChlamydia TTS during different stages in the infectious cycle ofChlamydia. TTS inhibitors have been screened such that they do not significantly affect thein vitro growth rate ofYersinia. In contrast, in the obligate intracellular pathogensC. trachomatis andC. pneumoniae (L.B., Å.G., C.S., S.M., M.E., P. Nordström, B.H.-N., A. Waldenström, H.W.-W., and S.B., unpublished data), those TTS inhibitors tested exhibited a dose-dependant antibacterial activity, suggesting that the TTS system ofChlamydia is required for the organism to undergo a productive infectious cycle.

The small molecule INP0400 partially inhibited uptake of EBs, suggesting an inhibitory effect on the internalization process. This inhibition, however, was not complete, meaning either that the compound did not completely inhibit translocation of Tarp from EBs or that some internalization may proceed also in the absence of this effector protein. Internalized EBs in the presence of drug converted to RBs, but RB multiplication was inhibited in a dose-dependant fashion, resulting in ultimately smaller inclusion bodies (SIB) containing just one or a few RBs.

We demonstrate that early drug treatment prevented the host protein 14-3-3β from localizing to the inclusion membrane of SIB, a process known to depend on translocation of the TTS effector IncG into the inclusion membrane. IncG is expressed already 2 h after infection (19). This correlates well with our finding that infected cells treated 8 h in the infectious cycle result in SIB able to recruit 14-3-3β to the membrane, indicative of a normal IncG translocation. Whether early treatment with INP0400 affects translocation of all early TTS effectors is not known. The antibacterial activity of INP0400, however, can not be dependent on an inhibition of TTS translocation in the early phase, because inhibition of RB multiplication was also seen when the drug was added during the chlamydial mid-cycle 3–18 h p.i. During this period, around 10–12 h p.i., IncA is transcribed and localized to the inclusion membrane. This effector protein is firmly linked to the ability of inclusion bodies to undergo homotypic fusions resulting in one single inclusion body in cells that are multiply infected byChlamydia. Using a high infection dose, we show that homotypic fusions are blocked by INP0400 given at 8 h, a time point in the chlamydial developmental cycle that precedes the onset of IncA production. Although these multiple SIB contained translocated IncG, an early-phase effector, we could not detect IncA in these nonfused inclusion bodies. Based on these data, we argue that INP0400 can inhibit TTS inC. trachomatis. It is tempting to suggest that the inhibitory effect of INP0400 on RB multiplication during the chlamydial mid-cycle is due to an inhibited translocation of other TTS effectors than IncA, becauseincA mutants ofC. trachomatis have been found among clinical isolates (21).

Little is known about TTS effectors secreted during the late phase of the infectious cycle apart from the chlamydial CADD protein that has been suggested to modulate apoptosis (17). Whether TTS is needed to trigger differentiation from RBs to EBs is not known. We could demonstrate here that INP0400 treatment during this phase had a dramatic effect on RB-to-EB terminal differentiation, resulting in a dramatic decrease of infectivity upon reinfection. This drug-mediated inhibition of RB-to-EB conversion was associated with a reversible detachment of the RBs from the inclusion membrane. It is not known whether RBs need to associate with the inclusion membrane for their multiplication and differentiation. The findings that host lipids are incorporated into the chlamydial membrane (24,25) are consistent with capture of host lipids through bacterial binding to the inclusion membrane. For enteropathogenicEscherichia coli, it has been firmly established that this pathogen, through its TTS system, inserts its own receptor into the mammalian cell membrane, to which it then adheres to trigger additional host signaling events (26). A similar mechanism could explain the attachment seen during normal chlamydial infection. The observed detachment seen in the presence of INP0400 is therefore compatible with an inhibition of chlamydial TTS.

TTS inhibitors represent a new paradigm for treatment of infectious diseases caused byYersinia spp.,Salmonellaspp.,Shigella spp., and other Gram-negative pathogens that depend on TTS for the dedicated delivery of effector proteins modulating and/or inhibiting host defenses. A major limitation for clinical use of TTS inhibitors on these extracellular and facultative intracellular pathogens, however, is likely to be due to difficulties in performingin vitro susceptibility testing, requiring the development also of novel diagnostic procedures. If, as our data suggest, TTS is essential for the obligate intracellular pathogenC. trachomatis to undergo a normal infectious cycle, susceptibility testing can be performed as is currently being done by conventional antibiotics. Also, more active TTS inhibitors can be directly screened for by usingChlamydia rather than the extracellular pathogenYersinia as the target organism. Finally, the exact mode of action for TTS inhibitors can more easily be worked out inChlamydia, by isolating and characterizing mutants resistant to the drugs.

Materials and Methods

Antibodies and Reagents.

INP0400, a small-molecule inhibitor isolated to block TTS ofY. pseudotuberculosis, was provided by Innate Pharmaceuticals. Rabbit polyclonal antibodies against mammalian 14-3-3β were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antiserum specific to IncA was generated by Innovagen (Lund, Sweden), using a peptide containing the following amino acids: CSQIRETLSSPRKSA (corresponding to amino acids 252–266). Cy3-conjugated goat anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The PathfinderChlamydia culture confirmation kit, containing genus-specific fluorescein-conjugated murine monoclonal antibodies toChlamydia-LPS, was obtained from Bio-Rad (Hercules, CA). The nucleic acid stain DAPI (4′,6′-diamino-2-phenylindole) was purchased from Sigma-Aldrich (St. Louis, MO).

Cell Culture and Propagation ofChlamydia.

C. trachomatis serovar L2 (ATCC VR-902B) was propagated in McCoy cells (ATCC CRL-1696). Cells were grown at 37°C with 5.0% CO₂ in RPMI medium 1640 supplemented with 10% (vol/vol) FBS, 25 mM Hepes, 2 mMl-glutamine and 10 μg/ml gentamicin. For propagation, McCoy cells were infected with serovar L2 and grown for 48 h in medium additionally containing 2 μg/ml cycloheximide. At 48 h p.i., cells were scraped off into medium and rigorously vortexed with glass beads to release bacteria from the cells. Whole lysates were centrifuged at 500 ×g to pellet cellular debris. Supernatant containingChlamydia was stored in aliquots at −70°C for later infection. If not otherwise stated, McCoy cell monolayers were infected with L2 at a MOI of 0.5–1 and centrifuged at 1,000 ×g for 45 min at 35°C. Cells were subsequently washed twice with Hank’s balanced salt solution (HBSS) and incubated in medium containing cycloheximide (see above) for the times indicated.

Cytotoxicity Assay.

Cytotoxicity of INP0400 was assessed by using a calceinAM assay (L.B., Å.G., C.S., S.M., M.E., P. Nordström, B.H.-N., A. Waldenström, H.W.-W., and S.B., unpublished data). Briefly, McCoy cells were grown in the presence of INP0400 at different concentrations and times. Cytotoxicity was then analyzed by the ability of living cells to convert nonfluorescent calceinAM to green fluorescent calcein. Fluorescence intensities were measured in a microplate reader, and the relative amount of living cells compared with dead cells was calculated.

Inhibitors andChlamydia.

McCoy cells were infected as described above and grown in the presence of TTS inhibitor INP0400 for the concentrations and times indicated. Effects of inhibitors on chlamydial growth were determined by immunofluorescence staining, infectivity assays, and electron microscopy.

Infectivity Assay.

Infected McCoy cells were cultured in the presence of INP0400 dissolved in medium at different concentrations (0–50 μM). At 48 h p.i., cells were washed twice with HBSS and harvested as described above. Bacterial suspensions obtained were used to reinfect fresh monolayers of McCoy cells. Infected cells were grown for 24 h in the absence of INP0400 and immunolabeled with anti-Chlamydia-LPS antibody. Immunofluorescence microscopy was performed to determine the number of infection-forming units. Data are represented as means ± SEM of three independent experiments, each with 2,000 single cells analyzed.

Immunofluorescence Microscopy.

McCoy cells were grown on sterile glass coverslips and infected as described above. For indirect immunofluorescence staining, coverslips were fixed in ice-cold methanol for 10 min at the times indicated. Coverslips were air-dried and washed three times with PBS. Permeabilization was performed with PBS/0.5% Triton X-100 for 5 min. Cells were washed three times with PBS and blocked overnight in 10% goat-serum/PBS. Incubation with first antibody (1:100–1:300) was performed for 45 min at 37°C in the dark. Coverslips were washed three times with PBS, and cells were subsequently incubated with secondary antibody (1:300) for 45 min at 37°C. Host-cell nuclei and intracellular bacteria were stained with the DNA-specific fluorochrome DAPI (1 μg/ml) for 2 min. Coverslips were mounted on Mowiol containing 10% wt/vol 1,4-diazabicyclo[2.2.2]octane (DABCO). When using the PathfinderChlamydia culture confirmation kit, coverslips were fixed in methanol as described above and directly stained forChlamydia. Images were acquired by using a Leica (Vienna, Austria) DMRE fluorescence microscope. Random areas were selected for analysis. Pictures were processed by using Photoshop 7 software (Adobe Systems, San Jose, CA).

Transmission Electron Microscopy.

McCoy cells infected withC. trachomatis L2 (MOI 0.5–1) were fixed at the indicated time points in 2% glutaraldehyde and 0.5% paraformaldehyde in 0.1 M sodium cacodylate buffer containing 0.1 M sucrose and 3 mM CaCl₂ (pH 7.4) at room temperature for 30 min and stored at 4°C until embedding. After fixation, cells were rinsed in 0.15 M sodium cacodylate buffer containing 3 mM CaCl₂ (pH 7.4) and centrifuged. The pellets were resuspended and postfixed in 2% osmium tetroxide in 0.07 M sodium cacodylate buffer containing 1.5 mM CaCl₂ (pH 7.4) at 4°C for 2 h, dehydrated in ethanol followed by acetone, and embedded in LX-112 (Ladd, Burlington, VT). Sections were contrasted with uranyl acetate followed by lead citrate and examined in a Tecnai 10 transmission electron microscope (FEI, Hillsboro, OR) at 80 kV. Digital images were captured by using a MegaView III digital camera (Soft Imaging System, Münster, Germany).

Acknowledgments

This work was partly funded by VINNOVA, the Swedish Foundation for Strategic Research, the Swedish Research Council and Torsten and Ragnar Söderbergs Foundation, and the European Marie Curie program European Initiative for basic research in Microbiology and Infectious Diseases.

Abbreviations

EB: elementary body
MOI: multiplicity of infection
p.i.: postinfection
RB: reticulate body
SIB: small inclusion bodies
TTS: type III secretion.

Footnotes

Conflict of interest statement: C.S. is employed by and M.E., H.W.-W., and S.N. are associated with Innate Pharmaceuticals.

References

1.Hung DT, Shakhnovich EA, Pierson E, Mekalanos JJ. Science. 2005;310:670–674. doi: 10.1126/science.1116739. [DOI] [PubMed] [Google Scholar]
2.Kauppi AM, Nordfelth R, Uvell H, Wolf-Watz H, Elofsson M. Chem Biol. 2003;10:241–249. doi: 10.1016/s1074-5521(03)00046-2. [DOI] [PubMed] [Google Scholar]
3.Nordfelth R, Kauppi AM, Norberg HA, Wolf-Watz H, Elofsson M. Infect Immun. 2005;73:3104–3114. doi: 10.1128/IAI.73.5.3104-3114.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
4.Schachter J. In: Chlamydia: Intracellular Biology, Pathogenesis, and Immunity. Stephens RS, editor. Washington, DC: Am Soc Microbiol; 1999. pp. 139–169. [Google Scholar]
5.AbdelRahman YM, Belland RJ. FEMS Microbiol Rev. 2005;29:949–959. doi: 10.1016/j.femsre.2005.03.002. [DOI] [PubMed] [Google Scholar]
6.Hackstadt T, Rockey DD, Heinzen RA, Scidmore MA. EMBO J. 1996;15:964–977. [PMC free article] [PubMed] [Google Scholar]
7.Beatty WL. J Cell Sci. 2006;119:350–359. doi: 10.1242/jcs.02733. [DOI] [PubMed] [Google Scholar]
8.Hsia RC, Pannekoek Y, Ingerowski E, Bavoil PM. Mol Microbiol. 1997;25:351–359. doi: 10.1046/j.1365-2958.1997.4701834.x. [DOI] [PubMed] [Google Scholar]
9.Hueck CJ. Microbiol Mol Biol Rev. 1998;62:379–433. doi: 10.1128/mmbr.62.2.379-433.1998. [DOI] [PMC free article] [PubMed] [Google Scholar]
10.Clifton DR, Fields KA, Grieshaber SS, Dooley CA, Fischer ER, Mead DJ, Carabeo RA, Hackstadt T. Proc Natl Acad Sci USA. 2004;101:10166–10171. doi: 10.1073/pnas.0402829101. [DOI] [PMC free article] [PubMed] [Google Scholar]
11.Rockey DD, Scidmore MA, Bannantine JP, Brown WJ. Microbes Infect. 2002;4:333–340. doi: 10.1016/s1286-4579(02)01546-0. [DOI] [PubMed] [Google Scholar]
12.Scidmore MA, Hackstadt T. Mol Microbiol. 2001;39:1638–1650. doi: 10.1046/j.1365-2958.2001.02355.x. [DOI] [PubMed] [Google Scholar]
13.Hackstadt T, Scidmore-Carlson MA, Shaw EI, Fischer ER. Cell Microbiol. 1999;1:119–130. doi: 10.1046/j.1462-5822.1999.00012.x. [DOI] [PubMed] [Google Scholar]
14.Fling SP, Sutherland RA, Steele LN, Hess B, D'Orazio SE, Maisonneuve J, Lampe MF, Probst P, Starnbach MN. Proc Natl Acad Sci USA. 2001;30:1160–1165. doi: 10.1073/pnas.98.3.1160. [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Fields KA, Hackstadt T. Mol Microbiol. 2000;38:1048–1060. doi: 10.1046/j.1365-2958.2000.02212.x. [DOI] [PubMed] [Google Scholar]
16.Subtil A, Delevoye C, Balana ME, Tastevin L, Perrinet S, Dautry-Varsat A. Mol Microbiol. 2005;56:1636–1647. doi: 10.1111/j.1365-2958.2005.04647.x. [DOI] [PubMed] [Google Scholar]
17.Stenner-Liewen F, Liewen H, Zapata JM, Pawlowski K, Godzik A, Reed JC. J Biol Chem. 2002;277:9633–9636. doi: 10.1074/jbc.C100693200. [DOI] [PubMed] [Google Scholar]
18.Zhong G, Fan P, Ji H, Dong F, Huang Y. J Exp Med. 2001;193:935–942. doi: 10.1084/jem.193.8.935. [DOI] [PMC free article] [PubMed] [Google Scholar]
19.Scidmore-Carlson MA, Shaw EI, Dooley CA, Fischer ER, Hackstadt T. Mol Microbiol. 1999;33:753–765. doi: 10.1046/j.1365-2958.1999.01523.x. [DOI] [PubMed] [Google Scholar]
20.Ho TD, Starnbach MN. Infect Immun. 2005;73:905–911. doi: 10.1128/IAI.73.2.905-911.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Suchland RJ, Rockey DD, Bannantine JP, Stamm WE. Infect Immun. 2000;68:360–361. doi: 10.1128/iai.68.1.360-367.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Rockey DD, Viratyosin W, Bannantine JP, Suchland RJ, Stamm WE. Microbiology. 2002;148:2497–2505. doi: 10.1099/00221287-148-8-2497. [DOI] [PubMed] [Google Scholar]
23.Hackstadt T, Fischer ER, Scidmore MA, Rockey DD, Heinzen RA. Trends Microbiol. 1997;5:305–306. doi: 10.1016/S0966-842X(97)01061-5. [DOI] [PubMed] [Google Scholar]
24.Wylie JL, Hatch GM, McClarty G. J Bacteriol. 1997;179:7233–7242. doi: 10.1128/jb.179.23.7233-7242.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Newhall WJ. In: Microbiology of Chlamydia. Barron AL, editor. Boca Raton, FL: CRC; 1988. pp. 47–70. [Google Scholar]
26.Kenny B, DeVinney R, Stein M, Reinscheid DJ, Frey EA, Finlay BB. Cell. 1997;91:511–520. doi: 10.1016/s0092-8674(00)80437-7. [DOI] [PubMed] [Google Scholar]

Movatterモバイル変換

PERMALINK

A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle ofChlamydia trachomatis

Sandra Muschiol

Leslie Bailey

Åsa Gylfe

Charlotta Sundin

Kjell Hultenby

Sven Bergström

Mikael Elofsson

Hans Wolf-Watz

Staffan Normark

Birgitta Henriques-Normark

Abstract

Results

The Small-Molecule TTS Inhibitor INP0400 Given at the Time of Infection Inhibits RB Multiplication Resulting in Small Inclusion Bodies (SIB).

Fig. 1.

Fig. 2.

Partial Inhibition of EB Entry.

Fig. 3.

Early Drug Treatment Prevents the IncG-Interacting Host Protein 14-3-3β from DecoratingChlamydia-Containing Inclusion Bodies.

Fig. 4.

INP0400 Treatment During the Chlamydial Mid-Cycle Blocks IncA Translocation and Homotypic Fusion Without Affecting Inclusion Membrane Targeting of the Early-Phase Effector Protein IncG.

Fig. 5.

Fig. 6.