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

Abstract

The intracellular pathogen Chlamydia 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 of Yersinia pseudotuberculosis. Here, we show that one of these compounds, INP0400, inhibits intracellular replication and infectivity of C. 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 14-3-3beta 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.

Fig. 1.

The small molecule INP0400 inhibits RB multiplication. (A) Dose-dependent growth inhibition of C. trachomatis serovar L2 by INP0400. McCoy cells were infected with C. 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 with C. 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).

Fig. 2.

Dose-dependent reduction of infectious bacteria. (A) Infectivity assay of INP0400-treated bacteria. McCoy cells infected with C. trachomatis serovar L2 (MOI 0.5) were cultured for 48 h in the presence of INP0400 at concentrations of 0–25 μM. At 48 h p.i., bacteria were harvested and inoculated undiluted onto fresh monolayers of McCoy cells. Infected cells were then grown in the absence of INP0400 and stained for Chlamydia 24 h p.i. (Scale bar, 50 μm.) Chlamydial titers of untreated cultures were too high to be used undiluted, and cells were lysed upon reinfection (data not shown). Similar results were observed for bacteria treated with 5 and 10 μM. Cells treated with 5 μM were already stained at 7 h p.i. for Chlamydia inclusions. (B) A dose-dependent reduction of infectious progeny was observed by immunofluorescence staining and quantification of infectious progeny. Data are represented as means ± SEM of three independent experiments.

Fig. 3.

Effect of INP0400 on chlamydial entry. McCoy cells were infected with C. 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.

Fig. 4.

Mammalian 14-3-3β is not localized to SIB. (A, D, and G) Uninfected control. McCoy cells were infected with C. trachomatis serovar L2 (MOI 1) and grown in the absence (B, E, and H) or presence (C, F, and I) 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, and I), 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.)

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, and E). At 8 h p.i., 50 μM INP0400 was added, and cells were allowed to grow for an additional 22 h (B, D, and F). Homotypic vesicle fusion was then analyzed by indirect immunofluorescence staining with anti-14-3-3β polyclonal antibodies (C and D). Host-cell nuclei and bacterial DNA were stained with DAPI (A and B). 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 with C. trachomatis serovar L2 (MOI 5) and grown in the absence of INP0400 for 8 h (A, C, and E). 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, and F) using IncA polyclonal antibodies (C and D). Host-cell nuclei and bacterial DNA were stained with DAPI (A and B). 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.)

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 with C. 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 of Chlamydia 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 with C. 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.