Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle

Abstract

The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) that is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the interdependence of the chlamydial T3SS and development. C1 treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.

Fig. 1

Dose-dependent inhibition of C. trachomatis but not C. burnetii development by C1. HeLa (C. trachomatis) or Vero (C. burnetii) cells were infected at an MOI of 0.5-1 with ca. 10⁶ C. trachomatis L2 or 10⁴ C. burnetii, respectively. Immediately after infection, DMSO was added as a control (mock) or C1 was added to 5, 10, 25, or 50 μM final. Cultures were maintained for 24 hr (Chlamydia) or 96 hrs (Coxiella) then disrupted for second passage on fresh cell culture monolayers lacking C1. Bacteria were stained with Chlamydia- or Coxiella-specific antibodies and C. trachomatis IFUs (white bars) or C. burnetii FFUs (dark grey bars) were enumerated by direct count of duplicate samples via indirect immunofluorescence microscopy. Data are represented as mean ± standard deviation.

Fig. 2

Indirect immunofluorescence analysis of C1-treated cultures. C. trachomatis L2-infected and DMSO (mock) or C1-treated cultures were examined to assess development of chlamydial inclusions. All cultures were fixed at 24 hr p.i., and chlamydiae were detected by probing with α-L2 followed by Alexa 488-conjugated secondary antibodies. Host-cell nuclei were visualized via DAPI staining of DNA. Epi-fluorescence images were acquired at 90X magnification and relative magnification of insets was maintained for each treatment. Bar = 5 μm.

Fig. 3

C1-treated Chlamydia are metabolically active. (A) C. trachomatis L2-infected cultures were either treated with DMSO (mock) or 50 μM C1 for 20 hr. Whole culture DNA and RNA was harvested and chlamydial genome content normalized by QC-PCR prior to RT-PCR analysis. RNA corresponding to ca. 10⁵ chlamydial genome equivalents was added to RT-PCR reactions in the presence (+) or absence (−) of reverse transcriptase (RT) and primers specific for representative genes from early-, mid-, and late-cycle development. Amplification products were resolved in 2.0% (wt/vol) agarose gels and visualized by staining with ethidium bromide. B) C. trachomatis L2-infected HeLa cultures were labeled with C₆-NBD-Ceramide and subjected to 1 hr of back-exchange. Uptake of fluorescent sphingomyelin by chlamydiae was detected in DMSO (mock) or C1-treated cultures by immunofluorescence microscopy at 90X magnification. Bar = 5 μm.

Fig. 4

C1-mediated inhibition of Chlamydia development is reversible. Duplicate C. trachomatis L2-infected HeLa monolayers were either mock-treated with DMSO, treated with 5, 10, 25, and 50 μM C1 or with 20 μg ml⁻¹ ampicillin (Amp) for 24 hr. C1 or Amp was then washed out of one replicate and all cultures were maintained for an additional 20 hr prior to cell lysis and re-plating for IFU quantitation on fresh HeLa monolayers. Dark grey bars correspond to cultures where C1 was washed out and white bars to cultures continuously maintained in C1. IFUs were directly enumerated in duplicate by indirect immunofluorescence microscopy. Data are represented as mean ± standard deviation.

Fig. 5

C1 treatment causes accumulation of T3SS substrates in RBs. DMSO (mock) or C1 (added to15 μM final) was added to HeLa monolayers immediately after infection at an MOI of 1 with C. trachomatis L2. (A) RBs were purified to homogeneity from disrupted cultures at 15 hr p.i. and proteins concentrated via TCA precipitation. SDS-PAGE-resolved proteins were probed in immunoblots with Chlamydia-specific antibodies (α-L2) generated against whole bacteria or individual proteins CdsJ, CopN, CopB2, CADD, IncA, and Tarp. (B) Whole-culture material from similarly treated cultures that were mock infected (M) or L2 infected and untreated (−C1) or C1 treated (+C1) was resolved via SDS-PAGE and probed with antibodies specific for CdsJ, CADD, Tarp. Respective proteins were visualized by probing with horseradish peroxidase-conjugated (A) or alkaline phosphatase-conjugated (B) secondary antibodies and development with appropriate chemical substrates.

Fig. 6

IncA secretion to the inclusion membrane is inhibited in C1 treated Chlamydia cultures. (A) HeLa monolayers were infected with C. trachomatis L2 at an MOI of ca. 5 and cultivated for 6 hr prior to addition of DMSO (−C1), 50 μM C1 (+C1), or 100 μg/ml ampicillin (AMP). At 20 hr p.i., cultures were paraformaldehyde fixed, permeablized and probed with Hsp60- and IncA-specific antibodies. Samples were stained with either Alexa 594-conjugated (Hsp60) or Alexa 488-conjugated (IncA) secondary antibodies and visualized via laser-scanning confocal microscopy. Bars = 5 μm. (B) Whole-culture lysates were prepared from similarly treated cultures infected at an MOI of 5, proteins resolved via SDS-PAGE, and probed in immunoblots with α-RFX5, α-Caspase-1, or α-Actin as a loading control. Respective proteins were visualized by probing with horseradish peroxidase-conjugated secondary antibodies and development with chemiluminescent reagent.

Fig. 7

Addition of C1 during mid-cycle development inhibits differentiation of RBs into EBs. C. trachomatis L2-infected HeLa monolayers were cultivated for 15 hr prior to addition of DMSO (mock), C1 to 50 μM, or chloramphenicol (Cm) to 200 μg ml⁻¹. Cultures were disrupted 24 hr later and EB levels determined by IFU assay performed on fresh HeLa monolayers. IFUs were directly enumerated in duplicate by indirect immunofluorescence microscopy, and data are represented as mean ± standard deviation.