Reagents and antibodies.
C6-NBD-ceramide was purchased from Life Technologies (Grand Island, NY; catalog number N-1154). The original high-throughput screen was conducted at SIGA Technologies (Corvallis, OR), who supplied micromolar amounts of the original compounds. Milligram amounts of (3-methoxyphenyl)-(4,4,7-trimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-C]quinolin-1-ylidene)amine (MDQA) were purchased from a commercial source (Chembridge, San Diego, CA; catalog number 5678481). Cells infected with Chlamydia
spp. or Coxiella burnetii
were labeled with monoclonal or polyclonal primary antibodies specific to chlamydial lipopolysaccharide (LPS) (34
) or whole C. burnetii
). Species- and isotype-specific secondary antibodies were purchased from Life Technologies (Alexa Fluor 488 [green] and 594 [red]).
Host cells, chlamydial strains, and cultures.
McCoy cells were propagated in minimum essential medium supplemented with 10% fetal bovine serum (MEM-10) and 10 μg/ml gentamicin. The following chlamydia strains were used in this study; C. caviae GPIC, C. trachomatis L2-434, C. trachomatis J6276, Chlamydia psittaci CP3, and Chlamydia muridarum MOPN. Other bacteria used to evaluate broad-spectrum activity of compounds were Coxiella burnetii Nine Mile phase II (obtained from Robert Heinzen, Rocky Mountain Laboratories), Staphylococcus aureus (ATCC number 25923), methicillin-resistant S. aureus (MRSA; ATCC 43300), and Escherichia coli (ATCC 25922).
Over 40,000 compounds were assayed from the SIGA chemical compound library for antichlamydial activity. The chemical library owned by SIGA Technologies consists of approximately 250,000 compounds, representing a broad chemical structural and property diversity space. Screening of this library has identified unique and potent drug-like compounds representing diverse and novel chemical structural classes that are selectively active against phylogenetically unrelated viral families.
We tested 4,500 compounds each week using 48 96-well clear-bottom, black-walled plates. A total of 5 × 104
McCoy cells were plated in 100 μl of MEM into wells and incubated overnight to a confluence of 100%. A Perkin-Elmer MultiPROBE II HT Plus robotic system was used to deliver individual structurally defined candidate inhibitory compounds from the SIGA library to 80 wells of each plate at a concentration of 5 μM. Wells in the first column of each plate were mock-infected controls, and the last column of each plate contained infected wells not treated with any compound. Controls in each assay also included wells incubated with chlamydiae plus tetracycline at 10 μg/ml. Gradient-purified (7
) C. caviae
GPIC cells were inoculated onto wells of the plates at a multiplicity of infection of between 1 and 2 inclusion-forming units per cell. Chlamydiae were centrifuged onto cells at 1,200 × g
for 1 h at 37°C and incubated under standard culture conditions for 24 h.
At 24 h postinfection, medium was aspirated from cells and replaced with 50 μl of a 10 μM C6-NBD-ceramide in phosphate-buffered saline (PBS). Plates were incubated for 1 h at 37°C, and then the fluorescent compound was aspirated from each well. Cells were then overlaid with MEM-10 and incubated at 37°C for an additional 3 h. At 28 h postinfection, the medium was replaced with PBS and fluorescence intensity was quantified on a Wallac Envision plate reader using an excitation wavelength of 485 nm and emission wavelength of 535 nm, with the instrument programmed to take readings from the bottom of the well. Positive inhibitor compounds were identified by a reduction in fluorescence intensity of 50% or greater relative to that of untreated but infected control wells. All wells identified as positive by the plate reader were evaluated visually on the fluorescence microscope in order to eliminate wells with culture artifacts and to identify compounds that were acutely toxic to the cells.
A total of 1 × 105 cells were plated onto 12-mm coverslips in 24-well trays, infected as described above, fixed with 100% methanol for 10 min, and washed twice with PBS. Fixed cells were labeled with monoclonal mouse anti-MOMP or anti-LPS primary antibodies at a 1:1,000 dilution followed by appropriate secondary antibodies at a 1:1,000 dilution.
A total of 4.5 × 105 cells were plated in 24-well trays and incubated overnight to a confluence of 100%. Bacterial strains were suspended in MEM-10 and infected onto cells at a multiplicity of infection of 0.5 to 1 bacteria per cell. Plates were centrifuged at 1,200 × g at 37°C for 1 h. Media were aspirated, and then compounds were suspended in fresh MEM-10 and added to plates in 0.5-ml aliquots and transferred to a 37°C incubator.
Quantification of bacteria from infected cell culture using TaqMan quantitative real-time PCR.
At the appropriate time points, infected cells were briefly sonicated to lyse and release bacteria. The lysates were collected in microcentrifuge tubes and stored at −20°C prior to analysis. DNA was extracted using the Qiagen DNeasy blood and tissue kit protocol with the addition of 5 mM dithiothreitol (DTT) to the initial lysis buffer. Quantitative PCR (qPCR) was run on all samples in triplicate using the TaqMan universal PCR master mix (Applied Biosystems), with an input of 5 μl of template DNA from each treatment for C. caviae GPIC and C. trachomatis L2-434-infected cells. Primers and probes were specific to C. caviae ompA (F-CCCTGCGCGGATGCT, R-CAGGCGATCCTTGTGATCCT, probe 6-carboxyfluorescein [FAM]-CATCACACCAAGTAGAGC-MGBNFQ) and C. trachomatis ompA (F-CATGGTATCTCCGAGCTGACC, R-ACTGTCTTTGATGTTACCACTCTGAAC, probe 6FAM-CTAGCTTTCACATCGCC-MGBNFQ). Tenfold serial dilutions of quantitated plasmid standards containing each targeted gene were included at copies ranging from 1 × 109 to 1 × 103. Genome copy number and standard error were calculated using an ABI StepOne real-time PCR machine with standard curve settings.
Acid sensitivity and cell cytotoxicity.
The acid lability of MDQA was assessed by suspending the compound at 10 mM in a citric acid buffer (10 mM citric acid, 10 mM KCl, 135 mM NaCl, pH 3) for 10 min prior to dilution to 10 μM and the addition to C. trachomatis L2-434-infected cells. Chlamydial growth was assayed 40 h postinoculation using qPCR. The MTT reagent was used as a measure of cytotoxicity for compounds, which were tested at concentrations up to 50 μM, using a standard assay system (Life Technologies). Rabbit kidney, murine McCoy, human HeLa, and bovine embryonic kidney immortalized cell lines were tested in these assays.
Secondary confirmation and MIC analysis.
MICs of each compound were determined using the Hewlett Packard D300 digital dispenser (Tecan Systems, San Jose, CA) that delivered compound in half-log dilutions to 96-well plates in concentrations that ranged from 20 μM to 0.0063 μM. Chlamydiae were then infected onto cells as described above under “Primary screen” and incubated until they were fixed for fluorescent antibody analysis (C. trachomatis) or labeled with C6-NBD-ceramide (C. caviae GPIC) to determine the MIC. When immunofluorescence was used to measure inhibition, the MIC was determined microscopically and scored as the inhibitory concentration that reduced observable inclusion formation by at least 90%. In assays where the C6-NBD-ceramide assay was used to examine inhibition, the MIC was determined by a quantitative measure of reduction of fluorescence to levels at or below the background. This generally translated to a reduction of fluorescence to below 12,500 on an Envision multiplate fluorescence spectrometer.
Selection for resistance.
Strains of C. trachomatis L2-434 were inoculated onto McCoy cells in 24-well trays using a multiplicity of infection between 1 and 10, depending on the strain. Infections were centrifuged at 1,200 × g for 1 h at 37°C, overlaid with subinhibitory concentrations (1/2 of MIC) of the appropriate drug, and incubated at 37°C for 24 to 48 h depending on the species inoculated. The cell monolayers were then disrupted by −80°C/37°C freeze-thaw or sonication, and then the lysate was centrifuged at 1,500 rpm for 10 min to pellet cell debris. Supernatants from disrupted cell monolayers were used to infect fresh McCoy cell monolayers in 24-well plates, and the concentration of compound was increased incrementally in sequential cultures until the MIC exceeded that of the original wild-type parental strains by 2-fold or greater. Emerging resistance was monitored using immunofluorescence. As resistant chlamydiae emerged in the treated population, they were cloned by limiting dilution.
Paired-end Illumina genome sequencing.
Purified elementary bodies were prepared for sequencing as previously described (35
). Draft genomes were assembled using the reference-guided assembly software Maq (http://maq.sourceforge.net/
) and aligned to the published C. trachomatis
L2-434 (GenBank accession no. AM884177
) or C. muridarum
(GenBank accession no. AE002160
) genome sequences. Single nucleotide polymorphisms (SNPs) identified by the genome alignment were confirmed by standard PCR sequencing. Any necessary manual sequence analysis was performed using MacVector sequence analysis software (MacVector, Cary, NC).
SecY structure prediction and modeling.
The amino acid sequence of C. trachomatis
SecY was submitted to the Phyre prediction server on the Web to predict the three-dimensional (3D) structure (17
). The mutated residues in the resistant SecY genes were mapped onto the predicted SecY three-dimensional structure using the UCSF Chimera modeling program (24