Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation

Strains, media, and chemicals.M. tuberculosis H37Rv, rv3852 deletion mutant (Δrv3852), and phoP deletion mutant (ΔphoP) (27) were grown at 37°C either in Middlebrook 7H9 broth (Difco) supplemented with 10% albumin-dextrose-catalase, 0.2% glycerol, and 0.05% Tween 80 or in Sauton's liquid medium supplemented with 0.005% Tween 80. Where necessary, the medium was buffered with 10% morpholineethanesulfonic acid (MES) (pH 5 or 6) or morpholinepropanesulfonic acid (MOPS) (pH 7). Cultures were plated on Middlebrook 7H10 (Difco) agar supplemented with 10% oleic acid-albumin-dextrose-catalase and 0.2% glycerol. Hygromycin (50 μg ml⁻¹), kanamycin (25 μg ml⁻¹), streptomycin (25 μg ml⁻¹), chloramphenicol (30 μg ml⁻¹), or 2.5% sucrose was added when needed. For cloning procedures, One Shot TOP10 chemically competent Escherichia coli (Invitrogen) was grown in Luria-Bertani (LB) broth or on LB agar with hygromycin (200 μg ml⁻¹), kanamycin (50 μg ml⁻¹), or spectinomycin (25 μg ml⁻¹). All chemicals were purchased from Sigma-Aldrich, unless otherwise stated.

Plasmid construction.Two 900-bp-long fragments corresponding to the upstream and downstream regions of rv3852 were generated by PCR amplification using the U-fwd (fwd stands for forward) and U-rev (rev stands for reverse) primers and the D-fwd and D-rev primers, listed in Table S2 in the supplemental material. Fragments were ligated with the AvrII restriction site and cloned into the PacI and AscI sites of pJG1100 (22, 23).

To complement the rv3852 deletion strain, pCS24 was constructed, where the rv3852 gene was amplified with primers rv3852 -F (F stands for forward) and rv3852 -R (R stands for reverse) and cloned in front of the ptr promoter into pGA44 (25), which stably integrates at the L5 attB site.

For plasmid generation purposes, all PCR products were ligated into the pCR-Blunt II-TOPO vector (Invitrogen) according to the manufacturer's recommendations. The plasmids and oligonucleotide sequences used in this study are given in Tables S1 and S2, respectively. All enzymes were purchased from New England BioLabs.

Construction of the unmarked in-frame Δrv3852 deletion strain.Deletion of rv3852 was accomplished by homologous recombination using the pJG1100-derived vector pCS21. After transformation of M. tuberculosis H37Rv, the first recombination event was selected on Middlebrook 7H10 medium containing hygromycin and kanamycin. Colonies were screened by colony PCR using primers 329-330 and 331-332 (Table S2). Positive clones were plated on Middlebrook 7H10 medium supplemented with sucrose to select for the second crossing over and loss of pJG1100. The resulting clones were tested by colony PCR with primers CS-351-rv3852F and CS-352-rv3852R by reverse transcription-PCR for loss of rv3852 expression and further confirmed by Southern blotting. The probe used for Southern blotting was amplified with primers U-fwd and D-rev.

Generation of the complemented strain was achieved by transformation of the rv3852 deletion strain with pCS24, providing the integrase in trans with pGA80 (25), selecting on streptomycin. The empty vector pGA44 was transformed in the same manner to generate the Δrv3852 empty vector control strain.

Growth curve measurements and MIC determination.To characterize the growth of the Δrv3852 mutant, the strains were grown to mid-logarithmic phase and then diluted to an optical density at 600 nm (OD₆₀₀) of 0.05 in Middlebrook 7H9 medium. The OD₆₀₀ was recorded at different time points to obtain the growth curves.

MIC determination using the resazurin reduction microplate assay (REMA) was performed as previously described (37). Briefly, M. tuberculosis was grown to mid-log phase, diluted to an OD₆₀₀ of 1 × 10⁻⁴, and added to a 96-well plate at a volume of 100 μl. The drug being tested was added at twofold serial dilutions to the bacteria, and the plates were then incubated at 37°C for 7 days before the addition of 0.025% resazurin. After overnight incubation, the fluorescence of the resazurin metabolite resorufin was determined (excitation at 560 nm and emission at 590 nm) by using a Tecan Infinite M200 microplate reader. Results were plotted in GraphPad Prism 5, and MIC was determined by the Gompertz equation.

Genomic DNA extraction and Southern blotting.Mycobacterial genomic DNA was extracted using standard protocols (38). To confirm successful allelic exchange of rv3852, genomic DNA was digested with NcoI or NcoI plus AvrII restriction enzymes. DNA fragments were separated by 0.8% agarose gel electrophoresis before capillary blotting onto a Hybond-N+ nylon membrane (GE Healthcare) and hybridization with a probe corresponding to the same upstream and downstream region of rv3852 cloned into pJG1100. Hybridization was carried out using the ECL direct nucleic acid labeling and detection system (GE Healthcare) as recommended by the manufacturer.

Total RNA extraction.M. tuberculosis cultures were harvested by centrifugation, and pellets were resuspended in TRIzol reagent (Thermo Fisher Scientific) and stored at −80° until further processing. Total RNA was extracted by bead beating as previously described (39). The integrity of RNA was checked by agarose gel electrophoresis, and the purity and amount of RNA were assessed using a Nanodrop instrument and Qubit fluorometric quantitation assay kit (Thermo Fisher Scientific), respectively. SuperScript III first-strand synthesis system (Invitrogen) was used to generate randomly primed cDNA from 500 ng of RNA, according to the manufacturer's recommendations.

Library preparation for RNA-seq analysis and Illumina high-throughput sequencing.A total of 300 ng of total RNA was mixed with 5× fragmentation buffer (Applied Biosystems), incubated at 70°C for 4 min, and then transferred immediately to ice. RNA was purified using RNAClean XP beads (Beckman Coulter), according to the manufacturer's recommendations, and subsequently treated with Antarctic phosphatase (New England BioLabs). RNA was then rephosphorylated at the 5′ end with polynucleotide kinase (New England BioLabs) and purified with RNeasy MinElute columns (Qiagen). To ensure strand specificity, v1.5 small RNA (sRNA) adapters (Illumina) were ligated at the 5′ and 3′ ends using RNA ligase. Reverse transcription was carried out using SuperScript III reverse transcriptase (Invitrogen) and SRA RT primer (Illumina). Twelve cycles of PCR amplification using Phusion DNA polymerase were then performed, and the resulting library was purified with AMPure beads (Beckman Coulter) per the manufacturer's instructions and sequenced on the Illumina HiSeq 2000 instrument using the TruSeq SR cluster generation kit v3 and TruSeq SBS kit v3. Data were processed with the Illumina Pipeline software v1.82. RNA-seq experiments were performed on two biological replicates per strain.

RNA-seq analysis.Reads were mapped against the H37Rv reference strain genome sequence (NC_000962.2) using Bowtie 2 (40). Read counting over features was done using htseq-count (41) and the annotation from Tuberculist. To take advantage of strand specificity of the libraries and to maximize detection of putative unknown transcriptionally active regions, we also included the reverse orientation of each feature, as well as intergenic regions. Differential gene expression analysis was done using DESeq (42).

Quantitative PCR.Primers listed in Table S2 were used for quantification of gene expression. Primers CS-057 and CS-058 for sigA were used to normalize the amount of cDNA template added to each sample. qRT-PCR was carried out in duplicate using 7900HT sequence detection system and Power SYBR green PR master mix (Applied Biosystems) according to the manufacturer's recommendations. The ΔΔC_T method was used for quantification.

5′ RACE.Two micrograms of M. tuberculosis H37Rv RNA and 1 μg of primer CS-397 were incubated at 70°C for 5 min and then at 55°C for 1 h in the presence of 1× cDNA synthesis buffer, 1 mM each deoxynucleoside triphosphate (dNTP), 40 U RNase inhibitor, 25 U Transcriptor reverse transcriptase (5′/3′ RACE kit, second generation; Roche). cDNA was then purified with the High Pure PCR product purification kit (Roche) and used in the subsequent poly(A) tailing reaction (30 min at 37°C in the presence of 0.2 mM dATP and 80 U terminal transferase; Roche). Seminested PCR amplification on poly(A)-tailed cDNA was performed using an oligo(dT) anchor primer (CS-81) and primer CS-395. Only one amplification product was obtained and directly sequenced.

Protein extraction, immunoblot analysis, and subcellular fractionation.M. tuberculosis cultures grown in Middlebrook 7H9 medium were pelleted at different time points by centrifugation, washed once in Tris-buffered saline (TBS) (20 mM Tris-HCl [pH 7.5], 150 mM NaCl), and stored at −80°C until further processing. The cells were sonicated in TBS supplemented with a protease inhibitor tablet (Roche) for 15 min, and the protein solution was then sterilized by filtration through a 0.2-μm filter to remove any residual intact cells. Protein samples were quantified using the Qubit fluorometric quantitation instrument (Thermo Fisher Scientific). Equal amounts of protein preparations were loaded on SDS-polyacrylamide 12 to 15% NuPAGE gels (Invitrogen) and transferred onto polyvinylidene difluoride (PVDF) membranes using a semidry electrophoresis transfer apparatus (Bio-Rad). The membranes were incubated in TBS-Tween blocking buffer (25 mM Tris [pH 7.5], 150 mM NaCl, 0.05% Tween 20) with 5% (wt/vol) skimmed milk powder for 3 h prior to overnight incubation with primary antibody. The membranes were washed in TBS-Tween blocking buffer three times and then incubated with secondary antibody for 2 h before washing. Signals were detected using chemiluminescent peroxidase substrate 1 (Sigma-Aldrich).

Primary anti-Rv3852 antibody was produced by Eurogentec against Rv3852 peptides and used at a concentration of 1:4,000 in immunoblots. Horseradish peroxidase (HRP)-conjugated anti-rabbit (Sigma-Aldrich) secondary antibody was used at a 1:150,000 dilution. Anti-RpoB antibodies (NeoClone) were used as an internal loading control. The band intensity of immunoblots was analyzed with Fiji/ImageJ and normalized to the intensity of the RpoB signal.

For cell fractionation, 50-ml cultures of H37Rv strain and Δrv3852 mutant were grown in Sauton's medium with 0.005% Tween 80. Fractions were obtained as described previously (43). Briefly, cells were collected by centrifugation, supernatant was filtered and concentrated 100 times to obtain the secreted fraction. The pellet was treated with 0.25% Genapol-X080 for 30 min followed by centrifugation at 14,000 × g for 10 min, and the proteins of the resulting supernatant were precipitated with trichloroacetic acid (TCA), yielding the capsular fraction. The remaining pellet was subjected to sonication to break the cells, sterilized by filtration through a 0.2-μm filter, and centrifuged at 45,000 rpm for 1 h in an ultracentrifuge. The supernatant contained the cytosolic fraction, while the pellet was enriched with membrane proteins.

Ex vivo virulence assay.THP-1 cells were grown in RPMI 1640 medium containing 10% fetal bovine serum (HyClone) and incubated at 37°C with 5% CO₂. To investigate the virulence of the Δrv3852 mutant compared to strain H37Rv, the strains were grown to mid-exponential phase and used to infect macrophages according to a previously published protocol (25, 44). Briefly, 100,000 THP-1 cells per well were seeded into 96-well plates, differentiated into macrophages by adding 100 nM phorbol myristate acetate (PMA) overnight and then infected with the various M. tuberculosis strains at a multiplicity of infection (MOI) of 2.5. Macrophage survival was measured 3 days postinfection by exposing the infected cells to PrestoBlue cell viability reagent (Life Technology) for 1 h. Fluorescence was read using a Tecan Infinite M200 microplate reader, and statistical significance was calculated by an unpaired Student's t test method.

In vivo virulence studies.Log-phase bacterial cultures of wild-type strain H37Rv, Δrv3852 deletion mutant, and the complemented strain were grown to mid-exponential phase and centrifuged briefly to exclude cell clumps, and the supernatant was diluted in phosphate-buffered saline (PBS) (supplemented with 0.05% Tween 80) to an OD₆₀₀ of 0.3. Seven-week-old female Fox Chase SCID mice (Charles River Laboratories) were infected via the aerosol route with the bacterial suspension by using a custom-built aerosol exposure chamber (Mechanical Engineering Shops, University of Wisconsin, Madison) to deliver approximately 300 CFU to the lungs of each mouse.

Three mice per group were sacrificed on day 1 postinfection, and lung homogenates were plated to assess the level of infection. Five mice per group and time point were sacrificed at week 2 and week 3.5 postinfection, and lung and spleen homogenates were plated on Middlebrook 7H10 medium plates supplemented with cycloheximide (10 μg ml⁻¹) and ampicillin (50 μg ml⁻¹). Spleen size was measured at the same time points. Experimental procedures involving animals were approved by the Swiss Cantonal and Federal Authorities (authorization 3082).

Fluorescence microscopy and image analysis.M. tuberculosis H37Rv and Δrv3852 mutant strain were grown to early logarithmic phase in Middlebrook 7H9 medium, washed once in TBS-Tween and stained with 10 μM SYTO9 (Thermo Fisher Scientific) for 25 min. Cells were mounted on an agarose pad and visualized with an Olympus IX81 fluorescence microscope under a 100× objective.

The resulting images were analyzed in Fiji/ImageJ. Each cell was visually inspected to be a single, straight cell before plotting a straight-line intensity profile in bright-field and fluorescence channels. Cell boundaries were determined as 10% decrease in intensity relative to the minimum value of the bright-field picture. Nucleoid boundaries were determined as 25% increase in fluorescence relative to the maximum value. All profiles were plotted in R, again visually analyzed to exclude any wrong boundary determination and used to define cell margins, nucleoid localization, and spread (as a percentage of nucleoid length relative to cell length). To assign a position to the nucleoid peaks relative to the individual cell length, each cell was divided into five even sections. The middle of the cell being the center, the two most polar sections were merged, as well as the two intermediary sections between the pole and the center (subpolar).

Scanning electron microscopy.Whole-cell scanning electron microscopy was done as previously published (25). Briefly, the Δrv3852 mutant and wild-type H37Rv strains were grown in Middlebrook 7H9 medium until mid-exponential phase, pelleted, washed in PBS, and resuspended to an OD₆₀₀ of 0.5. The samples were then fixed on a coverslip in a solution of 1.25% glutaraldehyde and 1% tannic acid in phosphate buffer (0.1 M; pH 7.4) for 1 to 2 h. The samples were washed in cacodylate buffer prior to fixation for 30 min in 1% osmium tetroxide in cacodylate buffer and washed again twice for 3 min each time in water. Gradual dehydration was carried out in ethanol. Finally, a metal coat (Au-Pd alloy) was applied to the critically dried samples. Images were obtained with a Zeiss Merlin scanning electron microscope.

Statistics.Statistical analysis was performed with unpaired t test in Prism version 5.0 (GraphPad, San Diego, CA).

Accession number(s).Raw sequencing data have been deposited in the GEO database under accession number GSE95181.