Research Article

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

Nina T. Odermatt, Claudia Sala, Andrej Benjak, Gaëlle S. Kolly, Anthony Vocat, Andréanne Lupien, Stewart T. Cole
Olaf Schneewind, Editor
DOI: 10.1128/JB.00129-17

ABSTRACT

A handful of nucleoid-associated proteins (NAPs) regulate the vast majority of genes in a bacterial cell. H-NS, the histone-like nucleoid-structuring protein, is one of these NAPs and protects Escherichia coli from foreign gene expression. Though lacking any sequence similarity with E. coli H-NS, Rv3852 was annotated as the H-NS ortholog in Mycobacterium tuberculosis, as it resembles human histone H1. The role of Rv3852 was thoroughly investigated by immunoblotting, subcellular localization, construction of an unmarked rv3852 deletion in the M. tuberculosis genome, and subsequent analysis of the resulting Δrv3852 strain. We found that Rv3852 was predominantly present in the logarithmic growth phase with a decrease in protein abundance in stationary phase. Furthermore, it was strongly associated with the cell membrane and not detected in the cytosolic fraction, nor was it secreted. The Δrv3852 strain displayed no growth defect or morphological abnormalities. Quantitative measurement of nucleoid localization in the Δrv3852 mutant strain compared to that in the parental H37Rv strain showed no difference in nucleoid position or spread. Infection of macrophages as well as severe combined immunodeficient (SCID) mice demonstrated that loss of Rv3852 had no detected influence on the virulence of M. tuberculosis. We thus conclude that M. tuberculosis Rv3852 is not involved in pathogenesis and is not a typical NAP. The existence of an as yet undiscovered Rv3852 ortholog cannot be excluded, although this role is likely played by the well-characterized Lsr2 protein.

IMPORTANCE Mycobacterium tuberculosis is the causative agent of the lung infection tuberculosis, claiming more than 1.5 million lives each year. To understand the mechanisms of latent infection, where M. tuberculosis can stay dormant inside the human host, we require deeper knowledge of the basic biology and of the regulatory networks. In our work, we show that Rv3852, previously annotated as H-NS, is not a typical nucleoid-associated protein (NAP) as expected from its initial annotation. Rv3852 from M. tuberculosis has neither influence on nucleoid shape or compaction nor a role in virulence. Our findings reduce the repertoire of identified nucleoid-associated proteins in M. tuberculosis to four transcription regulators and underline the importance of genetic studies to assign a function to bacterial genes.

INTRODUCTION

The chromosome must be heavily compacted to fit into a bacterial cell about 1,000 times smaller than the length of its DNA. Similarly to eukaryotes, where DNA is wrapped around histones for condensation, prokaryotes possess nucleoid-associated proteins (NAPs), which influence DNA topology. Typically, NAPs interact with the chromosome at several hundred binding sites in a sequence-specific or non-sequence-specific manner (1). As a result, NAPs can bridge, loop, and bend DNA (2), thus impacting global transcription regulation through activation or silencing of target genes (3). It is estimated that, together, a few NAPs control the expression of more than 50% of the genes in Escherichia coli (4).

In the well-characterized E. coli cell, at least 12 NAPs have been identified, among them the factor for inversion stimulation (Fis), the integration host factor (IHF), and the histone-like nucleoid-structuring protein (H-NS) (5). H-NS is also known as the “genome guardian” (2), as it silences xenogeneic genes acquired through horizontal gene transfer (6). E. coli H-NS predominantly forms dimers (7), has a preference for AT-rich bent DNA (8), and is expressed throughout the bacterial growth cycle (2).

Mycobacterium tuberculosis is considered to be the world's most successful pathogen, causing 1.5 million deaths each year (9). Once M. tuberculosis has entered the host, it can establish a latent infection by persisting inside macrophages for several decades before regrowth leads to active tuberculosis (10). Several transcription factors contribute to gene regulation in M. tuberculosis, and one of these transcription factors, EspR, proved to behave as a NAP by binding to more than 160 loci, including virulence genes (11). In addition to EspR, four NAPs have been reported so far in M. tuberculosis: HupB, Lsr2, mycobacterial IHF (mIHF), and Rv3852 (12). The latter was named H-NS due to its N-terminal similarity to human histone H1 (12).

Rv3852 is a small protein of 134 amino acids, with a proposed molecular mass of 13.8 kDa, and is predicted to be nonessential by transposon mutagenesis (13). Rv3852 from M. tuberculosis has a very low sequence similarity with E. coli H-NS, but it is highly conserved among pathogenic mycobacteria. Mycobacterium bovis (14), Mycobacterium marinum (15), and even Mycobacterium leprae with its downsized genome (16) possess a copy of the rv3852 gene, while it is absent from the nonpathogenic model organism Mycobacterium smegmatis (17). The mycobacterial proteins annotated as H-NS contain tetrapeptide repeats (PAKK and KAAK) that are critical for DNA binding by histones of the H1/H5 family, and this may explain the attribution of the hns gene name. The number of PAKK repeats varies between mycobacterial species (18), but all of the proteins contain a single conserved transmembrane domain at the C terminus.

Curiously, Werlang and colleagues reported that M. tuberculosis Rv3852 does not complement hns mutations in E. coli (19), whereas Lsr2, another NAP, does (20) and is therefore a functional homologue of E. coli H-NS. Ectopic expression of M. tuberculosisrv3852 in M. smegmatis led to a less-compacted nucleoid, altered biofilm formation, and decreased sliding motility (21). Unusually for a NAP, the C-terminal domain of Rv3852 was identified as a transmembrane helix, suggesting a role in anchoring DNA to the membrane. RNA profiling showed that expression of groEL1, a heat shock chaperone, and kasA, involved in fatty acid biosynthesis, are downregulated upon expression of rv3852 in M. smegmatis (21).

Here, we report the construction and in-depth characterization of an M. tuberculosisrv3852 deletion mutant. By means of biological, molecular, and microscopy techniques, we show that Rv3852 is not involved in nucleoid compaction or virulence and plays only a minor role in transcriptional control.

RESULTS

Generation of an rv3852 deletion mutant in M. tuberculosis.To probe the essentiality of the rv3852 gene in M. tuberculosis strain H37Rv, we planned the construction of a deletion mutant by a two-step allelic exchange method based on the suicide vector pJG1100 (22, 23). Since rv3852 was reported to be part of an operon with the downstream gene rraA (24), we first mapped the 5′ end of the bicistronic mRNA to ensure that the promoter region was not deleted and that rraA expression was not affected in the mutant. The 5′ rapid amplification of cDNA ends (5′-RACE) experiment identified a single 5′ end 47 bp upstream of the rv3852 start codon (see Fig. S1 in the supplemental material). An in-frame deletion of the rv3852 coding sequence was then designed. Homologous recombination with the pJG1100-derived plasmid pCS21 was induced in strain H37Rv and confirmed by colony PCR (data not shown). Deletion of rv3852 was demonstrated in two sucrose-resistant clones (clones 1 and 17), which were further analyzed by reverse transcription-PCR (RT-PCR) (data not shown) and by Southern blotting (Fig. S2), thus validating the deletion procedure. The resulting mutant (clone 1) was named Δrv3852 mutant and used for subsequent analyses.

The Δrv3852 mutant was transformed with plasmid pCS24 (Table S1), harboring the native rv3852 gene under the control of the constitutive ptr promoter, thereby obtaining the complemented mutant (Δrv3852/rv3852 [Fig. S3]). A control strain (Δrv3852/pGA44) was constructed by transforming Δrv3852 with the empty vector pGA44 (25).

In vitro phenotype of Δrv3852 deletion mutant.To assess the in vitro growth dynamics of the Δrv3852 deletion mutant compared to its H37Rv parent, growth curves were generated in Middlebrook 7H9 complete medium. None of the strains showed any growth defects, either during exponential phase or in early stationary phase (Fig. S4A). In addition, the mutant strain carrying either the empty vector or the complementing plasmid did not display any abnormal phenotype, indicating that the lack of Rv3852 or ectopic expression has no influence on growth of M. tuberculosisin vitro.

The effect of external pH was evaluated by comparing growth in low-pH media, buffered at pH 5 and pH 6. As PhoPR is required to slow growth at acidic pH (26), the ΔphoP strain (27) was used as a control. At neutral pH, the ΔphoP mutant showed a slight growth defect (Fig. S4B). At pH 6, all strains had lower growth rates, while the ΔphoP mutant grew slightly faster than the others (Fig. S4C). No difference was noticed for the Δrv3852 mutant relative to the wild-type H37Rv strain under any of the conditions tested. In more-acidic medium (pH 5), the optical density did not reach values higher than 0.1 for any of the strains after 10 days (data not shown).

Since Rv3852 had been hypothesized to play a role as a NAP (19), thus contributing to shaping the chromosome, the MICs of drugs that have an effect on the topology of DNA were measured (28). We used the fluoroquinolones moxifloxacin and novobiocin targeting the GyrA and GyrB subunits of DNA gyrase, respectively, for this purpose together with rifampin, inhibiting RNA synthesis, as a control. The resazurin microplate assay (REMA) was carried out in Middlebrook 7H9 medium at pH 7. Results showed that the MIC was the same for all three strains tested, namely, wild-type H37Rv, Δrv3852 mutant, and the complemented mutant (Fig. S5). We concluded that lack of H-NS does not noticeably influence the susceptibility of M. tuberculosis to DNA gyrase-targeting drugs.

Rv3852 is most abundant in exponential phase and localizes to the cell membrane.Some NAPs are present throughout the whole bacterial cell cycle, while others are present only at a specific growth stage. In order to analyze to which category M. tuberculosis Rv3852 belongs, total protein extracts were prepared at different time points from wild-type H37Rv and probed by immunoblotting. The extract from the Δrv3852 strain was used as a negative control, and the constitutively expressed RNA polymerase subunit β, RpoB, was used as an internal standard. Rv3852 was identified at approximately 19 kDa (Fig. 1A) on a denaturing gel. Figure 1A and B demonstrate that Rv3852 is present in early exponential phase and reaches its maximum level in mid-exponential phase on day 4. Its abundance declines at the onset of stationary phase and remains at about 30% of the initial level in late stationary phase. This pattern suggests that Rv3852 plays its main role during the logarithmic phase of growth.

FIG 1
FIG 1

Analysis of Rv3852 abundance in vitro. (A) Immunoblot analysis of total protein extracts from M. tuberculosis H37Rv and Δrv3852 mutant strain at different time points. (B) The corresponding graph representing Rv3852 band intensity, normalized to RpoB, and relative to the highest value at day 4. Samples were taken on day 2 (OD600 of 0.21) for Δrv3852 mutant and on days 2, 4, 7, and 14 (corresponding to OD600 values of 0.28, 0.36, 0.9, and 2.4, respectively) for strain H37Rv. The experiment was repeated twice. A representative image is shown.

To localize Rv3852 subcellularly, total proteins from the wild-type strain were fractionated into secreted (culture supernatant), capsular, membrane, and cytosolic fractions. RpoB was used as a lysis control and proved that cells did not lyse, as it was not detected in the culture supernatant. EsxB, which was used as a secretion control, was present in the cytosol and in the supernatant. Importantly, EsxB was not seen in the membrane and capsule, indicating a clear separation of these fractions from the cytosol. Interestingly, Rv3852 was found only in the cell membrane and in none of the other subcellular compartments (Fig. 2).

FIG 2
FIG 2

Localization of Rv3852 in subcellular fractions. Immunoblot of secreted (supernatant), capsular, membrane, and cytosolic protein fractions from strain H37Rv. RpoB was used as a lysis control for the supernatant, and EsxB was used as a control for the secreted fraction. Note that Rv3852 is found exclusively in the membrane fraction.

Effect of Rv3852 on the nucleoid.To evaluate the effects of Rv3852 on nucleoid compaction and shape, the Δrv3852 and H37Rv strains were stained with SYTO9, which specifically binds to DNA, and analyzed by fluorescence microscopy. Representative images are shown in Fig. 3A. Nucleoid position relative to the cell, the number of nucleoid peaks, and nucleoid spread were evaluated in 54 mutant cells and 74 wild-type cells. No significant difference was observed for any of the tested parameters (Fig. 3B) (Student's t test). As chloramphenicol is known to contract the nucleoid (29), H37Rv cells treated with chloramphenicol were used as a control to detect any change in nucleoid morphology (n = 50). Results confirmed the previous findings (29).

FIG 3
FIG 3

Impact of rv3852 deletion on nucleoid structure and shape. (A) Micrographs of the H37Rv strain, Δrv3852 mutant, and H37Rv treated with chloramphenicol (CAM) in bright-field (BF) channel, fluorescent channel (SYTO9), and overlay. Bars, 1 μm. (B) No difference was observed between the Δrv3852 mutant and H37Rv strain in the number of peaks per cell, peak position inside the cell, and nucleoid spread (Student's t test), while H37Rv treated with chloramphenicol showed a lower number of peaks and a reduced nucleoid spread relative to nontreated H37Rv cells. Dots indicate outliers. ns, not significant; ***, P < 0.001.

Δrv3852 mutant displays normal cell morphology by scanning electron microscopy.To evaluate a potential morphological difference between wild-type H37Rv and its Δrv3852 mutant, high-resolution scanning electron microscopy (SEM) analysis was performed. Representative images are shown in Fig. 4. No obvious morphological difference was observed. Wild-type and mutant bacteria were similar in size, shape, and thickness. Cell length was measured on 104 and 106 cells for Δrv3852 and H37Rv strains, respectively. The mean values were 1.93 ± 0.52 μm for the Δrv3852 mutant and 2.04 ± 0.55 μm for strain H37Rv (no significant difference by Student's t test).

FIG 4
FIG 4

Scanning electron micrographs of H37Rv and Δrv3852 bacterial cells. Bars, 1 μm.

Global transcriptomic analysis.Transcriptome sequencing (RNA-seq) studies were undertaken to examine the impact of rv3852 deletion on the global transcription profile. The analysis was performed on RNA extracted from exponential-phase cultures, where Rv3852 was shown to be most abundant. Between 16 and 18 million reads from each replicate mapped uniquely to the H37Rv genome and allowed quantification of gene expression as reported in Data Set S1. A false-discovery rate (FDR) of <1% and a cutoff fold change at log2 of 2 were applied to identify the deregulated genes. Surprisingly, only three features met these criteria. The most downregulated gene was rv3852 with 0.04-fold expression in the mutant compared to the wild type, confirming loss of the gene in the Δrv3852 strain. The rv3852 upstream intergenic region (5′ untranslated region [5′-UTR]) was also found to be highly downregulated with 0.05-fold expression, which suggests that Rv3852 controls its own expression. On the other hand, the leuC-leuD transcriptional unit was upregulated fourfold relative to the H37Rv parental strain.

We then relaxed the stringency conditions and set the cutoff at twofold change, resulting in identification of an additional 19 features (Table 1). Of the 22 deregulated features, most cluster at three different loci in the chromosome. Upstream of leuC, rv2989 (probable transcriptional regulatory protein) and rv2990 were expressed at a higher level in the mutant than in the wild type. Furthermore, the intergenic regions between mce1R and fadD5 (both strands) genes and mce1R (transcriptional regulator) and TB18.5 genes were found to be transcribed at a higher level in the mutant than in the wild type. A 2.2-fold change was observed for the fadD11 mRNA, similar to the downstream gene frdA, which was 2.7-fold upregulated. lipQ, encoding a putative carboxylesterase, was 2.2-fold upregulated. Interestingly, while the rv3852 5′-UTR was almost undetected, the downstream rraA gene was found to be slightly more expressed in the mutant than in the wild-type strain. Another downregulated gene was the initiator tRNA metU. Overall, three genes encode probable transcriptional regulators.

View this table:
TABLE 1

RNA-seq results with a false-discovery rate of <1% and a twofold change cutoff

It was reported that expression of kasA and groEL1 was reduced fourfold upon ectopic expression of M. tuberculosis Rv3852 in M. smegmatis (21). We did not observe the same deregulation in our experiments, where kasA, kasB, the intergenic region between the two genes and groEL1 were expressed at similar levels in the mutant and wild-type strains (Data Set S1). The same genes were tested by quantitative RT-PCR (qRT-PCR) on biologically different samples. Results confirmed the RNA-seq data, with an overall differential expression below 1.5-fold (data not shown).

Rv3852 does not influence virulence ex vivo.To test whether the lack of Rv3852 affected the ex vivo virulence of M. tuberculosis, macrophage infection assays were carried out. Differentiated THP-1 macrophages were infected at a multiplicity of infection (MOI) of 2.5 for 3 days. While the attenuated ΔRD1 strain (30), used as a control, was considerably less virulent, no significant difference was observed between the wild-type H37Rv strain, Δrv3852 mutant strain, and the complemented strain (Fig. 5). The Δrv3852 strain was as virulent as the wild type and effectively killed macrophages at the same level as H37Rv did.

FIG 5
FIG 5

Virulence of the Δrv3852 mutant compared to strain H37Rv in an ex vivo model. Macrophage survival was measured upon infection with H37Rv, Δrv3852, and control strains. The ΔRD1 strain carries a deletion of the RD1 region and is attenuated. Data were normalized to the noninfected condition and are presented as means plus standard deviations (SD) (error bars) from five independent replicates. ns, not significant (Student's t test).

Deletion of rv3852 has no impact on in vivo virulence.A more sensitive mouse infection model was chosen to investigate a potentially weak phenotype of the Δrv3852 mutant. Severe combined immunodeficient (SCID) mice were infected with strain H37Rv, Δrv3852 mutant strain, and the complemented strain by the aerosol route. The bacterial burden was evaluated by counting the CFU per lung and spleen after 1, 14 (acute infection), and 25 (onset of the chronic infection) days postinfection. Thirteen CFU for the mutant and the complemented strain and 33 CFU for the wild type were detected 1 day postinfection. Two weeks later, the number of bacteria reached 103 to 104 in the lungs but was below the detection limit of 20 CFU/organ in the spleens. After 25 days, the bacterial load in the lungs increased to approximately 106 CFU for all three strains, whereas 104 CFU were counted in the spleens (Fig. 6). Overall, no significant difference was observed between the strains. At days 14 and 25, spleen size and macroscopic lesions of the lungs were evaluated. As shown in Fig. S6, the spleen size increased marginally from 1 day postinfection to the 25-day time point. However, again, no difference was noted between the three strains (Fig. S6A). Similarly, macroscopic lesions in the lungs were visible at day 25 in animals infected with the wild-type strain and animals infected with the Δrv3852 strain (Fig. S6B).

FIG 6
FIG 6

Virulence of the Δrv3852 mutant compared to strain H37Rv in vivo. The bacterial burden in the lung and in the spleen 14 days (D14) and 25 days (D25) postinfection with strain H37Rv, Δrv3852 mutant, and the complemented Δrv3852/rv3852 strain. Values are means plus SD of log10 CFU for five SCID mice in each group at each time point. The values for the CFU level 14 days and 25 days postinfection for the Δrv3852 mutant or complemented Δrv3852/rv3852 strain relative to the H37Rv strain in lungs and spleens were not significantly different (ns) by Student's t test.

DISCUSSION

As part of our systematic investigation of the NAPs of M. tuberculosis, which began with the molecular characterization of EspR, a nonessential NAP that impacts virulence (11, 18, 31), we have extended the study to the Rv3852 protein. The rv3852 open reading frame was removed from the M. tuberculosis chromosome by means of allelic exchange, indicating that Rv3852 is not essential for growth. Extensive characterization of the Δrv3852 mutant was performed. Expression of the downstream gene rraA, which forms an operon with rv3852 (24), was analyzed by RNA-seq and was at the level of the parental strain. A wide range of phenotypic tests revealed no differences in the behavior of the Δrv3852 mutant compared to the parent strain be it in vitro, ex vivo, or during infection of mice.

With respect to NAP activity, loss of Rv3852 had no effect on nucleoid shape or size (Fig. 3), contrary to the findings of Ghosh and colleagues upon overexpression of M. tuberculosis H-NS in M. smegmatis (21). Moreover, unlike Ghosh et al. (21), who reported deregulation of the fatty acid biosynthetic genes kasA and kasB and of the major chaperone gene groEL1, we observed no change in expression of these genes when comparing the global transcriptome of the wild-type strain H37Rv to that of the Δrv3852 mutant by RNA-seq. Besides, we found only two genes involved in fatty acid metabolism (fadD11 and lipQ) and three coding for probable transcriptional regulators (rv0232, mce1R, and rv2989) that were slightly deregulated in expression. Overall, our experimental results rule out a role for Rv3852 as a global transcriptional regulator. The discrepancy between our findings and those of Ghosh et al. (21) could be ascribed to the ectopic expression of M. tuberculosis H-NS in M. smegmatis, a species that has no ortholog of rv3852 in its genome (17). The expression system employed by Ghosh and coworkers may have altered the regulatory network in the bacterium, thus generating an aberrant phenotype.

By immunoblotting (Fig. 1), we demonstrated that Rv3852 is mainly expressed during the logarithmic phase of growth, which is consistent with the findings of two recent investigations. In one investigation, the transcription of rv3852 was found to be reduced when M. tuberculosis entered a nonreplicating state (32), while in the other, rv3852 expression levels were sevenfold higher in exponential phase than in stationary phase (33). These findings are consistent with those of Sharadamma et al. (34), who showed that in vitro Rv3852 binds to DNA replication/repair intermediates and to Holliday junctions, which are mainly generated during replication and exponential growth. Another important feature of the M. tuberculosis Rv3852 protein is its subcellular localization, since it was detected only in the cell membrane and not in the other subcellular compartments or in the culture supernatant (Fig. 2), as reported earlier in a proteomic survey (35). This localization is compatible with a role for Rv3852 in chromosome segregation during cell division, as proposed by Ghosh et al. (21). However, although there is a clear transmembrane domain, encompassing amino acid residues 110 to 128, immediately preceding the C terminus of the Rv3852 protein, the bulk of the protein is predicted to be in the periplasm. Further topological investigation of Rv3852 is required.

Recently, during a screen for inhibitors that disrupt the intrabacterial pH in an acidic environment, the natural product agrimophol was found to show activity against M. tuberculosisin vitro (36). Agrimophol, which is used in traditional Chinese medicine to treat pulmonary infections, was thought to target Rv3852. However, deletion of the rv3852 gene in M. tuberculosis did not mimic the effect of agrimophol or impact virulence in the macrophage infection model (36). These results confirm our findings in vitro where we proved that Rv3852 does not respond to changes in external pH and demonstrated that Rv3852 does not play a role upon intracellular infection.

Taken together, our combined data indicate that Rv3852 does not act as a NAP in any of the experimental conditions tested, thus raising doubts about its functional attribution. Other investigators have proposed that Lsr2, a bona fide NAP (20), is the true ortholog of the E. coli H-NS, and this appears to be the case. Finally, the nature of the true function of rv3852 remains to be elucidated.

MATERIALS AND METHODS

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 ΔΔCT 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 .

ACKNOWLEDGMENTS

We thank the staff at the École Polytechnique Fédérale de Lausanne (EPFL) Electron Microscopy Core Facility, EPFL Bioimaging and Optics Platform, and Genomic Technologies Facility at the University of Lausanne for technical assistance and Jacques Rougemont for setting up a basic script for cell boundary determination.

This work was supported by the Swiss National Science Foundation (grant 31003A-162641).

FOOTNOTES

    • Received 28 February 2017.
    • Accepted 25 May 2017.
    • Accepted manuscript posted online 30 May 2017.

  • Supplemental material for this article may be found at https://doi.org/10.1128/JB.00129-17 .

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KEYWORDS

Bacterial Proteins
DNA-binding proteins
Gene Expression Regulation, Bacterial
Mycobacterium tuberculosis
virulence factors
Tuberculosis
Mycobacterium tuberculosis
H-NS
NAP
global regulation
rv3852

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