Cell envelope stress in mycobacteria is regulated by the novel signal transduction ATPase IniR in response to trehalose

<div><p>The cell envelope of mycobacteria is a highly unique and complex structure that is functionally equivalent to that of Gram-negative bacteria to protect the bacterial cell. Defects in the integrity or assembly of this cell envelope must be sensed to allow the induction of stress response systems. The promoter that is specifically and most strongly induced upon exposure to ethambutol and isoniazid, first line drugs that affect cell envelope biogenesis, is the <i>iniBAC</i> promoter. In this study, we set out to identify the regulator of the <i>iniBAC</i> operon in <i>Mycobacterium marinum</i> using an unbiased transposon mutagenesis screen in a constitutively <i>iniBAC</i>-expressing mutant background. We obtained multiple mutants in the <i>mce1</i> locus as well as mutants in an uncharacterized putative transcriptional regulator (<i>MMAR_0612</i>). This latter gene was shown to function as the <i>iniBAC</i> regulator, as overexpression resulted in constitutive <i>iniBAC</i> induction, whereas a knockout mutant was unable to respond to the presence of ethambutol and isoniazid. Experiments with the <i>M</i>. <i>tuberculosis</i> homologue (<i>Rv0339c</i>) showed identical results. RNAseq experiments showed that this regulatory gene was exclusively involved in the regulation of the <i>iniBAC</i> operon. We therefore propose to name this dedicated regulator <b><i>ini</i></b><i>BAC</i> <b>R</b>egulator (IniR). IniR belongs to the family of signal transduction ATPases with numerous domains, including a putative sugar-binding domain. Upon testing different sugars, we identified trehalose as an activator and metabolic cue for <i>iniBAC</i> activation, which could also explain the effect of the <i>mce1</i> mutations. In conclusion, cell envelope stress in mycobacteria is regulated by IniR in a cascade that includes trehalose.</p></div

ChIP sequencing shows a clear binding peak for IniR_Mtb upstream of the <i>iniBAC</i> operon.

<p>Chromatin immunoprecipitation (ChIP) sequencing was performed to identify binding events of IniR_Mtb-FLAG_. To do so <i>M</i>. <i>tuberculosis</i> containing a vector encoding the FLAG-tagged IniR_Mtb was induced with ATc and crosslinked after induction. After lysis, chromatin was sonicated to small fragments and immuno-precipitated with anti-FLAG antibody. And purified and sequenced. A clear binding peak of IniR_Mtb upstream of the <i>iniBAC</i> operon could be identified. The blue line indicates the (+) strand reads and the red line indicates the (-) strand reads. The number on the X-axis represent the nucleotide position in the H37Rv genome.</p

Overview of the three domains that can be found in <i>iniR</i>.

<p>Noted domains are the most significant hits using Phyre2 structure prediction [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007131#pgen.1007131.ref021" target="_blank">21</a>]. The number between brackets indicates the confidence with which the Phyre2 prediction was made. A combination of NCBI BLAST and the Phyre2 server was used to obtain domain predictions. Domain I (amino acid 24 to 247) is an AAA+ ATPase domain sharing 15% amino acid identity with sso_1545 in <i>S</i>. <i>solfataricus</i>. Domain II (470–731) is 16% identical to domain III of MalT in <i>E</i>. <i>coli</i> and the C-terminal domain (745–812) contains a DNA binding domain sharing 20% identity to <i>E</i>. <i>coli</i> SdiA. Displayed domains are proportional to actual domain size.</p

Immunoblot analysis of IniR_Mtb with α-Strep antibody.

<p><i>M</i>. <i>smegmatis</i> containing the construct pEXCF-<i>iniR</i>_<i>Mtb</i>-Strep vector was cultured and cultures were exposed for 18 hours to 10 ng/ml ATc/ Strep-tagged IniR_Mtb was isolated with StrepTactin beads. Purified protein from the elution fractions (elution 1–5, see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007131#pgen.1007131.s006" target="_blank">S6 Fig</a>) was exposed to either trehalose, ATP, both or neither. Presence is indicated with (+), absence is indicated with (-). Indicated on the image of the Western Blot are monomeric IniR (~90kDa) and possible dimers and tetramers.</p

Lipid profiles of <i>mce1</i>::<i>tn</i> mutants show an increase in free mycolic acid and a decrease in bound mycolic acids.

<p>TLC analysis of lipid extracts from <i>M</i>. <i>marinum</i> WT strain, <i>the cobC</i>::<i>tn</i> strain used for the transposon mutagenesis and three on/off mutants that showed decreased <i>iniBAC</i> induction, <i>i</i>.<i>e</i>. <i>mce1D</i>::<i>tn</i>, <i>yrbE1B</i>::<i>tn</i> and <i>cmaA2</i>::<i>tn</i>. TLC loading was normalized by OD and further adjusted according to pellet weight to ensure equal amounts of lipid were spotted. (A) TLC of polar lipids, triacylglycerols (TAG), free mycolic acids (FMAs) and diacylglycerols (DAG) are distinguished. A clear increase in FMAs can be seen for both <i>mce1</i> operon mutants. (B) TLC analysis of the different types of bound mycolic acids (MAMEs and FAMEs) where for MAMEs alpha (α), keto (k) and methoxy (m) species are indicated. There is a clear decrease in bound mycolic acids for <i>mce1D</i>::<i>tn</i> and <i>yrbE1B</i>::<i>tn</i>.</p

List of <i>M</i>. <i>marinum</i> mutants identified that showed high induction of <i>iniBAC</i> on 2% trehalose plates.

<p>List of <i>M</i>. <i>marinum</i> mutants identified that showed high induction of <i>iniBAC</i> on 2% trehalose plates.</p

Addition of trehalose is sufficient to induce <i>iniBAC</i> induction in <i>M</i>. <i>marinum</i>.

<p>The effect of additional trehalose is especially prominent when the outer membrane is more permeabilized by the expression of the MspA porin of <i>M</i>. <i>smegmatis</i>. (A) Flow cytometry histograms of induction levels of a WT <i>M</i>. <i>marinum</i> containing the <i>iniBAC</i> reporter upon treatment with either 1% trehalose (green), 1% maltose (red), 1% sucrose (blue) or no disachharide (black line). In the graph plots are shown for day 1, 2 and 3 after addition. The graphs are representative images of one experiment performed in triplicate. (B) Same as in (A) but this experiment the strains contained an additional plasmid encoding porin MspA. Fluorescence intensity of mEos3.1 (in arbitrary units) is plotted on the X-axis for all six histograms (C) A quantification of the fold induction on day 3 of three independent experiments. The fold induction was calculated by dividing the MFI of the treated sample to the MFI of the corresponding untreated control. The presence of MspA is indicated with + and -, as well as the different sugars tested; maltose (red), sucrose (blue) and trehalose (green). s.d. values are indicated by the error bars. Cultures were grown in 7H9 containing 0.2% glycerol and 0.05% Tween-80.</p

A Δ<i>iniR</i>_<i>Mm</i> mutant is unable to induce <i>iniBAC</i> upon addition of EMB or INH.

<p>(A) Gating strategy for this experiment. The bacterial population in the red box was selected to measure a population that is roughly equal in size and granularity (measured by forward scatter, FSC and side scatter, SSC). This gated population was used for all samples and a total of 30,000 cells were analyzed per sample (B) Histograms of mEos3.1 fluorescence intensity (in arbitrary units, on the X-axis) of untreated WT <i>M</i>. <i>marinum</i> (black line), an isogenic <i>iniR</i>_<i>Mm</i> knockout mutant and the <i>iniR</i>_<i>Mm</i> knockout mutant transformed with an integrative plasmid containing a copy of <i>iniR</i>_<i>Mm</i> with its native promoter are shown for day 3. (C) Fluorescence induction by EMB (1 μg/ml) is lost in the <i>iniR</i>_<i>Mm</i> mutant, but restored in the complemented strain to near-WT levels. (D) Fluorescence induction by INH (10 μg/ml) is lost in the <i>iniR</i>_<i>Mm</i> mutant, but also restored in the complemented strain to near-WT levels. The histograms are from one representative sample of a biological quadruplicate (E) Quantification of the fluorescence induction (in mean fluorescence intensity, MFI) measured on day 3. Measurements were performed in quadruplicate. Error bars indicate s.d. values.</p

An overview of the <i>iniR-</i>containing regions in mycobacteria.

<p>The species containing <i>iniR</i> (blue) are <i>M</i>. <i>marinum</i> (top), <i>M</i>. <i>tuberculosis</i> (middle) and <i>M</i>. <i>smegmatis</i> (lower). Conserved genes are indicated by identical colors. Insertions of genes in the genome of <i>M</i>. <i>smegmatis</i> are indicated with the black arrows. Distances are proportional to actual size. Red triangles indicate the approximate positions of the 3 independent transposon insertions in <i>iniR</i> that affect <i>iniBAC</i> induction in <i>M</i>. <i>marinum</i>.</p

Boosting PPE10 specific immune responses does not increase protection against <i>M</i>. <i>tuberculosis</i>.

<p>A) Graphical representation of the prime-boost vaccination protocol. Mice were immunized with either BCG or BCG38 (Green). 60 days post-infection (d.p.i.) C57BL/6 x CBA F1 mice were injected s.c. with a booster consisting of adjuvant CpG(DOTAP), alone or in combination with a mix of PPE10_221-235 and PPE10_381-395 peptides (blue). The same formulation was intranasally administered four weeks later. Nine days after the intranasal boost, mice were exposed to <i>M</i>. <i>tuberculosis</i> H37Rv aerosol infection (220 CFU/lung 1 d.p.i). Bacterial lung (B) and Spleen (C) burdens were assessed by dilution and counting 4 weeks post-infection (experimental end-point) after being photographed for macroscopic investigation (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007139#ppat.1007139.s002" target="_blank">S2C and S2D Fig</a>). Each data point represents the CFU value of one organ from a single mouse, error bars depict the standard deviation. No significant differences between the vaccination conditions were detected by ordinary one-way ANOVA followed by Tukey’s test of multiple comparisons. All vaccination conditions resulted in a significant (<i>p</i><0.01) reduction in lung burden compared to unimmunized controls (Ordinary one way ANOVA; Dunnett’s test of multiple comparisons against a single control). For simplicity, this latter information is not depicted in the figure. Reduction in spleen CFUs was not significant for any of the vaccination conditions. Statistical analyses were performed using PRISM software.</p