DrrS, a small non-coding Mycobacterium tuberculosis RNA, regulates the whole genome expression shifts consistent with adaptations for survival within host macrophages

Small non-coding RNAs play a significant role in regulation of bacterial transcription and translation. Their expression in response to external factors is important for the adaptation of bacteria to changing environmental conditions. We investigated the expression of DrrS, a small noncoding RNA of Mycobacterium tuberculosis, in the mouse model in vivo, in the ex vivo model based upon infected macrophages, and in bacterial cultures, and demonstrated its significant contribution to host-pathogen interactions. Activation of the host immune system triggers NO-inducible up-regulation of DrrS in macrophage-engulfed mycobacteria. Constitutive overexpression of DrrS in cultured mycobacteria launches a broad spectrum of shifts in the bacterial transcriptome profile very similar to those reported for M. tuberculosis adaptation to hostile intra-macrophage environment, and providing defense against oxidative and NO stresses. In addition, we observed dramatic up-regulation of genes for the PE/PPE proteins and proteins of the ESX-1 and ESX-5 secretion systems. Taken together, our results suggest a direct involvement on this small RNA in the interplay between mycobacteria and the host immune system during infectious process. Author summary Pathogenic mycobacteria, including Mycobacterium tuberculosis, are able to survive within host macrophages. In attempt to eliminate intracellular mycobacteria, innate and acquired immune responses of the host activate a number of effector reactions to achieve effective intracellular mycobacterial killing. Mycobacteria, in turn, evolved a plethora of molecular mechanism providing successful escape from host immunity, involving several metabolic pathways allowing transition to dormancy – the state of slow-to-no replicative activity and an increased resistance to external stresses. These mechanisms remain poorly characterized. Small non-coding bacterial RNAs are expressed in response to external factors and play an important role in adaptation of bacteria to changing environmental conditions and escape from host immune responses. We investigated DrrS, a small non-coding RNA of Mycobacterium tuberculosis, in the mouse TB model in vivo, in infected macrophages ex vivo and in bacterial culture, and demonstrated that DrrS up-regulation strictly follows activation of the host immune defense. We established the strain of M. tuberculosis overexpressing DrrS in culture and found that DrrS contributes to mycobacterial resistance to reactive intermediates and activation of dormancy-associated genes, thus participating in bacterial metabolic adaptations and interactions with the host immune system.


72
One of such RNAs, DrrS (DosR-associated sRNA, ncRv11733, MTS1338), is highly 73 expressed during the stationary phase of growth [13], and the dormancy state [14]. This small RNA 74 is present only in genomes of highly pathogenic mycobacteria and is highly conservative. In vitro 75 experiments demonstrated that its transcription is controlled by the transcriptional regulator DosR  Here, we characterize dynamic changes in the DrrS expression in mycobacteria obtained 82 from the lungs of genetically susceptible and resistant TB-infected mice and provide a direct 83 evidence that the level of expression is regulated by the IFN-γ-dependent NO production. Using 84 high-throughput technologies, we describe the changes in the genome transcription profile that 85 accompany an increased DrrS transcription by mycobacteria. Overexpression of DrrS has led to the 6 109 was isolated from the lungs, and the level of DrrS expression was determined using quantitative 110 real-time PCR (Fig 1B). The highest level of expression was observed at week 10 post-challenge. In 111 B6 mice, it remained high throughout the experiment, although slowly decreased at the very late 112 phase of infection. At week 10 of infection, when I/St mice start to lose control of the disease 113 progression, the level of DrrS expression in their lung mycobacterial population was significantly 114 higher (P < 0.01) than that in more resistant B6 mice (Fig 1B). This may reflect an attempt of   (Fig 2A). In IFN-γ-activated macrophages,

128
DrrS expression was significantly (P < 0.001, unpaired t-test) higher than in control macrophages at  [14], was not dependent upon the presence of L-NIL ( Fig 2C). 146 In the in vitro system, DrrS expression was shown to be induced by the transcription 147 regulatory protein DosR [11], thus we assessed the dynamics of DosR transcription in our co-culture 148 system ( Fig 2D). Remarkably, the level of DosR transcription in mycobacteria engulfed by activated   Mapping the processed reads against the reference M. tuberculosis genome (AL123456.3, 166 http://www.ncbi.nlm.nih.gov/), provided the following numbers of mapped reads: 22.6 x 10 6 for the 167 OVER strain (98% of all reads) and 11.8 x 10 6 for the pMV strain (98% of all reads). The 168 percentage of the protein-encoding part of the genome deduced from all reads mapped comprised 169 70% for pMV (8.2 x 10 6 reads) and 57% for OVER (12.8 x 10 6 reads). Statistical results were 170 visualized as transcription profiles using the Artemis genome browser [22].

171
Using the software package edgeR [23], we identified genes the expression of which 172 differed between the two strains. Overall, 235 genes were found to be differently expressed under 173 the DrrS overexpression condition (S1 Table), with 88 genes demonstrating a decreased and 147 an 174 increased expression. Further ascribing of genes to functional categories was performed using the    and cofactors [36]. In line with this general tendency, we observed a ~4-fold reduction in the 223 expression of genes involved in synthesis of several amino acids (tryptophan, arginine, cysteine, 224 leucine, and alanine) and vitamin B6 in the OVER strain (Fig 3).

225
Regulators. Overexpression of DrrS significantly changed transcription of regulatory genes 226 increasing 10 and decreasing 7 of them (Fig 3). Among up-regulated, was the Rv0079 (8-fold)     was performed using qPCRmix-HS SYBR (Evrogen, Russia) and the Light Cycler 480 real-time 358 PCR system (Roche, Switzerland); cycling conditions were as follows: 95C for20 s, 61C for 20 s, 359 72C for 30 s, repeat 40 times; primers are listed in S2 Table. In the end of amplification, a 360 dissociation curve was plotted to confirm specificity of the product. All real-time experiments were 361 repeated in triplicate. The results were normalized against the 16S rRNA gene.   To activate macrophages, monolayers were treated with murine rIFN-γ (100 U/ml, Sigma) 401 for 14 h before adding mycobacteria. To block iNOS, 100 µM L-NIL (Sigma) was added 1 h before 402 rIFN-γ administration.

403
To extract RNA, dishes with cell monolayers were gently shaken, culture medium was 404 completely aspirated and macrophages were lysed with 5 ml/dish of Trisol (Invitrogen, Carlsbad,