Proteomic and transcriptomic experiments reveal an essential role of RNA degradosome complexes in shaping the transcriptome of Mycobacterium tuberculosis.

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.

Mycobacterium tuberculosis MtrB sensor kinase interactions with FtsI and Wag31 proteins reveal a role for MtrB distinct from that regulating MtrA activities.

The septal association of Mycobacterium tuberculosis MtrB, the kinase partner of the MtrAB two-component signal transduction system, is necessary for the optimal expression of the MtrA regulon targets, including ripA, fbpB, and ftsI, which are involved in cell division and cell wall synthesis. Here, we show that MtrB, irrespective of its phosphorylation status, interacts with Wag31, whereas only phosphorylation-competent MtrB interacts with FtsI. We provide evidence that FtsI depletion compromises the MtrB septal assembly and MtrA regulon expression; likewise, the absence of MtrB compromises FtsI localization and, possibly, FtsI activity. We conclude from these results that FtsI and MtrB are codependent for their activities and that FtsI functions as a positive modulator of MtrB activation and MtrA regulon expression. In contrast to FtsI, Wag31 depletion does not affect MtrB septal assembly and MtrA regulon expression, whereas the loss of MtrB increased Wag31 localization and the levels of PknA/PknB (PknA/B) serine-threonine protein kinase-mediated Wag31 phosphorylation. Interestingly, we found that FtsI decreased levels of phosphorylated Wag31 (Wag31∼P) and that MtrB interacted with PknA/B. Overall, our results indicate that MtrB interactions with FtsI, Wag31, and PknA/B are required for its optimal localization, MtrA regulon expression, and phosphorylation of Wag31. Our results emphasize a new role for MtrB in cell division and cell wall synthesis distinct from that regulating the MtrA phosphorylation activities.

Septal localization of the Mycobacterium tuberculosis MtrB sensor kinase promotes MtrA regulon expression.

The mechanisms responsible for activation of the MtrAB two-component regulatory signal transduction system, which includes sensor kinase MtrB and response regulator MtrA, are unknown. Here, we show that an MtrB-GFP fusion protein localized to the cell membrane, the septa, and the poles in Mycobacterium tuberculosis and Mycobacterium smegmatis. This localization was independent of MtrB phosphorylation status but dependent upon the assembly of FtsZ, the initiator of cell division. The M. smegmatis mtrB mutant was filamentous, defective for cell division, and contained lysozyme-sensitive cell walls. The mtrB phenotype was complemented by either production of MtrB protein competent for phosphorylation or overproduction of MtrA(Y102C) and MtrA(D13A) mutant proteins exhibiting altered phosphorylation potential, indicating that either MtrB phosphorylation or MtrB independent expression of MtrA regulon genes, including those involved in cell wall processing, are necessary for regulated cell division. In partial support of this observation, we found that the essential cell wall hydrolase ripA is an MtrA target and that the expression of bona fide MtrA targets ripA, fbpB, and dnaA were compromised in the mtrB mutant and partially rescued upon MtrA(Y102C) and MtrA(D13A) overproduction. MtrB septal assembly was compromised upon FtsZ depletion and exposure of cells to mitomycin C, a DNA damaging agent, which interferes with FtsZ ring assembly. Expression of MtrA targets was also compromised under the above conditions, indicating that MtrB septal localization and MtrA regulon expression are linked. We propose that MtrB septal association is a necessary feature of MtrB activation that promotes MtrA phosphorylation and MtrA regulon expression.

The functional response of human monocyte-derived macrophages to serum amyloid A and Mycobacterium tuberculosis infection.

Introduction: In the course of tuberculosis (TB), the level of major acute phase protein, namely serum amyloid A (hSAA-1), increases up to a hundredfold in the pleural fluids of infected individuals. Tubercle bacilli infecting the human host can be opsonized by hSAA-1, which affects bacterial entry into human macrophages and their intracellular multiplication. Methods: We applied global RNA sequencing to evaluate the functional response of human monocyte-derived macrophages (MDMs), isolated from healthy blood donors, under elevated hSAA-1 conditions and during infection with nonopsonized and hSAA-1-opsonized Mycobacterium tuberculosis (Mtb). In the same infection model, we also examined the functional response of mycobacteria to the intracellular environment of macrophages in the presence and absence of hSAA-1. The RNASeq analysis was validated using qPCR. The functional response of MDMs to hSAA-1 and/or tubercle bacilli was also evaluated for selected cytokines at the protein level by applying the Milliplex system. Findings: Transcriptomes of MDMs cultured in the presence of hSAA-1 or infected with Mtb showed a high degree of similarity for both upregulated and downregulated genes involved mainly in processes related to cell division and immune response, respectively. Among the most induced genes, across both hSAA-1 and Mtb infection conditions, CXCL8, CCL15, CCL5, IL-1ß, and receptors for IL-7 and IL-2 were identified. We also observed the same pattern of upregulated pro-inflammatory cytokines (TNFα, IL-6, IL-12, IL-18, IL-23, and IL-1) and downregulated anti-inflammatory cytokines (IL-10, TGFß, and antimicrobial peptide cathelicidin) in the hSAA-1 treated-MDMs or the phagocytes infected with tubercle bacilli. At this early stage of infection, Mtb genes affected by the inside microenvironment of MDMs are strictly involved in iron scavenging, adaptation to hypoxia, low pH, and increasing levels of CO2. The genes for the synthesis and transport of virulence lipids, but not cholesterol/fatty acid degradation, were also upregulated. Conclusion: Elevated serum hSAA-1 levels in tuberculosis enhance the response of host phagocytes to infection, including macrophages that have not yet been in contact with mycobacteria. SAA induces antigen processing and presentation processes by professional phagocytes reversing the inhibition caused by Mtb infection.

The Orphan Response Regulator Rv3143 Modulates the Activity of the NADH Dehydrogenase Complex (Nuo) in Mycobacterium tuberculosis via Protein-Protein Interactions.

Two-component signal transduction systems enable mycobacterial cells to quickly adapt and adequately respond to adverse environmental conditions encountered at various stages of host infection. We attempted to determine the role of the Rv3143 "orphan" response regulator in the physiology of Mycobacterium tuberculosis and its orthologue Msmeg_2064 in Mycobacterium smegmatis. We identified the Rv3143 protein as an interaction partner for NuoD, a member of the type I NADH dehydrogenase complex involved in oxidative phosphorylation. The mutants Δrv3143 and Δmsmeg_2064 were engineered in M. tuberculosis and M. smegmatis cells, respectively. The Δmsmeg_2064 strain exhibited a significant reduction in growth and viability in the presence of reactive nitrogen species. The Rv3143-deficient strain was sensitive to valinomycin, which is known to reduce the electrochemical potential of the cell and overexpressed genes required for nitrate respiration. An increased level of reduction of the 2,3,5-triphenyltetrazolium chloride (TTC) electron acceptor in Δrv3143 and Δmsmeg_2064 cells was also evident. The silencing of ndh expression using CRISPRi/dCas9 affected cell survival under limited oxygen conditions. Oxygen consumption during entry to hypoxia was most severely affected in the double-mutant Δmsmeg_2064 ndhCRISPRi/dCas9 . We propose that the regulatory protein Rv3143 is a component of the Nuo complex and modulates its activity.

Mycobacterium tuberculosis Binds Human Serum Amyloid A, and the Interaction Modulates the Colonization of Human Macrophages and the Transcriptional Response of the Pathogen.

As a very successful pathogen with outstanding adaptive properties, Mycobacterium tuberculosis (Mtb) has developed a plethora of sophisticated mechanisms to subvert host defenses and effectively enter and replicate in the harmful environment inside professional phagocytes, namely, macrophages. Here, we demonstrated the binding interaction of Mtb with a major human acute phase protein, namely, serum amyloid A (SAA1), and identified AtpA (Rv1308), ABC (Rv2477c), EspB (Rv3881c), TB 18.6 (Rv2140c), and ThiC (Rv0423c) membrane proteins as mycobacterial effectors responsible for the pathogen-host protein interplay. SAA1-opsonization of Mtb prior to the infection of human macrophages favored bacterial entry into target phagocytes accompanied by a substantial increase in the load of intracellularly multiplying and surviving bacteria. Furthermore, binding of human SAA1 by Mtb resulted in the up- or downregulation of the transcriptional response of tubercle bacilli. The most substantial changes were related to the increased expression level of the genes of two operons encoding mycobacterial transporter systems, namely, mmpL5/mmpS5 (rv0676c), and rv1217c, rv1218c. Therefore, we postulate that during infection, Mtb-SAA1 binding promotes the infection of host macrophages by tubercle bacilli and modulates the functional response of the pathogen.

Two-component kinase TrcS complements Mycobacterium smegmatis mtrB kinase mutant.

MtrAB is one of the important two-component regulatory systems (2CRS) in mycobacteria and consists of MtrB sensor kinase and MtrA response regulator. Mycobacterium smegmatis mtrB mutant is filamentous and shows defects in cell division, cell shape and optimal expression of the MtrA-regulon. In an effort to identify M. tuberculosis sensor kinases that work with MtrA and/or bypass the need for MtrB, we attempted to overexpress other M. tuberculosis sensor kinases in M. smegmatis mtrB background and reverse the mtrB phenotype. Overexpression of trcS kinase, but not nine others tested, reversed the mtrB mutant phenotype including the expression of the MtrA-regulon. However, the overexpression of trcS kinase did not reverse the mutant phenotype of a mtrA mutant. Bacterial-two hybrid assays revealed that the TrcS kinase interacts with both MtrB kinase and the response regulator MtrA. Recombinant TrcS protein exhibits autophosphorylation and transphosphorylation of its cognate response regulator TrcR, and MtrA. Together, these results support a model involving cross-talk between the MtrAB and TrcRS two-component systems.

The NnaR orphan response regulator is essential for the utilization of nitrate and nitrite as sole nitrogen sources in mycobacteria.

Nitrogen is an essential component of biological molecules and an indispensable microelement required for the growth of cells. Nitrogen metabolism of Mycobacterium smegmatis is regulated by a number of transcription factors, with the glnR gene product playing a major role. Under nitrogen-depletion conditions, GlnR controls the expression of many genes involved in nitrogen assimilation, including the msmeg_0432 gene encoding NnaR, the homologue of a nitrite/nitrate transport regulator from Streptomyces coelicolor. In the present study, the role of NnaR in the nitrogen metabolism of M. smegmatis was evaluated. The ∆glnR and ∆nnaR mutant strains were generated and cultured under nitrogen-depletion conditions. Total RNA profiling was used to investigate the potential role of NnaR in the GlnR regulon under nitrogen-depletion and in nitrogen-rich media. We found that disruption of MSMEG_0432 affected the expression of genes involved in nitrite/nitrate uptake, and its removal rendered mycobacteria unable to assimilate nitrogen from those sources, leading to cell death. RNA-Seq results were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and electrophoretic mobility shift assays (EMSAs). The ability of mutants to grow on various nitrogen sources was evaluated using the BIOLOG Phenotype screening platform and confirmed on minimal Sauton's medium containing various sources of nitrogen. The ∆glnR mutant was not able to convert nitrates to nitrites. Interestingly, NnaR required active GlnR to prevent nitrogen starvation, and both proteins cooperated in the regulation of gene expression associated with nitrate/nitrite assimilation. The ∆nnaR mutant was able to convert nitrates to nitrites, but it could not assimilate the products of this conversion. Importantly, NnaR was the key regulator of the expression of the truncated haemoglobin trHbN, which is required to improve the survival of bacteria under nitrosative stress.

MtrA Response Regulator Controls Cell Division and Cell Wall Metabolism and Affects Susceptibility of Mycobacteria to the First Line Antituberculosis Drugs.

The biological processes regulated by the essential response regulator MtrA and the growth conditions promoting its activation in Mycobacterium tuberculosis, a slow grower and pathogen, are largely unknown. Here, using a gain-of-function mutant, MtrAY 102C, which functions in the absence of the cognate MtrB sensor kinase, we show that the MtrA regulon includes several genes involved in the processes of cell division and cell wall metabolism. The expression of selected MtrA targets and intracellular MtrA levels were compromised under replication arrest induced by genetic manipulation and under stress conditions caused by toxic radicals. The loss of the mtrA gene in M. smegmatis, a rapid grower and non-pathogen, produced filamentous cells with branches and bulges, indicating defects in cell division and cell shape. The ΔmtrA mutant was sensitized to rifampicin and vancomycin and became more resistant to isoniazid, the first line antituberculosis drug. Our data are consistent with the proposal that MtrA controls the optimal cell division, cell wall integrity, and susceptibility to some antimycobacterial drugs.

Mycobacterial DNA Replication as a Target for Antituberculosis Drug Discovery.

BACKGROUND: Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis, is a leading infectious disease organism, causing millions of deaths each year. This serious pathogen has been greatly spread worldwide and recent years have observed an increase in the number of multi-drug resistant and totally drug resistant M. tuberculosis strains (WHO report, 2014). The danger of tuberculosis becoming an incurable disease has emphasized the need for the discovery of a new generation of antimicrobial agents. The development of novel alternative medical strategies, new drugs and the search for optimal drug targets are top priority areas of tuberculosis research. FACTORS: Key characteristics of mycobacteria include: slow growth, the ability to transform into a metabolically silent - latent state, intrinsic drug resistance and the relatively rapid development of acquired drug resistance. These factors make finding an ideal antituberculosis drug enormously challenging, even if it is designed to treat drug sensitive tuberculosis strains. A vast majority of canonical antibiotics including antituberculosis agents target bacterial cell wall biosynthesis or DNA/RNA processing. Novel therapeutic approaches are being tested to target mycobacterial cell division, twocomponent regulatory factors, lipid synthesis and the transition between the latent and actively growing states. DISCUSSION AND CONCLUSION: This review discusses the choice of cellular targets for an antituberculosis therapy, describes putative drug targets evaluated in the recent literature and summarizes potential candidates under clinical and pre-clinical development. We focus on the key cellular process of DNA replication, as a prominent target for future antituberculosis therapy. We describe two main pathways: the biosynthesis of nucleic acids precursors - the nucleotides, and the synthesis of DNA molecules. We summarize data regarding replication associated proteins that are critical for nucleotide synthesis, initiation, unwinding and elongation of the DNA during the replication process. They are pivotal processes required for successful multiplication of the bacterial cells and hence they are extensively investigated for the development of antituberculosis drugs. Finally, we summarize the most potent inhibitors of DNA synthesis and provide an up to date report on their status in the clinical trials.

PdtaS Deficiency Affects Resistance of Mycobacteria to Ribosome Targeting Antibiotics.

Two-component regulatory systems (TCSSs) are key regulatory elements responsible for the adaptation of bacteria to environmental stresses. A classical TCSS is typically comprised of a sensory histidine kinase and a corresponding response regulator. Here, we used homologous recombination to construct a Mycobacterium smegmatis mutant defective in the synthesis of cytosolic histidine kinase PdtaS (Msmeg_1918). The resulting ΔpdtaS mutant strain was tested in the Phenotype Microarray screening system, which allowed us to identify aminoglycoside antibiotic sensitivity, tetracyclines antibiotic resistance as well as membrane transport and respiration, as the main processes affected by removal of pdtaS. The antibiotic sensitivity profiles were confirmed by survival assessment and complementation studies. To gain insight into the molecular mechanisms responsible for the observed phenotype, we compared ribosomal RNA and protein profiles of the mutant and wild-type strains. We carried out Northern blotting and qRT-PCR to compare rRNA levels and analyzed ribosome sedimentation patterns of the wild-type and mutant strains on sucrose gradients. Isolated ribosomes were further used to estimate relative abundance of individual proteins in the ribosomal subunits using label free mass spectrometry analysis. Additionally, the ΔpdtaS mutant revealed lower activity of the respiratory chain as measured by the rate of TTC (triphenyltetrazolium chloride) reduction, while at the same time showing only insignificant changes in the uptake of aminoglycosides. We postulate that deficiency of PdtaS affects the oxidative respiration rates and ribosomal composition causing relevant changes to intrinsic resistance or susceptibility to antibiotics targeting ribosomes, which are commonly used to treat mycobacterial infections.

Uptake and metabolism of fluorescent steroids by mycobacterial cells.

Fluorescent steroids BODIPY-cholesterol (BPCh) and 7-nitrobenzoxadiazole-4-amino-(NBD)-labeled 22-NBD-chelesterol (22NC) as well as synthesized 20-(NBD)-pregn-5-en-3ß-ol (20NP) were found to undergo bioconversions by Mycobacterium tuberculosis H37Rv and M. smegmatis mc2 155. The major fluorescent products were determined to be 4-en-3-one derivatives of the compounds. Degradation of NBD fluorophore was also detected in the cases of 22NC and 20NP, but neither NBD degradation nor steroidal part modification were observed for the synthesized 3-(NBD)-cholestane. Mycobacterial 3ß-hydroxysteroid dehydrogenases were concluded to be responsible for the formation of the 4-en-3-one derivatives. All the compounds tested were found to cause staining both membrane lipids and cytosolic lipid droplets when incubated with mycobacteria in different manner, demonstrating ability of the steroids to reside in the compartments. The findings reveal a potential of the compounds for monitoring of steroid interactions with mycobacteria and provide information for design of new probes for this purpose.

ChiZ levels modulate cell division process in mycobacteria.

We have previously shown that expression of chiZ (Rv2719c), encoding a cell wall hydrolase, is upregulated in response to DNA damaging agents and exposure to cephalexin. Furthermore, increased levels of ChiZ lead to decreased viability, loss of membrane integrity and defects in FtsZ-GFP localization and cell division. We now show that ChiZ N'-terminal 110 amino acid region, containing the cell wall hydrolase activity, is sufficient to modulate FtsZ-GFP localization. Further, we found that FtsZ-GFP rings are stabilized in a chiZ deletion strain indicating that ChiZ activity regulates FtsZ assembly. Overexpression of ftsZ did not reverse the reduction in viability caused by overproduction of ChiZ indicating that ChiZ neither interacts with nor directly influences FtsZ assembly. Bacterial two-hybrid assays revealed that ChiZ interacts with FtsI and FtsQ, two other septasomal proteins, but not with FtsZ. Finally, we show that ChiZ is not required for virulence of Mycobacterium tuberculosis in murine macrophages and mice. Our data suggest that optimal levels and activity of the cell wall hydrolase ChiZ are required for regulated cell division in mycobacteria.