Dynamic Transcriptional Landscape of Mycobacterium smegmatis under Cold Stress.

Bacterial adaptation to cold stress requires wide transcriptional reprogramming. However, the knowledge of molecular mechanisms underlying the cold stress response of mycobacteria is limited. We conducted comparative transcriptomic analysis of Mycobacterium smegmatis subjected to cold shock. The growth of M. smegmatis cultivated at 37 °C was arrested just after exposure to cold (acclimation phase) but later (by 24 h) was resumed at a much slower rate (adaptation phase). Transcriptomic analyses revealed distinct gene expression patterns corresponding to the two phases. During the acclimation phase, differential expression was observed for genes associated with cell wall remodeling, starvation response, and osmotic pressure stress, in parallel with global changes in the expression of transcription factors and the downregulation of ribosomal genes, suggesting an energy-saving strategy to support survival. At the adaptation phase, the expression profiles were recovered, indicating restoration of the processes repressed earlier. Comparison of transcriptional responses in M. smegmatis with those in other bacteria revealed unique adaptation strategies developed by mycobacteria. Our findings shed light on the molecular mechanisms underlying M. smegmatis survival under cold stress. Further research should clarify whether the discovered transcriptional mechanisms exist in other mycobacterial species, including pathogenic Mycobacterium tuberculosis, which could be important for transmission control.

Small RNA MTS1338 Configures a Stress Resistance Signature in Mycobacterium tuberculosis.

In the course of evolution, Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis, has developed sophisticated strategies to evade host immune response, including the synthesis of small non-coding RNAs (sRNAs), which regulate post-transcriptional pathways involved in the stress adaptation of mycobacteria. sRNA MTS1338 is upregulated in Mtb during its infection of cultured macrophages and in the model of chronic tuberculosis, suggesting involvement in host-pathogen interactions. Here, we analyzed the role of MTS1338 in the Mtb response to macrophage-like stresses in vitro. The Mtb strain overexpressing MTS1338 demonstrated enhanced survival ability under low pH, nitrosative, and oxidative stress conditions simulating the antimicrobial environment inside macrophages. Transcriptomic analysis revealed that in MTS1338-overexpressing Mtb, the stress factors led to the activation of a number of transcriptional regulators, toxin-antitoxin modules, and stress chaperones, about half of which coincided with the genes induced in Mtb phagocytosed by macrophages. We determined the MTS1338 "core regulon", consisting of 11 genes that were activated in all conditions under MTS1338 overexpression. Our findings indicate that MTS1338 is a stress-induced sRNA that promotes Mtb survival in macrophages by triggering adaptive transcriptional mechanisms in response to host antimicrobial defense reactions.

Red light-emitting short Mango-based system enables tracking a mycobacterial small noncoding RNA in infected macrophages.

Progress in RNA metabolism and function studies relies largely on molecular imaging systems, including those comprising a fluorogenic dye and an aptamer-based fluorescence-activating tag. G4 aptamers of the Mango family, typically combined with a duplex/hairpin scaffold, activate the fluorescence of a green light-emitting dye TO1-biotin and hold great promise for intracellular RNA tracking. Here, we report a new Mango-based imaging platform. Its key advantages are the tunability of spectral properties and applicability for visualization of small RNA molecules that require minimal tag size. The former advantage is due to an expanded (green-to-red-emitting) palette of TO1-inspired fluorogenic dyes, and the truncated duplex scaffold ensures the latter. To illustrate the applicability of the improved platform, we tagged Mycobacterium tuberculosis sncRNA with the shortened aptamer-scaffold tag. Then, we visualized it in bacteria and bacteria-infected macrophages using the new red light-emitting Mango-activated dye.

VapC toxin switches M. smegmatis cells into dormancy through 23S rRNA cleavage.

Mycobacterium tuberculosis is an extremely successful pathogen known for its ability to cause latent infection. The latter is connected with the bacterium resting state development and is considered to be based on the activity of toxin-antitoxin (TA) systems at least in part. Here we studied the physiological and proteomic consequences of VapC toxin overexpression together with the features of the protein synthesis apparatus and compared them with the characteristics of dormant mycobacterial cells in an M. smegmatis model. The findings allow suggesting the mechanism mycobacteria enter dormancy, which is realized through VapC-caused cleavage of the 23S rRNA Sarcin-Ricin loop followed by conservation of stalled ribosomes in a membrane-associated manner. The found features of resting mycobacteria protein synthesis apparatus hypothesize the mechanisms of resuscitation from dormancy through the ribosomes de-association off the membrane accompanied by the 23S rRNA break curing, and could be of value for the development of principally new antituberculosis agents.

Alterations in Proteostasis System Components in Peripheral Blood Mononuclear Cells in Parkinson Disease: Focusing on the HSP70 and p62 Levels.

Parkinson disease (PD) is attributed to a proteostasis disorder mediated by α-synuclein accumulating in a specific brain region. PD manifestation is often related to extraneuronal alterations, some of which could be used as diagnostic or prognostic PD biomarkers. In this work, we studied the shifts in the expression of proteostasis-associated chaperones of the HSP70 family and autophagy-dependent p62 protein values in the peripheral blood mononuclear cells (PBMC) of mild to moderate PD patients. Although we did not detect any changes in the intracellular HSP70 protein pool in PD patients compared to non-PD controls, an increase in the transcriptional activity of the stress-associated HSPA1A/B and HSPA6 genes was observed in these cells. Basal p62 content was found to be increased in PD patients' PBMC, similarly to the p62 level in substantia nigra neural cells in PD. Moreover, the spontaneous apoptosis level was increased among PBMC and positively correlated with the p62 intracellular level in the PD group. A combined HSPA6- and p62-based analysis among 26 PD patients and 36 age-matched non-PD controls pointed out the diagnostic significance of these markers, with intermediate sensitivity and high specificity of this combination when observing patients diagnosed with PD.

An In Vivo Model of Separate M. tuberculosis Phagocytosis by Neutrophils and Macrophages: Gene Expression Profiles in the Parasite and Disease Development in the Mouse Host.

The role of neutrophils in tuberculosis infection remains less well studied compared to that of the CD4+ T-lymphocytes and macrophages. Thus, alterations in Mycobacterium tuberculosis transcription profile following phagocytosis by neutrophils and how these shifts differ from those caused by macrophage phagocytosis remain unknown. We developed a mouse model that allows obtaining large amounts of either neutrophils or macrophages infected in vivo with M. tuberculosis for mycobacteria isolation in quantities sufficient for the whole genome RNA sequencing and aerosol challenge of mice. Here, we present: (i) the differences in transcription profiles of mycobacteria isolated from liquid cultures, neutrophils and macrophages infected in vivo; (ii) phenotypes of infection and lung inflammation (life span, colony forming units (CFU) counts in organs, lung pathology, immune cells infiltration and cytokine production) in genetically TB-susceptible mice identically infected via respiratory tract with neutrophil-passaged (NP), macrophage-passaged (MP) and conventionally prepared (CP) mycobacteria. Two-hour residence within neutrophils caused transcriptome shifts consistent with mycobacterial transition to dormancy and diminished their capacity to attract immune cells to infected lung tissue. Mycobacterial multiplication in organs did not depend upon pre-phagocytosis, whilst survival time of infected mice was shorter in the group infected with NP bacilli. We also discuss possible reasons for these phenotypic divergences.

Small RNA F6 Provides Mycobacterium smegmatis Entry into Dormancy.

Regulatory small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. Various stresses such as hypoxia and nutrient starvation cause a reduction in the metabolic activity of Mycobacterium smegmatis, leading to entry into dormancy. We investigated the functional role of F6, a small RNA of M. smegmatis, and constructed an F6 deletion strain of M. smegmatis. Using the RNA-seq approach, we demonstrated that gene expression changes that accompany F6 deletion contributed to bacterial resistance against oxidative stress. We also found that F6 directly interacted with 5'-UTR of MSMEG_4640 mRNA encoding RpfE2, a resuscitation-promoting factor, which led to the downregulation of RpfE2 expression. The F6 deletion strain was characterized by the reduced ability to enter into dormancy (non-culturability) in the potassium deficiency model compared to the wild-type strain, indicating that F6 significantly contributes to bacterial adaptation to non-optimal growth conditions.

Small Noncoding RNAs and Their Role in the Pathogenesis of Mycobacterium tuberculosis Infection.

Mycobacterium tuberculosis possesses a significant arsenal of strategies to combat immune defense of the host organism. Small noncoding RNAs, which constitute the largest group of regulatory RNAs, play an important role in the host-pathogen interactions and represent one of the levels of the regulation of interactions of microbial cells with their environment. The regulatory role of small RNAs in pathogenic bacteria is essential when rapid adaptation to the changing environmental conditions with further synchronization of metabolic reactions are required to ensure microbial survival and infection progression. During the past few years, eight small RNAs from M. tuberculosis have been functionally characterized, and targets for four of them have been identified. Small RNAs from M. tuberculosis and other pathogenic microorganisms were found to be one of the most important functional factors in the adaptive response to changing environmental conditions.

Mycobacterium tuberculosis Small RNA MTS1338 Confers Pathogenic Properties to Non-Pathogenic Mycobacterium smegmatis.

Small non-coding RNAs play a key role in bacterial adaptation to various stresses. Mycobacterium tuberculosis small RNA MTS1338 is upregulated during mycobacteria infection of macrophages, suggesting its involvement in the interaction of the pathogen with the host. In this study, we explored the functional effects of MTS1338 by expressing it in non-pathogenic Mycobacterium smegmatis that lacks the MTS1338 gene. The results indicated that MTS1338 slowed the growth of the recombinant mycobacteria in culture and increased their survival in RAW 264.7 macrophages, where the MTS1338-expressing strain significantly (p < 0.05) reduced the number of mature phagolysosomes and changed the production of cytokines IL-1ß, IL-6, IL-10, IL-12, TGF-ß, and TNF-α compared to those of the control strain. Proteomic and secretomic profiling of recombinant and control strains revealed differential expression of proteins involved in the synthesis of main cell wall components and in the regulation of iron metabolism (ESX-3 secretion system) and response to hypoxia (furA, whiB4, phoP). These effects of MTS1338 expression are characteristic for M. tuberculosis during infection, suggesting that in pathogenic mycobacteria MTS1338 plays the role of a virulence factor supporting the residence of M. tuberculosis in the host.

Long interspersed nuclear element-1 methylation status in the circulating DNA from blood of patients with malignant and chronic inflammatory lung diseases.

Along with other malignant diseases, lung cancer arises from the precancerous lung tissue state. Aberrant DNA methylation (hypermethylation of certain genes and hypomethylation of retrotransposons) is known as one of the driving forces of malignant cell transformation. Epigenetic changes were shown to be detectable in DNA, circulating in the blood (cirDNA) of cancer patients, indicating the possibility to use them as cancer markers. The current study is the first to compare the Long interspersed nuclear element-1 (LINE-1) methylation level in the blood from lung cancer patients before treatment versus different control groups as healthy subjects, patients with bronchitis and patients with chronic obstructive pulmonary disease (COPD). The concentration of LINE-1 methylated fragments, region 1 (LINE-1 methylated, LINE-1-met) was estimated by quantitative methyl-specific PCR. The total concentration of the circulating LINE-1 copies was measured by qPCR specific for LINE-1 region 2, which was selected due to its CpG methylation-independent sequence (LINE-1-Ind). Both LINE-1 methylation level and LINE-1 methylation index (LINE-1-met/LINE-1-Ind ratio) was decreased in lung cancer patients compared with the joint control group (healthy subjects + patients with bronchitis + COPD patients) (Mann-Whitney U-test, P = 0.016). We also found that the tendency of LINE-1 methylation index decreases in the cirDNA from lung cancer patients versus COPD patients (Mann-Whitney U-test, P = 0.07). Our data indicate that the quantitative analysis of the LINE-1 methylation level in the cirDNA is valuable for discrimination of lung cancer patients from patients with chronic inflammatory lung diseases.

Aberrant Methylation of LINE-1 Transposable Elements: A Search for Cancer Biomarkers.

Cancer remains one of the main causes of human mortality despite significant progress in its diagnostics and therapy achieved in the past decade. Massive hypomethylation of retrotransposons, in particular LINE-1, is considered a hallmark of most malignant transformations as it results in the reactivation of retroelements and subsequent genomic instability. Accumulating data on LINE-1 aberrant methylation in different tumor types indicates its significant role in cancer initiation and progression. However, direct evidence that LINE-1 activation can be used as a cancer biomarker is still limited. The objective of this review was to critically evaluate the published results regarding the diagnostic/prognostic potential of the LINE-1 methylation status in cancer. Our analysis indicates that LINE-1 hypomethylation is a promising candidate biomarker of cancer development, which, however, needs validation in both clinical and laboratory studies to confirm its applicability to different cancer types and/or stages. As LINE-1 is present in multiple cell-free copies in blood, it has advantages over single-copy genes regarding perspectives of using its methylation status as an epigenetic cancer biomarker for cell-free DNA liquid biopsy.

Mycolicibacterium smegmatis possesses operational agmatinase but contains no detectable polyamines.

Background: Polyamines are widespread intracellular molecules able to influence antibiotic susceptibility, but almost nothing is known on their occurrence and physiological role in mycobacteria. Methods: here, we analyzed transcriptomic, proteomic and biochemical data and obtained the first evidence for the post-transcriptional expression of some genes attributed to polyamine metabolism and polyamine transport in Mycolicibacterium smegmatis (basionym Mycobacterium smegmatis). Results: in our experiments, exponentially growing cells demonstrated transcription of 21 polyamine-associated genes and possessed 7 enzymes of polyamine metabolism and 2 polyamine transport proteins. Conclusion: Mycolicibacterium smegmatis putrescine synthesizing enzyme agmatinase SpeB was originally shown to catalyze agmatine conversion to putrescine in vitro. Nevertheless, we have not found any polyamines in mycobacterial cells.

New Insights into the Mechanism of Action of the Thienopyrimidine Antitubercular Prodrug TP053.

The thienopyrimidine TP053 is an antitubercular prodrug active against both replicating and nonreplicating Mycobacterium tuberculosis (M. tuberculosis) cells, which requires activation by the mycothiol-dependent nitroreductase Mrx2. The investigation of the mechanism of action of TP053 revealed that Mrx2 releases nitric oxide from this drug both in the enzyme assays with purified Mrx2 and in mycobacterial cultures, which can explain its activity against nonreplicating bacilli, similar to pretomanid activated by the nitroreductase Ddn. In addition, we identified a highly reactive metabolite, 2-(4-mercapto-6-(methylamino)-2-phenylpyrimidin-5-yl)ethan-1-ol, which can contribute to the antimycobacterial effects on replicating cells as well as on nonreplicating cells. In summary, we explain the mechanism of action of TP053 on both replicating and nonreplicating M. tuberculosis and report a novel activity for Mrx2, which in addition to Ddn, represents another example of nitroreductase releasing nitric oxide from its substrate. These findings are particularly relevant in the context of drugs targeting nonreplicating M. tuberculosis, which is shown to be killed by increased levels of nitric oxide.

MTS1338, A Small Mycobacterium tuberculosis RNA, Regulates Transcriptional Shifts Consistent With Bacterial Adaptation for Entering Into Dormancy and Survival Within Host Macrophages.

Small non-coding RNAs play a significant role in bacterial adaptation to changing environmental conditions. We investigated the dynamics of expression of MTS1338, a small non-coding RNA of Mycobacterium tuberculosis, in the mouse model in vivo, regulation of its expression in the infected macrophages, and the consequences of its overexpression in bacterial cultures. Here we demonstrate that MTS1338 significantly contributes to host-pathogen interactions. Activation of the host immune system triggered NO-inducible up-regulation of MTS1338 in macrophage-engulfed mycobacteria. Constitutive overexpression of MTS1338 in cultured mycobacteria improved their survival in vitro under low pH conditions. MTS1338 up-regulation launched a spectrum of shifts in the transcriptome profile similar to those reported for M. tuberculosis adaptation to hostile intra-macrophage environment. Using the RNA-seq approach, we demonstrate that gene expression changes accompanying MTS1338 overexpression indicate reduction in translational activity and bacterial growth. These changes indicate mycobacteria entering the dormant state. Taken together, our results suggest a direct involvement of this sRNA in the interplay between mycobacteria and the host immune system during infectious process.

A New Antisense Phosphoryl Guanidine Oligo-2'-O-Methylribonucleotide Penetrates Into Intracellular Mycobacteria and Suppresses Target Gene Expression.

The worldwide spread of multidrug-resistant Mycobacterium tuberculosis strains prompted the development of new strategies to combat tuberculosis, one of which is antisense therapy based on targeting bacterial mRNA by oligonucleotide derivatives. However, the main limitation of antisense antibacterials is poor cellular uptake because of electrostatic charge. Phosphoryl guanidine oligo-2'-O-methylribonucleotides (2'-OMe PGOs) are a novel type of uncharged RNA analogues with high RNA affinity, which penetrate through the bacterial cell wall more efficiently. In this study, we investigated the uptake and biological effects of 2'-OMe PGO in mycobacteria. The results indicated that 2'-OMe PGO specific for the alanine dehydrogenase-encoding ald gene inhibited the growth of Mycobacterium smegmatis and downregulated ald expression at both the transcriptional and translational levels through an RNase H-independent mechanism, showing higher biological activity than its phosphorothioate oligonucleotide counterpart. Confocal microscopy revealed that the anti-ald 2'-OMe PGO was taken up by intracellular mycobacteria residing in RAW 264.7 macrophages without exerting toxic effects on eukaryotic cells, indicating that 2'-OMe PGO was able to efficiently cross two cellular membranes. In addition, 2'-OMe PGO inhibited the transcription of the target ald gene in M. smegmatis-infected macrophages. Thus, we demonstrated, for the first time, a possibility of targeting gene expression and inhibiting growth of intracellular mycobacteria by antisense oligonucleotide derivatives. Strong antisense activity and efficient uptake of the new RNA analogue, 2'-OMe PGO, by intracellular microorganisms revealed here may promote the development of novel therapeutic strategies to treat TB and prevent the emergence of drug-resistant mycobacterial strains.

Resuscitation of Dormant "Non-culturable" Mycobacterium tuberculosis Is Characterized by Immediate Transcriptional Burst.

Under unfavorable conditions such as host immune responses and environmental stresses, human pathogen Mycobacterium tuberculosis may acquire the dormancy phenotype characterized by "non-culturability" and a substantial decrease of metabolic activity and global transcription rates. Here, we found that the transition of M. tuberculosis from the dormant "non-culturable" (NC) cells to fully replicating population in vitro occurred not earlier than 7 days after the start of the resuscitation process, with predominant resuscitation over this time interval evidenced by shortening apparent generation time up to 2.8 h at the beginning of resuscitation. The early resuscitation phase was characterized by constant, albeit low, incorporation of radioactive uracil, indicating de novo transcription immediately after the removal of the stress factor, which resulted in significant changes of the M. tuberculosis transcriptional profile already after the first 24 h of resuscitation. This early response included transcriptional upregulation of genes encoding enzymes of fatty acid synthase system type I (FASI) and type II (FASII) responsible for fatty acid/mycolic acid biosynthesis, and regulatory genes, including whiB6 encoding a redox-sensing transcription factor. The second resuscitation phase took place 4 days after the resuscitation onset, i.e., still before the start of active cell division, and included activation of central metabolism genes encoding NADH dehydrogenases, ATP-synthases, and ribosomal proteins. Our results demonstrate, for the first time, that the resuscitation of dormant NC M. tuberculosis is characterized by immediate activation of de novo transcription followed by the upregulation of genes controlling key metabolic pathways and then, cell multiplication.

Copper-related toxicity in replicating and dormant Mycobacterium tuberculosis caused by 1-hydroxy-5-R-pyridine-2(1H)-thiones.

With the emerging primary resistance of Mycobacterium tuberculosis to current drugs and wide distribution of latent tuberculosis infection, the need for new compounds with a novel mode of action is growing. Copper-mediated innate immunity and its antibacterial toxicity pose novel strategies for tuberculosis drug discovery and development. Transcriptome response to 1-hydroxy-5-R-pyridine-2(1H)-thiones, which were found to be highly active in vitro against actively growing and dormant nonculturable M. tuberculosis, revealed signs of copper toxicity. 1-Hydroxy-5-R-pyridine-2(1H)-thiones were found to form stable charged lipophilic complexes with Cu2+ ions that could transport into mycobacterial cells. Copper accumulated inside treated bacilli as subsequent metabolic destruction of the complex led to chemical transformation of 1-hydroxy-5-R-pyridine-2(1H)-thiones and release of free Cu2+ into the cytoplasm. 1-Hydroxy-5-R-pyridine-2(1H)-thiones are a potent class of Cu-dependent inhibitors of M. tuberculosis, and may control infection by impairment of copper homeostasis.

Assessment of piRNA biogenesis and function in testicular germ cell tumors and their precursor germ cell neoplasia in situ.

BACKGROUND: Aberrant overexpression of PIWI/piRNA pathway proteins is shown for many types of tumors. Interestingly, these proteins are downregulated in testicular germ cell tumors (TGCTs) compared to normal testis tissues. Here, we used germline and TGCT markers to assess the piRNA biogenesis and function in TGCTs and their precursor germ cell neoplasia in situ (GCNIS). METHODS: We used small RNA deep sequencing, qRT-PCR, and mining public RNAseq/small RNA-seq datasets to examine PIWI/piRNA gene expression and piRNA biogenesis at four stages of TGCT development: (i) germ cells in healthy testis tissues, (ii) germ cells in testis tissues adjacent to TGCTs, (iii) GCNIS cells and (iv) TGCT cells. To this end, we studied three types of samples: (a) healthy testis, (b) testis tissues adjacent to two types of TGCTs (seminomas and nonseminomas) and containing both germ cells and GCNIS cells, as well as (c) matching TGCT samples. RESULTS: Based on our analyses of small RNA-seq data as well as the presence/absence of expression correlation between PIWI/piRNA pathway genes and germline or TGCT markers, we can suggest that piRNA biogenesis is intact in germ cells present in healthy adult testes, and adjacent to TGCTs. Conversely, GCNIS and TGCT cells were found to lack PIWI/piRNA pathway gene expression and germline-like piRNA biogenesis. However, using an in vitro cell line model, we revealed a possible role for a short PIWIL2/HILI isoform expressed in TGCTs in posttranscriptional regulation of the youngest members of LINE and SINE classes of transposable elements. Importantly, this regulation is also implemented without involvement of germline-like biogenesis of piRNAs. CONCLUSIONS: Though further studies are warranted, these findings suggest that the conventional germline-like PIWI/piRNA pathway is lost in transition from germ cells to GCNIS cells.

Two modes of targeting transposable elements by piRNA pathway in human testis.

PIWI proteins and their partner small RNAs, termed piRNAs, are known to control transposable elements (TEs) in the germline. Here, we provide evidence that in humans this control is exerted in two different modes. On the one hand, production of piRNAs specifically targeting evolutionarily youngest TEs (L1HS, L1PA2-L1PA6, LTR12C, SVA) is present both at prenatal and postnatal stages of spermatogenesis and is performed without involvement of piRNA clusters. On the other hand, at postnatal stages, piRNAs deriving from pachytene clusters target "older" TEs and thus complement cluster-independent piRNA production to achieve relevant targeting of virtually all TEs expressed in postnatal testis. We also find that converging transcription of antisense-oriented genes contributes to the origin of genic postnatal prepachytene clusters. Finally, while a fraction of pachytene piRNAs was previously shown to arise from long intergenic noncoding RNAs (lincRNAs, i.e., pachytene piRNA cluster primary transcripts), we ascertain that these are a specific set of lincRNAs that both possess distinguishing epigenetic features and are expressed exclusively in testis.