RESUMO
The limitations of TB treatment are the long duration and immune-dampening effects of anti-tuberculosis therapy. The Cell wall plays a crucial role in survival and virulence; hence, enzymes involved in its biosynthesis are good therapeutic targets. Here, we identify Mycobacterium tuberculosis (Mtb) GlmM, (GlmMMtb) engaged in the UDP-GlcNAc synthesis pathway as an essential enzyme. We generated a conditional knockdown strain, Rv-glmMkD using the CRISPR interference-mediated gene silencing approach. Depletion of GlmMMtb affects the morphology and thickness of the cell wall. The Rv-glmMkD strain attenuated Mtb survival in vitro, in the host macrophages (ex vivo), and in a murine mice infection model (in vivo). Results suggest that the depletion of GlmMMtb induces M1 macrophage polarization, prompting a pro-inflammatory cytokine response, apparent from the upregulation of activation markers, including IFNÉ£ and IL-17 that resists the growth of Mtb. These observations provide a rationale for exploring GlmMMtb as a potential therapeutic target.
Assuntos
Proteínas de Bactérias , Macrófagos , Mycobacterium tuberculosis , Tuberculose , Mycobacterium tuberculosis/imunologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Animais , Camundongos , Tuberculose/imunologia , Tuberculose/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Macrófagos/imunologia , Macrófagos/microbiologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Feminino , Interações Hospedeiro-Patógeno/imunologia , Modelos Animais de Doenças , HumanosRESUMO
Mycobacterium tuberculosis's (Mtb) autarkic lifestyle within the host involves rewiring its transcriptional networks to combat host-induced stresses. With the help of RNA sequencing performed under various stress conditions, we identified that genes belonging to Mtb sulfur metabolism pathways are significantly upregulated during oxidative stress. Using an integrated approach of microbial genetics, transcriptomics, metabolomics, animal experiments, chemical inhibition, and rescue studies, we investigated the biological role of non-canonical L-cysteine synthases, CysM and CysK2. While transcriptome signatures of RvΔcysM and RvΔcysK2 appear similar under regular growth conditions, we observed unique transcriptional signatures when subjected to oxidative stress. We followed pool size and labelling (34S) of key downstream metabolites, viz. mycothiol and ergothioneine, to monitor L-cysteine biosynthesis and utilization. This revealed the significant role of distinct L-cysteine biosynthetic routes on redox stress and homeostasis. CysM and CysK2 independently facilitate Mtb survival by alleviating host-induced redox stress, suggesting they are not fully redundant during infection. With the help of genetic mutants and chemical inhibitors, we show that CysM and CysK2 serve as unique, attractive targets for adjunct therapy to combat mycobacterial infection.
Assuntos
Vias Biossintéticas , Cisteína Sintase , Cisteína , Inositol , Mycobacterium tuberculosis , Estresse Oxidativo , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Cisteína/metabolismo , Cisteína/biossíntese , Cisteína Sintase/metabolismo , Cisteína Sintase/genética , Vias Biossintéticas/genética , Inositol/metabolismo , Inositol/biossíntese , Animais , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Ergotioneína/biossíntese , Ergotioneína/metabolismo , Regulação Bacteriana da Expressão Gênica , Camundongos , Glicopeptídeos/metabolismo , Glicopeptídeos/biossíntese , Tuberculose/microbiologiaRESUMO
Mycobacterium tuberculosis encodes ~200 transcription factors that modulate gene expression under different microenvironments in the host. Even though high-throughput chromatin immunoprecipitation sequencing and transcriptome sequencing (RNA-seq) studies have identified the regulatory network for ~80% of transcription factors, many transcription factors remain uncharacterized. EmbR is one such transcription factor whose in vivo regulon and biological function are yet to be elucidated. Previous in vitro studies suggested that phosphorylation of EmbR by PknH upregulates the embCAB operon. Using a gene replacement mutant of embR, we investigated its role in modulating cellular morphology, antibiotic resistance, and survival in the host. Contrary to the prevailing hypothesis, under normal growth conditions, EmbR is neither phosphorylated nor impacted by ethambutol resistance through the regulation of the embCAB operon. The embR deletion mutant displayed attenuated M. tuberculosis survival in vivo. RNA-seq analysis suggested that EmbR regulates operons involved in the secretion pathway, lipid metabolism, virulence, and hypoxia, including well-known hypoxia-inducible genes devS and hspX. Lipidome analysis revealed that EmbR modulates levels of all lysophospholipids, several phospholipids, and M. tuberculosis-specific lipids, which is more pronounced under hypoxic conditions. We found that the EmbR mutant is hypersusceptible to hypoxic stress, and RNA sequencing performed under hypoxic conditions indicated that EmbR majorly regulates genes involved in response to acidic pH, hypoxia, and fatty acid metabolism. We observed condition-specific phosphorylation of EmbR, which contributes to EmbR-mediated transcription of several essential genes, ensuring enhanced survival. Collectively, the study establishes EmbR as a key modulator of hypoxic response that facilitates mycobacterial survival in the host. IMPORTANCE Mycobacterium tuberculosis modulates its transcriptional machinery in response to dynamic microenvironments encountered within the host. In this study, we identified that EmbR, a transcription factor, plays important roles in modulating cellular morphology, antibiotic resistance, and survival in the host. We found that EmbR undergoes condition-specific phosphorylation for its activation. Together, the study establishes a key role of EmbR as a transcriptional activator of genes belonging to multiple pathways, viz., virulence, secretion, or polyketide synthesis, that aid in mycobacterial survival during hypoxia and within the host.
Assuntos
Proteínas de Bactérias , Mycobacterium tuberculosis , Fatores de Transcrição , Fatores de Virulência , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Hipóxia , Mycobacterium tuberculosis/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Virulência/genética , Fatores de Virulência/metabolismoRESUMO
Mycobacterium tuberculosis (Mtb) has evolved diverse cellular processes in response to the multiple stresses it encounters within the infected host. We explored available TnSeq datasets to identify transcription factors (TFs) that are essential for Mtb survival inside the host. The analysis identified a single TF, Rv1332 (AosR), conserved across actinomycetes with a so-far uncharacterized function. AosR mitigates phagocyte-derived oxidative and nitrosative stress, thus promoting mycobacterial growth in the murine lungs and spleen. Oxidative stress induces formation of a single intrasubunit disulphide bond in AosR, which in turn facilitates AosR interaction with an extracytoplasmic-function sigma factor, SigH. This leads to the specific upregulation of the CysM-dependent non-canonical cysteine biosynthesis pathway through an auxiliary intragenic stress-responsive promoter, an axis critical in detoxifying host-derived oxidative and nitrosative radicals. Failure to upregulate AosR-dependent cysteine biosynthesis during the redox stress causes differential expression of 6% of Mtb genes. Our study shows that the AosR-SigH pathway is critical for detoxifying host-derived oxidative and nitrosative radicals to enhance Mtb survival in the hostile intracellular environment.
Assuntos
Actinobacteria/genética , Homeostase/genética , Mycobacterium tuberculosis/genética , Fatores de Transcrição/genética , Animais , Proteínas de Bactérias/genética , Feminino , Regulação Bacteriana da Expressão Gênica/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Oxirredução , Estresse Oxidativo/genética , Regiões Promotoras Genéticas/genética , Fator sigma/genética , Transcrição Gênica/genéticaRESUMO
Attenuated intracellular survival of Mycobacterium tuberculosis (Mtb) secretory gene mutants exemplifies their role as virulence factors. Mtb peptidyl prolyl isomerase A (PPiA) assists in protein folding through cis/trans isomerization of prolyl bonds. Here, we show that PPiA abets Mtb survival and aids in disease progression by exploiting host-associated factors. While the deletion of PPiA has no discernable effect on bacillary survival in a murine infection model, it compromises the formation of granuloma-like lesions and promotes host cell death through ferroptosis. Overexpression of PPiA enhances the bacillary load and exacerbates pathology in mice lungs. Importantly, PPiA interacts with the integrin α5ß1 receptor through a conserved surface-exposed RGD motif. The secretion of PPiA as well as interaction with integrin contributes to disease progression by upregulating multiple host matrix metalloproteinases. Collectively, we identified a novel nonchaperone role of PPiA that is critical in facilitating host-pathogen interaction and ensuing disease progression.
Assuntos
Interações Hospedeiro-Patógeno , Mycobacterium tuberculosis/enzimologia , Peptidilprolil Isomerase/metabolismo , Animais , Progressão da Doença , Integrinas , CamundongosRESUMO
Eradication of tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), has been a challenge due to its uncanny ability to survive in a dormant state inside host granulomas for decades. Mtb rewires its metabolic and redox regulatory networks to survive in the hostile hypoxic and nutrient-limiting environment, facilitating the formation of drug-tolerant persisters. Previously, we showed that protein kinase G (PknG), a virulence factor required for lysosomal escape, aids in metabolic adaptation, thereby promoting the survival of nonreplicating mycobacteria. Here, we sought to investigate the therapeutic potential of PknG against latent mycobacterium. We show that inhibition of PknG by AX20017 reduces mycobacterial survival in in vitro latency models such as hypoxia, persisters, and nutrient starvation. Targeting PknG enhances the bactericidal activity of the frontline anti-TB drugs in peritoneal macrophages. Deletion of pknG resulted in 5- to 15-fold-reduced survival of Mtb in chronically infected mice treated with anti-TB drugs. Importantly, in the Cornell mouse model of latent TB, the deletion of pknG drastically attenuated Mtb's ability to resuscitate after antibiotic treatment compared with wild-type and complemented strains. This is the first study to investigate the sterilizing activity of pknG deletion and inhibition for adjunct therapy against latent TB in a preclinical model. Collectively, these results suggest that PknG may be a promising drug target for adjunct therapy to shorten the treatment duration and reduce disease relapse.
Assuntos
Tuberculose Latente , Mycobacterium tuberculosis , Tuberculose , Animais , Antituberculosos/farmacologia , Tuberculose Latente/tratamento farmacológico , Camundongos , Mycobacterium tuberculosis/genética , Temefós , Tuberculose/tratamento farmacológicoRESUMO
(Mtb) produces inflections in the host signaling networks to create a favorable milieu for survival. The virulent Mtb strain, Rv caused double strand breaks (DSBs), whereas the non-virulent Ra strain triggered single-stranded DNA generation. The effectors secreted by SecA2 pathway were essential and adequate for the genesis of DSBs. Accumulation of DSBs mediated through Rv activates ATM-Chk2 pathway of DNA damage response (DDR) signaling, resulting in altered cell cycle. Instead of the classical ATM-Chk2 DDR, Mtb gains survival advantage through ATM-Akt signaling cascade. Notably, in vivo infection with Mtb led to sustained DSBs and ATM activation during chronic phase of tuberculosis. Addition of ATM inhibitor enhances isoniazid mediated Mtb clearance in macrophages as well as in murine infection model, suggesting its utility for host directed adjunct therapy. Collectively, data suggests that DSBs inflicted by SecA2 secretome of Mtb provides survival niche through activation of ATM kinase.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/metabolismo , Quebras de DNA de Cadeia Dupla , Interações Hospedeiro-Patógeno , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/patogenicidade , Transdução de Sinais , Adenosina Trifosfatases/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas de Bactérias/genética , Feminino , Humanos , Pulmão/efeitos dos fármacos , Pulmão/microbiologia , Masculino , Proteínas de Membrana Transportadoras/genética , Camundongos , Camundongos Endogâmicos BALB C , Morfolinas/administração & dosagem , Mycobacterium tuberculosis/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pironas/administração & dosagem , Células RAW 264.7 , Baço/efeitos dos fármacos , Baço/microbiologia , Células THP-1 , Tuberculose/microbiologiaRESUMO
Mycobacterium tuberculosis employs defense mechanisms to protect itself from reactive oxygen species (ROS)-mediated cytotoxicity inside macrophages. In the current study, we found that a secretory protein of M. tuberculosis PPE2 disrupted the assembly of NADPH oxidase complex. PPE2 inhibited NADPH oxidase-mediated ROS generation in RAW 264.7 macrophages and peritoneal macrophages from BALB/c mice. PPE2 interacted with the cytosolic subunit of NADPH oxidase, p67phox, and prevented translocation of p67phox and p47phox to the membrane, resulting in decreased NADPH oxidase activity. Trp236 residue present in the SH3-like domain of PPE2 was found to be critical for its interaction with p67phox Trp236Ala mutant of PPE2 did not interact with p67phox and thereby did not affect ROS generation. M. tuberculosis expressing PPE2 and PPE2-null mutants complemented with PPE2 survived better than PPE2-null mutants in infected RAW 264.7 macrophages. Altogether, this study suggests that PPE2 inhibits NADPH oxidase-mediated ROS production to favor M. tuberculosis survival in macrophages. The findings that M. tuberculosis PPE2 protein is involved in the modulation of oxidative response in macrophages will help us in improving our knowledge of host-pathogen interactions and the application of better therapeutics against tuberculosis.
Assuntos
Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/fisiologia , Fosfoproteínas/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Animais , Antígenos de Bactérias/química , Proteínas de Bactérias/química , Interações Hospedeiro-Patógeno , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Acetato de Tetradecanoilforbol/farmacologia , Domínios de Homologia de srcRESUMO
Extracytoplasmic function σ factors that are stress inducible are often sequestered in an inactive complex with a membrane-associated anti-σ factor. Mycobacterium tuberculosis membrane-associated anti-σ factors have a small, stable RNA gene A (ssrA)-like degron for targeted proteolysis. Interaction between the unfoldase, ClpX, and a substrate with an accessible degron initiates energy-dependent proteolysis. Four anti-σ factors with a mutation in the degron provided a set of natural substrates to evaluate the influence of the degron on degradation strength in ClpX-substrate processivity. We note that a point mutation in the degron (X-Ala-Ala) leads to an order-of-magnitude difference in the dwell time of the substrate on ClpX. Differences in ClpX/anti-σ interactions were correlated with changes in unfoldase activities. Green fluorescent protein (GFP) chimeras or polypeptides with a length identical to that of the anti-σ factor degron also demonstrate degron-dependent variation in ClpX activities. We show that degron-dependent ClpX activity leads to differences in anti-σ degradation, thereby regulating the release of free σ from the σ/anti-σ complex. M. tuberculosis ClpX activity thus influences changes in gene expression by modulating the cellular abundance of ECF σ factors.IMPORTANCE The ability of Mycobacterium tuberculosis to quickly adapt to changing environmental stimuli occurs by maintaining protein homeostasis. Extracytoplasmic function (ECF) σ factors play a significant role in coordinating the transcription profile to changes in environmental conditions. Release of the σ factor from the anti-σ is governed by the ClpXP2P1 assembly. M. tuberculosis ECF anti-σ factors have an ssrA-like degron for targeted degradation. A point mutation in the degron leads to differences in ClpX-mediated proteolysis and affects the cellular abundance of ECF σ factors. ClpX activity thus synchronizes changes in gene expression with environmental stimuli affecting M. tuberculosis physiology.
Assuntos
Endopeptidase Clp/metabolismo , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/metabolismo , Proteínas Repressoras/metabolismo , Fator sigma/metabolismo , Análise Mutacional de DNA , Mutação Puntual , Proteólise , Proteínas Repressoras/genéticaRESUMO
Identifying and characterizing the individual contributors to bacterial cellular elongation and division will improve our understanding of their impact on cell growth and division. Here, we delineated the role of ftsQ, a terminal gene of the highly conserved division cell wall (dcw) operon, in growth, survival, and cell length maintenance in the human pathogen Mycobacterium tuberculosis (Mtb). We found that FtsQ overexpression significantly increases the cell length and number of multiseptate cells. FtsQ depletion in Mtb resulted in cells that were shorter than WT cells during the initial growth stages (4 days after FtsQ depletion) but were longer than WT cells at later stages (10 days after FtsQ depletion) and compromised the survival in vitro and in differentiated THP1 macrophages. Overexpression of N- and C-terminal FtsQ regions altered the cell length, and the C-terminal domain alone complemented the FtsQ depletion phenotype. MS analyses suggested robust FtsQ phosphorylation on Thr-24, and although phosphoablative and -mimetic mutants rescued the FtsQ depletion-associated cell viability defects, they failed to complement the cell length defects. MS and coimmunoprecipitation experiments identified 63 FtsQ-interacting partners, and we show that the interaction of FtsQ with the recently identified cell division protein SepIVA is independent of FtsQ phosphorylation and suggests a role of FtsQ in modulating cell division. FtsQ exhibited predominantly septal localization in both the presence and absence of SepIVA. Our results suggest a role for FtsQ in modulating the length, division, and survival of Mtb cells both in vitro and in the host.
Assuntos
Proteínas de Bactérias/metabolismo , Divisão Celular , Macrófagos/citologia , Mycobacterium tuberculosis/fisiologia , Tuberculose/microbiologia , Proteínas de Bactérias/genética , Células Cultivadas , Humanos , Macrófagos/microbiologia , Mutação , Ligação ProteicaRESUMO
The cell wall of Mycobacterium tuberculosis (Mtb) is a complex structure that protects the pathogen in hostile environments. Peptidoglycan (PG), which helps determine the morphology of the cell envelope, undergoes substantial remodeling under stress. This meshwork of linear chains of sugars, cross-linked through attached peptides, is generated through the sequential action of enzymes termed transglycosylases and transpeptidases. The Mtb genome encodes two classical transglycosylases and four transpeptidases, the functions of which are not fully elucidated. Here, we present work on the yet uncharacterized transpeptidase PbpA and a nonclassical transglycosylase RodA. We elucidate their roles in regulating in vitro growth and in vivo survival of pathogenic mycobacteria. We find that RodA and PbpA are required for regulating cell length, but do not affect mycobacterial growth. Biochemical analyses show PbpA to be a classical transpeptidase, whereas RodA is identified to be a member of an emerging class of noncanonical transglycosylases. Phosphorylation of RodA at Thr-463 modulates its biological function. In a guinea pig infection model, RodA and PbpA are found to be required for both bacterial survival and formation of granuloma structures, thus underscoring the importance of these proteins in mediating mycobacterial virulence in the host. Our results emphasize the fact that whereas redundant enzymes probably compensate for the absence of RodA or PbpA during in vitro growth, the two proteins play critical roles for the survival of the pathogen inside its host.
Assuntos
Proteínas de Bactérias , Glicosiltransferases , Granuloma do Sistema Respiratório , Viabilidade Microbiana , Mycobacterium tuberculosis , Peptidil Transferases , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Feminino , Genoma Bacteriano , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Granuloma do Sistema Respiratório/enzimologia , Granuloma do Sistema Respiratório/genética , Granuloma do Sistema Respiratório/patologia , Cobaias , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Mycobacterium tuberculosis/patogenicidade , Peptidil Transferases/genética , Peptidil Transferases/metabolismo , Tuberculose/enzimologia , Tuberculose/genética , Tuberculose/patologiaRESUMO
Protein kinase G (PknG), a thioredoxin-fold-containing eukaryotic-like serine/threonine protein kinase, is a virulence factor in Mycobacterium tuberculosis, required for inhibition of phagolysosomal fusion. Here, we unraveled novel functional facets of PknG during latency-like conditions. We found that PknG mediates persistence under stressful conditions like hypoxia and abets drug tolerance. PknG mutant displayed minimal growth in nutrient-limited conditions, suggesting its role in modulating cellular metabolism. Intracellular metabolic profiling revealed that PknG is necessary for efficient metabolic adaptation during hypoxia. Notably, the PknG mutant exhibited a reductive shift in mycothiol redox potential and compromised stress response. Exposure to antibiotics and hypoxic environment resulted in higher oxidative shift in mycothiol redox potential of PknG mutant compared with the wild type. Persistence during latency-like conditions required kinase activity and thioredoxin motifs of PknG and is mediated through phosphorylation of a central metabolic regulator GarA. Finally, using a guinea pig model of infection, we assessed the in vivo role of PknG in manifestation of disease pathology and established a role for PknG in the formation of stable granuloma, hallmark structures of latent tuberculosis. Taken together, PknG-mediated GarA phosphorylation is important for maintenance of both mycobacterial physiology and redox poise, an axis that is dispensable for survival under normoxic conditions but is critical for non-replicating persistence of mycobacteria. In conclusion, we propose that PknG probably acts as a modulator of latency-associated signals.
Assuntos
Antígenos de Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Granuloma/etiologia , Tuberculose Latente/microbiologia , Mycobacterium tuberculosis/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Antibióticos Antituberculose/farmacologia , Antígenos de Bactérias/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Feminino , Deleção de Genes , Granuloma/metabolismo , Granuloma/microbiologia , Cobaias , Isoniazida/farmacologia , Cinética , Tuberculose Latente/metabolismo , Tuberculose Latente/fisiopatologia , Metabolômica/métodos , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/crescimento & desenvolvimento , Mycobacterium tuberculosis/fisiologia , Fosforilação/efeitos dos fármacos , Mutação Puntual , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Estresse FisiológicoRESUMO
Mycobacterium tuberculosis is known to modulate the host immune responses to facilitate its persistence inside the host cells. One of the key mechanisms includes repression of class-II transactivator (CIITA) and MHC-II expression in infected macrophages. However, the precise mechanism of CIITA and MHC-II down-regulation is not well studied. M. tuberculosis 6-kDa early secretory antigenic target (ESAT-6) is a known potent virulence and antigenic determinant. The M. tuberculosis genome encodes 23 such ESAT-6 family proteins. We herein report that M. tuberculosis and M. bovis bacillus Calmette-Guérin infection down-regulated the expression of CIITA/MHC-II by inducing hypermethylation in histone H3 lysine 9 (H3K9me2/3). Further, we showed that M. tuberculosis ESAT-6 family protein EsxL, encoded by Rv1198, is responsible for the down-regulation of CIITA/MHC-II by inducing H3K9me2/3. We further report that M. tuberculosis esxL induced the expression of nitric-oxide synthase, NO production, and p38 MAPK pathway, which in turn was responsible for the increased H3K9me2/3 in CIITA via up-regulation of euchromatic histone-lysine N-methyltransferase 2 (G9a). In contrast, inhibition of nitric-oxide synthase, p38 MAPK, and G9a abrogated H3K9me2/3, resulting in increased CIITA expression. A chromatin immunoprecipitation assay confirmed that hypermethylation at the promoter IV region of CIITA is mainly responsible for CIITA down-regulation and subsequent antigen presentation. We found that co-culture of macrophages infected with esxL-expressing M. smegmatis and mouse splenocytes led to down-regulation of IL-2, a key cytokine involved in T-cell proliferation. In summary, we demonstrate that M. tuberculosis EsxL inhibits antigen presentation by enhancing H3K9me2/3 at the CIITA promoter, thereby repressing its expression through NO and p38 MAPK activation.
Assuntos
Proteínas de Bactérias/fisiologia , Metilação de DNA , Macrófagos/metabolismo , Mycobacterium bovis/metabolismo , Mycobacterium tuberculosis/metabolismo , Proteínas Nucleares/genética , Transativadores/genética , Animais , Apresentação de Antígeno , Antígenos de Bactérias/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Genoma Bacteriano , Histonas/metabolismo , Humanos , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases , Camundongos , Mutação , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Células RAW 264.7 , Transdução de Sinais , Baço/citologia , Linfócitos T/citologia , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
A mammalian cell utilizes DNA methylation to modulate gene expression in response to environmental changes during development and differentiation. Aberrant DNA methylation changes as a correlate to diseased states like cancer, neurodegenerative conditions and cardiovascular diseases have been documented. Here we show genome-wide DNA methylation changes in macrophages infected with the pathogen M. tuberculosis. Majority of the affected genomic loci were hypermethylated in M. tuberculosis infected THP1 macrophages. Hotspots of differential DNA methylation were enriched in genes involved in immune response and chromatin reorganization. Importantly, DNA methylation changes were observed predominantly for cytosines present in non-CpG dinucleotide context. This observation was consistent with our previous finding that the mycobacterial DNA methyltransferase, Rv2966c, targets non-CpG dinucleotides in the host DNA during M. tuberculosis infection and reiterates the hypothesis that pathogenic bacteria use non-canonical epigenetic strategies during infection.
Assuntos
Proteínas de Bactérias/metabolismo , Metilação de DNA , Histonas/metabolismo , Mycobacterium tuberculosis/enzimologia , Tuberculose/genética , Mapeamento Cromossômico , Ilhas de CpG , Citosina/química , Epigênese Genética , Regulação da Expressão Gênica , Humanos , Imunidade , Células THP-1RESUMO
A major constituent of the nuclear basket region of the nuclear pore complex (NPC), nucleoporin Tpr, plays roles in regulating multiple important processes. We have previously established that Tpr is phosphorylated in both a MAP-kinase-dependent and MAP-kinase-independent manner, and that Tpr acts as both a substrate and as a scaffold for ERK2 (also known as MAPK1). Here, we report the identification of S2059 and S2094 as the major novel ERK-independent phosphorylation sites and T1677, S2020, S2023 and S2034 as additional ERK-independent phosphorylation sites found in the Tpr protein in vivo. Our results suggest that protein kinase A phosphorylates the S2094 residue and that the site is hyperphosphorylated during mitosis. Furthermore, we find that Tpr is phosphorylated at the S2059 residue by CDK1 and the phosphorylated form distinctly localizes with chromatin during telophase. Abrogation of S2059 phosphorylation abolishes the interaction of Tpr with Mad1, thus compromising the localization of both Mad1 and Mad2 proteins, resulting in cell cycle defects. The identification of novel phosphorylation sites on Tpr and the observations presented in this study allow better understanding of Tpr functions.
Assuntos
Mitose , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Motivos de Aminoácidos , Cromatina/genética , Cromatina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HeLa , Humanos , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Poro Nuclear/química , Poro Nuclear/genética , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fosforilação , Ligação Proteica , Transporte Proteico , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genéticaRESUMO
Nucleoporin Tpr is a component of the nuclear pore complex (NPC) that localizes exclusively to intranuclear filaments. Tpr functions as a scaffolding element in the nuclear phase of the NPC and plays a role in mitotic spindle checkpoint signalling. Export of intron-containing mRNA in Mason Pfizer Monkey Virus is regulated by direct interaction of cellular proteins with the cis-acting Constitutive Transport Element (CTE). In mammalian cells, the transport of Gag/Pol-CTE reporter construct is not very efficient, suggesting a regulatory mechanism to retain this unspliced RNA. Here we report that the knockdown of Tpr in mammalian cells leads to a drastic enhancement in the levels of Gag proteins (p24) in the cytoplasm, which is rescued by siRNA resistant Tpr. Tpr's role in the retention of unspliced RNA is independent of the functions of Sam68 and Tap/Nxf1 proteins, which are reported to promote CTE dependent export. Further, we investigated the possible role for nucleoporins that are known to function in nucleocytoplasmic transport in modulating unspliced RNA export. Results show that depletion of Nup153, a nucleoporin required for NPC anchoring of Tpr, plays a role in regulating the export, while depletion of other FG repeat-containing nucleoporins did not alter the unspliced RNA export. Results suggest that Tpr and Nup153 both regulate the export of unspliced RNA and they are most likely functioning through the same pathway. Importantly, we find that localization of Tpr to the NPC is necessary for Tpr mediated regulation of unspliced RNA export. Collectively, the data indicates that perinuclear localization of Tpr at the nucleopore complex is crucial for regulating intron containing mRNA export by directly or indirectly participating in the processing and degradation of aberrant mRNA transcripts.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Splicing de RNA , Transporte de RNA , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Sequência de Bases , Células COS , Chlorocebus aethiops , Proteínas de Ligação a DNA/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Proteína do Núcleo p24 do HIV/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Transporte Proteico , Interferência de RNA , Splicing de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Protein kinase G (PknG) in Mycobacterium tuberculosis has been shown to modulate phagosome-lysosome fusion. The protein has three distinct domains, an N-terminal Trx domain, a kinase domain, and a C-terminal TPR domain. The present study extensively analyzes the roles of these domains in regulating PknG kinase activity and function. We find that the kinase domain of PknG by itself is inactive, signifying the importance of the flanking domains. Although the deletion of the Trx domain severely impacts the activity of the protein, the C-terminal region also contributes significantly in regulating the activity of the kinase. Apart from this, PknG kinase activity is dependent on the presence of threonine 309 in the p + 1 loop of the activation segment. Mutating the conserved cysteine residues in the Trx motifs makes PknG refractory to changes in the redox environment. In vitro experiments identify threonine 63 as the major phosphorylation site of the protein. Importantly, we find that this is the only site in the protein that is phosphorylated in vivo. Macrophage infection studies reveal that the first 73 residues, the Trx motifs, and the threonine 63 residue are independently essential for modulating PknG-mediated survival of mycobacteria in its host. We have extended these studies to investigate the role of PknG and PknG mutants in the pathogenesis of mycobacteria in mice. Our results reinforce the findings from the macrophage infection experiments, and for the first time demonstrate that the expression of PknG in non-pathogenic mycobacteria allows the continued existence of these bacteria in host tissues.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/química , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas Quinases Dependentes de GMP Cíclico/genética , Regulação Bacteriana da Expressão Gênica , Lisossomos/metabolismo , Macrófagos/microbiologia , Camundongos , Dados de Sequência Molecular , Infecções por Mycobacterium não Tuberculosas , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/genética , Oxirredução , Fosforilação , Estrutura Terciária de Proteína , Deleção de SequênciaRESUMO
Monofunctional and bifunctional classes of Rel proteins catalyze pyrophosphoryl transfer from ATP to 3'-OH of GTP/GDP to synthesize (p)ppGpp, which is essential for normal microbial physiology and survival. Bifunctional proteins additionally catalyze the hydrolysis of (p)ppGpp. We have earlier demonstrated that although both catalyze identical the (p)ppGpp synthesis reaction, they exhibit a differential response to Mg(2+) due to a unique charge reversal in the synthesis domain; an RXKD motif in the synthesis domain of bifunctional protein is substituted by an EXDD motif in that of the monofunctional proteins. Here, we show that these motifs also determine substrate specificities (GTP/GDP), cooperativity, and regulation of catalytic activities at the N-terminal region through the C-terminal region. Most importantly, a mutant bifunctional Rel carrying an EXDD instigates a novel catalytic reaction, resulting in the synthesis of pGpp by an independent hydrolysis of the 5'P(alpha)-O-P(beta) bond of GTP/GDP or (p)ppGpp. Further experiments with RelA from Escherichia coli wherein EXDD is naturally present also revealed the presence of pGpp, albeit at low levels. This work brings out the biological significance of RXKD/EXDD motif conservation in Rel proteins and reveals an additional catalytic activity for the monofunctional proteins, prompting an extensive investigation for the possible existence and role of pGpp in the biological system.
Assuntos
Sequência Conservada , Ligases/química , Ligases/metabolismo , Motivos de Aminoácidos , Biocatálise , Difosfatos/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Ligases/genética , Dados de Sequência Molecular , Mycobacterium tuberculosis/enzimologia , Mycobacterium tuberculosis/genética , Nucleotídeos/metabolismo , Ligação Proteica , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Identifying direct substrates of mitogen-activated protein kinases (MAPKs) and understanding how those substrates are selected is central to understanding how these ubiquitously activated enzymes generate diverse biological responses. In previous work, we identified several new candidate substrates for the MAPK ERK2 (extracellular signal-regulated kinase 2), including the nuclear pore complex protein Tpr (translocated promoter region). In this report, we identify sites on Tpr for ERK2 phosphorylation and binding and demonstrate their functional interaction. ERK2 phosphorylation and dimerization are necessary for ERK2-Tpr binding, and this occurs through a DEF (docking site for ERK2, FXF) domain on Tpr. Surprisingly, the DEF domain and the phosphorylation sites displayed positive cooperativity to promote ERK2 binding to Tpr, in contrast to substrates where phosphorylation reduces binding. Ectopic expression or depletion of Tpr resulted in decreased movement of activated ERK2 from the cytoplasm to the nucleus, implying a role for Tpr in ERK2 translocation. Collectively, the data provide direct evidence that a component of the nuclear pore complex is a bona fide substrate of ERK2 in vivo and that activated ERK2 stably associates with this substrate after phosphorylation, where it could play a continuing role in nuclear pore function. We propose that Tpr is both a substrate and a scaffold for activated ERKs.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Humanos , MAP Quinase Quinase 1/genética , MAP Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/química , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Fosforilação , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Serina/metabolismo , Treonina/metabolismoRESUMO
A major regulatory mechanism evolved by microorganisms to combat stress is the regulation mediated by (p)ppGpp (the stringent response molecule), synthesized and hydrolyzed by Rel proteins. These are divided into bifunctional and monofunctional proteins based on the presence or absence of the hydrolysis activity. Although these proteins require Mg(2+) for (p)ppGpp synthesis, high Mg(2+) was shown to inhibit this reaction in bifunctional Rel proteins from Mycobacterium tuberculosis and Streptococcus equisimilis. This is not a characteristic feature in enzymes that use a dual metal ion mechanism, such as DNA polymerases that are known to carry out a similar pyrophosphate transfer reaction. Comparison of polymerase Polbeta and Rel(Seq) structures that share a common fold led to the proposal that the latter would follow a single metal ion mechanism. Surprisingly, in contrast to bifunctional Rel, we did not find inhibition of guanosine 5'-triphosphate, 3'-diphosphate (pppGpp) synthesis at higher Mg(2+) in the monofunctional RelA from Escherichia coli. We show that a charge reversal in a conserved motif in the synthesis domains explains this contrast; an RXKD motif in the bifunctional proteins is reversed to an EXDD motif. The differential response of these proteins to Mg(2+) could also be noticed in fluorescent nucleotide binding and circular dichroism experiments. In mutants where the motifs were reversed, the differential effect could also be reversed. We infer that although a catalytic Mg(2+) is common to both bifunctional and monofunctional proteins, the latter would utilize an additional metal binding site formed by EXDD. This work, for the first time, brings out differences in (p)ppGpp synthesis by the two classes of Rel proteins.