Host cholesterol modulates the generation and enrichment of persister population during Mycobacterium tuberculosis infection.
Ontology highlight
ABSTRACT:
INSTRUMENT(S): TripleTOF 5600
ORGANISM(S): Mycobacterium Tuberculosis M24
DISEASE(S): Tuberculosis
SUBMITTER: 
Renu Goel
LAB HEAD: Dr Amit Pandey
PROVIDER: PXD022028 | Pride | 2020-10-16
REPOSITORIES: Pride
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Project description:We previously reported that the utilization of host cholesterol is essential for mycobacterial persistence. In this study, we demonstrated that cholesterol-induced activation of a ribonuclease toxin (VapC12) inhibits translation by targeting proT tRNA in Mtb. The resulting cholesterol-specific growth modulation increases the frequency of the generation of persisters in a heterogeneous Mtb population. A vapC12-null mutant strain (ΔvapC12) failed to persist and demonstrated a hypervirulent phenotype in a guinea pig model of Mtb infection. This is the first study to identify a novel strategy through which cholesterol-specific activation of a toxin–antitoxin (TA) module in Mtb enhances persister formation during infection. In addition to identifying the mechanism, the study provides opportunity for targeting persisters, a new paradigm facilitating tuberculosis drug development.
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Project description:Cholesterol of the host macrophage membrane is vital for mycobacterial infection, replication, and persistence. During chronic infection within host lung tissues, cholesterol facilitates the phagocytosis of mycobacteria into macrophages. Cholesterol degradation leads to increased flux of acetyl-coenzyme A (CoA) and propionyl-CoA, providing energy and building blocks for virulence macromolecules as well as donors for global protein acylation. Potential functions of lysine acylation are gradually revealed in bacterial survival and pathogenesis. However, the mycobacterial proteome and posttranslational modification (PTM) changes involved in the cholesterol catabolism bioprocess remain unclear. Here, we used nonpathogenic Mycobacterium smegmatis as a model and simultaneously monitored mycobacterial proteome and acetylome changes in the presence of glucose and cholesterol. We discovered that cholesterol metabolic enzymes were upregulated with respect to both protein expression levels and lysine acylation levels during the metabolic shift from glucose to cholesterol. After that, adenylating enzymes related to cholesterol metabolism were proven to be precisely regulated at the propionylation level by mycobacterial acyltransferase M. smegmatis Kat (MsKat) in response to cellular propionyl-CoA accumulation. Furthermore, the kinase expression and phosphorylation levels were also changed along with fluctuations in cholesterol levels. Our results expanded current knowledge of acylation regulation in the cholesterol catabolism of mycobacteria and provided references for possible antimycobacterium strategy.IMPORTANCE Cholesterol assimilation is a critical step in mycobacterial chronic infection. However, knowledge from the dynamic characterization of cholesterol metabolism in mycobacteria at the protein expression and PTM levels remains limited. Our study uncovered the landscape of protein expression, lysine acetylation, lysine propionylation, and S/T/Y phosphorylation during the metabolic changes from glucose to cholesterol in mycobacteria. The data showed that cholesterol-induced carbon shift resulted in the elevation of protein expression and lysine acylation in diverse metabolic enzymes involved in cholesterol degradation and that the presence of cholesterol also promoted the perturbations at the phosphorylation level in the kinase system in mycobacteria. This study systematically characterized the regulation of cholesterol catabolism at several different levels, which provided the detailed references in mycobacterial proteome and potential antimycobacterial strategies.
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Project description:Histone acetylation and deacetylation are among the principal mechanisms by which chromatin is regulated during transcription, DNA silencing, and DNA repair. We analyzed patterns of genetic interactions uncovered during comprehensive genome-wide analyses in yeast to probe how histone acetyltransferase (HAT) and histone deacetylase (HDAC) protein complexes interact. The genetic interaction data unveil an underappreciated role of HDACs in maintaining cellular viability, and led us to show that deacetylation of the histone variant Htz1p at lysine 14 is mediated by Hda1p. Studies of the essential nucleosome acetyltransferase of H4 (NuA4) revealed acetylation-dependent protein stabilization of Yng2p, a potential nonhistone substrate of NuA4 and Rpd3C, and led to a new functional organization model for this critical complex. We also found that DNA double-stranded breaks (DSBs) result in local recruitment of the NuA4 complex, followed by an elaborate NuA4 remodeling process concomitant with Rpd3p recruitment and histone deacetylation. These new characterizations of the HDA and NuA4 complexes demonstrate how systematic analyses of genetic interactions may help illuminate the mechanisms of intricate cellular processes. Keywords: genetic modification The 44 datasets in this Series profiled the genome-wide genetic interactions for query genes encoding either HAT and HDAC catalytic subunits or subunits of the associated protein complexes. Of the 32 query genes, 5 were essential and were tested as temperature-sensitive (ts) alleles at three or more temperatures. (ESA1 was also tested as a hypomorphic allele.) The other query genes were tested as null deletion alleles derived from the Yeast Knockout strain collection.
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Project description:Bacterial persistence, the ability to survive antibiotic treatment by entering a physiologically dormant state, is a serious biomedical problem. Protein Ser/Thr kinase HipA, the first toxin connected to bacterial multidrug tolerance (persistence), exerts its function by phosphorylating glutamate--tRNA ligase GltX, leading to a halt in translation, accumulation of ppGpp and induction of persistence. Intriguingly, its variant HipA7 is able to induce significantly higher levels of persistence despite being less toxic for the cell. We postulated that this phenotypic difference may be driven by diverse substrate pools of the two kinase variants. To address this, we ectopically expressed hipA and hipA7 in E. coli and monitored their in vivo substrates during growth inhibition and resuscitation using SILAC-based quantitative phosphoproteomics. Our assays confirmed that both forms of the kinase phosphorylate GltX as the main substrate. Importantly, HipA phosphorylated several additional substrates involved in protein synthesis, transcription and replication, such as ribosomal protein L11 and SeqA. Conversely, HipA7 had a lower kinase activity, no additional substrates under tested conditions and showed a similar substrate pool only when expressed at significantly higher levels. The two forms of the kinase also differed in autophosphorylation level, which was significantly lower in HipA7. Initial testing of the novel HipA substrate L11 did not reveal a connection between HipA-induced phosphorylation and RelA-dependent persistence, opening the possibility that the novel HipA substrates are predominantly responsible for the toxic phenotype of the kinase. Our results contribute to understanding of HipA7 action and present a resource for studies of HipA-related persistence.
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Project description:A conserved aminopeptidase PepN, exhibits divergent traits in pathogenic and non- pathogenic mycobacteria
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Project description:Both genetic and environmental factors are implicated in Type 1 Diabetes (T1D). Since environmental factors can trigger epigenetic changes, we hypothesized that variations in histone posttranslational modifications (PTMs) at the promoter/enhancer regions of T1D susceptible genes may be associated with T1D. We therefore evaluated histone PTM variations at known T1D susceptible genes in blood cells from T1D patients versus healthy non-diabetic controls, and explored their connections to T1D. We used the chromatin-immunoprecipitation-linked-to-microarray approach to profile key histone PTMs, including H3-lysine-4 trimethylation (H3K4me3), H3K27me3, H3K9me3, H3K9 acetylation (H3K9Ac) and H4K16Ac at genes within the T1D susceptible loci in lymphocytes, and H3K4me3, H3K9me2, H3K9Ac and H4K16Ac at the IDDM1 region in monocytes of T1D patients and healthy controls separately. We screened for potential variations in histone PTMs using computational methods to compare datasets from T1D and controls. Interestingly, we observed marked variations in H3K9Ac levels at the upstream regions of HLA-DRB1 and HLA-DQB1 within the IDDM1 locus in T1D monocytes relative to controls. Additional experiments with THP-1 monocytes demonstrated increased expression of HLA-DRB1 and HLA-DQB1 in response to interferon- and TNF-treatment that were accompanied by changes in H3K9Ac at the same promoter regions as that seen in the patient monocytes. These results suggest that the H3K9Ac status of HLA-DRB1 and HLA-DQB1, two genes highly associated with T1D, may be relevant to their regulation and transcriptional response towards external stimuli. Thus, the promoter/enhancer architecture and chromatin status of key susceptible loci could be important determinants in their functional association to T1D susceptibility. We evaluated histone PTM variations at known T1D susceptible genes in blood cells from T1D patients versus healthy non-diabetic controls, and explored their connections to T1D. We used the ChIP-chip approach to profile key histone PTMs, including histone H3K4me3, H3K27me3, H3K9me3, H3K9 acetylation (H3K9Ac) and H4K16Ac, at genes within the T1D susceptible loci in lymphocytes.
2012-05-31 | E-GEOD-36402 | biostudies-arrayexpress
Project description:Persisters are cells which evade stresses like antibiotics and which are characterized by reduced metabolism and a lack of genetic alterations required to achieve this state. We showed previously that MqsR and MqsA of Escherichia coli are a toxin-antitoxin pair that influence cell physiology (e.g., biofilm formation and motility) via RNase activity as well as through regulation of toxin CspD. Here, we show that deletion of the mqsRA locus decreases persister cell formation and, consistent with this result, overexpression of MqsR increases persister cell formation. Furthermore, toxins Hha, CspD, and HokA increase persister cell formation. In addition, by overproducing MqsR in a series of isogenic mutants, we show that Hha and CspD are necessary for persister cell formation via MqsR overexpression. Surprisingly, Hfq, a small RNA chaperone, decreases persistence. A whole-transcriptome study shows that Hfq induces transport-related genes (oppA, oppB, oppC, oppD, oppF, and dppA), outer membrane protein-related genes (ybfM and ybfN), toxins (hha), and proteases (clpX, clpP, and lon). Taken together, these results indicate that toxins CspD and Hha influence persister cell formation via MqsR and that Hfq plays an important role in the regulation of persister cell formation via regulation of transport or outer membrane proteins. Strains: E. coli BW25113 K-12 hfq deleted mutant vs. wild-type Medium: LB Culture: Planktonic cell grown OD=0.5, adjusted OD=1.0, and then exposed to 100 ug/mL ampicillin for 2 h.
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Project description:BCG strain overexpressing the toxin (vapC12)–antitoxin (vapB12)-His(6X) locus was used for sample preparation. Log phase culture of the overexpressed strain was washed with PBS and inoculated in minimal media with 0.1% glycerol and minimal media with 0.01% cholesterol. The cultures were allowed to grow for 48 hrs and cell lysate was prepared. Immunoprecipitation was performed using an anti-His antibody (BTL1010, Biospecs).
In-solution digestion was carried out for 10 µg of proteins from each condition. The samples were subjected to reduction and alkylation using 5 mM dithiothreitol (DTT) (60°C for 45 minutes) and alkylation using 10 mM Iodoacetamide (IAA). Trypsin (Gold mass-spectrometry trypsin; Promega, Madison, WI) digestion was carried out at 37°C for 10-12 hrs. The peptides were vacuum-dried and stored at -80°C until LC-MS/MS analysis.
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Project description:Background: Histone post-translational modifications (PTMs) constitute a branch of epigenetic mechanisms that can control the expression of eukaryotic genes in a heritable manner. Recent studies have identified several PTM-binding proteins containing diverse specialized domains whose recognition of specific PTM sites leads to gene activation or repression. Here, we present a high-throughput proteogenomic platform designed to characterize the nucleosomal make-up of chromatin enriched with a set of histone PTM-binding proteins known as histone PTM readers. We support our findings with gene expression data correlating to PTM distribution. Results: We isolated human mononucleosomes bound by the bromodomain-containing proteins Brd2, Brd3 and Brd4, and by the chromodomain-containing heterochromatin proteins HP1alpha and HP1beta. Histone PTMs were quantified by mass spectrometry (ChIP-qMS), and their associated DNAs were mapped using deep sequencing. Our results reveal that Brd- and HP1-bound nucleosomes are enriched in histone PTMs consistent with actively transcribed euchromatin and silent heterochromatin, respectively. Data collected using RNA-Seq (GSM301568) show that Brd-bound sites correlate with highly expressed genes. In particular, Brd3 and Brd4 are most enriched on nucleosomes located within HOX gene clusters, whose expression is reduced upon Brd4 depletion by shRNA. Conclusions: Proteogenomic mapping of histone PTM readers, alongside the characterization of their local chromatin environments and transcriptional information, should prove useful for determining how histone PTMs are bound by these readers and how they contribute to distinct transcriptional states. Comparison of Brd2 and HP1b shRNA knockdown HEK293 cells to control knockdown HEK293 cells.
2012-07-30 | E-GEOD-39580 | biostudies-arrayexpress
Project description:Cholesterol synthesis is a tightly regulated process, both transcriptionally and post-translationally. Transcriptional control of cholesterol synthesis is relatively well understood. However, of the ~20 enzymes in cholesterol biosynthesis, post-translational regulation has only been examined for a small number. Three of the four sterol reductases, DHCR7, DHCR14 and LBR, share evolutionary ties with a high level of sequence homology and predicted structural homology. Despite their homology and that they uniquely share the same 14 reductase activity in cholesterol biosynthesis, little is known about the post-translational regulation of DHCR14 and LBR. Using CHO-7 cells stably expressing epitope tagged DHCR14 or LBR we investigated the post-translational regulation of these enzymes. We found that DHCR14 and LBR undergo differential post translational regulation, with DHCR14 being rapidly turned over, triggered by cholesterol and other sterol intermediates while LBR remained stable. DHCR14 is degraded via the ubiquitin-proteasome system and we identified several DHCR14 and DHCR7 putative interaction partners including the E3 ligase WWP2, which plays a role in the basal and cholesterol-mediated regulation of DHCR14. Interestingly, we found that gene expression across an array of human tissues showed that the C14-SRs gene expression is negatively related; one enzyme or the other tends to be predominately expressed in each tissue. Overall, our findings indicate that while LBR tends to be the constitutively active C14-SR, DHCR14 levels are tuneable, responding to the local cellular demands for cholesterol.
2020-01-15 | PXD016417 | Pride